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Thiele, Dennis James

Overview:


Stress genes in cellular homeostasis and disease

All organisms are exposed to stressful conditions including elevated temperatures, reactive oxygen species and other metabolites generated by normal biochemical reactions, rapid cellular proliferation, infection, inflammation, pharmacological agents or other pathophysiological states. These stressful conditions can lead to protein misfolding and aggregation, disruption of cellular signaling pathways, cellular dysfunction and cell death. Recent studies strongly suggest that the ability to sense and respond to stress signals, through the activation of signal transduction pathways, transcription factors and gene products that function in protein homeostasis is critical for normal growth, development and in the protection against diseases that include cancer, cardiovascular disease and protein folding diseases such as Alzheimer’s, Huntington’s and prion based disease. Furthermore, studies in model systems have established a strong correlation between longevity and the ability to mount robust stress responses for healthy immune systems, normal nutrient sensing and metabolism, protection from infectious disease and the prevention of cardiovascular disease.

Our laboratory explores how organisms sense stress and the mechanisms by which gene expression is activated by Heat Shock Transcription Factors (HSF), a family of central stress-responsive proteins conserved from yeast to humans. Furthermore, we have identified HSF target genes on a genome-wide scale and we are exploring the role these gene products play in protection from disease states such as cardiovascular disease, amyloidoses, prion disease and polyglutamine-based disease. Moreover, we are interested in how distinct stress-responsive pathways communicate. Understanding how HSF is activated, and the role HSF target genes play in cellular homeostasis, is of great importance in the treatment of cardiovascular, neurological and other diseases characterized by stress damage and aberrant protein folding.


Regulation of Copper and Iron acquisition in health and disease

The nutrients copper (Cu) and iron (Fe) serve as essential catalytic co-factors to drive biochemical reactions involved in oxygen transport, neuropeptide hormone maturation, DNA replication, oxidative phosphorylation, blood vessel formation, blood clotting, oxidative stress protection and a variety of other biological processes pivotal to normal growth and development. Our laboratory explores how organisms sense, acquire, distribute and utilize Cu and Fe for these essential processes, and we use yeast, fruit flies, mice and cultured human cells to understand these mechanisms. Given that Cu deficiency causes irreversible developmental and cognitive defects, and Fe deficiency anemia is thought to affect approximately 2 billion people around the globe, understanding how organisms establish homeostatic control of Cu and Fe are of great importance. We study two families of high affinity Cu transporters. The Ctr1 family functions in the delivery of Cu across cell membranes, and mouse knock out studies have established an essential role for Ctr1 in embryonic development and tissue-specific Cu-dependent functions. We are interested in the biochemical function and regulation of Ctr1, and the role of Ctr1 in metazoan development and disease. The Ctr2 family is localized to intracellular membranes and mobilizes intracellular Cu stores. We are exploring the function and mechanism of action of Ctr2, as well as the regulatory mechanisms by which the machinery for extracellular Cu uptake communicates and coordinates with intracellular Cu mobilization.

We have recently discovered a post-transcriptional regulatory process, controlled by Fe deficiency, which drives genome-wide metabolic reprogramming. This coordinated global metabolic reprogramming in response to Fe deficiency is accomplished by targeting specific mRNA molecules for degradation, thereby facilitating the utilization of limited cellular Fe levels. We are investigating the biochemical mechanisms by which specific mRNA molecules are targeted for degradation and the metabolic networks that are reprogrammed as a consequence of Fe deficiency.

Positions:

George Barth Geller Professor

Pharmacology & Cancer Biology
School of Medicine

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1983

Ph.D. — Rutgers University

News:

Grants:

Genetics Training Grant

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 1979
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Molecular Mycology and Pathogenesis Training Program

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
August 08, 2014
End Date
July 31, 2019

Sensing and Responding to Cellular Protein Misfolding

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2016
End Date
March 31, 2019

Duke University Program in Environmental Health

Administered By
Environmental Sciences and Policy
AwardedBy
National Institute of Environmental Health Sciences
Role
Mentor
Start Date
July 01, 2013
End Date
June 30, 2018

Metal Homeostasis in Yeast

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 16, 2003
End Date
March 31, 2018

Wilson's Disease Research Collaboration

Administered By
Pharmacology & Cancer Biology
AwardedBy
Alnylam Pharmaceuticals
Role
Principal Investigator
Start Date
January 11, 2017
End Date
January 10, 2018

Prevention of abnormal degradationof the neuronal protective factor HSF1 in Huntington Disease

Administered By
Pharmacology & Cancer Biology
AwardedBy
Huntington's Disease Society of America
Role
Principal Investigator
Start Date
January 01, 2017
End Date
December 31, 2017

Copper Homeostasis in Mammals

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2004
End Date
July 31, 2017

Development of Chemical Tools to Manipulate Copper at the Host/Pathogen Interface

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
June 01, 2008
End Date
May 31, 2017

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

HSF1 as a therapeutic target in neurodegenerative disease

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 15, 2010
End Date
January 31, 2016

High sensitivity multi-purpose electron paramagnetic resonance spectroscopy for biotechnological and biomedical research

Administered By
Biochemistry
AwardedBy
North Carolina Biotechnology Center
Role
Collaborating Investigator
Start Date
May 01, 2014
End Date
April 30, 2015

Automated detection of protein crystals in high-throughput crystallography experiments

Administered By
Duke Human Vaccine Institute
AwardedBy
North Carolina Biotechnology Center
Role
Major User
Start Date
April 01, 2014
End Date
April 30, 2015

Targeting copper homeostasis in the fungal pathogen, Cryptococcus neoformans

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2012
End Date
August 31, 2014

Identification of pharmacological chaperones for misfolded proteins

Administered By
Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
March 01, 2008
End Date
February 28, 2011

Molecular mechanisms of iron homeostasis in Saccharomyces cerevisiae

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2008
End Date
March 31, 2010

Heat Shock Transcription Factor Function and Regulation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2004
End Date
March 31, 2009

Stress regulation of RNA metabolism

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2006
End Date
December 31, 2008

Mechanism by which a single transcription factor responds to multiple distinct stressors

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
June 01, 2005
End Date
May 31, 2006
Show More

Publications:

Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain.

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition.

Authors
Öhrvik, H; Logeman, B; Turk, B; Reinheckel, T; Thiele, DJ
MLA Citation
Öhrvik, H, Logeman, B, Turk, B, Reinheckel, T, and Thiele, DJ. "Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain." The Journal of biological chemistry 291.27 (July 2016): 13905-13916.
PMID
27143361
Source
epmc
Published In
The Journal of biological chemistry
Volume
291
Issue
27
Publish Date
2016
Start Page
13905
End Page
13916
DOI
10.1074/jbc.m116.731281

Structures of HSF2 reveal mechanisms for differential regulation of human heat-shock factors.

Heat-shock transcription factor (HSF) family members function in stress protection and in human diseases including proteopathies, neurodegeneration and cancer. The mechanisms that drive distinct post-translational modifications, cofactor recruitment and target-gene activation for specific HSF paralogs are unknown. We present crystal structures of the human HSF2 DNA-binding domain (DBD) bound to DNA, revealing an unprecedented view of HSFs that provides insights into their unique biology. The HSF2 DBD structures resolve a new C-terminal helix that directs wrapping of the coiled-coil domain around DNA, thereby exposing paralog-specific sequences of the DBD surface for differential post-translational modifications and cofactor interactions. We further demonstrate a direct interaction between HSF1 and HSF2 through their coiled-coil domains. Together, these features provide a new model for HSF structure as the basis for differential and combinatorial regulation, which influences the transcriptional response to cellular stress.

Authors
Jaeger, AM; Pemble, CW; Sistonen, L; Thiele, DJ
MLA Citation
Jaeger, AM, Pemble, CW, Sistonen, L, and Thiele, DJ. "Structures of HSF2 reveal mechanisms for differential regulation of human heat-shock factors." Nature structural & molecular biology 23.2 (February 2016): 147-154.
PMID
26727490
Source
epmc
Published In
Nature Structural & Molecular Biology
Volume
23
Issue
2
Publish Date
2016
Start Page
147
End Page
154
DOI
10.1038/nsmb.3150

Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese.

Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.

Authors
Shawki, A; Anthony, SR; Nose, Y; Engevik, MA; Niespodzany, EJ; Barrientos, T; Öhrvik, H; Worrell, RT; Thiele, DJ; Mackenzie, B
MLA Citation
Shawki, A, Anthony, SR, Nose, Y, Engevik, MA, Niespodzany, EJ, Barrientos, T, Öhrvik, H, Worrell, RT, Thiele, DJ, and Mackenzie, B. "Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese." American journal of physiology. Gastrointestinal and liver physiology 309.8 (October 2015): G635-G647.
PMID
26294671
Source
epmc
Published In
American journal of physiology. Gastrointestinal and liver physiology
Volume
309
Issue
8
Publish Date
2015
Start Page
G635
End Page
G647
DOI
10.1152/ajpgi.00160.2015

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

Authors
Öhrvik, H; Logeman, B; Noguchi, G; Eriksson, I; Kjellén, L; Thiele, DJ; Pejler, G
MLA Citation
Öhrvik, H, Logeman, B, Noguchi, G, Eriksson, I, Kjellén, L, Thiele, DJ, and Pejler, G. "Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans." Journal of immunology (Baltimore, Md. : 1950) 195.8 (October 2015): 3654-3664.
PMID
26342034
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
195
Issue
8
Publish Date
2015
Start Page
3654
End Page
3664
DOI
10.4049/jimmunol.1500283

Copper at the Fungal Pathogen-Host Axis.

Fungal infections are responsible for millions of human deaths annually. Copper, an essential but toxic trace element, plays an important role at the host-pathogen axis during infection. In this review, we describe how the host uses either Cu compartmentalization within innate immune cells or Cu sequestration in other infected host niches such as in the brain to combat fungal infections. We explore Cu toxicity mechanisms and the Cu homeostasis machinery that fungal pathogens bring into play to succeed in establishing an infection. Finally, we address recent approaches that manipulate Cu-dependent processes at the host-pathogen axis for antifungal drug development.

Authors
García-Santamarina, S; Thiele, DJ
MLA Citation
García-Santamarina, S, and Thiele, DJ. "Copper at the Fungal Pathogen-Host Axis." The Journal of biological chemistry 290.31 (July 2015): 18945-18953. (Review)
PMID
26055724
Source
epmc
Published In
The Journal of biological chemistry
Volume
290
Issue
31
Publish Date
2015
Start Page
18945
End Page
18953
DOI
10.1074/jbc.r115.649129

Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer.

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. In vivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Authors
Allensworth, JL; Evans, MK; Bertucci, F; Aldrich, AJ; Festa, RA; Finetti, P; Ueno, NT; Safi, R; McDonnell, DP; Thiele, DJ; Van Laere, S; Devi, GR
MLA Citation
Allensworth, JL, Evans, MK, Bertucci, F, Aldrich, AJ, Festa, RA, Finetti, P, Ueno, NT, Safi, R, McDonnell, DP, Thiele, DJ, Van Laere, S, and Devi, GR. "Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer." Molecular oncology 9.6 (June 2015): 1155-1168.
PMID
25769405
Source
epmc
Published In
Molecular Oncology
Volume
9
Issue
6
Publish Date
2015
Start Page
1155
End Page
1168
DOI
10.1016/j.molonc.2015.02.007

Preface

Authors
Crumbliss, AL; Franz, KJ; Thiele, DJ
MLA Citation
Crumbliss, AL, Franz, KJ, and Thiele, DJ. "Preface." BioMetals 28.3 (June 2015): 431-431.
Source
crossref
Published In
BioMetals
Volume
28
Issue
3
Publish Date
2015
Start Page
431
End Page
431
DOI
10.1007/s10534-015-9854-8

Preface. Biometals 2014--Proceedings of the 9th International Symposium Biometals 2014 at Duke University, Durham, NC, USA.

Authors
Crumbliss, AL; Franz, KJ; Thiele, DJ
MLA Citation
Crumbliss, AL, Franz, KJ, and Thiele, DJ. "Preface. Biometals 2014--Proceedings of the 9th International Symposium Biometals 2014 at Duke University, Durham, NC, USA." Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 28.3 (June 2015): 431-.
PMID
25929460
Source
epmc
Published In
BioMetals
Volume
28
Issue
3
Publish Date
2015
Start Page
431
DOI
10.1007/s10534-015-9854-8

Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer

© 2015 Federation of European Biochemical Societies.Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both invitro and invivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. Invivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBCtumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Authors
Allensworth, JL; Evans, MK; Bertucci, F; Aldrich, AJ; Festa, RA; Finetti, P; Ueno, NT; Safi, R; McDonnell, DP; Thiele, DJ; Van Laere, S; Devi, GR
MLA Citation
Allensworth, JL, Evans, MK, Bertucci, F, Aldrich, AJ, Festa, RA, Finetti, P, Ueno, NT, Safi, R, McDonnell, DP, Thiele, DJ, Van Laere, S, and Devi, GR. "Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer." Molecular Oncology 9.6 (January 1, 2015): 1155-1168.
Source
scopus
Published In
Molecular Oncology
Volume
9
Issue
6
Publish Date
2015
Start Page
1155
End Page
1168
DOI
10.1016/j.molonc.2015.02.007

The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy.

Copper (Cu) is an essential metal for growth and development that has the potential to be toxic if levels accumulate beyond the ability of cells to homeostatically balance uptake with detoxification. One system for Cu acquisition is the integral membrane Cu(+) transporter, Ctr1, which has been quite well characterized in terms of its function and physiology. The mammalian Ctr2 protein has been a conundrum for the copper field, as it is structurally closely related to the high affinity Cu transporter Ctr1, sharing important motifs for Cu transport activity. However, in contrast to mammalian Ctr1, Ctr2 fails to suppress the Cu-dependent growth phenotype of yeast cells defective in Cu(+) import, nor does it appreciably stimulate Cu acquisition when over-expressed in mammalian cells, underscoring important functional dissimilarities between the two proteins. Several roles for the mammalian Ctr2 have been suggested both in vitro and in vivo. Here, we summarize and discuss current insights into the Ctr2 protein and its interaction with Ctr1, its functions in mammalian Cu homeostasis and platinum-based chemotherapy.

Authors
Öhrvik, H; Thiele, DJ
MLA Citation
Öhrvik, H, and Thiele, DJ. "The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy." Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS) 31 (January 2015): 178-182. (Review)
PMID
24703712
Source
epmc
Published In
Journal of Trace Elements in Medicine and Biology
Volume
31
Publish Date
2015
Start Page
178
End Page
182
DOI
10.1016/j.jtemb.2014.03.006

The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy

© 2014 Elsevier GmbH.Copper (Cu) is an essential metal for growth and development that has the potential to be toxic if levels accumulate beyond the ability of cells to homeostatically balance uptake with detoxification. One system for Cu acquisition is the integral membrane Cu<sup>+</sup> transporter, Ctr1, which has been quite well characterized in terms of its function and physiology. The mammalian Ctr2 protein has been a conundrum for the copper field, as it is structurally closely related to the high affinity Cu transporter Ctr1, sharing important motifs for Cu transport activity. However, in contrast to mammalian Ctr1, Ctr2 fails to suppress the Cu-dependent growth phenotype of yeast cells defective in Cu<sup>+</sup> import, nor does it appreciably stimulate Cu acquisition when over-expressed in mammalian cells, underscoring important functional dissimilarities between the two proteins. Several roles for the mammalian Ctr2 have been suggested both in vitro and in vivo. Here, we summarize and discuss current insights into the Ctr2 protein and its interaction with Ctr1, its functions in mammalian Cu homeostasis and platinum-based chemotherapy.

Authors
Öhrvik, H; Thiele, DJ
MLA Citation
Öhrvik, H, and Thiele, DJ. "The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy." Journal of Trace Elements in Medicine and Biology 31 (2015): 178-182.
Source
scival
Published In
Journal of Trace Elements in Medicine and Biology
Volume
31
Publish Date
2015
Start Page
178
End Page
182
DOI
10.1016/j.jtemb.2014.03.006

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70

©2014 Elsevier Ltd. All rights reserved.Summary Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

Authors
Howe, MK; Bodoor, K; Carlson, DA; Hughes, PF; Alwarawrah, Y; Loiselle, DR; Jaeger, AM; Darr, DB; Jordan, JL; Hunter, LM; Molzberger, ET; Gobillot, TA; Thiele, DJ; Brodsky, JL; Spector, NL; Haystead, TAJ
MLA Citation
Howe, MK, Bodoor, K, Carlson, DA, Hughes, PF, Alwarawrah, Y, Loiselle, DR, Jaeger, AM, Darr, DB, Jordan, JL, Hunter, LM, Molzberger, ET, Gobillot, TA, Thiele, DJ, Brodsky, JL, Spector, NL, and Haystead, TAJ. "Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70." Chemistry and Biology 21.12 (December 18, 2014): 1648-1659.
Source
scopus
Published In
Chemistry & Biology
Volume
21
Issue
12
Publish Date
2014
Start Page
1648
End Page
1659
DOI
10.1016/j.chembiol.2014.10.016

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

Authors
Howe, MK; Bodoor, K; Carlson, DA; Hughes, PF; Alwarawrah, Y; Loiselle, DR; Jaeger, AM; Darr, DB; Jordan, JL; Hunter, LM; Molzberger, ET; Gobillot, TA; Thiele, DJ; Brodsky, JL; Spector, NL; Haystead, TAJ
MLA Citation
Howe, MK, Bodoor, K, Carlson, DA, Hughes, PF, Alwarawrah, Y, Loiselle, DR, Jaeger, AM, Darr, DB, Jordan, JL, Hunter, LM, Molzberger, ET, Gobillot, TA, Thiele, DJ, Brodsky, JL, Spector, NL, and Haystead, TAJ. "Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70." Chemistry & biology 21.12 (December 11, 2014): 1648-1659.
PMID
25500222
Source
epmc
Published In
Chemistry & Biology
Volume
21
Issue
12
Publish Date
2014
Start Page
1648
End Page
1659
DOI
10.1016/j.chembiol.2014.10.016

A direct regulatory interaction between chaperonin TRiC and stress-responsive transcription factor HSF1.

Heat shock transcription factor 1 (HSF1) is an evolutionarily conserved transcription factor that protects cells from protein-misfolding-induced stress and apoptosis. The mechanisms by which cytosolic protein misfolding leads to HSF1 activation have not been elucidated. Here, we demonstrate that HSF1 is directly regulated by TRiC/CCT, a central ATP-dependent chaperonin complex that folds cytosolic proteins. A small-molecule activator of HSF1, HSF1A, protects cells from stress-induced apoptosis, binds TRiC subunits in vivo and in vitro, and inhibits TRiC activity without perturbation of ATP hydrolysis. Genetic inactivation or depletion of the TRiC complex results in human HSF1 activation, and HSF1A inhibits the direct interaction between purified TRiC and HSF1 in vitro. These results demonstrate a direct regulatory interaction between the cytosolic chaperone machine and a critical transcription factor that protects cells from proteotoxicity, providing a mechanistic basis for signaling perturbations in protein folding to a stress-protective transcription factor.

Authors
Neef, DW; Jaeger, AM; Gomez-Pastor, R; Willmund, F; Frydman, J; Thiele, DJ
MLA Citation
Neef, DW, Jaeger, AM, Gomez-Pastor, R, Willmund, F, Frydman, J, and Thiele, DJ. "A direct regulatory interaction between chaperonin TRiC and stress-responsive transcription factor HSF1." Cell reports 9.3 (November 2014): 955-966.
PMID
25437552
Source
epmc
Published In
Cell Reports
Volume
9
Issue
3
Publish Date
2014
Start Page
955
End Page
966
DOI
10.1016/j.celrep.2014.09.056

Genomic heat shock element sequences drive cooperative human heat shock factor 1 DNA binding and selectivity.

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.

Authors
Jaeger, AM; Makley, LN; Gestwicki, JE; Thiele, DJ
MLA Citation
Jaeger, AM, Makley, LN, Gestwicki, JE, and Thiele, DJ. "Genomic heat shock element sequences drive cooperative human heat shock factor 1 DNA binding and selectivity." The Journal of biological chemistry 289.44 (October 2014): 30459-30469.
PMID
25204655
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
44
Publish Date
2014
Start Page
30459
End Page
30469
DOI
10.1074/jbc.m114.591578

Exploiting innate immune cell activation of a copper-dependent antimicrobial agent during infection.

Recalcitrant microbial infections demand new therapeutic options. Here we present an approach that exploits two prongs of the host immune cell antimicrobial response: the oxidative burst and the compartmentalization of copper (Cu) within phagolysosomes. The prochelator QBP is a nontoxic protected form of 8-hydroxyquinoline (8HQ) in which a pinanediol boronic ester blocks metal ion coordination by 8HQ. QBP is deprotected via reactive oxygen species produced by activated macrophages, creating 8HQ and eliciting Cu-dependent killing of the fungal pathogen Cryptococcus neoformans in vitro and in mouse pulmonary infection. 8HQ ionophoric activity increases intracellular Cu, overwhelming the Cu-resistance mechanisms of C. neoformans to elicit fungal killing. The Cu-dependent antimicrobial activity of 8HQ against a spectrum of microbial pathogens suggests that this strategy may have broad utility. The conditional activation of Cu ionophores by innate immune cells intensifies the hostile antimicrobial environment and represents a promising approach to combat infectious disease.

Authors
Festa, RA; Helsel, ME; Franz, KJ; Thiele, DJ
MLA Citation
Festa, RA, Helsel, ME, Franz, KJ, and Thiele, DJ. "Exploiting innate immune cell activation of a copper-dependent antimicrobial agent during infection." Chemistry & biology 21.8 (August 2014): 977-987.
PMID
25088681
Source
epmc
Published In
Chemistry & Biology
Volume
21
Issue
8
Publish Date
2014
Start Page
977
End Page
987
DOI
10.1016/j.chembiol.2014.06.009

Copper is required for oncogenic BRAF signalling and tumorigenesis.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

Authors
Brady, DC; Crowe, MS; Turski, ML; Hobbs, GA; Yao, X; Chaikuad, A; Knapp, S; Xiao, K; Campbell, SL; Thiele, DJ; Counter, CM
MLA Citation
Brady, DC, Crowe, MS, Turski, ML, Hobbs, GA, Yao, X, Chaikuad, A, Knapp, S, Xiao, K, Campbell, SL, Thiele, DJ, and Counter, CM. "Copper is required for oncogenic BRAF signalling and tumorigenesis." Nature 509.7501 (May 2014): 492-496.
PMID
24717435
Source
epmc
Published In
Nature
Volume
509
Issue
7501
Publish Date
2014
Start Page
492
End Page
496
DOI
10.1038/nature13180

How copper traverses cellular membranes through the mammalian copper transporter 1, Ctr1.

The copper transporter 1, Ctr1, is part of a major pathway for cellular copper (Cu) uptake in the intestinal epithelium, in hepatic and cardiac tissue, and likely in many other mammalian cells and tissues. Here, we summarize what is currently known about how extracellular Cu travels across the plasma membrane to enter the cytoplasm for intracellular distribution and for use by proteins and enzymes, the physiological roles of Ctr1, and its regulation. As a critical Cu importer, Ctr1 occupies a strategic position to exert a strong modifying influence on diseases and pathophysiological states caused by imbalances in Cu homeostasis. A more thorough understanding of the mechanisms that regulate Ctr1 abundance, trafficking, and function will provide new insights and opportunities for disease therapies.

Authors
Ohrvik, H; Thiele, DJ
MLA Citation
Ohrvik, H, and Thiele, DJ. "How copper traverses cellular membranes through the mammalian copper transporter 1, Ctr1." Annals of the New York Academy of Sciences 1314 (May 2014): 32-41.
PMID
24697869
Source
epmc
Published In
Annals of the New York Academy of Sciences
Volume
1314
Publish Date
2014
Start Page
32
End Page
41
DOI
10.1111/nyas.12371

Introduction to Human Disorders of Copper Metabolism.

Authors
Graper, ML; Huster, D; Kaler, SG; Lutsenko, S; Schilsky, ML; Thiele, DJ
MLA Citation
Graper, ML, Huster, D, Kaler, SG, Lutsenko, S, Schilsky, ML, and Thiele, DJ. "Introduction to Human Disorders of Copper Metabolism." Annals of the New York Academy of Sciences 1314 (May 2014): v-vi.
PMID
24820197
Source
epmc
Published In
Annals of the New York Academy of Sciences
Volume
1314
Publish Date
2014
Start Page
v
End Page
vi
DOI
10.1111/nyas.12448

Innate immune cell activation of a copper-dependent anti-cryptococcal agent

Authors
Festa, RA; Helsel, ME; Franz, KJ; Thiele, DJ
MLA Citation
Festa, RA, Helsel, ME, Franz, KJ, and Thiele, DJ. "Innate immune cell activation of a copper-dependent anti-cryptococcal agent." MYCOSES 57 (May 2014): 56-57.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
56
End Page
57

Full characterization of the Cu-, Zn-, and Cd-binding properties of CnMT1 and CnMT2, two metallothioneins of the pathogenic fungus Cryptococcus neoformans acting as virulence factors.

We report here the full characterization of the metal binding abilities of CnMT1 and CnMT2, two Cryptococcus neoformans proteins recently identified as metallothioneins (MTs), which have been shown to play a crucial role in the virulence and pathogenicity of this human-infecting fungus. In this work, we first performed a thorough in silico study of the CnMT1 and CnMT2 genes, cDNAs and corresponding encoded products. Subsequently, the Zn(II)-, Cd(II)- and Cu(I) binding abilities of both proteins were fully determined through the analysis of the metal-to-protein stoichiometries and the structural features (determined by ESI-MS, CD, ICP-AES and UV-vis spectroscopies) of the corresponding recombinant Zn-, Cd- and Cu-MT preparations synthesized in metal-enriched media. Finally, the analysis of the Zn/Cd and Zn/Cu replacement processes of the respective Zn-MT complexes when allowed to react with Cd(II) or Cu(I) aqueous solutions was performed. Comprehensive consideration of all gathered results allows us to consider both isoforms as genuine copper-thioneins, and led to the identification of unprecedented Cu5-core clusters in MTs. CnMT1 and CnMT2 polypeptides appear to be evolutionarily related to the small fungal MTs, probably by ancient tandem-duplication events responding to a highly selective pressure to chelate copper, and far from the properties of Zn- and Cd-thioneins. Finally, we propose a modular structure of the Cu-CnMT1 and Cu-CnMT2 complexes on the basis of Cu5 clusters, concordantly with the modular structure of the sequence of CnMT1 and CnMT2, constituted by three and five Cys-rich units, respectively.

Authors
Palacios, Ò; Espart, A; Espín, J; Ding, C; Thiele, DJ; Atrian, S; Capdevila, M
MLA Citation
Palacios, Ò, Espart, A, Espín, J, Ding, C, Thiele, DJ, Atrian, S, and Capdevila, M. "Full characterization of the Cu-, Zn-, and Cd-binding properties of CnMT1 and CnMT2, two metallothioneins of the pathogenic fungus Cryptococcus neoformans acting as virulence factors." Metallomics : integrated biometal science 6.2 (February 2014): 279-291.
PMID
24317230
Source
epmc
Published In
Metallomics
Volume
6
Issue
2
Publish Date
2014
Start Page
279
End Page
291
DOI
10.1039/c3mt00266g

Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis.

Copper homeostasis is important for virulence of the fungus Cryptococcus neoformans, which can cause lethal meningoencephalitis in humans. Cryptococcus cells encounter high copper levels in the lung, where infection is initiated, and low copper levels in the brain. Here we demonstrate that two Cryptococcus copper transporters, Ctr1 and Ctr4, differentially influence fungal survival during pulmonary infection and the onset of meningoencephalitis. Protein Ctr1 is rapidly degraded under the high-copper conditions found in infected lungs, and its loss has no effect in fungal virulence in mice. By contrast, deleting CTR4 results in a hypervirulent phenotype. Overexpressing either Ctr1 or Ctr4 leads to profound reductions in fungal burden in the lung. However, during the onset of meningoencephalitis, expression of the copper transporters is induced and is critical for Cryptococcus virulence. Our work demonstrates that the fungal cells switch between copper detoxification and acquisition to address different copper stresses in the host.

Authors
Sun, T-S; Ju, X; Gao, H-L; Wang, T; Thiele, DJ; Li, J-Y; Wang, Z-Y; Ding, C
MLA Citation
Sun, T-S, Ju, X, Gao, H-L, Wang, T, Thiele, DJ, Li, J-Y, Wang, Z-Y, and Ding, C. "Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis." Nature communications 5 (January 2014): 5550-.
PMID
25417972
Source
epmc
Published In
Nature Communications
Volume
5
Publish Date
2014
Start Page
5550
DOI
10.1038/ncomms6550

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain.

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2(-/-) mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.

Authors
Öhrvik, H; Nose, Y; Wood, LK; Kim, B-E; Gleber, S-C; Ralle, M; Thiele, DJ
MLA Citation
Öhrvik, H, Nose, Y, Wood, LK, Kim, B-E, Gleber, S-C, Ralle, M, and Thiele, DJ. "Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain." Proc Natl Acad Sci U S A 110.46 (November 12, 2013): E4279-E4288.
PMID
24167251
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
46
Publish Date
2013
Start Page
E4279
End Page
E4288
DOI
10.1073/pnas.1311749110

Genetic selection for constitutively trimerized human HSF1 mutants identifies a role for coiled-coil motifs in DNA binding.

Human heat shock transcription factor 1 (HSF1) promotes the expression of stress-responsive genes and is a critical factor for the cellular protective response to proteotoxic and other stresses. In response to stress, HSF1 undergoes a transition from a repressed cytoplasmic monomer to a homotrimer, accumulates in the nucleus, binds DNA, and activates target gene transcription. Although these steps occur as sequential and highly regulated events, our understanding of the full details of the HSF1 activation pathway remains incomplete. Here we describe a genetic screen in humanized yeast that identifies constitutively trimerized HSF1 mutants. Surprisingly, constitutively trimerized HSF1 mutants do not bind to DNA in vivo in the absence of stress and only become DNA binding competent upon stress exposure, suggesting that an additional level of regulation beyond trimerization and nuclear localization may be required for HSF1 DNA binding. Furthermore, we identified a constitutively trimerized and nuclear-localized HSF1 mutant, HSF1 L189P, located in LZ3 of the HSF1 trimerization domain, which in response to proteotoxic stress is strongly compromised for DNA binding at the Hsp70 and Hsp25 promoters but readily binds to the interleukin-6 promoter, suggesting that HSF1 DNA binding is in part regulated in a locus-dependent manner, perhaps via promoter-specific differences in chromatin architecture. Furthermore, these results implicate the LZ3 region of the HSF1 trimerization domain in a function beyond its canonical role in HSF1 trimerization.

Authors
Neef, DW; Jaeger, AM; Thiele, DJ
MLA Citation
Neef, DW, Jaeger, AM, and Thiele, DJ. "Genetic selection for constitutively trimerized human HSF1 mutants identifies a role for coiled-coil motifs in DNA binding. (Published online)" G3 (Bethesda) 3.8 (August 7, 2013): 1315-1324.
PMID
23733891
Source
pubmed
Published In
G3 (Bethesda, Md.)
Volume
3
Issue
8
Publish Date
2013
Start Page
1315
End Page
1324
DOI
10.1534/g3.113.006692

The 62nd Annual Meeting of the Congress of Neurological Surgeons was held in Chicago, Illinois from October 6-10, 2012. Preface.

Authors
Grant, GA; Hankinson, T; Muh, C; Dumont, A
MLA Citation
Grant, GA, Hankinson, T, Muh, C, and Dumont, A. "The 62nd Annual Meeting of the Congress of Neurological Surgeons was held in Chicago, Illinois from October 6-10, 2012. Preface." Neurosurgery 60 Suppl 1 (August 2013): v-.
PMID
23839472
Source
epmc
Published In
Neurosurgery
Volume
60 Suppl 1
Publish Date
2013
Start Page
v
DOI
10.1227/neu.0000000000000006

Negative feedback regulation of the yeast CTH1 and CTH2 mRNA binding proteins is required for adaptation to iron deficiency and iron supplementation.

Iron (Fe) is an essential element for all eukaryotic organisms because it functions as a cofactor in a wide range of biochemical processes. Cells have developed sophisticated mechanisms to tightly control Fe utilization in response to alterations in cellular demands and bioavailability. In response to Fe deficiency, the yeast Saccharomyces cerevisiae activates transcription of the CTH1 and CTH2 genes, which encode proteins that bind to AU-rich elements (AREs) within the 3' untranslated regions (3'UTRs) of many mRNAs, leading to metabolic reprogramming of Fe-dependent pathways and decreased Fe storage. The precise mechanisms underlying Cth1 and Cth2 function and regulation are incompletely understood. We report here that the Cth1 and Cth2 proteins specifically bind in vivo to AREs located at the 3'UTRs of their own transcripts in an auto- and cross-regulated mechanism that limits their expression. By mutagenesis of the AREs within the CTH2 transcript, we demonstrate that a Cth2 negative-feedback loop is required for the efficient decline in Cth2 protein levels observed upon a rapid rise in Fe availability. Importantly, Cth2 autoregulation is critical for the appropriate recovery of Fe-dependent processes and resumption of growth in response to a change from Fe deficiency to Fe supplementation.

Authors
Martínez-Pastor, M; Vergara, SV; Puig, S; Thiele, DJ
MLA Citation
Martínez-Pastor, M, Vergara, SV, Puig, S, and Thiele, DJ. "Negative feedback regulation of the yeast CTH1 and CTH2 mRNA binding proteins is required for adaptation to iron deficiency and iron supplementation." Mol Cell Biol 33.11 (June 2013): 2178-2187.
PMID
23530061
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
33
Issue
11
Publish Date
2013
Start Page
2178
End Page
2187
DOI
10.1128/MCB.01458-12

Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence.

Copper (Cu) is an essential metal that is toxic at high concentrations. Thus, pathogens often rely on host Cu for growth, but host cells can hyperaccumulate Cu to exert antimicrobial effects. The human fungal pathogen Cryptococcus neoformans encodes many Cu-responsive genes, but their role in infection is unclear. We determined that pulmonary C. neoformans infection results in Cu-specific induction of genes encoding the Cu-detoxifying metallothionein (Cmt) proteins. Mutant strains lacking CMTs or expressing Cmt variants defective in Cu-coordination exhibit severely attenuated virulence and reduced pulmonary colonization. Consistent with the upregulation of Cmt proteins, C. neoformans pulmonary infection results in increased serum Cu concentrations and increases and decreases alveolar macrophage expression of the Cu importer (Ctr1) and ATP7A, a transporter implicated in phagosomal Cu compartmentalization, respectively. These studies indicate that the host mobilizes Cu as an innate antifungal defense but C. neoformans senses and neutralizes toxic Cu to promote infection.

Authors
Ding, C; Festa, RA; Chen, Y-L; Espart, A; Palacios, Ò; Espín, J; Capdevila, M; Atrian, S; Heitman, J; Thiele, DJ
MLA Citation
Ding, C, Festa, RA, Chen, Y-L, Espart, A, Palacios, Ò, Espín, J, Capdevila, M, Atrian, S, Heitman, J, and Thiele, DJ. "Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence." Cell Host Microbe 13.3 (March 13, 2013): 265-276.
PMID
23498952
Source
pubmed
Published In
Cell Host and Microbe
Volume
13
Issue
3
Publish Date
2013
Start Page
265
End Page
276
DOI
10.1016/j.chom.2013.02.002

Yeast protective response to arsenate involves the repression of the high affinity iron uptake system

Arsenic is a double-edge sword. On the one hand it is powerful carcinogen and on the other it is used therapeutically to treat acute promyelocytic leukemia. Here we report that arsenic activates the iron responsive transcription factor, Aft1, as a consequence of a defective high-affinity iron uptake mediated by Fet3 and Ftr1, whose mRNAs are drastically decreased upon arsenic exposure. Moreover, arsenic causes the internalization and degradation of Fet3. Most importantly, fet3ftr1 mutant exhibits increased arsenic resistance and decreased arsenic accumulation over the wild-type suggesting that Fet3 plays a role in arsenic toxicity. Finally we provide data suggesting that arsenic also disrupts iron uptake in mammals and the link between Fet3, arsenic and iron, can be relevant to clinical applications. © 2013 Elsevier B.V.

Authors
Batista-Nascimento, L; Toledano, MB; Thiele, DJ; Rodrigues-Pousada, C
MLA Citation
Batista-Nascimento, L, Toledano, MB, Thiele, DJ, and Rodrigues-Pousada, C. "Yeast protective response to arsenate involves the repression of the high affinity iron uptake system." Biochimica et Biophysica Acta - Molecular Cell Research 1833.5 (2013): 997-1005.
PMID
23295455
Source
scival
Published In
BBA - Molecular Cell Research
Volume
1833
Issue
5
Publish Date
2013
Start Page
997
End Page
1005
DOI
10.1016/j.bbamcr.2012.12.018

Copper at the front line of the host-pathogen battle.

Authors
Festa, RA; Thiele, DJ
MLA Citation
Festa, RA, and Thiele, DJ. "Copper at the front line of the host-pathogen battle." PLoS Pathog 8.9 (September 2012): e1002887-.
PMID
23028306
Source
pubmed
Published In
PLoS pathogens
Volume
8
Issue
9
Publish Date
2012
Start Page
e1002887
DOI
10.1371/journal.ppat.1002887

Charting the travels of copper in eukaryotes from yeast to mammals.

Throughout evolution, all organisms have harnessed the redox properties of copper (Cu) and iron (Fe) as a cofactor or structural determinant of proteins that perform critical functions in biology. At its most sobering stance to Earth's biome, Cu biochemistry allows photosynthetic organisms to harness solar energy and convert it into the organic energy that sustains the existence of all nonphotosynthetic life forms. The conversion of organic energy, in the form of nutrients that include carbohydrates, amino acids and fatty acids, is subsequently released during cellular respiration, itself a Cu-dependent process, and stored as ATP that is used to drive a myriad of critical biological processes such as enzyme-catalyzed biosynthetic processes, transport of cargo around cells and across membranes, and protein degradation. The life-supporting properties of Cu incur a significant challenge to cells that must not only exquisitely balance intracellular Cu concentrations, but also chaperone this redox-active metal from its point of cellular entry to its ultimate destination so as to avert the potential for inappropriate biochemical interactions or generation of damaging reactive oxidative species (ROS). In this review we chart the travels of Cu from the extracellular milieu of fungal and mammalian cells, its path within the cytosol as inferred by the proteins and ligands that escort and deliver Cu to intracellular organelles and protein targets, and its journey throughout the body of mammals. This article is part of a Special Issue entitled: Cell Biology of Metals.

Authors
Nevitt, T; Ohrvik, H; Thiele, DJ
MLA Citation
Nevitt, T, Ohrvik, H, and Thiele, DJ. "Charting the travels of copper in eukaryotes from yeast to mammals." Biochim Biophys Acta 1823.9 (September 2012): 1580-1593. (Review)
PMID
22387373
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1823
Issue
9
Publish Date
2012
Start Page
1580
End Page
1593
DOI
10.1016/j.bbamcr.2012.02.011

A novel role for copper in Ras/mitogen-activated protein kinase signaling.

Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.

Authors
Turski, ML; Brady, DC; Kim, HJ; Kim, B-E; Nose, Y; Counter, CM; Winge, DR; Thiele, DJ
MLA Citation
Turski, ML, Brady, DC, Kim, HJ, Kim, B-E, Nose, Y, Counter, CM, Winge, DR, and Thiele, DJ. "A novel role for copper in Ras/mitogen-activated protein kinase signaling." Mol Cell Biol 32.7 (April 2012): 1284-1295.
PMID
22290441
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
32
Issue
7
Publish Date
2012
Start Page
1284
End Page
1295
DOI
10.1128/MCB.05722-11

Copper in microbial pathogenesis: Meddling with the metal

Transition metals such as iron, zinc, copper, and manganese are essential for the growth and development of organisms ranging from bacteria to mammals. Numerous studies have focused on the impact of iron availability during bacterial and fungal infections, and increasing evidence suggests that copper is also involved in microbial pathogenesis. Not only is copper an essential cofactor for specific microbial enzymes, but several recent studies also strongly suggest that copper is used to restrict pathogen growth in vivo. Here, we review evidence that animals use copper as an antimicrobial weapon and that, in turn, microbes have developed mechanisms to counteract the toxic effects of copper. © 2012 Elsevier Inc.

Authors
Samanovic, MI; Ding, C; Thiele, DJ; Darwin, KH
MLA Citation
Samanovic, MI, Ding, C, Thiele, DJ, and Darwin, KH. "Copper in microbial pathogenesis: Meddling with the metal." Cell Host and Microbe 11.2 (2012): 106-115.
PMID
22341460
Source
scival
Published In
Cell Host & Microbe
Volume
11
Issue
2
Publish Date
2012
Start Page
106
End Page
115
DOI
10.1016/j.chom.2012.01.009

Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases.

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and prion-based neurodegeneration are associated with the accumulation of misfolded proteins, resulting in neuronal dysfunction and cell death. However, current treatments for these diseases predominantly address disease symptoms, rather than the underlying protein misfolding and cell death, and are not able to halt or reverse the degenerative process. Studies in cell culture, fruitfly, worm and mouse models of protein misfolding-based neurodegenerative diseases indicate that enhancing the protein-folding capacity of cells, via elevated expression of chaperone proteins, has therapeutic potential. Here, we review advances in strategies to harness the power of the natural cellular protein-folding machinery through pharmacological activation of heat shock transcription factor 1--the master activator of chaperone protein gene expression--to treat neurodegenerative diseases.

Authors
Neef, DW; Jaeger, AM; Thiele, DJ
MLA Citation
Neef, DW, Jaeger, AM, and Thiele, DJ. "Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases. (Published online)" Nat Rev Drug Discov 10.12 (December 1, 2011): 930-944. (Review)
PMID
22129991
Source
pubmed
Published In
Nature Reviews Drug Discovery
Volume
10
Issue
12
Publish Date
2011
Start Page
930
End Page
944
DOI
10.1038/nrd3453

Copper: an essential metal in biology.

Authors
Festa, RA; Thiele, DJ
MLA Citation
Festa, RA, and Thiele, DJ. "Copper: an essential metal in biology." Curr Biol 21.21 (November 8, 2011): R877-R883.
PMID
22075424
Source
pubmed
Published In
Current Biology
Volume
21
Issue
21
Publish Date
2011
Start Page
R877
End Page
R883
DOI
10.1016/j.cub.2011.09.040

The copper regulon of the human fungal pathogen Cryptococcus neoformans H99.

Cryptococcus neoformans is a human fungal pathogen that is the causative agent of cryptococcosis and fatal meningitis in immuno-compromised hosts. Recent studies suggest that copper (Cu) acquisition plays an important role in C. neoformans virulence, as mutants that lack Cuf1, which activates the Ctr4 high affinity Cu importer, are hypo-virulent in mouse models. To understand the constellation of Cu-responsive genes in C. neoformans and how their expression might contribute to virulence, we determined the transcript profile of C. neoformans in response to elevated Cu or Cu deficiency. We identified two metallothionein genes (CMT1 and CMT2), encoding cysteine-rich Cu binding and detoxifying proteins, whose expression is dramatically elevated in response to excess Cu. We identified a new C. neoformans Cu transporter, CnCtr1, that is induced by Cu deficiency and is distinct from CnCtr4 and which shows significant phylogenetic relationship to Ctr1 from other fungi. Surprisingly, in contrast to other fungi, we found that induction of both CnCTR1 and CnCTR4 expression under Cu limitation, and CMT1 and CMT2 in response to Cu excess, are dependent on the CnCuf1 Cu metalloregulatory transcription factor. These studies set the stage for the evaluation of the specific Cuf1 target genes required for virulence in C. neoformans.

Authors
Ding, C; Yin, J; Tovar, EMM; Fitzpatrick, DA; Higgins, DG; Thiele, DJ
MLA Citation
Ding, C, Yin, J, Tovar, EMM, Fitzpatrick, DA, Higgins, DG, and Thiele, DJ. "The copper regulon of the human fungal pathogen Cryptococcus neoformans H99." Mol Microbiol 81.6 (September 2011): 1560-1576.
PMID
21819456
Source
pubmed
Published In
Molecular Microbiology
Volume
81
Issue
6
Publish Date
2011
Start Page
1560
End Page
1576
DOI
10.1111/j.1365-2958.2011.07794.x

Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1.

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

Authors
Haas, KL; Putterman, AB; White, DR; Thiele, DJ; Franz, KJ
MLA Citation
Haas, KL, Putterman, AB, White, DR, Thiele, DJ, and Franz, KJ. "Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1." J Am Chem Soc 133.12 (March 30, 2011): 4427-4437.
PMID
21375246
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
133
Issue
12
Publish Date
2011
Start Page
4427
End Page
4437
DOI
10.1021/ja108890c

Host iron withholding demands siderophore utilization for Candida glabrata to survive macrophage killing.

The fungal pathogen Candida glabrata has risen from an innocuous commensal to a major human pathogen that causes life-threatening infections with an associated mortality rate of up to 50%. The dramatic rise in the number of immunocompromised individuals from HIV infection, tuberculosis, and as a result of immunosuppressive regimens in cancer treatment and transplant interventions have created a new and hitherto unchartered niche for the proliferation of C. glabrata. Iron acquisition is a known microbial virulence determinant and human diseases of iron overload have been found to correlate with increased bacterial burden. Given that more than 2 billion people worldwide suffer from iron deficiency and that iron overload is one of the most common single-gene inherited diseases, it is important to understand whether host iron status may influence C. glabrata infectious disease progression. Here we identify Sit1 as the sole siderophore-iron transporter in C. glabrata and demonstrate that siderophore-mediated iron acquisition is critical for enhancing C. glabrata survival to the microbicidal activities of macrophages. Within the Sit1 transporter, we identify a conserved extracellular SIderophore Transporter Domain (SITD) that is critical for siderophore-mediated ability of C. glabrata to resist macrophage killing. Using macrophage models of human iron overload disease, we demonstrate that C. glabrata senses altered iron levels within the phagosomal compartment. Moreover, Sit1 functions as a determinant for C. glabrata to survive macrophage killing in a manner that is dependent on macrophage iron status. These studies suggest that host iron status is a modifier of infectious disease that modulates the dependence on distinct mechanisms of microbial Fe acquisition.

Authors
Nevitt, T; Thiele, DJ
MLA Citation
Nevitt, T, and Thiele, DJ. "Host iron withholding demands siderophore utilization for Candida glabrata to survive macrophage killing." PLoS Pathog 7.3 (March 2011): e1001322-.
PMID
21445236
Source
pubmed
Published In
PLoS pathogens
Volume
7
Issue
3
Publish Date
2011
Start Page
e1001322
DOI
10.1371/journal.ppat.1001322

Early recruitment of AU-rich element-containing mRNAs determines their cytosolic fate during iron deficiency.

The yeast Cth2 protein is a CX(8)CX(5)CX(3)H tandem zinc finger protein that binds AU-rich element (ARE)-containing transcripts to enhance their decay in response to iron (Fe) deficiency. Mammalian members of this family of proteins are known to undergo nucleocytoplasmic shuttling, but little is known about the role of shuttling in the mechanism of ARE-dependent mRNA decay. Here we demonstrate that, like its mammalian homologues, Cth2 is a nucleocytoplasmic shuttling protein whose nuclear export depends on mRNA transport to the cytosol. The nuclear import information of Cth2 is contained within its tandem zinc finger domain, but it is independent of mRNA-binding function. Moreover, we also demonstrate that nucleocytoplasmic shuttling of Cth2 requires active transcription and that disruption of shuttling leads to defects in Cth2 function in mRNA decay under Fe deficiency. Taken together, our data suggest that under conditions of Fe deficiency Cth2 travels into the nucleus to recruit target mRNAs, perhaps cotranscriptionally, that are destined for cytosolic degradation as part of the mechanism of adaptation to growth under Fe limitation. These data also suggest an important role for nucleocytoplasmic shuttling in this conserved family of proteins in the mechanism of ARE-mediated mRNA decay.

Authors
Vergara, SV; Puig, S; Thiele, DJ
MLA Citation
Vergara, SV, Puig, S, and Thiele, DJ. "Early recruitment of AU-rich element-containing mRNAs determines their cytosolic fate during iron deficiency." Mol Cell Biol 31.3 (February 2011): 417-429.
PMID
21135132
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
31
Issue
3
Publish Date
2011
Start Page
417
End Page
429
DOI
10.1128/MCB.00754-10

Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions

The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4-Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X 3-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4-Ctr5 complex, whereas the Met-X 3- Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface. © 2011 SGM.

Authors
Beaudoin, J; Thiele, DJ; Labbé, S; Puig, S
MLA Citation
Beaudoin, J, Thiele, DJ, Labbé, S, and Puig, S. "Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions." Microbiology 157.4 (2011): 1021-1031.
PMID
21273250
Source
scival
Published In
Microbiology (Reading, England)
Volume
157
Issue
4
Publish Date
2011
Start Page
1021
End Page
1031
DOI
10.1099/mic.0.046854-0

Deciphering human heat shock transcription factor 1 regulation via post-translational modification in yeast

Heat shock transcription factor 1 (HSF1) plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE) are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAPkinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications. © 2011 Batista-Nascimento et al.

Authors
Batista-Nascimento, L; Neef, DW; Liu, PCC; Rodrigues-Pousada, C; Thiele, DJ
MLA Citation
Batista-Nascimento, L, Neef, DW, Liu, PCC, Rodrigues-Pousada, C, and Thiele, DJ. "Deciphering human heat shock transcription factor 1 regulation via post-translational modification in yeast." PLoS ONE 6.1 (2011).
PMID
21253609
Source
scival
Published In
PloS one
Volume
6
Issue
1
Publish Date
2011
DOI
10.1371/journal.pone.0015976

Ctr1 is an apical copper transporter in mammalian intestinal epithelial cells in vivo that is controlled at the level of protein stability.

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu(+) importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.

Authors
Nose, Y; Wood, LK; Kim, B-E; Prohaska, JR; Fry, RS; Spears, JW; Thiele, DJ
MLA Citation
Nose, Y, Wood, LK, Kim, B-E, Prohaska, JR, Fry, RS, Spears, JW, and Thiele, DJ. "Ctr1 is an apical copper transporter in mammalian intestinal epithelial cells in vivo that is controlled at the level of protein stability." J Biol Chem 285.42 (October 15, 2010): 32385-32392.
PMID
20699218
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
42
Publish Date
2010
Start Page
32385
End Page
32392
DOI
10.1074/jbc.M110.143826

Cardiac copper deficiency activates a systemic signaling mechanism that communicates with the copper acquisition and storage organs.

Copper (Cu) is an essential cofactor for a variety of metabolic functions, and the regulation of systemic Cu metabolism is critical to human health. Dietary Cu is absorbed through the intestine, stored in the liver, and mobilized into the circulation; however, systemic Cu homeostasis is poorly understood. We generated mice with a cardiac-specific knockout of the Ctr1 Cu transporter (Ctr1(hrt/hrt)), resulting in cardiac Cu deficiency and severe cardiomyopathy. Unexpectedly, Ctr1(hrt/hrt) mice exhibited increased serum Cu levels and a concomitant decrease in hepatic Cu stores. Expression of the ATP7A Cu exporter, thought to function predominantly in intestinal Cu acquisition, was strongly increased in liver and intestine of Ctr1(hrt/hrt) mice. These studies identify ATP7A as a candidate for hepatic Cu mobilization in response to peripheral tissue demand, and illuminate a systemic regulation in which the Cu status of the heart is signaled to organs that take up and store Cu.

Authors
Kim, B-E; Turski, ML; Nose, Y; Casad, M; Rockman, HA; Thiele, DJ
MLA Citation
Kim, B-E, Turski, ML, Nose, Y, Casad, M, Rockman, HA, and Thiele, DJ. "Cardiac copper deficiency activates a systemic signaling mechanism that communicates with the copper acquisition and storage organs." Cell Metab 11.5 (May 5, 2010): 353-363.
PMID
20444417
Source
pubmed
Published In
Cell Metabolism
Volume
11
Issue
5
Publish Date
2010
Start Page
353
End Page
363
DOI
10.1016/j.cmet.2010.04.003

Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease.

Neurodegenerative diseases such as Huntington disease are devastating disorders with no therapeutic approaches to ameliorate the underlying protein misfolding defect inherent to poly-glutamine (polyQ) proteins. Given the mounting evidence that elevated levels of protein chaperones suppress polyQ protein misfolding, the master regulator of protein chaperone gene transcription, HSF1, is an attractive target for small molecule intervention. We describe a humanized yeast-based high-throughput screen to identify small molecule activators of human HSF1. This screen is insensitive to previously characterized activators of the heat shock response that have undesirable proteotoxic activity or that inhibit Hsp90, the central chaperone for cellular signaling and proliferation. A molecule identified in this screen, HSF1A, is structurally distinct from other characterized small molecule human HSF1 activators, activates HSF1 in mammalian and fly cells, elevates protein chaperone expression, ameliorates protein misfolding and cell death in polyQ-expressing neuronal precursor cells and protects against cytotoxicity in a fly model of polyQ-mediated neurodegeneration. In addition, we show that HSF1A interacts with components of the TRiC/CCT complex, suggesting a potentially novel regulatory role for this complex in modulating HSF1 activity. These studies describe a novel approach for the identification of new classes of pharmacological interventions for protein misfolding that underlies devastating neurodegenerative disease.

Authors
Neef, DW; Turski, ML; Thiele, DJ
MLA Citation
Neef, DW, Turski, ML, and Thiele, DJ. "Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease. (Published online)" PLoS Biol 8.1 (January 19, 2010): e1000291-.
Website
http://hdl.handle.net/10161/4442
PMID
20098725
Source
pubmed
Published In
PLoS biology
Volume
8
Issue
1
Publish Date
2010
Start Page
e1000291
DOI
10.1371/journal.pbio.1000291

The Drosophila copper transporter Ctr1C functions in male fertility

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Steiger, D; Fetchko, M; Vardanyan, A; Atanesyan, L; Steiner, K; Turski, ML; Thiele, DJ; Georgiev, O; Schaffner, W
MLA Citation
Steiger, D, Fetchko, M, Vardanyan, A, Atanesyan, L, Steiner, K, Turski, ML, Thiele, DJ, Georgiev, O, and Schaffner, W. "The Drosophila copper transporter Ctr1C functions in male fertility." Journal of Biological Chemistry 285.22 (2010): 17089-17097.
PMID
20351114
Source
scival
Published In
The Journal of biological chemistry
Volume
285
Issue
22
Publish Date
2010
Start Page
17089
End Page
17097
DOI
10.1074/jbc.M109.090282

Copper Metabolism and the Liver

Copper plays a critical biochemical role in the function of many enzymes and proteins that contain this essential element. Consequently, copper deficiency leads to loss of the catalytic function of copper-dependent enzymes and structural changes in proteins with prosthetic copper, while excess metal is toxic and leads to cell injury and death. Homeostatic control mechanisms have evolved to coordinate a healthy balance for copper, allowing cells to accumulate sufficient copper to support essential biochemical reactions and yet prevent toxicity due to an excess of this metal. Much of our knowledge of these metabolic pathways is derived from the study of the inherited metabolic disorders of copper metabolism and from microbial and mouse models that have deciphered new copper transporters, metallochaperones, and signaling pathways. This in turn has led to a better understanding of the pathophysiology of each of the metabolic disorders of copper metabolism, aiding in diagnosis and treatment and in the design of future therapies. © 2009, John Wiley & Sons Ltd.

Authors
Schilsky, ML; Thiele, DJ
MLA Citation
Schilsky, ML, and Thiele, DJ. "Copper Metabolism and the Liver." (October 6, 2009): 221-233. (Chapter)
Source
scopus
Publish Date
2009
Start Page
221
End Page
233
DOI
10.1002/9780470747919.ch15

Enhancer of decapping proteins 1 and 2 are important for translation during heat stress in Saccharomyces cerevisiae.

In mammalian and Drosophila cells, heat stress strongly reduces general protein translation while activating cap-independent translation mechanisms to promote the expression of stress-response proteins. In contrast, in Saccharomyces cerevisiae general translation is only mildly and transiently reduced by heat stress and cap-independent translation mechanisms have not been correlated with the heat stress response. Recently we have identified direct target genes of the heat shock transcription factor (HSF), including genes encoding proteins thought to be important for general translation. One gene activated by HSF during heat stress encodes the enhancer of decapping protein, Edc2, previously shown to enhance mRNA decapping under conditions when the decapping machinery is limited. In this report we show that strains lacking Edc2, as well as the paralogous protein Edc1, are compromised for growth under persistent heat stress. This growth deficiency can be rescued by expression of a mutant Edc1 protein deficient in mRNA decapping indicative of a decapping independent function during heat stress. Yeast strains lacking Edc1 and Edc2 are also sensitive to the pharmacological inhibitor of translation paromomycin and exposure to heat stress and paromomycin functions synergistically to reduce yeast viability, suggesting that in the absence of Edc1 and Edc2 translation is compromised under heat stress conditions. Strains lacking Edc1 and Edc2 have significantly reduced rates of protein translation during growth under heat stress conditions, but not under normal growth conditions. We propose that Edc1 and the stress responsive isoform Edc2 play important roles in protein translation during stress.

Authors
Neef, DW; Thiele, DJ
MLA Citation
Neef, DW, and Thiele, DJ. "Enhancer of decapping proteins 1 and 2 are important for translation during heat stress in Saccharomyces cerevisiae." Mol Microbiol 73.6 (September 2009): 1032-1042.
PMID
19682251
Source
pubmed
Published In
Molecular Microbiology
Volume
73
Issue
6
Publish Date
2009
Start Page
1032
End Page
1042
DOI
10.1111/j.1365-2958.2009.06827.x

Ironing out a midlife crisis.

There is a strong correlation between age, genomic instability, and the development of cancer. Working in yeast, Veatch et al. (2009) now propose that defects in the biogenesis of iron-sulfur clusters arising as a consequence of mitochondrial dysfunction contribute to the increase in genomic instability as cells age.

Authors
Vergara, SV; Thiele, DJ
MLA Citation
Vergara, SV, and Thiele, DJ. "Ironing out a midlife crisis." Cell 137.7 (June 26, 2009): 1179-1181.
PMID
19563748
Source
pubmed
Published In
Cell
Volume
137
Issue
7
Publish Date
2009
Start Page
1179
End Page
1181
DOI
10.1016/j.cell.2009.06.005

New roles for copper metabolism in cell proliferation, signaling, and disease.

Authors
Turski, ML; Thiele, DJ
MLA Citation
Turski, ML, and Thiele, DJ. "New roles for copper metabolism in cell proliferation, signaling, and disease." J Biol Chem 284.2 (January 9, 2009): 717-721. (Review)
PMID
18757361
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
2
Publish Date
2009
Start Page
717
End Page
721
DOI
10.1074/jbc.R800055200

Transcriptional activation in yeast in response to copper deficiency involves copper-zinc superoxide dismutase.

Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated.

Authors
Wood, LK; Thiele, DJ
MLA Citation
Wood, LK, and Thiele, DJ. "Transcriptional activation in yeast in response to copper deficiency involves copper-zinc superoxide dismutase." J Biol Chem 284.1 (January 2, 2009): 404-413.
PMID
18977757
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
1
Publish Date
2009
Start Page
404
End Page
413
DOI
10.1074/jbc.M807027200

New roles for copper meabolism in cell proliferation,signaling, and disease

Authors
Turski, ML; Thiele, DJ
MLA Citation
Turski, ML, and Thiele, DJ. "New roles for copper meabolism in cell proliferation,signaling, and disease." Journal of Biological Chemistry 284.2 (2009): 723-727.
Source
scival
Published In
The Journal of biological chemistry
Volume
284
Issue
2
Publish Date
2009
Start Page
723
End Page
727
DOI
10.1074/jbc.R800045200

Post-transcriptional regulation of gene expression in response to iron deficiency: co-ordinated metabolic reprogramming by yeast mRNA-binding proteins.

Saccharomyces cerevisiae (baker's yeast) is an excellent model for understanding fundamental biological mechanisms that are conserved in Nature and that have an impact on human disease. The metal iron is a redox-active cofactor that plays critical biochemical roles in a broad range of functions, including oxygen transport, mitochondrial oxidative phosphorylation, chromatin remodelling, intermediary metabolism and signalling. Although iron deficiency is the most common nutritional disorder on the planet, little is known about the metabolic adjustments that cells undergo in response to iron deficit and the regulatory mechanisms that allow these adaptive responses. In the present article, we summarize recent work on genome-wide metabolic reprogramming in response to iron deficiency, mediated by specific mRNA degradation mechanisms that allow S. cerevisiae cells to adapt to iron deficiency.

Authors
Vergara, SV; Thiele, DJ
MLA Citation
Vergara, SV, and Thiele, DJ. "Post-transcriptional regulation of gene expression in response to iron deficiency: co-ordinated metabolic reprogramming by yeast mRNA-binding proteins." Biochem Soc Trans 36.Pt 5 (October 2008): 1088-1090. (Review)
PMID
18793194
Source
pubmed
Published In
Biochemical Society transactions
Volume
36
Issue
Pt 5
Publish Date
2008
Start Page
1088
End Page
1090
DOI
10.1042/BST0361088

Mechanisms for copper acquisition, distribution and regulation.

Copper (Cu) is a redox-active metal ion essential for most aerobic organisms. Cu serves as a catalytic and structural cofactor for enzymes that function in energy generation, iron acquisition, oxygen transport, cellular metabolism, peptide hormone maturation, blood clotting, signal transduction and a host of other processes. The inability to control Cu balance is associated with genetic diseases of overload and deficiency and has recently been tied to neurodegenerative disorders and fungal virulence. The essential nature of Cu, the existence of human genetic disorders of Cu metabolism and the potential impact of Cu deposition in the environment have been driving forces for detailed investigations in microbial and eukaryotic model systems. Here we review recent advances in the identification and function of cellular and systemic molecules that drive Cu accumulation, distribution and sensing.

Authors
Kim, B-E; Nevitt, T; Thiele, DJ
MLA Citation
Kim, B-E, Nevitt, T, and Thiele, DJ. "Mechanisms for copper acquisition, distribution and regulation." Nat Chem Biol 4.3 (March 2008): 176-185. (Review)
PMID
18277979
Source
pubmed
Published In
Nature Chemical Biology
Volume
4
Issue
3
Publish Date
2008
Start Page
176
End Page
185
DOI
10.1038/nchembio.72

Assembling the pieces.

Transition metals function as cofactors in specific proteins, catalyzing electron exchange reactions, binding substrates and stabilizing protein structure. Studies of human diseases and of model organisms have defined many of the molecular details of metal uptake, trafficking, and excretion. The current challenge is to integrate these details into a systematic view of metal content, speciation, localization and use within organisms and ecosystems.

Authors
Thiele, DJ; Gitlin, JD
MLA Citation
Thiele, DJ, and Gitlin, JD. "Assembling the pieces." Nat Chem Biol 4.3 (March 2008): 145-147.
PMID
18277968
Source
pubmed
Published In
Nature Chemical Biology
Volume
4
Issue
3
Publish Date
2008
Start Page
145
End Page
147
DOI
10.1038/nchembio0308-145

The Cth2 ARE-binding protein recruits the Dhh1 helicase to promote the decay of succinate dehydrogenase SDH4 mRNA in response to iron deficiency

Iron is an essential nutrient that participates as a redox cofactor in a broad range of cellular processes. In response to iron deficiency, the budding yeast Saccharomyces cerevisiae induces the expression of the Cth1 and Cth2 mRNA-binding proteins to promote a genome-wide remodeling of cellular metabolism that contributes to the optimal utilization of iron. Cth1 and Cth2 proteins bind to specific AU-rich elements within the 3′-untranslated region of many mRNAs encoding proteins involved in iron-dependent pathways, thereby promoting their degradation. Here, we show that the DEAD box Dhh1 helicase plays a crucial role in the mechanism of Cth2-mediated mRNA turnover. Yeast two-hybrid experiments indicate that Cth2 protein interacts in vivo with the carboxyl-terminal domain of Dhh1. We demonstrate that the degradation of succinate dehydrogenase SDH4 mRNA, a known target of Cth2 on iron-deficient conditions, depends on Dhh1. In addition, we localize the Cth2 protein to cytoplasmic processing bodies in strains defective in the 5′ to 3′ mRNA decay pathway. Finally, the degradation of trapped SDH4 mRNA intermediates by Cth2 supports the 5′ to 3′ directionality of mRNA turnover. Taken together, these results suggest that Cth2 protein recruits the Dhh1 helicase to ARE-containing mRNAs to promote mRNA decay. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Pedro-Segura, E; Vergara, SV; Rodríguez-Navarro, S; Parker, R; Thiele, DJ; Puig, S
MLA Citation
Pedro-Segura, E, Vergara, SV, Rodríguez-Navarro, S, Parker, R, Thiele, DJ, and Puig, S. "The Cth2 ARE-binding protein recruits the Dhh1 helicase to promote the decay of succinate dehydrogenase SDH4 mRNA in response to iron deficiency." Journal of Biological Chemistry 283.42 (2008): 28527-28535.
PMID
18715869
Source
scival
Published In
The Journal of biological chemistry
Volume
283
Issue
42
Publish Date
2008
Start Page
28527
End Page
28535
DOI
10.1074/jbc.M804910200

Cooperation of Two mRNA-Binding Proteins Drives Metabolic Adaptation to Iron Deficiency

Iron (Fe) is an essential cofactor for a wide range of cellular processes. We have previously demonstrated in yeast that Cth2 is expressed during Fe deficiency and promotes degradation of a battery of mRNAs leading to reprogramming of Fe-dependent metabolism and Fe storage. We report here that the Cth2-homologous protein Cth1 is transiently expressed during Fe deprivation and participates in the response to Fe deficiency through the degradation of mRNAs primarily involved in mitochondrially localized activities including respiration and amino acid biosynthesis. In parallel, wild-type cells, but not cth1Δcth2Δ cells, accumulate mRNAs encoding proteins that function in glucose import and storage and store high levels of glycogen. In addition, Fe deficiency leads to phosphorylation of Snf1, an AMP-activated protein kinase family member required for the cellular response to glucose starvation. These studies demonstrate a metabolic reprogramming as a consequence of Fe starvation that is dependent on the coordinated activities of two mRNA-binding proteins. © 2008 Elsevier Inc. All rights reserved.

Authors
Puig, S; Vergara, SV; Thiele, DJ
MLA Citation
Puig, S, Vergara, SV, and Thiele, DJ. "Cooperation of Two mRNA-Binding Proteins Drives Metabolic Adaptation to Iron Deficiency." Cell Metabolism 7.6 (2008): 555-564.
PMID
18522836
Source
scival
Published In
Cell Metabolism
Volume
7
Issue
6
Publish Date
2008
Start Page
555
End Page
564
DOI
10.1016/j.cmet.2008.04.010

How reliable and robust are current biomarkers for copper status? - Reply by Danzeisen

Authors
Danzeisen, R; Araya, M; Harrison, B; Keen, C; Solioz, M; Thiele, D; Mcardle, HJ
MLA Citation
Danzeisen, R, Araya, M, Harrison, B, Keen, C, Solioz, M, Thiele, D, and Mcardle, HJ. "How reliable and robust are current biomarkers for copper status? - Reply by Danzeisen." British Journal of Nutrition 100.6 (2008): 1343-1344.
Source
scival
Published In
The British journal of nutrition
Volume
100
Issue
6
Publish Date
2008
Start Page
1343
End Page
1344
DOI
10.1017/S0007114508958025

Drosophila Ctr1A functions as a copper transporter essential for development.

Copper is an essential trace element required by all aerobic organisms as a cofactor for enzymes involved in normal growth, development, and physiology. Ctr1 proteins are members of a highly conserved family of copper importers responsible for copper uptake across the plasma membrane. Mice lacking Ctr1 die during embryogenesis from widespread developmental defects, demonstrating the need for adequate copper acquisition in the development of metazoan organisms via as yet uncharacterized mechanisms. Whereas the fruit fly, Drosophila melanogaster, expresses three Ctr1 genes, ctr1A, ctr1B, and ctr1C, little is known about their protein isoform-specific roles. Previous studies demonstrated that Ctr1B localizes to the plasma membrane and is not essential for development unless flies are severely copper-deficient or are subjected to copper toxicity. Here we demonstrate that Ctr1A also resides on the plasma membrane and is the primary Drosophila copper transporter. Loss of Ctr1A results in copper-remedial developmental arrest at early larval stages. Ctr1A mutants are deficient in the activity of copper-dependent enzymes, including cytochrome c oxidase and tyrosinase. Amidation of Phe-Met-Arg-Phe-amides, a group of cardiomodulatory neuropeptide hormones that are matured via the action of peptidylglycine alpha-hydroxylating monooxygenase, is defective in neuroendocrine cells of Ctr1A mutant larvae. Moreover, both the Phe-Met-Arg-Phe-amide maturation and heart beat rate defects observed in Ctr1A mutant larvae can be partially rescued by exogenous copper. These studies establish clear physiological distinctions between two Drosophila plasma membrane copper transport proteins and demonstrate that copper import by Ctr1A is required to drive neuropeptide maturation during normal growth and development.

Authors
Turski, ML; Thiele, DJ
MLA Citation
Turski, ML, and Thiele, DJ. "Drosophila Ctr1A functions as a copper transporter essential for development." J Biol Chem 282.33 (August 17, 2007): 24017-24026.
PMID
17573340
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
33
Publish Date
2007
Start Page
24017
End Page
24026
DOI
10.1074/jbc.M703792200

Identification of a vacuole-associated metalloreductase and its role in Ctr2-mediated intracellular copper mobilization.

Copper is an essential trace metal whose biological utility is derived from its ability to cycle between oxidized Cu(II) and reduced Cu(I). Ctr1 is a high affinity plasma membrane copper permease, conserved from yeast to humans, that mediates the physiological uptake of Cu(I) from the extracellular environment. In the baker's yeast Saccharomyces cerevisiae, extracellular Cu(II) is reduced to Cu(I) via the action of the cell surface metalloreductase Fre1, similar to the human gp91(phox) subunit of the NADPH oxidase complex, which utilizes heme and flavins to catalyze electron transfer. The S. cerevisiae Ctr2 protein is structurally similar to Ctr1, localizes to the vacuole membrane, and mobilizes vacuolar copper stores to the cytosol via a mechanism that is not well understood. Here we show that Ctr2-1, a mutant form of Ctr2 that mislocalizes to the plasma membrane, requires the Fre1 plasma membrane metalloreductase for Cu(I) import. The conserved methionine residues that are essential for Ctr1 function at the plasma membrane are also essential for Ctr2-1-mediated Cu(I) uptake. We demonstrate that Fre6, a member of the yeast Fre1 metalloreductase protein family, resides on the vacuole membrane and functions in Ctr2-mediated vacuolar copper export, and cells lacking Fre6 phenocopy the Cu-deficient growth defect of ctr2Delta cells. Furthermore, both CTR2 and FRE6 mRNA levels are regulated by iron availability. Taken together these studies suggest that copper movement across intracellular membranes is mechanistically similar to that at the plasma membrane. This work provides a model for communication between the extracellular Cu(I) uptake and the intracellular Cu(I) mobilization machinery.

Authors
Rees, EM; Thiele, DJ
MLA Citation
Rees, EM, and Thiele, DJ. "Identification of a vacuole-associated metalloreductase and its role in Ctr2-mediated intracellular copper mobilization." J Biol Chem 282.30 (July 27, 2007): 21629-21638.
PMID
17553781
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
30
Publish Date
2007
Start Page
21629
End Page
21638
DOI
10.1074/jbc.M703397200

How reliable and robust are current biomarkers for copper status?

Cu is an essential nutrient for man, but can be toxic if intakes are too high. In sensitive populations, marginal over- or under-exposure can have detrimental effects. Malnourished children, the elderly, and pregnant or lactating females may be susceptible for Cu deficiency. Cu status and exposure in the population can currently not be easily measured, as neither plasma Cu nor plasma cuproenzymes reflect Cu status precisely. Some blood markers (such as ceruloplasmin) indicate severe Cu depletion, but do not inversely respond to Cu excess, and are not suitable to indicate marginal states. A biomarker of Cu is needed that is sensitive to small changes in Cu status, and that responds to Cu excess as well as deficiency. Such a marker will aid in monitoring Cu status in large populations, and will help to avoid chronic health effects (for example, liver damage in chronic toxicity, osteoporosis, loss of collagen stability, or increased susceptibility to infections in deficiency). The advent of high-throughput technologies has enabled us to screen for potential biomarkers in the whole proteome of a cell, not excluding markers that have no direct link to Cu. Further, this screening allows us to search for a whole group of proteins that, in combination, reflect Cu status. The present review emphasises the need to find sensitive biomarkers for Cu, examines potential markers of Cu status already available, and discusses methods to identify a novel suite of biomarkers. © 2007 The Authors.

Authors
Danzeisen, R; Araya, M; Harrison, B; Keen, C; Solioz, M; Thiele, D; Mcardle, HJ
MLA Citation
Danzeisen, R, Araya, M, Harrison, B, Keen, C, Solioz, M, Thiele, D, and Mcardle, HJ. "How reliable and robust are current biomarkers for copper status?." British Journal of Nutrition 98.4 (2007): 676-683.
PMID
17666147
Source
scival
Published In
The British journal of nutrition
Volume
98
Issue
4
Publish Date
2007
Start Page
676
End Page
683
DOI
10.1017/S0007114507798951

Higher plants possess two different types of ATX1-like copper chaperones

Copper (Cu) chaperones constitute a family of small Cu+-binding proteins required for Cu homeostasis in eukaryotes. The ATX1 family of Cu chaperones specifically delivers Cu to heavy metal P-type ATPases. The plant Arabidopsis thaliana expresses the ATX1-like Cu chaperone CCH, which exhibits a plant-specific carboxy-terminal domain (CTD) with unique structural properties. We show that CCH homologues from other higher plants contain CTDs with structural properties similar to Arabidopsis CCH. Furthermore, we identify a new ATX1-like Cu chaperone in Arabidopsis, AtATX1, which functionally complements yeast atx1Δ and sod1Δ associated phenotypes, and localizes to the cytosol of Arabidopsis cells. Interestingly, AtATX1, but not full-length CCH, interacts in vivo with the Arabidopsis RAN1 Cu-transporting P-type ATPase by yeast two-hybrid. We propose that higher plants express two types of ATX1-like Cu chaperones: the ATX1-type with a predominant function in Cu delivery to P-type ATPases, and the CCH-type with additional CTD-mediated plant-specific functions. © 2007 Elsevier Inc. All rights reserved.

Authors
Puig, S; Mira, H; Dorcey, E; Sancenón, V; Andrés-Colás, N; Garcia-Molina, A; Burkhead, JL; Gogolin, KA; Abdel-Ghany, SE; Thiele, DJ; Ecker, JR; Pilon, M; Peñarrubia, L
MLA Citation
Puig, S, Mira, H, Dorcey, E, Sancenón, V, Andrés-Colás, N, Garcia-Molina, A, Burkhead, JL, Gogolin, KA, Abdel-Ghany, SE, Thiele, DJ, Ecker, JR, Pilon, M, and Peñarrubia, L. "Higher plants possess two different types of ATX1-like copper chaperones." Biochemical and Biophysical Research Communications 354.2 (2007): 385-390.
PMID
17223078
Source
scival
Published In
Biochemical and Biophysical Research Communications
Volume
354
Issue
2
Publish Date
2007
Start Page
385
End Page
390
DOI
10.1016/j.bbrc.2006.12.215

Structure of the Ctr1 copper trans'PORE'ter reveals novel architecture.

Copper is essential for biological processes such as free radical detoxification, mitochondrial respiration and iron metabolism. A central player in copper homeostasis is the high-affinity integral plasma membrane copper transporter Ctr1. However, the precise mechanisms by which Ctr1 functions are not known. Here, we highlight an important breakthrough in our understanding of how Ctr1 facilitates Cu(I) movement across membranes: the publication of structural details for human Ctr1 obtained from 2D crystallography and electron microscopy.

Authors
Nose, Y; Rees, EM; Thiele, DJ
MLA Citation
Nose, Y, Rees, EM, and Thiele, DJ. "Structure of the Ctr1 copper trans'PORE'ter reveals novel architecture." Trends Biochem Sci 31.11 (November 2006): 604-607.
PMID
16982196
Source
pubmed
Published In
Trends in Biochemical Sciences
Volume
31
Issue
11
Publish Date
2006
Start Page
604
End Page
607
DOI
10.1016/j.tibs.2006.09.003

Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function.

The trace element copper (Cu) is a cofactor for biochemical functions ranging from energy generation to iron (Fe) acquisition, angiogenesis, and free radical detoxification. While Cu is essential for life, the molecules that mediate dietary Cu uptake have not been identified. Ctr1 is a homotrimeric protein, conserved from yeast to humans, that transports Cu across the plasma membrane with high affinity and specificity. Here we describe the generation of intestinal epithelial cell-specific Ctr1 knockout mice. These mice exhibit striking neonatal defects in Cu accumulation in peripheral tissues, hepatic Fe overload, cardiac hypertrophy, and severe growth and viability defects. Consistent with an intestinal Cu absorption block, the growth and viability defects can be partially rescued by a single postnatal Cu administration, indicative of a critical neonatal metabolic requirement for Cu that is provided by intestinal Ctr1. These studies identify Ctr1 as the major factor driving intestinal Cu absorption in mammals.

Authors
Nose, Y; Kim, B-E; Thiele, DJ
MLA Citation
Nose, Y, Kim, B-E, and Thiele, DJ. "Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function." Cell Metab 4.3 (September 2006): 235-244.
PMID
16950140
Source
pubmed
Published In
Cell Metabolism
Volume
4
Issue
3
Publish Date
2006
Start Page
235
End Page
244
DOI
10.1016/j.cmet.2006.08.009

A stress regulatory network for co-ordinated activation of proteasome expression mediated by yeast heat shock transcription factor

Heat shock transcription factor (HSF) mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. Although genes encoding heat shock proteins (HSPs) are the best characterized targets of HSF, recent genome-wide localization of Saccharomyces cerevisiae HSF revealed novel HSF targets involved in a wide range of cellular functions. One such target, the RPN4 gene, encodes a transcription factor that directly activates expression of a number of genes encoding proteasome subunits. Here we demonstrate that HSF co-ordinates a feed-forward gene regulatory circuit for RPN4 activation. We show that HSF activates expression of PDR3, encoding a multidrug resistance (MDR) transcription factor that also directly activates RPN4 gene expression. We demonstrate that the HSF binding site (HSE) in the RPN4 promoter is primarily responsible for heat- or methyl methanesulphonate induction of RPN4, with a minor contribution of Pdr3 binding sites (PDREs), while a Yap1 binding site (YRE) is responsible for RPN4 induction in response to oxidative stress. Furthermore, heat-induced expression of Rpn4 protein leads to expression of Rpn4 targets at later stages of heat stress, providing a temporal controlling mechanism for proteasome synthesis upon stress conditions that could result in irreversibly damaged proteins. In addition, the overlapping transcriptional regulatory networks involving HSF, Yap1 and Pdr3 suggest a close linkage between stress responses and pleiotropic drug resistance. © 2006 Blackwell Publishing Ltd.

Authors
Hahn, J-S; Neef, DW; Thiele, DJ
MLA Citation
Hahn, J-S, Neef, DW, and Thiele, DJ. "A stress regulatory network for co-ordinated activation of proteasome expression mediated by yeast heat shock transcription factor." Molecular Microbiology 60.1 (2006): 240-251.
PMID
16556235
Source
scival
Published In
Molecular Microbiology
Volume
60
Issue
1
Publish Date
2006
Start Page
240
End Page
251
DOI
10.1111/j.1365-2958.2006.05097.x

De novo appearance and "strain" formation of yeast prion [PSI+] are regulated by the heat-shock transcription factor

Yeast prions are non-Mendelian genetic elements that are conferred by altered and self-propagating protein conformations. Such a protein conformation-based transmission is similar to that of PrPSc, the infectious protein responsible for prion diseases. Despite recent progress in understanding the molecular nature and epigenetic transmission of prions, the underlying mechanisms governing prion conformational switch and determining prion "strains" are not understood. We report here that the evolutionarily conserved heat-shock transcription factor (HSF) strongly influences yeast prion formation and strain determination. An hsf1 mutant lacking the amino-terminal activation domain inhibits the yeast prion [PSI +] formation whereas a mutant lacking the carboxyl-terminal activation domain promotes [PSI+] formation. Moreover, specific [PSI+] strains are preferentially formed in these mutants, demonstrating the importance of genetic makeup in determining de novo appearance of prion strains. Although these hsf1 mutants preferentially support the formation of certain [PSI+] strains, they are capable of receiving and faithfully propagating nonpreferable strains, suggesting that prion initiation and propagation are distinct processes requiring different cellular components. Our findings establish the importance of HSF in prion initiation and strain determination and imply a similar regulatory role of mammalian HSFs in the complex etiology of prion disease. Copyright © 2006 by the Genetics Society of America.

Authors
Park, K-W; Hahn, J-S; Fan, Q; Thiele, DJ; Li, L
MLA Citation
Park, K-W, Hahn, J-S, Fan, Q, Thiele, DJ, and Li, L. "De novo appearance and "strain" formation of yeast prion [PSI+] are regulated by the heat-shock transcription factor." Genetics 173.1 (2006): 35-47.
PMID
16452152
Source
scival
Published In
Genetics
Volume
173
Issue
1
Publish Date
2006
Start Page
35
End Page
47
DOI
10.1534/genetics.105.054221

Inhibition of DNA binding by differential sumoylation of heat shock factors

Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions. Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, we have utilized the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, we also demonstrate that sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Anckar, J; Hietakangas, V; Denessiouk, K; Thiele, DJ; Johnson, MS; Sistonen, L
MLA Citation
Anckar, J, Hietakangas, V, Denessiouk, K, Thiele, DJ, Johnson, MS, and Sistonen, L. "Inhibition of DNA binding by differential sumoylation of heat shock factors." Molecular and Cellular Biology 26.3 (2006): 955-964.
PMID
16428449
Source
scival
Published In
Molecular and Cellular Biology
Volume
26
Issue
3
Publish Date
2006
Start Page
955
End Page
964
DOI
10.1128/MCB.26.3.955-964.2006

The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots

Since copper (Cu) is essential in key physiological oxidation reactions, organisms have developed strategies for handling Cu while avoiding its potentially toxic effects. Among the tools that have evolved to cope with Cu is a network of Cu homeostasis factors such as Cu-transporting P-type ATPases that play a key role in transmembrane Cu transport. In this work we present the functional characterization of an Arabidopsis Cu-transporting P-type ATPase, denoted heavy metal ATPase 5 (HMA5), and its interaction with Arabidopsis metallochaperones. HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu in whole plants. We have identified and characterized plants carrying two independent T-DNA insertion alleles, hma5-1 and hma5-2. Both mutants are hypersensitive to Cu but not to other metals such as iron, zinc or cadmium. Interestingly, root tips from Cu-treated hma5 mutants exhibit a wave-like phenotype at early stages and later on main root growth completely arrests whereas lateral roots emerge near the crown. Accordingly, these lines accumulate Cu in roots to a greater extent than wild-type plants under Cu excess. Finally, yeast two-hybrid experiments demonstrate that the metal-binding domains of HMA5 interact with Arabidopsis ATX1-like Cu chaperones, and suggest a regulatory role for the plant-specific domain of the CCH Cu chaperone. Based on these findings, we propose a role for HMA5 in Cu compartmentalization and detoxification. © 2005 Blackwell Publishing Ltd.

Authors
Andrés-Colás, N; Sancenón, V; Rodríguez-Navarro, S; Mayo, S; Thiele, DJ; Ecker, JR; Puig, S; Peñarrubia, L
MLA Citation
Andrés-Colás, N, Sancenón, V, Rodríguez-Navarro, S, Mayo, S, Thiele, DJ, Ecker, JR, Puig, S, and Peñarrubia, L. "The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots." Plant Journal 45.2 (2006): 225-236.
PMID
16367966
Source
scival
Published In
The Plant Journal
Volume
45
Issue
2
Publish Date
2006
Start Page
225
End Page
236
DOI
10.1111/j.1365-313X.2005.02601.x

Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation.

Iron (Fe) is an essential micronutrient for virtually all organisms and serves as a cofactor for a wide variety of vital cellular processes. Although Fe deficiency is the primary nutritional disorder in the world, cellular responses to Fe deprivation are poorly understood. We have discovered a posttranscriptional regulatory process controlled by Fe deficiency, which coordinately drives widespread metabolic reprogramming. We demonstrate that, in response to Fe deficiency, the Saccharomyces cerevisiae Cth2 protein specifically downregulates mRNAs encoding proteins that participate in many Fe-dependent processes. mRNA turnover requires the binding of Cth2, an RNA binding protein conserved in plants and mammals, to specific AU-rich elements in the 3' untranslated region of mRNAs targeted for degradation. These studies elucidate coordinated global metabolic reprogramming in response to Fe deficiency and identify a mechanism for achieving this by targeting specific mRNA molecules for degradation, thereby facilitating the utilization of limited cellular Fe levels.

Authors
Puig, S; Askeland, E; Thiele, DJ
MLA Citation
Puig, S, Askeland, E, and Thiele, DJ. "Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation." Cell 120.1 (January 14, 2005): 99-110.
PMID
15652485
Source
pubmed
Published In
Cell
Volume
120
Issue
1
Publish Date
2005
Start Page
99
End Page
110
DOI
10.1016/j.cell.2004.11.032

Metal-responsive transcription factor (MTF-1) handles both extremes, copper load and copper starvation, by activating different genes

From insects to mammals, metallothionein genes are induced in response to heavy metal load by the transcription factor MTF-1, which binds to short DNA sequence motifs, termed metal response elements (MREs). Here we describe a novel and seemingly paradoxical role for MTF-1 in Drosophila in that it also mediates transcriptional activation of Ctr1B, a copper importer, upon copper depletion. Activation depends on the same type of MRE motifs in the upstream region of the Ctr1B gene as are normally required for metal induction. Thus, a single transcription factor, MTF-1, plays a direct role in both copper detoxification and acquisition by inducing the expression of metallothioneins and of a copper importer, respectively. © 2005 by Cold Spring Harbor Laboratory Press.

Authors
Selvaraj, A; Balamurugan, K; Yepiskoposyan, H; Zhou, H; Egli, D; Georgiev, O; Thiele, DJ; Schaffner, W
MLA Citation
Selvaraj, A, Balamurugan, K, Yepiskoposyan, H, Zhou, H, Egli, D, Georgiev, O, Thiele, DJ, and Schaffner, W. "Metal-responsive transcription factor (MTF-1) handles both extremes, copper load and copper starvation, by activating different genes." Genes and Development 19.8 (2005): 891-896.
PMID
15833915
Source
scival
Published In
Genes and Development
Volume
19
Issue
8
Publish Date
2005
Start Page
891
End Page
896
DOI
10.1101/gad.1301805

Mobilization of intracellular copper stores by the ctr2 vacuolar copper transporter.

Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores in a manner dependent on amino acid residues conserved between the Ctr1 and Ctr2 copper transport family and that ctr2 Delta mutants hyper-accumulate vacuolar copper. Furthermore, a Ctr2 mutant that is mislocalized to the plasma membrane stimulates extracellular copper uptake, supporting a direct role for Ctr2 in copper transport across membranes. These studies identify a novel mechanism for copper mobilization and suggest that organisms cope with copper deprivation via the use of intracellular vesicular stores.

Authors
Rees, EM; Lee, J; Thiele, DJ
MLA Citation
Rees, EM, Lee, J, and Thiele, DJ. "Mobilization of intracellular copper stores by the ctr2 vacuolar copper transporter." J Biol Chem 279.52 (December 24, 2004): 54221-54229.
PMID
15494390
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
52
Publish Date
2004
Start Page
54221
End Page
54229
DOI
10.1074/jbc.M411669200

Cti6 is an Rpd3-Sin3 histone deacetylase-associated protein required for growth under iron-limiting conditions in Saccharomyces cerevisiae.

Iron and copper are redox active metals essential for life. In the budding yeast Saccharomyces cerevisiae, expression of iron and copper genes involved in metal acquisition and utilization is tightly regulated at the transcriptional level. In addition iron and copper metabolism are inextricably linked because of the dependence on copper as a co-factor for iron uptake or mobilization. To further identify genes that function in iron and copper homeostasis, we screened for novel yeast mutants defective for iron limiting growth and thereby identified the CTI6 gene. Cti6 is a PHD finger-containing protein that has been shown to participate in the interaction of the Ssn6-Tup1 co-repressor with the Gcn5-containing SAGA chromatin-remodeling complex. In this report we show that CTI6 mRNA levels are increased under iron-limiting conditions, and that cti6 mutants display a growth defect under conditions of iron deprivation. Furthermore, we demonstrate that Cti6 is a nuclear protein that functionally associates with the Rpd3-Sin3 histone deacetylase complex involved in transcriptional repression. Cti6 demonstrates Rpd3-dependent transcriptional repression, and cti6 mutants exhibit an enhanced silencing of telomeric, rDNA and HMR loci, similar to mutants in genes encoding other Rpd3-Sin3-associated proteins. Microarray experiments with cti6 mutants grown under iron-limiting conditions show a down-regulation of telomeric genes and an up-regulation of Aft1 and Tup1 target genes involved in iron and oxygen regulation. Taken together, these data suggest a specific role for Cti6 in the regulation of gene expression under conditions of iron limitation.

Authors
Puig, S; Lau, M; Thiele, DJ
MLA Citation
Puig, S, Lau, M, and Thiele, DJ. "Cti6 is an Rpd3-Sin3 histone deacetylase-associated protein required for growth under iron-limiting conditions in Saccharomyces cerevisiae." J Biol Chem 279.29 (July 16, 2004): 30298-30306.
PMID
15133041
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
29
Publish Date
2004
Start Page
30298
End Page
30306
DOI
10.1074/jbc.M313463200

Genome-wide analysis of the biology of stress responses through heat shock transcription factor.

Heat shock transcription factor (HSF) and the promoter heat shock element (HSE) are among the most highly conserved transcriptional regulatory elements in nature. HSF mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. While HSF is essential for cell viability in Saccharomyces cerevisiae, oogenesis and early development in Drosophila melanogaster, extended life span in Caenorhabditis elegans, and extraembryonic development and stress resistance in mammals, little is known about its full range of biological target genes. We used whole-genome analyses to identify virtually all of the direct transcriptional targets of yeast HSF, representing nearly 3% of the genomic loci. The majority of the identified loci are heat-inducibly bound by yeast HSF, and the target genes encode proteins that have a broad range of biological functions including protein folding and degradation, energy generation, protein trafficking, maintenance of cell integrity, small molecule transport, cell signaling, and transcription. This genome-wide identification of HSF target genes provides novel insights into the role of HSF in growth, development, disease, and aging and in the complex metabolic reprogramming that occurs in all cells in response to stress.

Authors
Hahn, J-S; Hu, Z; Thiele, DJ; Iyer, VR
MLA Citation
Hahn, J-S, Hu, Z, Thiele, DJ, and Iyer, VR. "Genome-wide analysis of the biology of stress responses through heat shock transcription factor." Mol Cell Biol 24.12 (June 2004): 5249-5256.
PMID
15169889
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
24
Issue
12
Publish Date
2004
Start Page
5249
End Page
5256
DOI
10.1128/MCB.24.12.5249-5256.2004

Identification of methionine-rich clusters that regulate copper-stimulated endocytosis of the human Ctr1 copper transporter.

Copper uptake and subsequent delivery to copper-dependent enzymes are essential for many cellular processes. However, the intracellular levels of this nutrient must be controlled because of its potential toxicity. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. Recent studies have shown that elevated copper stimulates the endocytosis and degradation of the hCtr1 protein, and this response is likely an important homeostatic mechanism that prevents the overaccumulation of copper. The domains of hCtr1 involved in copper-stimulated endocytosis and degradation are unknown. In this study we examined the importance of potential copper-binding sequences in the extracellular domain and a conserved transmembrane (150)MXXXM(154) motif for copper-stimulated endocytosis and degradation of hCtr1. The endocytic response of hCtr1 to low copper concentrations required an amino-terminal methionine cluster ((40)MMMMPM(45)) closest to the transmembrane region. However, this cluster was not required for the endocytic response to higher copper levels, suggesting this motif may function as a high affinity copper-sensing domain. Moreover, the transmembrane (150)MXXXM(154) motif was absolutely required for copper-stimulated endocytosis and degradation of hCtr1 even under high copper concentrations. Together with previous studies demonstrating a role for these motifs in high affinity copper transport activity, our findings suggest common biochemical mechanisms regulate both transport and trafficking functions of hCtr1.

Authors
Guo, Y; Smith, K; Lee, J; Thiele, DJ; Petris, MJ
MLA Citation
Guo, Y, Smith, K, Lee, J, Thiele, DJ, and Petris, MJ. "Identification of methionine-rich clusters that regulate copper-stimulated endocytosis of the human Ctr1 copper transporter." J Biol Chem 279.17 (April 23, 2004): 17428-17433.
PMID
14976198
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
17
Publish Date
2004
Start Page
17428
End Page
17433
DOI
10.1074/jbc.M401493200

The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development.

Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of (64)Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters.

Authors
Sancenón, V; Puig, S; Mateu-Andrés, I; Dorcey, E; Thiele, DJ; Peñarrubia, L
MLA Citation
Sancenón, V, Puig, S, Mateu-Andrés, I, Dorcey, E, Thiele, DJ, and Peñarrubia, L. "The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development." J Biol Chem 279.15 (April 9, 2004): 15348-15355.
PMID
14726516
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
15
Publish Date
2004
Start Page
15348
End Page
15355
DOI
10.1074/jbc.M313321200

From aging to virulence: forging connections through the study of copper homeostasis in eukaryotic microorganisms.

Recent years have witnessed an explosion in the breadth of investigations on transition metal homeostasis and the subsequent depth of our understanding of metals in biology. Many genes and proteins that serve in the uptake, distribution, sensing and detoxification of one such transition metal, copper, have been identified. Through genetic and biochemical studies, the molecular details of copper uptake are being elucidated, and evidence suggests a largely conserved mechanism for copper acquisition and distribution from yeast to humans. Investigations of the mitochondrial copper pathway reveal the complexity surrounding copper delivery to cytochrome oxidase and highlight additional roles for some of the participants in copper homeostasis, such as a copper chaperone that influences the subcellular distribution of its target for copper incorporation. Furthermore, our understanding of the structure and function of copper transporters, chaperones and cupro-proteins, coupled with the emergence of additional model systems, is providing surprising examples of the integration of copper homeostasis with other physiological and pathophysiological processes and states, such as cancer, aging and virulence.

Authors
Rees, EM; Thiele, DJ
MLA Citation
Rees, EM, and Thiele, DJ. "From aging to virulence: forging connections through the study of copper homeostasis in eukaryotic microorganisms." Curr Opin Microbiol 7.2 (April 2004): 175-184. (Review)
PMID
15063856
Source
pubmed
Published In
Current Opinion in Microbiology
Volume
7
Issue
2
Publish Date
2004
Start Page
175
End Page
184
DOI
10.1016/j.mib.2004.02.004

Activation of the Saccharomyces cerevisiae heat shock transcription factor under glucose starvation conditions by Snf1 protein kinase.

Heat shock transcription factor (HSF) is an evolutionarily conserved protein that mediates eukaryotic transcriptional responses to stress. Although the mammalian stress-responsive HSF1 isoform is activated in response to a wide array of seemingly unrelated stresses, including heat shock, pharmacological agents, infection and inflammation, little is known about the precise mechanisms or pathways by which this factor is activated by many stressors. The baker's yeast Saccharomyces cerevisiae encodes a single HSF protein that responds to heat stress and glucose starvation and provides a simple model system to investigate how a single HSF is activated by multiple stresses. Although induction of the HSF target gene CUP1 by glucose starvation is dependent on the Snf1 kinase, HSF-dependent heat shock induction of CUP1 is Snf1-independent. Approximately 165 in vivo targets for HSF have been identified in S. cerevisiae using chromatin immunoprecipitation combined with DNA microarrays. Interestingly, approximately 30% of the HSF direct target genes are also induced by the diauxic shift, in which glucose levels begin to be depleted. We demonstrate that HSF and Snf1 kinase interact in vivo and that HSF is a direct substrate for phosphorylation by Snf1 kinase in vitro. Furthermore, glucose starvation-dependent, but not heat shock-dependent HSF phosphorylation, and enhanced chromosomal HSF DNA binding to low affinity target promoters such as SSA3 and HSP30, occurred in a Snf1-dependent manner. Consistent with a more global role for HSF and Snf1 in activating gene expression in response to changes in glucose availability, expression of a subset of HSF targets by glucose starvation was dependent on Snf1 and the HSF carboxyl-terminal activation domain.

Authors
Hahn, J-S; Thiele, DJ
MLA Citation
Hahn, J-S, and Thiele, DJ. "Activation of the Saccharomyces cerevisiae heat shock transcription factor under glucose starvation conditions by Snf1 protein kinase." J Biol Chem 279.7 (February 13, 2004): 5169-5176.
PMID
14612437
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
7
Publish Date
2004
Start Page
5169
End Page
5176
DOI
10.1074/jbc.M311005200

Metal accumulation and transport in the ovary of the lizard Podarcis sicula

The molecular characterisation of a CTR protein is reported in the ovary of the lizard Podarcis sicula; this protein proves to be homologous with the mammalian high affinity copper transporter CTRL Gene expression assessed by Northern blot hybridisation of RNA from growing ovarian follicles and eggs demonstrated that the transcript accumulated during the oocyte growth and reached the highest levels in ovulated eggs. Analysis of the copper content paralleled the profile of CTR1 mRNA, with a rapid increase of the metal concentration during oocyte maturation. These data suggest that the P. sicula CTR1 protein may function in the uptake and storage of the copper to be used during embryonic development.

Authors
Riggio, M; Scudiero, R; Lee, J; Thiele, DJ; Parisi, E; Filosa, S
MLA Citation
Riggio, M, Scudiero, R, Lee, J, Thiele, DJ, Parisi, E, and Filosa, S. "Metal accumulation and transport in the ovary of the lizard Podarcis sicula." Italian Journal of Zoology 71.SUPPL.2 (2004): 59-62.
Source
scival
Published In
Italian Journal of Zoology
Volume
71
Issue
SUPPL.2
Publish Date
2004
Start Page
59
End Page
62
DOI
10.1080/11250000409356607

Erratum: A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster (Journal of Biological Chemistry (2003) 278 (48210-48218))

Authors
Zhou, H; Cadigan, KM; Thiele, DJ
MLA Citation
Zhou, H, Cadigan, KM, and Thiele, DJ. "Erratum: A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster (Journal of Biological Chemistry (2003) 278 (48210-48218))." Journal of Biological Chemistry 279.3 (2004): 2332--.
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
3
Publish Date
2004
Start Page
2332-

A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster.

The trace element copper is required for normal growth and development, serving as an essential catalytic co-factor for enzymes involved in energy generation, oxidative stress protection, neuropeptide maturation, and other fundamental processes. In yeast and mammals copper acquisition occurs through the action of the Ctr1 family of high affinity copper transporters. Here we describe studies using Drosophila melanogaster to investigate the role of copper acquisition through Ctr1 in normal growth and development. Three distinct Drosophila Ctr1 genes (Ctr1A, Ctr1B, and Ctr1C) have been identified, which have unique expression patterns over the course of development. Interestingly, Ctr1B, which is expressed exclusively during the late embryonic and larval stages of development, is transcriptionally activated in response to nutritionally induced copper deprivation and down-regulated in response to copper adequacy. The generation of Ctr1B mutant flies results in decreased larval copper accumulation, marked body pigmentation defects that parallel defects in tyrosinase activity, and specific developmental arrest under conditions of both nutritional copper limitation and excess. These studies establish that copper acquisition through the Drosophila Ctr1B transporter is crucial for normal growth and in early and specific stages of metazoan development.

Authors
Zhou, H; Cadigan, KM; Thiele, DJ
MLA Citation
Zhou, H, Cadigan, KM, and Thiele, DJ. "A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster." J Biol Chem 278.48 (November 28, 2003): 48210-48218.
PMID
12966081
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
48
Publish Date
2003
Start Page
48210
End Page
48218
DOI
10.1074/jbc.M309820200

Integrating trace element metabolism from the cell to the whole organism.

The redox chemistry of copper (Cu) makes this both a powerful enzyme catalyst and a dangerous reactant that generates hydroxyl radical. Although virtually all cells from microbes to mammals must acquire Cu to drive important biochemical reactions, the potential toxicity of Cu demands an exquisite level of vectorial transport and homeostatic control. Our laboratory is interested in how organisms acquire Cu through the action of high-affinity plasma membrane Cu transporters of the copper transport protein (Ctr) class of proteins. We have isolated Ctr Cu transporters from baker's yeast and fission yeast and from flies, mice and mammals. This review will focus on understanding how the Ctr high-affinity Cu transport proteins function, from their biochemical mechanism of action in yeast and cultured metazoan cells to their roles in Cu delivery and mammalian embryonic development.

Authors
Thiele, DJ
MLA Citation
Thiele, DJ. "Integrating trace element metabolism from the cell to the whole organism." J Nutr 133.5 Suppl 1 (May 2003): 1579S-1580S.
PMID
12730470
Source
pubmed
Published In
The Journal of nutrition
Volume
133
Issue
5 Suppl 1
Publish Date
2003
Start Page
1579S
End Page
1580S

Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1.

Copper uptake at the plasma membrane and subsequent delivery to copper-dependent enzymes is essential for many cellular processes, including mitochondrial oxidative phosphorylation, free radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. However, intracellular levels of this nutrient must be controlled because it is potentially toxic in excess concentrations. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. In this study, we demonstrate that levels of the hCtr1 protein at the plasma membrane of HEK293 cells were reduced when cells were exposed to elevated copper. This decrease in surface hCtr1 levels was associated with an increased rate of endocytosis, and low micromolar concentrations of copper were sufficient to stimulate this process. Inhibitors of clathrin-dependent endocytosis prevented the trafficking of hCtr1 from the plasma membrane, and newly internalized hCtr1 and transferrin were co-localized. Significantly, elevated copper concentrations also resulted in the degradation of the hCtr1 protein. Our findings suggest that hCtr1-mediated copper uptake into mammalian cells is regulated by a post-translational mechanism involving copper-stimulated endocytosis and degradation of the transporter.

Authors
Petris, MJ; Smith, K; Lee, J; Thiele, DJ
MLA Citation
Petris, MJ, Smith, K, Lee, J, and Thiele, DJ. "Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1." J Biol Chem 278.11 (March 14, 2003): 9639-9646.
PMID
12501239
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
11
Publish Date
2003
Start Page
9639
End Page
9646
DOI
10.1074/jbc.M209455200

Identification of a copper transporter family in Arabidopsis thaliana.

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1-5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1-5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.

Authors
Sancenón, V; Puig, S; Mira, H; Thiele, DJ; Peñarrubia, L
MLA Citation
Sancenón, V, Puig, S, Mira, H, Thiele, DJ, and Peñarrubia, L. "Identification of a copper transporter family in Arabidopsis thaliana." Plant Mol Biol 51.4 (March 2003): 577-587.
PMID
12650623
Source
pubmed
Published In
Plant Molecular Biology
Volume
51
Issue
4
Publish Date
2003
Start Page
577
End Page
587

Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress.

The activation of eukaryotic heat shock protein (Hsp) gene expression occurs in response to a wide variety of cellular stresses including heat shock, hydrogen peroxide, uncoupled oxidative phosphorylation, infection, and inflammation. Biochemical and genetic studies have clearly demonstrated critical roles for mammalian heat shock factor 1 (HSF1) in stress-inducible Hsp gene expression, resistance to stress-induced programmed cell death, extra-embryonic development, and other biological functions. Activation of mammalian Hsp gene expression involves the stress-inducible conversion of HSF1 from the inactive monomer to the DNA-binding competent homotrimer. Although Hsp activation is a central conserved process in biology, the precise mechanisms for stress sensing and signaling to activate HSF1, and the mechanisms by which many distinct stresses activate HSF1, are poorly understood. In this report we demonstrate that recombinant mammalian HSF1 directly senses both heat and hydrogen peroxide to assemble into a homotrimer in a reversible and redox-regulated manner. The sensing of both stresses requires two cysteine residues within the HSF1 DNA-binding domain that are engaged in redox-sensitive disulfide bonds. HSF1 derivatives in which either or both cysteines were mutated are defective in stress-inducible trimerization and DNA binding, stress-inducible nuclear translocation and Hsp gene trans-activation, and in the protection of mouse cells from stress-induced apoptosis. This redox-dependent activation of HSF1 by heat and hydrogen peroxide establishes a common mechanism in the stress activation of Hsp gene expression by mammalian HSF1.

Authors
Ahn, S-G; Thiele, DJ
MLA Citation
Ahn, S-G, and Thiele, DJ. "Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress." Genes Dev 17.4 (February 15, 2003): 516-528.
PMID
12600944
Source
pubmed
Published In
Genes & development
Volume
17
Issue
4
Publish Date
2003
Start Page
516
End Page
528
DOI
10.1101/gad.1044503

Integrating trace element metabolism from the cell to the whole organism

The redox chemistry of copper (Cu) makes this both a powerful enzyme catalyst and a dangerous reactant that generates hydroxyl radical. Although virtually all cells from microbes to mammals must acquire Cu to drive important biochemical reactions, the potential toxicity of Cu demands an exquisite level of vectorial transport and homeostatic control. Our laboratory is interested in how organisms acquire Cu through the action of high-affinity plasma membrane Cu transporters of the copper transport protein (Ctr) class of proteins. We have isolated Ctr Cu transporters from baker's yeast and fission yeast and from flies, mice and mammals. This review will focus on understanding how the Ctr high-affinity Cu transport proteins function, from their biochemical mechanism of action in yeast and cultured metazoan cells to their roles in Cu delivery and mammalian embryonic development.

Authors
Thiele, DJ
MLA Citation
Thiele, DJ. "Integrating trace element metabolism from the cell to the whole organism." Journal of Nutrition 133.5 SUPPL. 2 (2003): 1579S-1580S.
Source
scival
Published In
Journal of Nutrition
Volume
133
Issue
5 SUPPL. 2
Publish Date
2003
Start Page
1579S
End Page
1580S

Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals.

Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug.

Authors
Ishida, S; Lee, J; Thiele, DJ; Herskowitz, I
MLA Citation
Ishida, S, Lee, J, Thiele, DJ, and Herskowitz, I. "Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals." Proc Natl Acad Sci U S A 99.22 (October 29, 2002): 14298-14302.
PMID
12370430
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
22
Publish Date
2002
Start Page
14298
End Page
14302
DOI
10.1073/pnas.162491399

Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system.

The trace metal copper is an essential cofactor for a number of enzymes that have critical roles in biological processes, but it is highly toxic when allowed to accumulate in excess of cellular needs. Consequently, homeostatic copper metabolism is maintained by molecules involved in copper uptake, distribution, excretion, and incorporation into copper-requiring enzymes. Previously, we reported that overexpression of the human or mouse Ctr1 copper transporter stimulates copper uptake in mammalian cells, and deletion of one Ctr1 allele in mice gives rise to tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. To investigate the physiological roles for mammalian Ctr1 protein in cellular copper metabolism, we characterized wild type, Ctr1 heterozygous, and Ctr1 homozygous knock-out cells isolated from embryos obtained by the inter-cross of Ctr1 heterozygous mice. Ctr1-deficient mouse embryonic cells are viable but exhibit significant defects in copper uptake and accumulation and in copper-dependent enzyme activities. Interestingly, Ctr1-deficient cells exhibit approximately 30% residual copper transport activity that is saturable, with a K(m) of approximately 10 microm, with biochemical features distinct from that of Ctr1. These observations demonstrate that, although Ctr1 is critical for both cellular copper uptake and embryonic development, mammals possess additional biochemically distinct functional copper transport activities.

Authors
Lee, J; Petris, MJ; Thiele, DJ
MLA Citation
Lee, J, Petris, MJ, and Thiele, DJ. "Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system." J Biol Chem 277.43 (October 25, 2002): 40253-40259.
PMID
12177073
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
43
Publish Date
2002
Start Page
40253
End Page
40259
DOI
10.1074/jbc.M208002200

The ABCDs of periplasmic copper trafficking.

The structure of the CopC protein of Pseudomonas syringae pathovar tomato provides fascinating clues, not only to its role in the periplasmic space in copper resistance, but also to features important for copper trafficking and homeostasis that may be conserved in a variety of biological systems.

Authors
Puig, S; Rees, EM; Thiele, DJ
MLA Citation
Puig, S, Rees, EM, and Thiele, DJ. "The ABCDs of periplasmic copper trafficking." Structure 10.10 (October 2002): 1292-1295.
PMID
12377116
Source
pubmed
Published In
Structure
Volume
10
Issue
10
Publish Date
2002
Start Page
1292
End Page
1295

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake.

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.

Authors
Puig, S; Lee, J; Lau, M; Thiele, DJ
MLA Citation
Puig, S, Lee, J, Lau, M, and Thiele, DJ. "Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake." J Biol Chem 277.29 (July 19, 2002): 26021-26030.
PMID
11983704
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
29
Publish Date
2002
Start Page
26021
End Page
26030
DOI
10.1074/jbc.M202547200

Regulation of the Saccharomyces cerevisiae Slt2 kinase pathway by the stress-inducible Sdp1 dual specificity phosphatase.

The Slt2/Mpk1 mitogen-activated protein kinase (MAPK) cell integrity pathway is involved in maintenance of cell shape and integrity during vegetative growth and mating in Saccharomyces cerevisiae. Slt2 is activated by dual phosphorylation of a threonine and tyrosine residue in response to several environmental stresses that perturb cell integrity. Negative regulation of Slt2 is achieved via dephosphorylation by two protein-tyrosine phosphatases, Ptp2 and Ptp3, and a dual specificity phosphatase, Msg5. In this study, we provide genetic and biochemical evidence that the stress-inducible dual specificity phosphatase, Sdp1, negatively regulates Slt2 by direct dephosphorylation. Deletion of SDP1 exacerbated growth defects due to overexpression of Mkk1(p386), a constitutively active mutant of Slt2 MAPK kinase, whereas overexpression of Sdp1 suppressed lethality caused by Mkk1(p386) overexpression. The heat shock-induced phosphorylation level of Slt2 was elevated in an sdp1Delta strain compared with that of the wild type, and heat shock-activated phospho-Slt2 was dephosphorylated by recombinant Sdp1 in vitro. Under normal growth conditions, an Sdp1-GFP fusion protein was localized to both the nucleus and cytoplasm. However, the Sdp1-GFP protein translocated to punctate spots throughout the cell after heat shock. SDP1 transcription was induced by several stress conditions in an Msn2/4-dependent manner but independent of the Rlm1 transcription factor, a downstream target activated by Slt2. Induction of SLT2 by high osmolarity was dependent on Rlm1 transcription factor and Hog1 kinase, suggesting cross-talk between Slt2 and Hog1 MAPK pathways. These studies demonstrate regulation of Slt2 activity and gene expression in coordination with other stress signaling pathways.

Authors
Hahn, J-S; Thiele, DJ
MLA Citation
Hahn, J-S, and Thiele, DJ. "Regulation of the Saccharomyces cerevisiae Slt2 kinase pathway by the stress-inducible Sdp1 dual specificity phosphatase." J Biol Chem 277.24 (June 14, 2002): 21278-21284.
PMID
11923319
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
24
Publish Date
2002
Start Page
21278
End Page
21284
DOI
10.1074/jbc.M202557200

High affinity copper transport protein in the lizard Podarcis sicula: molecular cloning, functional characterization and expression in somatic tissues, follicular oocytes and eggs.

Copper (Cu) is an essential element required in many biological processes including cellular growth and development. The molecular mechanisms involved in copper homeostasis include proteins that play a role in Cu uptake. Genes encoding high affinity copper transporters (Ctr) have been identified in yeast, plant and mammalian cells. Analysis of copper and zinc content in growing ovarian follicles and ovulated eggs of the reptilian Podarcis sicula demonstrated that the levels of both metals rise during oocyte growth, reaching the maximum in ovulated eggs. By exploiting the remarkable evolutionary conservation of the primary structure of Ctr proteins, cDNA encoding a Ctr was isolated from the liver of the lizard P. sicula by reverse transcriptase PCR and RACE strategy by using primers designed based on consensus motifs present in mammalian Ctr. The predicted protein sequence contains three transmembrane domains and a putative hydrophilic extracellular amino-terminal domain. Besides complementing the respiratory deficiency of yeast cells defective in high affinity Cu transport, expression of lizard Ctr1(1) in Hek293 cells stimulates Cu uptake.Gene expression assessed by Northern blot hybridization of RNA from different tissues of P. sicula shows the highest levels of transcript in both intestine and liver. The profile of Ctr1 mRNA in growing ovarian follicles and eggs demonstrates that the transcript accumulates during the oocyte growth and reaches the highest levels in ovulated eggs. These results suggest that lizard Ctr1 protein may function in Cu acquisition in growing oocytes and eggs.

Authors
Riggio, M; Lee, J; Scudiero, R; Parisi, E; Thiele, DJ; Filosa, S
MLA Citation
Riggio, M, Lee, J, Scudiero, R, Parisi, E, Thiele, DJ, and Filosa, S. "High affinity copper transport protein in the lizard Podarcis sicula: molecular cloning, functional characterization and expression in somatic tissues, follicular oocytes and eggs." Biochim Biophys Acta 1576.1-2 (June 7, 2002): 127-135.
PMID
12031492
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1576
Issue
1-2
Publish Date
2002
Start Page
127
End Page
135

Molecular mechanisms of copper uptake and distribution.

In the past few years, exciting advances have been made toward understanding how copper is transported into and distributed to cupro-proteins within cells. Recent work has identified high-affinity copper transporters at the plasma membrane in a number of organisms. The elucidation of the three-dimensional structure of copper chaperones and target cupro-proteins has shown that highly specific interactions between homologous domains foster copper transfer between conserved copper ligands, and facilitate a detailed understanding of vectorial copper-transfer reactions. Furthermore, the recent generation of mouse-knockout models, deficient in a high-affinity copper transporter, or in copper chaperones, has demonstrated the importance of copper uptake and targeted distribution in both predicted and fascinating unanticipated ways in growth and development.

Authors
Puig, S; Thiele, DJ
MLA Citation
Puig, S, and Thiele, DJ. "Molecular mechanisms of copper uptake and distribution." Curr Opin Chem Biol 6.2 (April 2002): 171-180. (Review)
PMID
12039001
Source
pubmed
Published In
Current Opinion in Chemical Biology
Volume
6
Issue
2
Publish Date
2002
Start Page
171
End Page
180

Biochemical characterization of the human copper transporter Ctr1.

The trace metal copper is an essential cofactor for a number of biological processes including mitochondrial oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal physiology. Little is known about the molecules and biochemical mechanisms responsible for copper uptake at the plasma membrane in mammals. Here, we demonstrate that human Ctr1 (hCtr1) is a component of the copper transport machinery at the plasma membrane. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated (64)Cu transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. This is the first report on the biochemical characterization of the human copper transporter hCtr1, which is important for understanding mechanisms for mammalian copper transport at the plasma membrane.

Authors
Lee, J; Peña, MMO; Nose, Y; Thiele, DJ
MLA Citation
Lee, J, Peña, MMO, Nose, Y, and Thiele, DJ. "Biochemical characterization of the human copper transporter Ctr1." J Biol Chem 277.6 (February 8, 2002): 4380-4387.
PMID
11734551
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
6
Publish Date
2002
Start Page
4380
End Page
4387
DOI
10.1074/jbc.M104728200

Novel stress-responsive genes EMG1 and NOP14 encode conserved, interacting proteins required for 40S ribosome biogenesis.

Under stressful conditions organisms adjust the synthesis, processing, and trafficking of molecules to allow survival from and recovery after stress. In baker's yeast Saccharomyces cerevisiae, the cellular production of ribosomes is tightly matched with environmental conditions and nutrient availability through coordinate transcriptional regulation of genes involved in ribosome biogenesis. On the basis of stress-responsive gene expression and functional studies, we have identified a novel, evolutionarily conserved gene, EMG1, that has similar stress-responsive gene expression patterns as ribosomal protein genes and is required for the biogenesis of the 40S ribosomal subunit. The Emg1 protein is distributed throughout the cell; however, its nuclear localization depends on physical interaction with a newly characterized nucleolar protein, Nop14. Yeast depleted of Nop14 or harboring a temperature-sensitive allele of emg1 have selectively reduced levels of the 20S pre-rRNA and mature18S rRNA and diminished cellular levels of the 40S ribosomal subunit. Neither Emg1 nor Nop14 contain any characterized functional motifs; however, isolation and functional analyses of mammalian orthologues of Emg1 and Nop14 suggest that these proteins are functionally conserved among eukaryotes. We conclude that Emg1 and Nop14 are novel proteins whose interaction is required for the maturation of the 18S rRNA and for 40S ribosome production.

Authors
Liu, PC; Thiele, DJ
MLA Citation
Liu, PC, and Thiele, DJ. "Novel stress-responsive genes EMG1 and NOP14 encode conserved, interacting proteins required for 40S ribosome biogenesis." Mol Biol Cell 12.11 (November 2001): 3644-3657.
PMID
11694595
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
12
Issue
11
Publish Date
2001
Start Page
3644
End Page
3657

The loop domain of heat shock transcription factor 1 dictates DNA-binding specificity and responses to heat stress.

Eukaryotic heat shock transcription factors (HSF) regulate an evolutionarily conserved stress-response pathway essential for survival against a variety of environmental and developmental stresses. Although the highly similar HSF family members have distinct roles in responding to stress and activating target gene expression, the mechanisms that govern these roles are unknown. Here we identify a loop within the HSF1 DNA-binding domain that dictates HSF isoform specific DNA binding in vitro and preferential target gene activation by HSF family members in both a yeast transcription assay and in mammalian cells. These characteristics of the HSF1 loop region are transposable to HSF2 and sufficient to confer DNA-binding specificity, heat shock inducible HSP gene expression and protection from heat-induced apoptosis in vivo. In addition, the loop suppresses formation of the HSF1 trimer under basal conditions and is required for heat-inducible trimerization in a purified system in vitro, suggesting that this domain is a critical part of the HSF1 heat-stress-sensing mechanism. We propose that this domain defines a signature for HSF1 that constitutes an important determinant for how cells utilize a family of transcription factors to respond to distinct stresses.

Authors
Ahn, SG; Liu, PC; Klyachko, K; Morimoto, RI; Thiele, DJ
MLA Citation
Ahn, SG, Liu, PC, Klyachko, K, Morimoto, RI, and Thiele, DJ. "The loop domain of heat shock transcription factor 1 dictates DNA-binding specificity and responses to heat stress." Genes Dev 15.16 (August 15, 2001): 2134-2145.
PMID
11511544
Source
pubmed
Published In
Genes & development
Volume
15
Issue
16
Publish Date
2001
Start Page
2134
End Page
2145
DOI
10.1101/gad.894801

Identification of a novel high affinity copper transport complex in the fission yeast Schizosaccharomyces pombe.

Copper is an essential nutrient that serves as a co-factor for enzymes involved in critical cellular processes including energy generation, peptide hormone maturation, oxidative stress protection, and iron homeostasis. Although genes have been identified from yeast and mammals encoding a homologous subunit of a plasma membrane high affinity copper transporter, the presence of additional subunits that function as part of a copper transport complex has not been reported. We observed that ctr4(+), a previously identified copper transport protein from the fission yeast Schizosaccharomyces pombe, fails to complement bakers' yeast cells defective in high affinity copper transport and fails to be targeted to the plasma membrane. However, selection for S. pombe genes, which, when co-expressed with Ctr4, confer high affinity copper transport to S. cerevisiae cells resulted in the identification of ctr5(+). Both Ctr4 and Ctr5 are integral membrane proteins, are co-regulated by copper levels and the copper-sensing transcription factor Cuf1, physically associate in vivo, are interdependent for secretion to the plasma membrane, and are each essential for high affinity copper transport. These studies in S. pombe identify Ctr4 and Ctr5 as components of a novel eukaryotic heteromeric plasma membrane complex that is essential for high affinity copper transport.

Authors
Zhou, H; Thiele, DJ
MLA Citation
Zhou, H, and Thiele, DJ. "Identification of a novel high affinity copper transport complex in the fission yeast Schizosaccharomyces pombe." J Biol Chem 276.23 (June 8, 2001): 20529-20535.
PMID
11274192
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
23
Publish Date
2001
Start Page
20529
End Page
20535
DOI
10.1074/jbc.M102004200

Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development.

The trace metal copper (Cu) plays an essential role in biology as a cofactor for many enzymes that include Cu, Zn superoxide dismutase, cytochrome oxidase, ceruloplasmin, lysyl oxidase, and dopamine beta-hydroxylase. Consequently, Cu transport at the cell surface and the delivery of Cu to intracellular compartments are critical events for a wide variety of biological processes. The components that orchestrate intracellular Cu trafficking and their roles in Cu homeostasis have been elucidated by the studies of model microorganisms and by the characterizations of molecular basis of Cu-related genetic diseases, including Menkes disease and Wilson disease. However, little is known about the mechanisms for Cu uptake at the plasma membrane and the consequences of defects in this process in mammals. Here, we show that the mouse Ctr1 gene encodes a component of the Cu transport machinery and that mice heterozygous for Ctr1 exhibit tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. Mice completely deficient for Ctr1 exhibit profound growth and developmental defects and die in utero in mid-gestation. These results demonstrate a crucial role for Cu acquisition through the Ctr1 transporter for mammalian Cu homeostasis and embryonic development.

Authors
Lee, J; Prohaska, JR; Thiele, DJ
MLA Citation
Lee, J, Prohaska, JR, and Thiele, DJ. "Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development." Proc Natl Acad Sci U S A 98.12 (June 5, 2001): 6842-6847.
PMID
11391005
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
12
Publish Date
2001
Start Page
6842
End Page
6847
DOI
10.1073/pnas.111058698

The Candida glabrata Amt1 copper-sensing transcription factor requires Swi/Snf and Gcn5 at a critical step in copper detoxification.

The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper-inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome. Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE. In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels. We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific. Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE. Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation.

Authors
Koch, KA; Allard, S; Santoro, N; Côté, J; Thiele, DJ
MLA Citation
Koch, KA, Allard, S, Santoro, N, Côté, J, and Thiele, DJ. "The Candida glabrata Amt1 copper-sensing transcription factor requires Swi/Snf and Gcn5 at a critical step in copper detoxification." Mol Microbiol 40.5 (June 2001): 1165-1174.
PMID
11401720
Source
pubmed
Published In
Molecular Microbiology
Volume
40
Issue
5
Publish Date
2001
Start Page
1165
End Page
1174

Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3

Copper is an essential nutrient required for the activity of a number of enzymes with diverse biological roles. In the bakers' yeast Saccharomyces cerevisiae, copper is transported into cells by two high affinity copper transport proteins, Ctr1 and Ctr3. Although Ctr1 and Ctr3 are functionally redundant, they bear little homology at the amino acid sequence level. In this report, we characterize Ctr3 with respect to its localization, assembly, and post-transcriptional regulation. Ctr3 is an integral membrane protein that assembles as a trimer to form a competent copper uptake permease at the plasma membrahe. Whereas the CTR1 and CTR3 genes are similarly regulated at the transcriptional level in response to copper, post-transcriptional regulation of these proteins is distinct. Unlike Ctr1, the Ctr3 transporter is neither regulated at the level of protein degradation nor endocytosis as a function of elevated copper levels. Our studies suggest that Ctr3 constitutes a fundamental module found in all eukaryotic high affinity copper transporters to date, which is sufficient for copper uptake but lacks elements for post-transcriptional regulation by copper.

Authors
Pena, MMO; Puig, S; Thiele, DJ
MLA Citation
Pena, MMO, Puig, S, and Thiele, DJ. "Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3." Journal of Biological Chemistry 275.43 (2000): 33244-33251.
PMID
10924521
Source
scival
Published In
Journal of Biological Chemistry
Volume
275
Issue
43
Publish Date
2000
Start Page
33244
End Page
33251
DOI
10.1074/jbc.M005392200

Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant

A polymerase chain reaction (PCR)-based strategy was used to isolate a mouse cDNA (mCtr1) encoding a Cu transport protein. The deduced mCtr1 protein sequence exhibits 92% identity to human Ctr1, and has structural features in common with known high affinity Cu transporters from yeast. The expression of mouse Ctr1 functionally complements baker's yeast cells defective in high affinity Cu transport. Characterization of the mCtr1 genomic clone showed that the mCtr1 coding sequence is encompassed within four exons and that the mCtr1 locus maps to chromosome band 4C1-2. RNA blotting analysis demonstrated that mCtr1 is ubiquitously expressed, with high levels in liver and kidney, and early in embryonic development. Steady state mammalian Ctr1 mRNA levels were not changed in response to cellular Cu availability, which is distinct from the highly Cu-regulated transcription of genes encoding yeast high affinity Cu transporters. These studies provide fundamental information for further investigations on the function and regulation of Ctr1 in Cu acquisition in mammals. (C) 2000 Elsevier Science B.V.

Authors
Lee, J; Prohaska, JR; Dagenais, SL; Glover, TW; Thiele, DJ
MLA Citation
Lee, J, Prohaska, JR, Dagenais, SL, Glover, TW, and Thiele, DJ. "Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant." Gene 254.1-2 (2000): 87-96.
PMID
10974539
Source
scival
Published In
Gene
Volume
254
Issue
1-2
Publish Date
2000
Start Page
87
End Page
96
DOI
10.1016/S0378-1119(00)00287-0

Effects of nitric oxide on the copper-responsive transcription factor Ace1 in Saccharomyces cerevisiae: Cytotoxic and cytoprotective actions of nitric oxide

Previous studies indicate that nitric oxide (NO) can serve as a regulator/disrupter of metal-metabolizing systems in cells and, indeed, this function may represent an important physiological and/or pathophysiological role for NO. In order to address possible mechanisms of this aspect of NO biology, the effect of NO on copper metabolism and toxicity in the yeast Saccharomyces cerevisiae was examined. Exposure of S. cerevisiae to NO resulted in an alteration of the activity of the copper-responsive transcription factor Ace1. Low concentrations of the NO donor DEA/NO were found to slightly enhance copper-mediated activation of Ace1. Since Ace1 regulates the expression of genes responsible for the protection of S. cerevisiae from metal toxicity, the effect of NO on the toxicity of copper toward S. cerevisiae was also examined. Interestingly, low concentrations of NO were also found to protect S. cerevisiae against the toxicity of copper. The effect of NO at high concentrations was, however, opposite. High concentrations of DEA/NO inhibited copper-mediated Ace1 activity. Correspondingly, high concentrations of DEA/NO (1 mM) dramatically enhanced copper toxicity. An intermediate concentration of DEA/NO (0.5 mM) exhibited a dual effect, enhancing toxicity at lower copper concentrations (< 0.5 mM) and protecting at higher (≥0.5 mM) copper concentrations. Thus, it is proposed that the ability of NO to both protect against (at low concentrations) and enhance (at high concentration) copper toxicity in S. cerevisiae is, at least partially, a result of its effect on Ace1. The results of this study have implications for the role of NO as a mediator of metal metabolism. (C) 2000 Academic Press.

Authors
Chiang, KT; Shinyashiki, M; Switzer, CH; Valentine, JS; Gralla, EB; Thiele, DJ; Fukuto, JM
MLA Citation
Chiang, KT, Shinyashiki, M, Switzer, CH, Valentine, JS, Gralla, EB, Thiele, DJ, and Fukuto, JM. "Effects of nitric oxide on the copper-responsive transcription factor Ace1 in Saccharomyces cerevisiae: Cytotoxic and cytoprotective actions of nitric oxide." Archives of Biochemistry and Biophysics 377.2 (2000): 296-303.
PMID
10845707
Source
scival
Published In
Archives of Biochemistry and Biophysics
Volume
377
Issue
2
Publish Date
2000
Start Page
296
End Page
303
DOI
10.1006/abbi.2000.1785

The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A model system for NO-protein thiol interactions with implications to metal metabolism

Nitric oxide (NO) was found to inhibit the copper-dependent induction of the yeast CUP1 gene. This effect is attributable to an inhibition of the copper-responsive CUP1 transcriptional activator Ace1. A mechanism is proposed whereby the metal binding thiols of Ace1 are chemically modified via NO- and O2-dependent chemistry, thereby diminishing the ability of Ace1 to bind and respond to copper. Moreover, it is proposed that demetallated Ace1 is proteolytically degraded in the cell, resulting in a prolonged inhibition of copper-dependent CUP1 induction. These findings indicate that NO may serve as a disrupter of yeast copper metabolism. More importantly, considering the similarity of Ace1 to other mammalian metal-binding proteins, this work lends support to the hypothesis that NO may regulate/disrupt metal homeostasis under both normal physiological and pathophysiological circumstances.

Authors
Shinyashiki, M; Chiang, KT; Switzer, CH; Gralla, EB; Valentine, JS; Thiele, DJ; Fukuto, JM
MLA Citation
Shinyashiki, M, Chiang, KT, Switzer, CH, Gralla, EB, Valentine, JS, Thiele, DJ, and Fukuto, JM. "The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A model system for NO-protein thiol interactions with implications to metal metabolism." Proceedings of the National Academy of Sciences of the United States of America 97.6 (2000): 2491-2496.
PMID
10694579
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
6
Publish Date
2000
Start Page
2491
End Page
2496
DOI
10.1073/pnas.050586597

Pipes and wiring: The regulation of copper uptake and distribution in yeast

Copper is required for processes as conserved as respiration and as specialized as protein modification. Recent exciting findings from studies in yeast cells have revealed the presence of specific pathways for copper transport, trafficking and signal transduction that maintain the delicate balance of this essential yet toxic metal ion.

Authors
Labbé, S; Thiele, DJ
MLA Citation
Labbé, S, and Thiele, DJ. "Pipes and wiring: The regulation of copper uptake and distribution in yeast." Trends in Microbiology 7.12 (1999): 500-505.
PMID
10603486
Source
scival
Published In
Trends in Microbiology
Volume
7
Issue
12
Publish Date
1999
Start Page
500
End Page
505
DOI
10.1016/S0966-842X(99)01638-8

A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe

Copper and iron serve essential functions as catalytic co-factors in a wide variety of critical cellular enzymes. Studies in yeast have demonstrated an absolute dependence upon copper acquisition for proper assembly and function of the iron transport machinery. We have cloned genes for a high affinity copper transporter (Ctr4) and copper-sensing transcription factor (Cuf1) from Schizosaccharomyces pombe. Interestingly, the primary structure of Ctr4 and a putative human high affinity copper transport protein, hCtr1, suggests that they are derived from a fusion of the functionally redundant but structurally distinct Ctr1 and Ctr3 copper transporters from Saccharomyces cerevisiae. Furthermore, although Cuf1 activates ctr4+ gene expression under copper starvation conditions, under these same conditions Cuf1 directly represses expression of genes encoding components of the iron transport machinery. These studies have identified an evolutionary step in which copper transport modules have been fused, and describe a mechanism by which a copper-sensing factor directly represses expression of the iron uptake genes under conditions in which the essential copper co-factor is scarce.

Authors
Labbé, S; Peña, MMO; Fernandes, AR; Thiele, DJ
MLA Citation
Labbé, S, Peña, MMO, Fernandes, AR, and Thiele, DJ. "A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe." Journal of Biological Chemistry 274.51 (1999): 36252-36260.
PMID
10593913
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
51
Publish Date
1999
Start Page
36252
End Page
36260
DOI
10.1074/jbc.274.51.36252

The mammalian HSF4 gene generates both an activator and a repressor of heat shock genes by alternative splicing

The expression of heat shock genes is controlled at the level of transcription by members of the heat shock transcription factor family in vertebrates. HSF4 is a mammalian factor characterized by its lack of a suppression domain that modulates formation of DNA-binding homotrimer. Here, we have determined the exon structure of the human HSF4 gene and identified a major new isoform, HSF4b, derived by alternative RNA splicing events, in addition to a previously reported HSF4a isoform. In mouse tissues HSF4b mRNA was more abundant than HSF4a as examined by reverse transcription-polymerase chain reaction, and its protein was detected in the brain and lung. Although both mouse HSF4a and HSF4b form trimers in the absence of stress, these two isoforms exhibit different transcriptional activity; HSF4a acts as an inhibitor of the constitutive expression of heat shock genes, and hHSF4b acts as a transcriptional activator. Furthermore HSF4b but not HSF4a complements the viability defect of yeast cells lacking HSF. Moreover, heat shock and other stresses stimulate transcription of target genes by HSF4b in both yeast and mammalian cells. These results suggest that differential splicing of HSF4 mRNA gives rise to both an inhibitor and activator of tissue-specific heat shock gene expression.

Authors
Tanabe, M; Sasai, N; Nagata, K; Liu, X-D; Liu, PCC; Thiele, DJ; Nakai, A
MLA Citation
Tanabe, M, Sasai, N, Nagata, K, Liu, X-D, Liu, PCC, Thiele, DJ, and Nakai, A. "The mammalian HSF4 gene generates both an activator and a repressor of heat shock genes by alternative splicing." Journal of Biological Chemistry 274.39 (1999): 27845-27856.
PMID
10488131
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
39
Publish Date
1999
Start Page
27845
End Page
27856
DOI
10.1074/jbc.274.39.27845

The Sch9 protein kinase regulates Hsp90 chaperone complex signal transduction activity in vivo

Basal and stress-induced synthesis of the components of the highly conserved heat shock protein Hsp90 chaperone complex require the heat shock transcription factor (HSF); Saccharomyces cerevisiae cells expressing the HSF allele HSF(1-583) reversibly arrest growth at 37°C in the G2/M phase of the cell cycle due to diminished expression of these components. A suppressor mutant capable of restoring high-temperature growth to HSF(1-583) cells was identified, harboring a disruption of the SCH9 protein kinase gene, homologous to the protein kinase A and protein kinase B/Akt families of mammalian growth control kinases. Loss of Sch9 in HSF(1-583) cells derepresses Hsp90 signal transduction functions as demonstrated by restoration of transcriptional activity by the mammalian glucocorticoid receptor and the heme-dependent transcription factor Hap1, and by enhanced pheromone-dependent signaling through the Ste11 mitogen-activated protein kinase (MAPK). Moreover, Sch9-deficient cells with normal levels of Hsp90 chaperone complex components display hyperactivation of the pheromone response MAPK pathway in the absence of pheromone. These results demonstrate that the evolutionarily conserved function of the Hsp90 chaperone complex as a signal transduction facilitator is modulated by a growth regulatory kinase.

Authors
Morano, KA; Thiele, DJ
MLA Citation
Morano, KA, and Thiele, DJ. "The Sch9 protein kinase regulates Hsp90 chaperone complex signal transduction activity in vivo." EMBO Journal 18.21 (1999): 5953-5962.
PMID
10545107
Source
scival
Published In
EMBO Journal
Volume
18
Issue
21
Publish Date
1999
Start Page
5953
End Page
5962
DOI
10.1093/emboj/18.21.5953

Functional analysis of a homopolymeric (dA-dT) element that provides nucleosomal access to yeast and mammalian transcription factors

Eukaryotic organisms ranging from yeast to humans maintain a large amount of genetic information in the highly compact folds of chromatin, which poses a large DNA accessibility barrier to rapid changes in gene expression. The ability of the yeast Candida glabrata to survive copper insult requires rapid transcriptional autoactivation of the AMT1 copper-metalloregulatory transcription factor gene. The kinetics of AMT1 autoactivation is greatly enhanced by homopolymeric (dA-dT) element (A16)-mediated nucleosomal accessibility for Amt1p to a metal response element in this promoter. Analysis of the nucleosomal positional requirements for the A16 element reveal an impaired ability of the A16 element to stimulate AMT1 autoregulation when positioned downstream of the metal response element within the nucleosome, implicating an inherent asymmetry to the nucleosome positioned within the AMT1 promoter. Importantly, we demonstrate that the A16 element functions to enhance nucleosomal access and hormone-stimulated transcriptional activation for the mammalian glucocorticoid receptor, in a rotational phase-dependent manner. These data provide compelling evidence that nucleosomal homopolymeric (dA-dT) elements provide enhanced DNA access to diverse classes of transcription factors and suggest that these elements may function in this manner to elicit rapid transcriptional responses in higher eukaryotic organisms.

Authors
Koch, KA; Thiele, DJ
MLA Citation
Koch, KA, and Thiele, DJ. "Functional analysis of a homopolymeric (dA-dT) element that provides nucleosomal access to yeast and mammalian transcription factors." Journal of Biological Chemistry 274.34 (1999): 23752-23760.
PMID
10446135
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
34
Publish Date
1999
Start Page
23752
End Page
23760
DOI
10.1074/jbc.274.34.23752

Modulation of human heat shock factor trimerization by the linker domain

Heat shock transcription factors (HSFs) are stress-responsive proteins that activate the expression of heat shock genes and are highly conserved from bakers' yeast to humans. Under basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a homotrimer that binds to its cognate DNA binding site with high affinity. To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system. Whereas wild-type human HSF1 is restrained as an inactive monomer in yeast that is unable to substitute for the essential yeast HSF protein, mutations in the linker region between the DNA binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue the viability defect of a hsfΔ strain. Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells. Although linker regions in transcription factors are known to modulate DNA binding specificity, our studies suggest that the human HSF1 linker plays no role in determining HSF1 binding preferences in vivo but is a critical determinant in regulating the HSF1 monomer-trimer equilibrium.

Authors
Liu, PCC; Thiele, DJ
MLA Citation
Liu, PCC, and Thiele, DJ. "Modulation of human heat shock factor trimerization by the linker domain." Journal of Biological Chemistry 274.24 (1999): 17219-17225.
PMID
10358080
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
24
Publish Date
1999
Start Page
17219
End Page
17225
DOI
10.1074/jbc.274.24.17219

The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone

In eukaryotes, production of the diverse repertoire of molecular chaperones during normal growth and in response to stress is governed by the heat shock transcription factor HSF. The HSC82 and HSP82 genes, encoding isoforms of the yeast Hsp90 molecular chaperone, were recently identified as targets of the HSF carboxyl-terminal activation domain (CTA), whose expression is required for cell cycle progression during prolonged heat stress conditions. In the present study, we have identified additional target genes of the HSF CTA, which include nearly all of the heat shock-inducible members of the Hsp90 chaperone complex, demonstrating coordinate regulation of these components by HSF. Heat shock induction of SSE1, encoding a member of the Hsp110 family of heat shock proteins, was also dependent on the HSF CTA. Disruption of SSE1 along with STI1, encoding an established subunit of the Hsp90 chaperone complex, resulted in a severe synthetic growth phenotype. Sse1 associated with partially purified Hsp90 complexes and deletion of the SSE1 gene rendered cells susceptible to the Hsp90 inhibitors macbecin and geldanamycin, suggesting functional interaction between Sse1 and Hsp90. Sse1 is required for function of the glucocorticoid receptor, a model substrate of the Hsp90 chaperone machinery, and Hsp90-based repression of HSF under nonstress conditions. Taken together, these data establish Sse1 as an integral new component of the Hsp90 chaperone complex in yeast.

Authors
Liu, X-D; Morano, KA; Thiele, DJ
MLA Citation
Liu, X-D, Morano, KA, and Thiele, DJ. "The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone." Journal of Biological Chemistry 274.38 (1999): 26654-26660.
PMID
10480867
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
38
Publish Date
1999
Start Page
26654
End Page
26660
DOI
10.1074/jbc.274.38.26654

A trans-activation domain in yeast heat shock transcription factor is essential for cell cycle progression during stress

Gene expression in response to heat shock is mediated by the heat shock transcription factor (HSF), which in yeast harbors both amino- and carboxyl- terminal transcriptional activation domains. Yeast cells bearing a truncated form of HSF in which the carboxyl-terminal transcriptional activation domain has been deleted [HSF(1-583)] are temperature sensitive for growth at 37°C, demonstrating a requirement for this domain for sustained viability during thermal stress. Here we demonstrate that HSF(1-583) cells undergo reversible cell cycle arrest at 37°C in the G2/M phase of the cell cycle and exhibit marked reduction in levels of the molecular chaperone Hsp90. As in higher eukaryotes, yeast possesses two nearly identical isoforms of Hsp90: one constitutively expressed and one highly heat inducible. When expressed at physiological levels in HSF(1-583) cells, the inducible Hsp90 isoform encoded by HSP82 more efficiently suppressed the temperature sensitivity of this strain than the constitutively expressed gene HSC82, suggesting that different functional roles may exist for these chaperones. Consistent with a defect in Hsp90 production, HSF(1-583) cells also exhibited hypersensitivity to the Hsp90-binding ansamycin antibiotic geldanamycin. Depletion of Hsp90 from yeast cells wild type for HSF results in cell cycle arrest in both G1/S and G2/M phases, suggesting a complex requirement for chaperone function in mitotic division during stress.

Authors
Morano, KA; Santoro, N; Koch, KA; Thiele, DJ
MLA Citation
Morano, KA, Santoro, N, Koch, KA, and Thiele, DJ. "A trans-activation domain in yeast heat shock transcription factor is essential for cell cycle progression during stress." Molecular and Cellular Biology 19.1 (1999): 402-411.
PMID
9858564
Source
scival
Published In
Molecular and Cellular Biology
Volume
19
Issue
1
Publish Date
1999
Start Page
402
End Page
411

A delicate balance: Homeostatic control of copper uptake and distribution

The cellular uptake and intracellular distribution of the essential but highly toxic nutrient, copper, is a precisely orchestrated process. Copper homeostasis is coordinated by several proteins to ensure that it is delivered to specific subcellular compartments and copper-requiring proteins without releasing free copper ions that will cause damage to cellular components. Genetic studies in prokaryotic organisms and yeast have identified membrane- associated proteins that mediate the uptake or export of copper from cells. Within cells, small cytosolic proteins, called copper chaperones, have been identified that bind copper ions and deliver them to specific compartments and copper-requiring proteins. The identification of mammalian homologues of these proteins reveal a remarkable structural and functional conservation of copper metabolism between bacteria, yeast and humans. Furthermore, studies on the function and localization of the products of the Menkes and Wilson's disease genes, which are defective in patients afflicted with these diseases, have provided valuable insight into the mechanisms of copper balance and their role in maintaining appropriate copper distribution in mammals.

Authors
Peña, MMO; Lee, J; Thiele, DJ
MLA Citation
Peña, MMO, Lee, J, and Thiele, DJ. "A delicate balance: Homeostatic control of copper uptake and distribution." Journal of Nutrition 129.7 (1999): 1251-1260.
PMID
10395584
Source
scival
Published In
The Journal of nutrition
Volume
129
Issue
7
Publish Date
1999
Start Page
1251
End Page
1260

Copper ion inducible and repressible promoter systems in yeast

Authors
Labbé, S; Thiele, DJ
MLA Citation
Labbé, S, and Thiele, DJ. "Copper ion inducible and repressible promoter systems in yeast." Methods in Enzymology 306 (1999): 145-153.
PMID
10432452
Source
scival
Published In
Methods in Enzymology
Volume
306
Publish Date
1999
Start Page
145
End Page
153
DOI
10.1016/S0076-6879(99)06010-3

Heat shock factor function and regulation in response to cellular stress, growth, and differentiation signals

Heat shock factors (HSF) activate the transcription of genes encoding products required for protein folding, processing, targeting, degradation, and function. Although HSFs have been extensively studied with respect to their role in thermotolerance and the activation of gene expression in response to environmental stress, the involvement of HSFs in response to stresses associated with cell growth and differentiation, and in response to normal physiological processes is becoming increasingly clear. In this work, we review recent advances toward understanding how cells transmit growth control and developmental signals, and interdigitate cellular physiology, to regulate HSF function.

Authors
Morano, KA; Thiele, DJ
MLA Citation
Morano, KA, and Thiele, DJ. "Heat shock factor function and regulation in response to cellular stress, growth, and differentiation signals." Gene Expression 7.4-6 (1999): 271-282.
PMID
10440228
Source
scival
Published In
Gene expression
Volume
7
Issue
4-6
Publish Date
1999
Start Page
271
End Page
282

Protein chaperones and the heat shock response in Saccharomyces cerevisiae

Recent studies have shed new light on the complexities of the heat shock response in yeast. Multiple pathways for transcriptional induction of both classic and novel heat shock proteins are emerging together with a more detailed understanding of the interactions between protein chaperones and their physiological targets. New roles for heat shock proteins in defense and recovery from the impacts of thermal stress on critical cellular processes have expanded our understanding of these elaborate and ubiquitous proteins.

Authors
Morano, KA; Liu, PCC; Thiele, DJ
MLA Citation
Morano, KA, Liu, PCC, and Thiele, DJ. "Protein chaperones and the heat shock response in Saccharomyces cerevisiae." Current Opinion in Microbiology 1.2 (1998): 197-203.
PMID
10066474
Source
scival
Published In
Current Opinion in Microbiology
Volume
1
Issue
2
Publish Date
1998
Start Page
197
End Page
203

Heat shock element architecture is an important determinant in the temperature and transactivation domain requirements for heat shock transcription factor

The baker's yeast Saccharomyces cerevisiae possesses a single gene encoding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability. Yeast HSF (yHSF) contains two distinct transcriptional activation regions located at the amino and carboxyl termini. Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF. The results described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1 HSE (HSE1) functions to limit the magnitude of CUP1 transcriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA. Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the transcriptional activation of CUP1, via HSE1, in response to stress. In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1. Limited proteolysis assays show that HSF adopts a distinct protease-sensitive conformation when bound to the CUP1 HSE1, providing evidence that the HSE influences DNA-bound HSF conformation. Together, these results suggest that CUP1 regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs. Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those of CUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.

Authors
Santoro, N; Johansson, N; Thiele, DJ
MLA Citation
Santoro, N, Johansson, N, and Thiele, DJ. "Heat shock element architecture is an important determinant in the temperature and transactivation domain requirements for heat shock transcription factor." Molecular and Cellular Biology 18.11 (1998): 6340-6352.
PMID
9774650
Source
scival
Published In
Molecular and Cellular Biology
Volume
18
Issue
11
Publish Date
1998
Start Page
6340
End Page
6352

Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae

The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity. These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.

Authors
Peña, MMO; Koch, KA; Thiele, DJ
MLA Citation
Peña, MMO, Koch, KA, and Thiele, DJ. "Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae." Molecular and Cellular Biology 18.5 (1998): 2514-2523.
PMID
9599102
Source
scival
Published In
Molecular and Cellular Biology
Volume
18
Issue
5
Publish Date
1998
Start Page
2514
End Page
2523

Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor

Copper is an essential metal ion that is toxic when accumulated to high intracellular concentrations. The yeast Mac1 protein is a copper-sensing transcription factor that is essential for both the activation and inactivation of genes required for high affinity copper ion transport. Here we demonstrate that in response to low copper ion concentrations Mac1 protein is rendered inactive for copper transporter gene transcription. Under high copper ion concentrations Mac1 is degraded in a rapid, copper-specific manner. This degradation is critical to prevent copper toxicity that would otherwise result from sustained expression of the copper transport genes. These results demonstrate that nutritional and toxic copper concentrations elicit distinct fates for the Mac1 copper-sensing transcription factor and establish a new mechanism by which trace metals regulate gene expression.

Authors
Zhu, Z; Labbé, S; Peña, MMO; Thiele, DJ
MLA Citation
Zhu, Z, Labbé, S, Peña, MMO, and Thiele, DJ. "Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor." Journal of Biological Chemistry 273.3 (1998): 1277-1280.
PMID
9430656
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
3
Publish Date
1998
Start Page
1277
End Page
1280
DOI
10.1074/jbc.273.3.1277

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription.

The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly for acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution of redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions.

Authors
Szczypka, MS; Zhu, Z; Silar, P; Thiele, DJ
MLA Citation
Szczypka, MS, Zhu, Z, Silar, P, and Thiele, DJ. "Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription." Yeast (Chichester, England) 13.15 (December 1997): 1423-1435.
PMID
9434348
Source
epmc
Published In
Yeast
Volume
13
Issue
15
Publish Date
1997
Start Page
1423
End Page
1435
DOI
10.1002/(sici)1097-0061(199712)13:15<1423::aid-yea190>3.3.co;2-3

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription

The metal ions, CU(2+/+) and Fe(3+/2+), are essential co-factors for a wide variety of enzymatic reactions. However, bath metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on CU2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly or acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis, Yeast cells lacking a functional PEP3 or PEPS gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA. levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution or redox states of Fe(3+/2+) in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions.

Authors
Szczypka, MS; Zhu, Z; Silar, P; Thiele, DJ
MLA Citation
Szczypka, MS, Zhu, Z, Silar, P, and Thiele, DJ. "Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription." Yeast 13.15 (1997): 1423-1435.
Source
scival
Published In
Yeast
Volume
13
Issue
15
Publish Date
1997
Start Page
1423
End Page
1435
DOI
10.1002/(SICI)1097-0061(199712)13:15<1423::AID-YEA190>3.0.CO;2-C

Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway

Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1 and CTR3- encoded membrane-associated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down- regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down- or up- regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

Authors
Labbé, S; Zhu, Z; Thiele, DJ
MLA Citation
Labbé, S, Zhu, Z, and Thiele, DJ. "Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway." Journal of Biological Chemistry 272.25 (1997): 15951-15958.
PMID
9188496
Source
scival
Published In
The Journal of biological chemistry
Volume
272
Issue
25
Publish Date
1997
Start Page
15951
End Page
15958
DOI
10.1074/jbc.272.25.15951

Copper-binding motifs in catalysis, transport, detoxification and signaling

Copper is required for many biological processes but is toxic at high cellular concentrations, so levels in the cell must be strictly controlled. Copper-binding motifs have been identified and characterized in many proteins. The way in which copper is coordinated by these motifs is important for the transport and distribution of intrecellular copper and for the effective functioning of copper-dependent enzymes.

Authors
Koch, KA; Peña, MMO; Thiele, DJ
MLA Citation
Koch, KA, Peña, MMO, and Thiele, DJ. "Copper-binding motifs in catalysis, transport, detoxification and signaling." Chemistry and Biology 4.8 (1997): 549-560.
PMID
9281528
Source
scival
Published In
Chemistry & Biology
Volume
4
Issue
8
Publish Date
1997
Start Page
549
End Page
560

Yeast metallothionein gene expression in response to metals and oxidative stress

Metals and oxygen are chemically linked in biological systems. Metals and oxygen play important roles in enzymatic reactions, metabolism, and signal transduction; however, metals and oxygen react to form highly toxic oxygen-derived free radical species. In this review we focus on the use of yeast cells, as unicellular eukaryotic model systems, to conduct studies aimed at understanding fundamental mechanisms for the sensation and protective responses to toxic metals and oxygen-derived radicals via the activation of yeast metallothionein gene expression.

Authors
Liu, X-D; Thiele, DJ
MLA Citation
Liu, X-D, and Thiele, DJ. "Yeast metallothionein gene expression in response to metals and oxidative stress." Methods: A Companion to Methods in Enzymology 11.3 (1997): 289-299.
PMID
9073572
Source
scival
Published In
Methods
Volume
11
Issue
3
Publish Date
1997
Start Page
289
End Page
299
DOI
10.1006/meth.1996.0423

Conservation of a stress response: Human heat shock transcription factors functionally substitute for yeast HSF

Heat shock factors (HSF) are important eukaryotic stress responsive transcription factors which are highly structurally conserved from yeast to mammals. HSFs bind as homotrimers to conserved promoter DNA recognition sites called HSEs. The baker's yeast Saccharomyces cerevisiae possesses a single essential HSF gene, while distinct HSF isoforms have been identified in humans. To ascertain the degree of functional similarity between the yeast and human HSF proteins, human HSF1 and HSF2 were expressed in yeast cells lacking the endogenous HSF gene. We demonstrate that human HSF2, but not HSF1, homotrimerizes and functionally complements the viability defect associated with a deletion of the yeast HSF gene. However, derivatives of hHSF1 that give rise to a trimerized protein, through disruption of a carboxyl- or aminoterminal coiled-coil domain thought to engage in intramolecular interactions that maintain the protein in a monomeric state, functionally substitute for yeast HSF. Surprisingly, hHSF2 expressed in yeast activates target gene transcription in response to thermal stress. Moreover, hHSF1 and hHSF2 exhibit selectivity for transcriptional activation of two distinct yeast heat shock responsive genes, which correlate with previously established mammalian HSF DNA binding preferences in vitro. These results provide new insight into the function of human HSF isoforms, and demonstrate the remarkable functional conservation between yeast and human HSFs, critical transcription factors required for responses to physiological, pharmacological and environmental stresses.

Authors
Liu, X-D; Liu, PCC; Santoro, N; Thiele, DJ
MLA Citation
Liu, X-D, Liu, PCC, Santoro, N, and Thiele, DJ. "Conservation of a stress response: Human heat shock transcription factors functionally substitute for yeast HSF." EMBO Journal 16.21 (1997): 6466-6477.
PMID
9351828
Source
scival
Published In
EMBO Journal
Volume
16
Issue
21
Publish Date
1997
Start Page
6466
End Page
6477
DOI
10.1093/emboj/16.21.6466

A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium

The yeast cadmium factor (YCF1) gene encodes an MgATP-energized glutathione S-conjugate transporter responsible for the vacuolar sequestration of organic compounds after their S-conjugation with glutathione. However, while YCF1 was originally isolated according to its ability to confer resistance to cadmium salts, neither its mode of interaction with Cd2+ nor the relationship between this process and organic glutathione-conjugate transport are known. Here we show through direct comparisons between vacuolar membrane vesicles purified from Saccharomyces cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild-type strain DTY165 that YCF1 mediates the MgATP-energized vacuolar accumulation of Cd·glutathione complexes. The substrate requirements, kinetics and Cd2+/glutathione stoichiometry of cadmium uptake and the molecular weight of the transport-active complex demonstrate that YCF1 selectively catalyzes the transport of bis(glutathionato)cadmium (Cd·GS2). On the basis of these results-the Cd2+ hypersensitivity of DTY167, versus DTY165, cells, the inducibility of YCF1-mediated transport, and the rapidity and spontaneity of Cd·GS2 formation-this new pathway is concluded to contribute substantially to Cd2+ detoxification.

Authors
Li, Z-S; Lu, Y-P; Zhen, R-G; Szczypka, M; Thiele, DJ; Rea, PA
MLA Citation
Li, Z-S, Lu, Y-P, Zhen, R-G, Szczypka, M, Thiele, DJ, and Rea, PA. "A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium." Proceedings of the National Academy of Sciences of the United States of America 94.1 (1997): 42-47.
PMID
8990158
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
1
Publish Date
1997
Start Page
42
End Page
47
DOI
10.1073/pnas.94.1.42

High affinity copper transport in yeast

Kacchawmyces ccrevisiae has proven to be an ideal model organism to study trace metal metabolism in eukaryotes. Here we describe a protein (Ctr3p) that restores; Cu and Fe uptake, growth on non-fermentable carbon sources, and Cu, Zn Superoxide d ismutase activity to strains lacking the copper transport protein, Ctrlp. CTR3 gene expression is inactivated in the majority of yeast strains by a transposable element within the CTR3 gene promoter. In yeast cells expressing both the CTR2 and CTR3 genes the inactivation of both genes is required to generate a Cu-deficient phonotype. In these cells CTR3 mRNA and Ctr3p protein levels are repressed by Cu. CtrSp is a 241 amino acid membrane protein with eleven cysteine residues that is located in the endoplasmic reticulum and secretory compartments of the cell. The functional independence of Ctrlp and Ctr3p suggest that yeast have two high affinity Cu uptake pathways. Funding: NIH fellowship F32 GM 15662-01 to S.A.B.K., NIH grants UP1 GM46787 to D.|.K. and ROI CM41840 to D.J.T.

Authors
Knight, SAB; Kwon, LF; Kosman, DI; Thiele, DI
MLA Citation
Knight, SAB, Kwon, LF, Kosman, DI, and Thiele, DI. "High affinity copper transport in yeast." FASEB Journal 10.3 (1996): A293-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
3
Publish Date
1996
Start Page
A293

A specialized nucleosome modulates transcription factor access to a C. glabrata metal responsive promoter

The ability of DNA binding transcription factors to access cis-acting promoter elements is critical for transcriptional responses. We demonstrate that rapid transcriptional autoactivation by the Amt1 Cu metalloregulatory transcription factor from the opportunistic pathogenic yeast Candida glabrata is dependent on rapid metal-induced DNA binding to a single metal response element (MRE). In vive footprinting and chromatin-mapping experiments demonstrate that the MRE and a homopolymeric (dA · dT) element adjacent to the MRE are packaged into a positioned nucleosome that exhibits homopolymeric (dA · dT)-dependent localized distortion. This distortion is critical for rapid Amt1 binding to the MRE, for Cu-dependent AMT1 gene transcription, and for C. glabrata cells to mount a rapid transcriptional response to Cu for normal metal detoxification. The AMT1 promoter represents a novel class of specialized nucleosomal structures that links rapid transcriptional responses to the biology of metal homeostasis.

Authors
Zhu, Z; Thiele, DJ
MLA Citation
Zhu, Z, and Thiele, DJ. "A specialized nucleosome modulates transcription factor access to a C. glabrata metal responsive promoter." Cell 87.3 (1996): 459-470.
PMID
8898199
Source
scival
Published In
Cell
Volume
87
Issue
3
Publish Date
1996
Start Page
459
End Page
470
DOI
10.1016/S0092-8674(00)81366-5

Oxidative stress induces heat shock factor phosphorylation and HSF-dependent activation of yeast metallothionein gene transcription

Metallothioneins (MTs) are a class of low-molecular-weight, cysteine-rich metal-binding proteins that function in metal detoxification and oxidative stress protection. We demonstrate that transcription of the Saccharomyces cerevisiae MT gene CUP1 is strongly activated by the superoxide anion generator menadione. This activation is exacerbated in a strain lacking the gene encoding Cu, Zn superoxide dismutase (SOD1). CUP1 transcriptional activation by oxidative stress is dependent on a functional CUP1 promoter heat shock element (HSE) and the carboxy-terminal trans-activation domain of heat shock transcription factor (HSF). Furthermore, protection against oxidative stress conferred by CUP1 in a sod1Δ strain requires HSF-mediated CUP1 transcription. Although in response to heat, HSF-mediated CUP1 transcription and HSF phosphorylation are transient, both CUP1 gene expression and HSF phosphorylation are sustained in response to oxidative stress. Moreover, the patterns of tryptic phosphopeptides resolved from HSF derived from cells subjected to heat shock or oxidative stress are distinct. These results demonstrate that transcription of the S. cerevisiae metallothionein gene under conditions of oxidative stress is mediated by HSF and that in response to distinct activation stimuli, HSF is differentially phosphorylated in a manner that parallels metallothionein gene transcription.

Authors
Liu, X-D; Thiele, DJ
MLA Citation
Liu, X-D, and Thiele, DJ. "Oxidative stress induces heat shock factor phosphorylation and HSF-dependent activation of yeast metallothionein gene transcription." Genes and Development 10.5 (1996): 592-603.
PMID
8598289
Source
scival
Published In
Genes and Development
Volume
10
Issue
5
Publish Date
1996
Start Page
592
End Page
603

Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions

Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing- nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu- activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.

Authors
Koch, KA; Thiele, DJ
MLA Citation
Koch, KA, and Thiele, DJ. "Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions." Molecular and Cellular Biology 16.2 (1996): 724-734.
PMID
8552101
Source
scival
Published In
Molecular and Cellular Biology
Volume
16
Issue
2
Publish Date
1996
Start Page
724
End Page
734

The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump

The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, which was isolated according to its ability to confer cadmium resistance, encodes a 1,515-amino acid ATP-binding cassette (ABC) protein with extensive sequence homology to the human multidrug resistance-associated protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, D. J. (1994) J. Biol. Chem. 269, 22853-22857). Direct comparisons between S. cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild type strain, DTY165, demonstrate that YCF1 is required for increased resistance to the toxic effects of the exogenous glutathione S-conjugate precursor, 1-chloro-2,4-dinitrobenzene, as well as cadmium. Whereas membrane vesicles isolated from DTY165 cells contain two major pathways for transport of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS), an MgATP-dependent, uncoupler-insensitive pathway and an electrically driven pathway, the corresponding membrane fraction from DTY167 cells is more than 90% impaired for MgATP-dependent, uncoupler-insensitive DNP-GS transport. Of the two DNP-GS transport pathways identified, only the MgATP-dependent, uncoupler-insensitive pathway is subject to inhibition by glutathione disulfide, vanadate, verapamil, and vinblastine. The capacity for MgATP-dependent, uncoupler-insensitive conjugate transport in vitro strictly copurifies with the vacuolar membrane fraction. Intact DTY165 cells, but not DTY167 cells, mediate vacuolar accumulation of the fluorescent glutathione-conjugate, monochlorobimane-GS. Introduction of plasmid borne, epitope-tagged gene encoding functional YCF1 into DTY167 cells alleviates the 1-chloro-2,4-dinitrobenzene-hypersensitive pheno-type concomitant with restoration of the capacity of vacuolar membrane vesicles isolated from these cells for MgATP-dependent, uncoupler-insensitive DNP-GS transport. On the basis of these findings, the YCF1 gene of S. cerevisiae is inferred to encode an MgATP-energized, uncoupler-insensitive vacuolar glutathione S-conjugate transporter. The energy requirements, kinetics, substrate specificity, and inhibitor profile of YCF1-mediated transport demonstrate that the vacuolar glutathione conjugate pump of yeast bears a strong mechanistic resemblance to the MRP1-encoded transporter of mammalian cells and the cognate, but as yet molecularly undefined, function of plant cells.

Authors
Li, Z-S; Szczypka, M; Lu, Y-P; Thiele, DJ; Rea, PA
MLA Citation
Li, Z-S, Szczypka, M, Lu, Y-P, Thiele, DJ, and Rea, PA. "The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump." Journal of Biological Chemistry 271.11 (1996): 6509-6517.
PMID
8626454
Source
scival
Published In
Journal of Biological Chemistry
Volume
271
Issue
11
Publish Date
1996
Start Page
6509
End Page
6517

Trace metal uptake by Pichia spartinae, an endosymbiotic yeast in the salt marsh cord grass Spartina alterniflora

Authors
Catallo, WJ; Henk, W; Younger, L; Mills, O; Thiele, DJ; Meyers, SP
MLA Citation
Catallo, WJ, Henk, W, Younger, L, Mills, O, Thiele, DJ, and Meyers, SP. "Trace metal uptake by Pichia spartinae, an endosymbiotic yeast in the salt marsh cord grass Spartina alterniflora." Chemistry and Ecology 13.2 (1996): 113-131.
Source
scival
Published In
Chemistry and Ecology
Volume
13
Issue
2
Publish Date
1996
Start Page
113
End Page
131

A widespread transposable element masks expression of a yeast copper transport gene

The trace element copper (Cu) is essential lot cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intracellular cysteine-rich integral membrane protein that restores high- affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTR1 and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctr1p and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

Authors
Knight, SAB; Labbé, S; Kwon, LF; Kosman, DJ; Thiele, DJ
MLA Citation
Knight, SAB, Labbé, S, Kwon, LF, Kosman, DJ, and Thiele, DJ. "A widespread transposable element masks expression of a yeast copper transport gene." Genes and Development 10.15 (1996): 1917-1929.
PMID
8756349
Source
scival
Published In
Genes & development
Volume
10
Issue
15
Publish Date
1996
Start Page
1917
End Page
1929

Toxic metal-responsive gene transcription.

Metals play a dual role in biological systems, serving as essential co-factors for a wide range of biochemical reactions yet these same metals may be extremely toxic to cells. To cope with the stress of increases in environmental metal concentrations, eukaryotic cells have developed sophisticated toxic metal sensing proteins which respond to elevations in metal concentrations. This signal is transmitted to stimulate the cellular transcriptional machinery to activate expression of metal detoxification and homeostasis genes. This review summarizes our current understanding of the biochemical and genetic mechanisms which underlie cellular responses to toxic metals via metalloregulatory transcription factors.

Authors
Zhu, Z; Thiele, DJ
MLA Citation
Zhu, Z, and Thiele, DJ. "Toxic metal-responsive gene transcription." EXS 77 (1996): 307-320.
PMID
8856982
Source
scival
Published In
EXS
Volume
77
Publish Date
1996
Start Page
307
End Page
320

Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1

Elevations in gene dosage of the transcriptional regulatory protein yAP-1 in Saccharomyces cerevisiae can elicit pronounced phenotypic increases in tolerance of a variety of drugs including the toxic heavy metal cadmium. While a large elevation in cadmium tolerance occurs in response to overproduction of yAP-1, the target genes under yAP-1 control have not yet been identified that are responsible for this increase. We show here that the YCF1 gene, encoding a likely integral membrane protein, is required for yAP- 1 to exert its normal effects on cadmium tolerance. Mutant strains of yeast that lack the YCF1 gene are hypersensitive to cadmium and this hypersensitivity is epistatic to yAP-1 overexpression. YCF1 mRNA levels and the expression of a YCF1-lacZ reporter construct positively correlates with changes in YAP1 gene dosage. A set of 5' truncation derivatives of the YCF1- lacZ fusion gene identified the region from -201 to +47 as being sufficient for the yAP-1-dependent increase in expression. DNase I footprinting using a probe from this segment of the YCF1 promoter showed that bacterially-produced yAP-1 protein was capable of binding a novel DNA element we have designated the yAP-1 response element. Insertion of the yAP-1 response element upstream of a CYC1-lacZ gene fusion led to the production of β-galactosidase in a yAP-1-dependent fashion. These data establish that an important physiological target of yAP-1 transcriptional regulation is the YCF1 structural gene.

Authors
Wemmie, JA; Szczypka, MS; Thiele, DJ; Moye-Rowley, WS
MLA Citation
Wemmie, JA, Szczypka, MS, Thiele, DJ, and Moye-Rowley, WS. "Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1." Journal of Biological Chemistry 269.51 (1994): 32592-32597.
PMID
7798263
Source
scival
Published In
Journal of Biological Chemistry
Volume
269
Issue
51
Publish Date
1994
Start Page
32592
End Page
32597

A system for gene cloning and manipulation in the yeast Candida glabrata

The opportunistic pathogenic yeast, Candida (Torulopsis) glabrata, is an asexual imperfect fungus that exists largely as a haploid. Besides being a clinically important pathogen, this yeast also provides a model system for understanding basic biological mechanisms such as metal-activated metallothionein-encoding gene transcription. To facilitate molecular genetic studies in C. glabrata, we isolated a strain auxotrophic for uracil biosynthesis. The ura- mutation could be functionally complemented by the URA3 gene of Saccharomyces cerevisiae, consistent with a defect in the C. glabrata URA3 gene in this strain. We also found that the centromere-based S. cerevisiae plasmid pRS316 could stably transform and replicate in multiple copies m C. glabrata. In contrast, high-copy-number S. cerevisiae plasmids containing the 2μ. circle autonomous replication sequence were not able to replicate productively in C. glabrata. We cloned the C. glabrata URA3 gene, encoding orotidine-5'-phosphate decarboxylase, by complementation of a ura3- strain of S. cerevisiae. The deduced amino-acid sequence is highly similar to that of the URA3 protein from S. cerevisiae. C. glabrata URA3 provides a genetic locus for targeted gene integration in C. glabrata. Integrative plasmids were constructed based on the cloned C. glabrata URA3 and are applicable for directed insertions of genes of interest at the ura3 locus through homologous recombination. © 1994.

Authors
Zhou, P; Szczypka, MS; Young, R; Thiele, DJ
MLA Citation
Zhou, P, Szczypka, MS, Young, R, and Thiele, DJ. "A system for gene cloning and manipulation in the yeast Candida glabrata." Gene 142.1 (1994): 135-140.
PMID
8181748
Source
scival
Published In
Gene
Volume
142
Issue
1
Publish Date
1994
Start Page
135
End Page
140

Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein

Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measurement of intracellular copper levels of the wild-type and cup9-1 mutant demonstrated that total intracellular copper concentrations were unaffected by CUP9. CUP9 mRNA levels were, however, down regulated by copper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was independent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino acids in which a 55-amino-acid sequence showed 47% identity with the homeobox domain of the human proto-oncogene PBX1, suggesting that CUP9 is a DNA-binding protein which regulates the expression of important copper homeostatic genes.

Authors
Knight, SAB; Tamai, KT; Kosman, DJ; Thiele, DJ
MLA Citation
Knight, SAB, Tamai, KT, Kosman, DJ, and Thiele, DJ. "Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein." Molecular and Cellular Biology 14.12 (1994): 7792-7804.
PMID
7969120
Source
scival
Published In
Molecular and Cellular Biology
Volume
14
Issue
12
Publish Date
1994
Start Page
7792
End Page
7804

Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways

Metallothioneins constitute a class of low-molecular-weight, cysteine- rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses. In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP1 promoter upstream regulatory region. CUP1 gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP1 gene expression transiently, whereas glucose starvation activates CUP1 gene expression in a sustained manner for at least 2.5 h. Although a carboxyl-terminal HSF transcriptional activation domain is critical for the activation of CUP1 transcription in response to both heat shock stress and glucose starvation, this region is dispensable for transient heat shock activation of at least two genes encoding members of the S. cerevisiae hsp70 family. Furthermore, inactivation of the chromosomal SNF1 gene, encoding a serine-threonine protein kinase, or the SNF4 gene, encoding a SNF1 cofactor, abolishes CUP1 transcriptional activation in response to glucose starvation without altering heat shock- induced transcription. These studies demonstrate that the S. cerevisiae HSF responds to multiple, distinct stimuli to activate yeast metallothionein gene transcription and that these stimuli elicit responses through nonidentical, genetically separable signalling pathways.

Authors
Tamai, KT; Liu, X; Silar, P; Sosinowski, T; Thiele, DJ
MLA Citation
Tamai, KT, Liu, X, Silar, P, Sosinowski, T, and Thiele, DJ. "Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways." Molecular and Cellular Biology 14.12 (1994): 8155-8165.
PMID
7969152
Source
scival
Published In
Molecular and Cellular Biology
Volume
14
Issue
12
Publish Date
1994
Start Page
8155
End Page
8165

A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein

Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides. The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). S. cerevisiae cells harboring a deletion of the YCF1 gene are hypersensitive to cadmium compared with wild type cells. Mutagenesis experiments demonstrate that conserved amino acid residues, functionally critical in hCFTR, play a vital role in YCF1-mediated cadmium resistance. Mutagenesis of phenylalanine 713 in the YCF1 nucleotide binding fold 1, which correlates with the ΔF508 mutation found in the most common form of cystic fibrosis, completely abolished YCF1 function in cadmium detoxification. Furthermore, substitution of a serine to alanine residue in a potential protein kinase A phosphorylation site in a central region of YCF1, which displays sequence similarity to the central regulatory domain of hCFTR, also rendered YCF1 nonfunctional. These results suggest that YCF1 is composed of modular domains found in human proteins which function in drug and ion transport.

Authors
Szczypka, MS; Wemmie, JA; Moye-Rowley, WS; Thiele, DJ
MLA Citation
Szczypka, MS, Wemmie, JA, Moye-Rowley, WS, and Thiele, DJ. "A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein." Journal of Biological Chemistry 269.36 (1994): 22853-22857.
PMID
7521334
Source
scival
Published In
Journal of Biological Chemistry
Volume
269
Issue
36
Publish Date
1994
Start Page
22853
End Page
22857

Old Yellow Enzyme: The discovery of multiple isozymes and a family of related proteins

Using fast protein liquid chromatography, we have separated native Old Yellow Enzyme from Brewer's Bottom Yeast into three distinct fractions. Two of these fractions are homodimeric forms of the enzyme while the third is the corresponding heterodimeric form. One of these homodimeric fractions is identical in every respect to OYE1, originally cloned from Brewer's Bottom Yeast (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). We have cloned, sequenced, and expressed a second Old Yellow Enzyme gene from Saccharomyces cerevisiae, showing close similarity, but not identity, with OYE1. Native Old Yellow Enzyme samples were also affinity-purified from a strain of S. cerevisiae and an OYE deletion mutant constructed from it. A total of at least seven isozymes of Old Yellow Enzyme have been discovered, each having slightly different characteristics ranging from surface charge to NADPH dehydrogenase activities with different electron acceptors, as well as N-terminal amino acid sequence. In addition, both recombinant enzymes showed considerable similarity to two proteins in the GenBank/EMBL data bank, a 60,000-dalton bile acid-inducible polypeptide in Eubacterium sp. (Mallonee, D. H., White, W. B., and Hylemon, P. B. (1990) J. Lipid Res. 172, 7011-7019) and a 72,000-dalton NADH oxidase in Thermoanaerobium brockii.

Authors
Stott, K; Saito, K; Thiele, DJ; Massey, V
MLA Citation
Stott, K, Saito, K, Thiele, DJ, and Massey, V. "Old Yellow Enzyme: The discovery of multiple isozymes and a family of related proteins." Journal of Biological Chemistry 268.9 (1993): 6097-6106.
PMID
8454584
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
9
Publish Date
1993
Start Page
6097
End Page
6106

Copper and gene regulation in yeast

Authors
Zhou, P; Thiele, DJ
MLA Citation
Zhou, P, and Thiele, DJ. "Copper and gene regulation in yeast." BioFactors 4.2 (1993): 105-115.
PMID
8347274
Source
scival
Published In
BioFactors
Volume
4
Issue
2
Publish Date
1993
Start Page
105
End Page
115

Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase

Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1-deletion strains, indicating that copper metallothioneins substitute for copper-zinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.

Authors
Tamai, KT; Gralla, EB; Ellerby, LM; Valentine, JS; Thiele, DJ
MLA Citation
Tamai, KT, Gralla, EB, Ellerby, LM, Valentine, JS, and Thiele, DJ. "Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase." Proceedings of the National Academy of Sciences of the United States of America 90.17 (1993): 8013-8017.
PMID
8367458
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
90
Issue
17
Publish Date
1993
Start Page
8013
End Page
8017

Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for nigh-level copper detoxification

Copper detoxification in the yeast Candida glabrata is carried out in large part by a family of metallothionein (MT) genes: a unique MT-I gene, a tandemly amplified MT-IIa gene, and a single unlinked MT-IIb gene. In response to elevated environmental copper levels, members of this MT gene family are transcriptionally activated by a copper-dependent, sequence-specific DNA-binding transcription factor, AMT1, AMT1 shares several structural and functional features with the Saccharomyces cerevisiae copper metalloregulatory transcription factor ACE1, which is constitutively expressed and poised for rapid transcriptional responses to the toxic metal copper. In this paper, we demonstrate that AMT1 is subject to positive transcriptional autoregulation, which is exerted through binding of copper-activated AMT1 to a single copper responsive element in the AMT1 promoter. A nonautoregulatory amt1 mutant displayed a marked decrease in both copper tolerance and expression of the MT-II genes, which are critical for high-level copper detoxification in Candida glabrata. Kinetic analysis demonstrated the remarkably rapid AMT1 mRNA accumulation in the presence of copper, which is followed by increased expression of the metallothionein gene products. These results demonstrate that AMT1-positive autoregulation plays a critical role in metal detoxification and suggest that the rapid autoactivation of the AMT1 metalloregulatory transcription factor biosynthesis is essential for C. glabrata to quickly build up a cellular defense line to protect cells upon exposure to high environmental copper levels.

Authors
Zhou, P; Thiele, DJ
MLA Citation
Zhou, P, and Thiele, DJ. "Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for nigh-level copper detoxification." Genes and Development 7.9 (1993): 1824-1835.
PMID
8370529
Source
scival
Published In
Genes and Development
Volume
7
Issue
9
Publish Date
1993
Start Page
1824
End Page
1835

Expression of a Yeast Metallothionein Gene Family Is Activated by a Single Metalloregulatory Transcription Factor

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (γ-Glu-CyS)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.

Authors
Zhou, P; Szczypka, MS; Sosinowski, T; Thiele, DJ
MLA Citation
Zhou, P, Szczypka, MS, Sosinowski, T, and Thiele, DJ. "Expression of a Yeast Metallothionein Gene Family Is Activated by a Single Metalloregulatory Transcription Factor." Molecular and Cellular Biology 12.9 (1992): 3766-3775.
PMID
1508182
Source
scival
Published In
Molecular and Cellular Biology
Volume
12
Issue
9
Publish Date
1992
Start Page
3766
End Page
3775

Parallel pathways of gene regulation: Homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase

Two independent pathways of transcriptional regulation that show functional homology have been identified in yeast. It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated. We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle-regulated transcription and nuclear localization similar to those seen previously for SWI5. Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation. SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not. In contrast, ACE2 is a transcriptional activator of the CTS1 gene (which encodes chitinase), whereas SWI5 is not. An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTS1 both are cell cycle regulated in the same way, and HO and CTS1 both require the SWI4 and SWI6 transcriptional activators. Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate. Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain.

Authors
Dohrmann, PR; Butler, G; Tamai, K; Borland, S; Greene, JR; Thiele, DJ; Stillman, DJ
MLA Citation
Dohrmann, PR, Butler, G, Tamai, K, Borland, S, Greene, JR, Thiele, DJ, and Stillman, DJ. "Parallel pathways of gene regulation: Homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase." Genes and Development 6.1 (1992): 93-104.
PMID
1730413
Source
scival
Published In
Genes & development
Volume
6
Issue
1
Publish Date
1992
Start Page
93
End Page
104

Metal-regulated transcription in eukaryotes

Authors
Thiele, DJ
MLA Citation
Thiele, DJ. "Metal-regulated transcription in eukaryotes." Nucleic Acids Research 20.6 (1992): 1183-1191.
PMID
1561077
Source
scival
Published In
Nucleic Acids Research
Volume
20
Issue
6
Publish Date
1992
Start Page
1183
End Page
1191

ACE2, an Activator of Yeast Metallothionein Expression Which Is Homologous to SWI5

Transcription of the Saccharomyces cerevisiae metallothionein gene CUP1 is induced in response to high environmental levels of copper. Induction requires the ACE1 gene product, which binds to specific sites in the promoter region of the CUP1 gene. In this study, we found that deleting the entire coding sequence of the ACE1 gene resulted in a decrease in basal-level transcription of CUP1 to low but detectable levels and conferred a copper-sensitive phenotype to the cells. We have isolated a gene, designated ACE2, which when present on a high-copy-number plasmid suppresses the copper-sensitive phenotype of an ace1-deletion strain. The presence of multiple copies of the ACE2 gene enhanced expression of an unlinked CUP1-lacZ fusion integrated in the yeast genome and resulted in an increase in the steady-state levels of CUP1 mRNA in an ace1-deletion background. A large deletion of the coding region of the genomic copy of ACE2 resulted in a decrease in steady-state levels of CUP1 mRNA, indicating that ACE2 plays a role in regulating basal-level expression of CUP1. The ACE2 open reading frame encodes a polypeptide of 770 amino acids, with putative zinc finger structures near the carboxyl terminus. This protein is 37% identical to the SW15 gene product, an activator of HO gene transcription in S. cerevisiae, suggesting that ACE2 and SW15 may have functional similarities.

Authors
Butler, G; Thiele, DJ
MLA Citation
Butler, G, and Thiele, DJ. "ACE2, an Activator of Yeast Metallothionein Expression Which Is Homologous to SWI5." Molecular and Cellular Biology 11.1 (1991): 476-485.
PMID
1986241
Source
scival
Published In
Molecular and Cellular Biology
Volume
11
Issue
1
Publish Date
1991
Start Page
476
End Page
485

Heat shock transcription factor activates transcription of the yeast metallothionein gene

In the yeast Saccharomyces cerevisiae, transcription of the metallothionein gene CUP1 is induced by copper and silver. Strains with a complete deletion of the ACE1 gene, the copper-dependent activator of CUP1 transcription, are hypersensitive to copper. These strains have a low but significant basal level of CUP1 transcription. To identify genes which mediate basal transcription of CUP1 or which activate CUP1 in response to other stimuli, we isolated an extragenic suppressor of an ace1 deletion. We demonstrate that a single amino acid substitution in the heat shock transcription factor (HSF) DNA-binding domain dramatically enhances CUP1 transcription while reducing transcription of the SSA3 gene, a member of the yeast hsp70 gene family. These results indicate that yeast metallothionein transcription is under HSF control and that metallothionein biosynthesis is important in response to heat shock stress. Furthermore, our results suggest that WSF may modulate the magnitude of individual heat shock gene transcription by subtle differences in its interaction with heat shock elements and that a single-amino-acid change can dramatically alter the activity of the factor for different target genes.

Authors
Silar, P; Butler, G; Thiele, DJ
MLA Citation
Silar, P, Butler, G, and Thiele, DJ. "Heat shock transcription factor activates transcription of the yeast metallothionein gene." Molecular and Cellular Biology 11.3 (1991): 1232-1238.
PMID
1996089
Source
scival
Published In
Molecular and Cellular Biology
Volume
11
Issue
3
Publish Date
1991
Start Page
1232
End Page
1238

ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene

Copper, zinc superoxide dismutase (SOD1 gene product) (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a copper-containing enzyme that functions to prevent oxygen toxicity. In the yeast Saccharomyces cerevisiae, copper levels exert some control over the level of SOD1 expression. We show that the ACE1 transcriptional activator protein, which is responsible for the induction of yeast metallothionein (CUP1) in response to copper, also controls the SOD1 response to copper. A single binding site for ACE1 is present in the SODl promoter region, as demonstrated by DNase I protection and methylation interference experiments, and is highly homologous to a high-affinity ACE1 binding site in the CUP1 promoter. The functional importance of this DNA-protein interaction is demonstrated by the facts that (i) copper induction of SOD1 mRNA does not occur in a strain lacking ACE1 and (ii) it does not occur in a strain containing a genetically engineered SOD1 promoter that lacks a functional ACE1 binding site.

Authors
Gralla, EB; Thiele, DJ; Silar, P; Valentine, JS
MLA Citation
Gralla, EB, Thiele, DJ, Silar, P, and Valentine, JS. "ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene." Proceedings of the National Academy of Sciences of the United States of America 88.19 (1991): 8558-8562.
PMID
1924315
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
88
Issue
19
Publish Date
1991
Start Page
8558
End Page
8562
DOI
10.1073/pnas.88.19.8558

The cloning and expression of a gene encoding old yellow enzyme from Saccharomyces carlsbergensis

We have identified a gene that encodes Old Yellow Enzyme in brewer's bottom yeast. The open reading frame encodes a polypeptide of 400 amino acids with Mr = 45,021. Using the T7 RNA polymerase system, recombinant enzyme was expressed in Escherichia coli. 17 mg of Old Yellow Enzyme was obtained from a 3-liter cell culture, and the recombinant enzyme had NADPH oxidase activity. On fast protein liquid chromatography separation, the recombinant enzyme showed a single large peak, while native enzyme from brewer's bottom yeast separated into five fractions on fast protein liquid chromatography. Southern blot analysis showed that there are at least two Old Yellow Enzyme genes in brewer's bottom yeast genomic DNA. These results suggest that the heterogeneity of Old Yellow Enzyme in Saccharomyces carlsbergensis is due to the presence of multiple genes.

Authors
Saito, K; Thiele, DJ; Davio, M; Lockridge, O; Massey, V
MLA Citation
Saito, K, Thiele, DJ, Davio, M, Lockridge, O, and Massey, V. "The cloning and expression of a gene encoding old yellow enzyme from Saccharomyces carlsbergensis." Journal of Biological Chemistry 266.31 (1991): 20720-20724.
PMID
1939123
Source
scival
Published In
Journal of Biological Chemistry
Volume
266
Issue
31
Publish Date
1991
Start Page
20720
End Page
20724

Isolation of a metal-activated transcription factor gene from Candida glabrata by complementation in Saccharomyces cerevisiae

Metal-inducible transcription of metallothionein (MT) genes involves the interaction of metal-responsive trans-acting factors with specific promoter DNA sequence elements. In this report, we present a genetic selection using the baker's yeast, Saccharomyces cerevisiae, to clone a gene from Candida glabrata encoding a metal-activated DNA-binding protein denoted AMT1. This selection is based on the ability of the AMT1 gene product to activate expression of the C. glabrata MT-I gene in a copper-sensitive S. cerevisiae host strain. DNA-binding studies using AMT1 protein expressed in Escherichia coli demonstrate that AMT1 is activated by copper or silver to bind to both the MT-I and MT-II promoters of C. glabrata. Sequence comparison of AMT1 protein to the S. cerevisiae copper- or silver-activated DNA-binding protein, ACE1, indicates that AMT1 contains the 11 amino terminal cysteine residues known to be critical for the metal-activated DNA-binding activity of ACE1. In contrast, the carboxyl-terminal portion of AMT1 bears only slight similarity at the primary structure level to the same region of ACE1 known to be important for transcriptional activation. These results suggest that the amino-terminal cysteines, and other conserved residues, play an important role in the ability of AMT1 and ACE1 to sense intracellular copper levels and assume a metal-activated DNA-binding structure.

Authors
Zhou, P; Thiele, DJ
MLA Citation
Zhou, P, and Thiele, DJ. "Isolation of a metal-activated transcription factor gene from Candida glabrata by complementation in Saccharomyces cerevisiae." Proceedings of the National Academy of Sciences of the United States of America 88.14 (1991): 6112-6116.
PMID
2068090
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
88
Issue
14
Publish Date
1991
Start Page
6112
End Page
6116

New shuttle vectors for direct cloning in Saccharomyces cerevisiae

We have constructed new shuttle vectors to facilitate the screening of recombinant plasmids after direct transformation of yeast cells. The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene. DNA fragments are inserted in a Polylinker located after the beginning of the URA3 coding sequence. Transformants are selected either by Trp or Leu prototrophy. Plasmids bearing an insert are selected by growth on 5-fluoro-orotic acid (5-FOA), a uracil analog toxic to cells containing a functional URA3 + gene (thus, this method requires the recipient strain to be ura3 -); only cells containing a plasmid with an insert that disrupts the functional continuity of the URA3 gene can grow on medium containing 5-FOA. Using these plasmids, we were able to directly redone the ACE 1 gene from genomic DNA by directly transforming a strain deleted for ACE 1. These vectors can be used for a variety of purposes including rapid cloning of genes by complementation or expression of fusion genes driven from the URA3 promoter. © 1991.

Authors
Silar, P; Thiele, DJ
MLA Citation
Silar, P, and Thiele, DJ. "New shuttle vectors for direct cloning in Saccharomyces cerevisiae." Gene 104.1 (1991): 99-102.
PMID
1916284
Source
scival
Published In
Gene
Volume
104
Issue
1
Publish Date
1991
Start Page
99
End Page
102
DOI
10.1016/0378-1119(91)90472-N

Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae, with the of the copper-inducible CUP1 promoter

The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) in Saccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and the ADH1 promoter, is described. Strains 50.L4 and PP1002 were compared for optimal levels of expressed protein. Immunoblot analysis showed that a much higher level of expression of cytochrome P-450 3a is obtained with strain 50.L4, and that the uninduced levels of expressed protein are similar with the two promoters. With the CUP1 promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed is increased 20-fold in strain 50.L4, such that it constitutes 0.8% of the total cellular protein. The cytochrome P-450 holoenzyme content of these cells, calculated from the reduced CO difference spectrum, is about 0.02 nmole/mg of protein, or 0.1% of the total cellular protein. The holoenzyme content of microsomes prepared from these cells is up to 0.06 nmole/mg of protein, or 0.4% of the microsomal protein. Microsomal assays for ethylene formation from N-nitrosodiethylamide and for aniline p-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions. Furthermore, polyclonal anti-3a IgG completely inhibits the reactions with both substrates in yeast microsomes. A comparison of the product ratios from these substrates showed that the cytochrome P-450 3a expressed in yeast has catalytic activities similar to those of the authentic rabbit protein.

Authors
Fujita, VS; Thiele, DJ; Coon, MJ
MLA Citation
Fujita, VS, Thiele, DJ, and Coon, MJ. "Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae, with the of the copper-inducible CUP1 promoter." DNA and Cell Biology 9.2 (1990): 111-118.
PMID
2188656
Source
scival
Published In
DNA and Cell Biology
Volume
9
Issue
2
Publish Date
1990
Start Page
111
End Page
118

ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5′-TC(T)4-6GCTG-3′, which we propose constitutes an important part of the ACE1 consensus recognition sequence.

Authors
Evans, CF; Engelke, DR; Thiele, DJ
MLA Citation
Evans, CF, Engelke, DR, and Thiele, DJ. "ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences." Molecular and Cellular Biology 10.1 (1990): 426-429.
PMID
2403647
Source
scival
Published In
Molecular and Cellular Biology
Volume
10
Issue
1
Publish Date
1990
Start Page
426
End Page
429

A cysteine-rich nuclear protein activates yeast metallothionein gene transcription.

The ACE1 gene of the yeast Saccharomyces cerevisiae is required for copper-inducible transcription of the metallothionein gene (CUP1). The sequence of the cloned ACE1 gene predicted an open reading frame for translation of a 225-amino-acid polypeptide. This polypeptide was characterized by an amino-terminal half rich in cysteine residues and positively charged amino acids. The arrangement of many of the 12 cysteines in the configuration Cys-X-Cys or Cys-X-X-Cys suggested that the ACE1 protein may bind metal ions. The carboxyl-terminal half of the ACE1 protein was devoid of cysteines but was highly acidic in nature. The ability of a bifunctional ACE1-beta-galactosidase fusion protein to accumulate in yeast cell nuclei was consistent with the possibility that ACE1 plays a direct role in the regulation of copper-inducible transcription of the yeast metallothionein gene.

Authors
Szczypka, MS; Thiele, DJ
MLA Citation
Szczypka, MS, and Thiele, DJ. "A cysteine-rich nuclear protein activates yeast metallothionein gene transcription." Molecular and Cellular Biology 9.2 (1989): 421-429.
PMID
2651899
Source
scival
Published In
Molecular and Cellular Biology
Volume
9
Issue
2
Publish Date
1989
Start Page
421
End Page
429

Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences

Copper-inducible transcription of the yeast metallothionein gene (CUP1) occurs by means of cis-acting upstream activation sequences (UAS) and trans-acting cellular factors. We have used a high-resolution chromosomal foot-printing technique to detect the interaction of cellular factors with UAS(CUP1). Our results demonstrate that one or more cellular factors bind to UAS(CUP1) in a copper-inducible fashion. This copper-inducible binding is enhanced in a yeast strain that harbors several copies of the positive regulatory gene ACE1 and is not detectable in yeast cells that contain a nonfunctional (ace1-Δ1) locus. The correlation between yeast metallothionein gene activation and copper-inducible occupation of UAS(CUP1) sequences suggests that the binding of metallothionein transcriptional regulatory factors to cis-acting control sequences is copper-inducible.

Authors
Huibregtse, JM; Engelke, DR; Thiele, DJ
MLA Citation
Huibregtse, JM, Engelke, DR, and Thiele, DJ. "Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences." Proceedings of the National Academy of Sciences of the United States of America 86.1 (1989): 65-69.
PMID
2643107
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
86
Issue
1
Publish Date
1989
Start Page
65
End Page
69

ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene.

Copper resistance in Saccharomyces cerevisiae is mediated, in large part, by the CUP1 locus, which encodes a low-molecular-weight, cysteine-rich metal-binding protein. Expression of the CUP1 gene is regulated at the level of transcriptional induction in response to high environmental copper levels. This report describes the isolation of a yeast mutant, ace1-1, which is defective in the activation of CUP1 expression upon exposure to exogenous copper. The ace1-1 mutation is recessive and lies in a genetic element that encodes a trans-acting CUP1 regulatory factor. The wild-type ACE1 gene was isolated by in vivo complementation and restores copper inducibility of CUP1 expression and copper resistance to the otherwise copper-sensitive ace1-1 mutant. Linkage analysis and gene deletion experiments verified that this gene represents the authentic ACE1 locus. ACE1 maps to the left arm of chromosome VII, 9 centimorgans centromere distal to lys5. The ACE1 gene appears to play a direct or indirect positive role in activation of CUP1 expression in response to elevated copper concentrations.

Authors
Thiele, DJ
MLA Citation
Thiele, DJ. "ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene." Molecular and Cellular Biology 8.7 (1988): 2745-2752.
PMID
3043194
Source
scival
Published In
Molecular and Cellular Biology
Volume
8
Issue
7
Publish Date
1988
Start Page
2745
End Page
2752

Function and regulation of yeast copperthionein.

A functional copperthionein (CUP1) gene in Saccharomyces cerevisiae is essential to prevent copper-mediated cytotoxicity, but is dispensable for cell growth in the absence of exogenous copper. The CUP1 gene is negatively autoregulated, as observed by the necessity for a functional CUP1 gene in order to repress basal level transcription from the CUP1 promoter. Both the copper protection and transcriptional autoregulatory functions can be complemented by expression in yeast of either of two monkey metallothionein isoform cDNAs. The expression of the CUP1 gene is induced at the level of transcription by copper via cis-dominant upstream control sequences which are tandemly repeated. Synthetic CUP1 upstream control sequences confer copper inducibility on a heterologous yeast promoter in a manner similar to that observed for the authentic upstream control region.

Authors
Thiele, DJ; Wright, CF; Walling, MJ; Hamer, DH
MLA Citation
Thiele, DJ, Wright, CF, Walling, MJ, and Hamer, DH. "Function and regulation of yeast copperthionein." Experientia. Supplementum 52 (1987): 423-429.
PMID
2959531
Source
scival
Published In
Experientia. Supplementum
Volume
52
Publish Date
1987
Start Page
423
End Page
429

Tandemly duplicated upstream control sequences mediate copper- induced transcription of the Saccharomyces cerevisiae copper- metallothionein gene

Authors
Thiele, DJ; Hamer, DH
MLA Citation
Thiele, DJ, and Hamer, DH. "Tandemly duplicated upstream control sequences mediate copper- induced transcription of the Saccharomyces cerevisiae copper- metallothionein gene." Molecular and Cellular Biology 6.4 (1986): 1158-1163.
PMID
3537699
Source
scival
Published In
Molecular and Cellular Biology
Volume
6
Issue
4
Publish Date
1986
Start Page
1158
End Page
1163

Mammalian metallothionein is functional in yeast

Expression of two monkey metallothioneins in yeast leads to complementation of both known functions of the endogenous yeast copperthionein gene, namely copper detoxification and autoregulation of transcription. The metallothionein-like proteins of higher and lower eukaryotes are therefore functionally analogous despite their dissimilar primary sequences.

Authors
Thiele, DJ; Walling, MJ; Hamer, DH
MLA Citation
Thiele, DJ, Walling, MJ, and Hamer, DH. "Mammalian metallothionein is functional in yeast." Science 231.4740 (1986): 854-856.
PMID
3080806
Source
scival
Published In
Science
Volume
231
Issue
4740
Publish Date
1986
Start Page
854
End Page
856

Function and autoregulation of yeast copperthionein

The CUP1 gene of yeast encodes a small, metallothionein-like protein that binds to and is inducible by copper. A gene replacement experiment shows that this protein protects cells against copper poisoning but is dispensable for normal cellular growth and development throughout the yeast life cycle. The transcription of CUP1 is negatively autoregulated. This feedback mechanism, which is mediated through upstream control sequences, may play an important role in heavy metal homeostasis.

Authors
Hamer, DH; Thiele, DJ; Lemontt, JE
MLA Citation
Hamer, DH, Thiele, DJ, and Lemontt, JE. "Function and autoregulation of yeast copperthionein." Science 228.4700 (1985): 685-690.
PMID
3887570
Source
scival
Published In
Science
Volume
228
Issue
4700
Publish Date
1985
Start Page
685
End Page
690

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

The L double-stranded (ds) RNA component of Saccharomyces cerevisiae may contain up to three dsRNA species, each with a distinct sequence but with identical molecular weights. These dsRNAs have been separated from each other by denaturation and polyacrylamide gel electrophoresis. The 3' terminal sequences of the major species, LA dsRNA, were determined. Secondary structural analysis supported the presence of two stem and loop structures at the 3' terminus of the LA positive strand. In strain T132B NK-3, both the LA and LC species are virion encapsidated. Two distinct classes of virions were purified from this strain, each with a different RNA polymerase activity and with distinct protein components. The heavy virions harbored LA dsRNA, whereas the LC dsRNA species co purified with the light virion peak. Thus, LA and LC dsRNAs, when present in the same cell, may be separately encapsidated.

Authors
Thiele, DJ; Hannig, EM; Leibowitz, MJ
MLA Citation
Thiele, DJ, Hannig, EM, and Leibowitz, MJ. "Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation." Molecular and Cellular Biology 4.1 (1984): 92-100.
PMID
6366515
Source
scival
Published In
Molecular and Cellular Biology
Volume
4
Issue
1
Publish Date
1984
Start Page
92
End Page
100

Saccharomyces cerevisiae killer virus transcripts contain template-coded polyadenylate tracts.

The M double-stranded RNA component of type 1 killer strains of the yeast Saccharomyces cerevisiae contains an internal 200-base pair adenine- and uracil-rich region. The plus strands of this viral genomic RNA contain an internal adenine-rich region which allows these strands to bind to polyuridylate-Sepharose as tightly as do polyadenylated RNAs with 3'-terminal polyadenylated tracts of 70 to 100 residues. Internal template coding of an adenine-rich tract in positive polarity in vivo and in vitro transcripts of M double-stranded RNA may serve as an alternate method of transcript polyadenylation. The 3'-terminal residue of the in vitro m transcript is a non-template-encoded purine residue. The 5' terminus of this transcript is involved in a stem-and-loop structure which includes an AUG initiation codon, along with potential 18S and 5.8S rRNA binding sites. Except for the 3'-terminal residue, transcription in in vitro shows complete fidelity.

Authors
Hannig, EM; Thiele, DJ; Leibowitz, MJ
MLA Citation
Hannig, EM, Thiele, DJ, and Leibowitz, MJ. "Saccharomyces cerevisiae killer virus transcripts contain template-coded polyadenylate tracts." Molecular and Cellular Biology 4.1 (1984): 101-109.
PMID
6199660
Source
scival
Published In
Molecular and Cellular Biology
Volume
4
Issue
1
Publish Date
1984
Start Page
101
End Page
109

Genome structure and expression of a defective interfering mutant of the killer virus of yeast

A large internal deletion in M1 double-stranded (ds) RNA from the killer virus of Saccharomyces cerevisiae generates a suppressive (S3) dsRNA molecule. Strains which harbor S3 dsRNA are defective in toxin production and immunity to the toxin. The biochemical defect in expression has been investigated and is apparently due to truncation of the protoxin polypeptide translation reading frame on S3 dsRNA. Transcription in vivo, and in isolated virions in vitro, results in the synthesis of a full-length positive polarity messenger RNA, denoted s. The s transcript contains no long poly(A) tracts as determined by its lack of affinity for oligo(dT)-cellulose, and as inferred by sequence analysis of approximately 87% of the S3 dsRNA genome. These data support a model for template coding of polyadenylate in transcripts derived from the wild-type M1 dsRNA. The orientation of the sequences conserved on S3 dsRNA with respect to M1 dsRNA has been determined. Some of the conserved sequences are likely to be required for the maintenance and replication of these viral dsRNA genomes in S. cerevisiae. © 1984.

Authors
Thiele, DJ; Hannig, EM; Leibowitz, MJ
MLA Citation
Thiele, DJ, Hannig, EM, and Leibowitz, MJ. "Genome structure and expression of a defective interfering mutant of the killer virus of yeast." Virology 137.1 (1984): 20-31.
PMID
6382788
Source
scival
Published In
Virology
Volume
137
Issue
1
Publish Date
1984
Start Page
20
End Page
31

Structural and functional analysis of separated strands of killer double-stranded RNA of yeast

The two strands of the M double-stranded RNA species from a killer strain of Saccharomyces cerevisiae have been separated, and the 3′-terminal sequences of these strands have been determined. The positive strand programs the synthesis of the putative killer toxin precursor (M-p32) in a rabbit reticulocyte in vitro translation system. Only the negative strand hybridizes to the positive polarity transcript (m) synthesized in vitro by the virion-associated transcriptase activity. Secondary structural analysis of the extreme 3′-terminus of the negative strand using S1 nuclease is consistent with the presence of a large stem and loop structure previously proposed on the basis of RNA sequence data. This structure, and a similar structure at the corresponding 5′-terminus of the positive strand, may have functional significance in vivo. © 1982 IRL Press Limited.

Authors
Thiele, DJ; Leibowitz, MJ
MLA Citation
Thiele, DJ, and Leibowitz, MJ. "Structural and functional analysis of separated strands of killer double-stranded RNA of yeast." Nucleic Acids Research 10.21 (1982): 6903-6918.
PMID
6757869
Source
scival
Published In
Nucleic Acids Research
Volume
10
Issue
21
Publish Date
1982
Start Page
6903
End Page
6918
DOI
10.1093/nar/10.21.6903

Separation and sequence of the 3′ termini of M double-stranded RNA from killer yeast

Four subspecies of M double-stranded RNA from a killer strain of Saccharomyces cerevisiae were isolated. Each subspecies was susceptible to heat cleavage, presumably at an internal 190 base pair A,U-rich region, generating two discrete fragments corresponding to each side of the A, U-rich region. Enzymatic and chemical RNA sequence analysis defined the 3′-terminal 175 bases for the larger fragment (M-1) and 231 bases for the smaller fragment (M-2). All four subspecies of M have identical size and 3′-terminal sequences. Potential translation initiation codons are present on the corresponding 5′ termini of both fragments, and a possible 18S ribosomal RHA binding site is also present on the 5′ terminus of M-1. Stem and loop structures for the 5′ and 3′ termini of M-1 may function as recognition sites for replication, transcription, and translation. © 1982 IRL Press Limited.

Authors
Thiele, DJ; Wang, RW; Leibowitz, MJ
MLA Citation
Thiele, DJ, Wang, RW, and Leibowitz, MJ. "Separation and sequence of the 3′ termini of M double-stranded RNA from killer yeast." Nucleic Acids Research 10.5 (1982): 1661-1678.
PMID
7041093
Source
scival
Published In
Nucleic Acids Research
Volume
10
Issue
5
Publish Date
1982
Start Page
1661
End Page
1678
DOI
10.1093/nar/10.5.1661
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