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Cardenas-Corona, Maria Elena

Positions:

Research Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1988

Ph.D. — University of North Texas

Grants:

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

Activation of rapamycin-sensitive TORC1 by endomembrane amino acid transporters

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2011
End Date
June 30, 2016

Instrumentation for Quantitative Phosphoproteomics and Acetylomics

Administered By
Duke Center for Genomic and Computational Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2014
End Date
May 14, 2015

Novel Antifungal Therapeutic Approaches

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
July 01, 2001
End Date
December 31, 2014

Signaling mechanisms by the rapamycin target: Tor kinase

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 17, 2005
End Date
January 31, 2011

Role of Calcineurin in Fungal Virulence

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
May 15, 2004
End Date
April 30, 2010

Function of the Rapamycin Targets: The TOR Kinases

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2002
End Date
August 31, 2005

Structure and Function of the Targets of Rapamycin: The TQR Kinanse Homologs

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1997
End Date
August 31, 2002

Novel Antifungal Drug Targets in Cryptococcus Neoformans

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
September 01, 1997
End Date
June 30, 2001

Novel Antifungal Drug Targets In Cryptococcus Neoformans

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1997
End Date
August 31, 1999

Immunosuppressant Targets In Cryptococcus Neoformans

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
May 01, 1997
End Date
April 30, 1999

The Barrier To Xenotransplantation

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
February 01, 1996
End Date
January 31, 1999

The Barrier To Xenotransplantation

Administered By
Surgery
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
February 01, 1993
End Date
January 31, 1999
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Awards:

AAAS Fellows. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2010

Publications:

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

Authors
Park, H-S; Chow, EWL; Fu, C; Soderblom, EJ; Moseley, MA; Heitman, J; Cardenas, ME
MLA Citation
Park, H-S, Chow, EWL, Fu, C, Soderblom, EJ, Moseley, MA, Heitman, J, and Cardenas, ME. "Calcineurin Targets Involved in Stress Survival and Fungal Virulence." PLoS pathogens 12.9 (September 9, 2016): e1005873-.
Website
http://hdl.handle.net/10161/13053
PMID
27611567
Source
epmc
Published In
PLoS pathogens
Volume
12
Issue
9
Publish Date
2016
Start Page
e1005873
DOI
10.1371/journal.ppat.1005873

Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability.

A major advance in understanding the progression and prognostic outcome of certain cancers, such as low-grade gliomas, acute myeloid leukaemia, and chondrosarcomas, has been the identification of early-occurring mutations in the NADP+-dependent isocitrate dehydrogenase genes IDH1 and IDH2 These mutations result in the production of the onco-metabolite D-2-hydroxyglutarate (2HG), thought to contribute to disease progression. To better understand the mechanisms of 2HG pathophysiology, we introduced the analogous glioma-associated mutations into the NADP+ isocitrate dehydrogenase genes (IDP1, IDP2, IDP3) in Saccharomyces cerevisiae Intriguingly, expression of the mitochondrial IDP1R148H mutant allele results in high levels of 2HG production as well as extensive mtDNA loss and respiration defects. We find no evidence for a reactive oxygen-mediated mechanism mediating this mtDNA loss. Instead, we show that 2HG production perturbs the iron sensing mechanisms as indicated by upregulation of the Aft1-controlled iron regulon and a concomitant increase in iron levels. Accordingly, iron chelation, or overexpression of a truncated AFT1 allele that dampens transcription of the iron regulon, suppresses the loss of respirative capacity. Additional suppressing factors include overexpression of the mitochondrial aldehyde dehydrogenase gene ALD5 or disruption of the retrograde response transcription factor RTG1 Furthermore, elevated α-ketoglutarate levels also suppress 2HG-mediated respiration loss; consistent with a mechanism by which 2HG contributes to mtDNA loss by acting as a toxic α-ketoglutarate analog. Our findings provide insight into the mechanisms that may contribute to 2HG oncogenicity in glioma and acute myeloid leukaemia progression, with the promise for innovative diagnostic and prognostic strategies and novel therapeutic modalities.

Authors
Kingsbury, JM; Shamaprasad, N; Billmyre, RB; Heitman, J; Cardenas, ME
MLA Citation
Kingsbury, JM, Shamaprasad, N, Billmyre, RB, Heitman, J, and Cardenas, ME. "Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability." Human molecular genetics 25.16 (August 2016): 3524-3538.
PMID
27427385
Source
epmc
Published In
Human Molecular Genetics
Volume
25
Issue
16
Publish Date
2016
Start Page
3524
End Page
3538
DOI
10.1093/hmg/ddw195

Vesicular trafficking systems impact TORC1-controlled transcriptional programs in Saccharomyces cerevisiae

© 2016 Kingsbury and Cardenas.The Target of Rapamycin Complex I (TORC1) orchestrates global reprogramming of transcriptional programs in response to myriad environmental conditions, yet, despite the commonality of the TORC1 complex components, different TORC1-inhibitory conditions do not elicit a uniform transcriptional response. In Saccharomyces cerevisiae, TORC1 regulates the expression of nitrogen catabolite repressed (NCR) genes by controlling the nuclear translocation of the NCR transactivator Gln3. Moreover, Golgi-to-endosome trafficking was shown to be required for nuclear translocation of Gln3 upon a shift from rich medium to the poor nitrogen source proline, but not upon rapamycin treatment. Here, we employed microarray profiling to survey the full impact of the vesicular trafficking system on yeast TORC1-orchestrated transcriptional programs. In addition to the NCR genes, we found that ribosomal protein, ribosome biogenesis, phosphate-responsive, and sulfur-containing amino acid metabolism genes are perturbed by disruption of Golgi-to-endosome trafficking following a nutritional shift from rich to poor nitrogen source medium, but not upon rapamycin treatment. Similar to Gln3, defects in Golgi-to-endosome trafficking significantly delayed cytoplasmic-nuclear translocation of Sfp1, but did not detectably affect the cytoplasmic-nuclear or nuclear-cytoplasmic translocation of Met4, which are the transactivators of these genes. Thus, Golgi-to-endosome trafficking defects perturb TORC1 transcriptional programs via multiple mechanisms. Our findings further delineate the downstream transcriptional responses of TORC1 inhibition by rapamycin compared with a nitrogen quality downshift. Given the conservation of both TORC1 and endomembrane networks throughout eukaryotes, our findings may also have implications for TORC1-mediated responses to nutritional cues in mammals and other eukaryotes.

Authors
Kingsbury, JM; Cardenas, ME
MLA Citation
Kingsbury, JM, and Cardenas, ME. "Vesicular trafficking systems impact TORC1-controlled transcriptional programs in Saccharomyces cerevisiae." G3: Genes, Genomes, Genetics 6.3 (January 1, 2016): 641-652.
Source
scopus
Published In
G3 (Bethesda, Md.)
Volume
6
Issue
3
Publish Date
2016
Start Page
641
End Page
652

Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae

Authors
Kingsbury, JM; Sen, ND; Cardenas, ME
MLA Citation
Kingsbury, JM, Sen, ND, and Cardenas, ME. "Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae." Ed. GP Copenhaver. PLOS Genetics 11.12 (December 11, 2015): e1005714-e1005714.
Source
crossref
Published In
PLoS genetics
Volume
11
Issue
12
Publish Date
2015
Start Page
e1005714
End Page
e1005714
DOI
10.1371/journal.pgen.1005714

L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS.

Authors
Xu, B; Sowa, N; Cardenas, ME; Gerton, JL
MLA Citation
Xu, B, Sowa, N, Cardenas, ME, and Gerton, JL. "L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome." Human molecular genetics 24.6 (March 2015): 1540-1555.
PMID
25378554
Source
epmc
Published In
Human Molecular Genetics
Volume
24
Issue
6
Publish Date
2015
Start Page
1540
End Page
1555
DOI
10.1093/hmg/ddu565

Antifungal drug resistance evoked via RNAi-dependent epimutations.

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

Authors
Calo, S; Shertz-Wall, C; Lee, SC; Bastidas, RJ; Nicolás, FE; Granek, JA; Mieczkowski, P; Torres-Martínez, S; Ruiz-Vázquez, RM; Cardenas, ME; Heitman, J
MLA Citation
Calo, S, Shertz-Wall, C, Lee, SC, Bastidas, RJ, Nicolás, FE, Granek, JA, Mieczkowski, P, Torres-Martínez, S, Ruiz-Vázquez, RM, Cardenas, ME, and Heitman, J. "Antifungal drug resistance evoked via RNAi-dependent epimutations." Nature 513.7519 (September 2014): 555-558.
PMID
25079329
Source
epmc
Published In
Nature
Volume
513
Issue
7519
Publish Date
2014
Start Page
555
End Page
558
DOI
10.1038/nature13575

Endolysosomal membrane trafficking complexes drive nutrient-dependent TORC1 signaling to control cell growth in Saccharomyces cerevisiae.

The rapamycin-sensitive and endomembrane-associated TORC1 pathway controls cell growth in response to nutrients in eukaryotes. Mutations in class C Vps (Vps-C) complexes are synthetically lethal with tor1 mutations and confer rapamycin hypersensitivity in Saccharomyces cerevisiae, suggesting a role for these complexes in TORC1 signaling. Vps-C complexes are required for vesicular trafficking and fusion and comprise four distinct complexes: HOPS and CORVET and their minor intermediaries (i)-CORVET and i-HOPS. We show that at least one Vps-C complex is required to promote TORC1 activity, with the HOPS complex having the greatest input. The vps-c mutants fail to recover from rapamycin-induced growth arrest and show low levels of TORC1 activity. TORC1 promotes cell growth via Sch9, a p70(S6) kinase ortholog. Constitutively active SCH9 or hyperactive TOR1 alleles restored rapamycin recovery and TORC1 activity of vps-c mutants, supporting a role for the Vps-C complexes upstream of TORC1. The EGO GTPase complex Exit from G0 Complex (EGOC) and its homologous Rag-GTPase complex convey amino acid signals to TORC1 in yeast and mammals, respectively. Expression of the activated EGOC GTPase subunits Gtr1(GTP) and Gtr2(GDP) partially suppressed vps-c mutant rapamycin recovery defects, and this suppression was enhanced by increased amino acid concentrations. Moreover, vps-c mutations disrupted EGOC-TORC1 interactions. TORC1 defects were more severe for vps-c mutants than those observed in EGOC mutants. Taken together, our results support a model in which distinct endolysosomal trafficking Vps-C complexes promote rapamycin-sensitive TORC1 activity via multiple inputs, one of which involves maintenance of amino acid homeostasis that is sensed and transmitted to TORC1 via interactions with EGOC.

Authors
Kingsbury, JM; Sen, ND; Maeda, T; Heitman, J; Cardenas, ME
MLA Citation
Kingsbury, JM, Sen, ND, Maeda, T, Heitman, J, and Cardenas, ME. "Endolysosomal membrane trafficking complexes drive nutrient-dependent TORC1 signaling to control cell growth in Saccharomyces cerevisiae." Genetics 196.4 (April 2014): 1077-1089.
PMID
24514902
Source
epmc
Published In
Genetics
Volume
196
Issue
4
Publish Date
2014
Start Page
1077
End Page
1089
DOI
10.1534/genetics.114.161646

Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.

Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold.Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

Authors
Butts, A; Koselny, K; Chabrier-Roselló, Y; Semighini, CP; Brown, JCS; Wang, X; Annadurai, S; DiDone, L; Tabroff, J; Childers, WE; Abou-Gharbia, M; Wellington, M; Cardenas, ME; Madhani, HD; Heitman, J; Krysan, DJ
MLA Citation
Butts, A, Koselny, K, Chabrier-Roselló, Y, Semighini, CP, Brown, JCS, Wang, X, Annadurai, S, DiDone, L, Tabroff, J, Childers, WE, Abou-Gharbia, M, Wellington, M, Cardenas, ME, Madhani, HD, Heitman, J, and Krysan, DJ. "Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo." mBio 5.1 (February 11, 2014): e00765-e00713.
PMID
24520056
Source
epmc
Published In
mBio
Volume
5
Issue
1
Publish Date
2014
Start Page
e00765
End Page
e00713
DOI
10.1128/mbio.00765-13

Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis

Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level.Weidentified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A.Wefurther present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linkedMATloci. Importance Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets. © 2013 Gioti et al.

Authors
Gioti, A; Nystedt, B; Li, W; Xu, J; Andersson, A; Averette, AF; Münch, K; Wang, X; Kappauf, C; Kingsbury, JM; Kraak, B; Walker, LA; Johansson, HJ; Holm, T; Lehtiö, J; Stajich, JE; Mieczkowski, P; Kahmann, R; Kennell, JC; Cardenas, ME; Lundeberg, J; Saunders, CW; Boekhout, T; Dawson, TL; Munro, CA; Groot, PWJD; Butler, G; Heitman, J; Scheynius, A
MLA Citation
Gioti, A, Nystedt, B, Li, W, Xu, J, Andersson, A, Averette, AF, Münch, K, Wang, X, Kappauf, C, Kingsbury, JM, Kraak, B, Walker, LA, Johansson, HJ, Holm, T, Lehtiö, J, Stajich, JE, Mieczkowski, P, Kahmann, R, Kennell, JC, Cardenas, ME, Lundeberg, J, Saunders, CW, Boekhout, T, Dawson, TL, Munro, CA, Groot, PWJD, Butler, G, Heitman, J, and Scheynius, A. "Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis." mBio 4.1 (2013).
PMID
23341551
Source
scival
Published In
mBio
Volume
4
Issue
1
Publish Date
2013
DOI
10.1128/mBio.00572-12

Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis.

ABSTRACT: Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

Authors
Gioti, A; Nystedt, B; Li, W; Xu, J; Andersson, A; Averette, AF; Münch, K; Wang, X; Kappauf, C; Kingsbury, JM; Kraak, B; Walker, LA; Johansson, HJ; Holm, T; Lehtiö, J; Stajich, JE; Mieczkowski, P; Kahmann, R; Kennell, JC; Cardenas, ME; Lundeberg, J; Saunders, CW; Boekhout, T; Dawson, TL; Munro, CA; Groot, PWJD; Butler, G; Heitman, J; Scheynius, A
MLA Citation
Gioti, A, Nystedt, B, Li, W, Xu, J, Andersson, A, Averette, AF, Münch, K, Wang, X, Kappauf, C, Kingsbury, JM, Kraak, B, Walker, LA, Johansson, HJ, Holm, T, Lehtiö, J, Stajich, JE, Mieczkowski, P, Kahmann, R, Kennell, JC, Cardenas, ME, Lundeberg, J, Saunders, CW, Boekhout, T, Dawson, TL, Munro, CA, Groot, PWJD, Butler, G, Heitman, J, and Scheynius, A. "Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis." mBio 4.1 (2013): e00572-e00512.
Source
scival
Published In
mBio
Volume
4
Issue
1
Publish Date
2013
Start Page
e00572
End Page
e00512
DOI
10.1128/mBio.00572-12

Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor.

The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.

Authors
Bastidas, RJ; Shertz, CA; Lee, SC; Heitman, J; Cardenas, ME
MLA Citation
Bastidas, RJ, Shertz, CA, Lee, SC, Heitman, J, and Cardenas, ME. "Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor." Eukaryotic cell 11.3 (March 2012): 270-281.
PMID
22210828
Source
epmc
Published In
Eukaryotic cell
Volume
11
Issue
3
Publish Date
2012
Start Page
270
End Page
281
DOI
10.1128/ec.05284-11

Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics.

Calcineurin is a calcium-calmodulin-dependent serine/threonine specific protein phosphatase operating in key cellular processes governing responses to extracellular cues. Calcineurin is essential for growth at high temperature and virulence of the human fungal pathogen Cryptococcus neoformans but the underlying mechanism is unknown. We performed a mass spectrometry analysis to identify proteins that associate with the calcineurin A catalytic subunit (Cna1) in C. neoformans cells grown under non-stress and high temperature stress conditions. A novel prioritization strategy for mass spectrometry data from immunoprecipitation experiments identified putative substrates and proteins potentially operating with calcineurin in common pathways. Cna1 co-purified with proteins involved in membrane trafficking including the COPI component Sec28 and the COPII component Sec13. The association of Cna1 with Sec28 and Sec13 was confirmed by co-immunoprecipitation. Cna1 exhibited a dramatic change in subcellular localization during high temperature stress from diffuse cytoplasmic to ER-associated puncta and the mother-bud neck and co-localized with Sec28 and Sec13.

Authors
Kozubowski, L; Thompson, JW; Cardenas, ME; Moseley, MA; Heitman, J
MLA Citation
Kozubowski, L, Thompson, JW, Cardenas, ME, Moseley, MA, and Heitman, J. "Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics." PloS one 6.10 (January 2011): e25280-.
PMID
21984910
Source
epmc
Published In
PloS one
Volume
6
Issue
10
Publish Date
2011
Start Page
e25280
DOI
10.1371/journal.pone.0025280

Calcineurin colocalizes with P-bodies and stress granules during thermal stress in cryptococcus neoformans

Calcineurin is a calcium-calmodulin-activated serine/threonine-specific phosphatase that operates during cellular responses to stress and plays a prominent role in transcriptional control, whereas regulatory events beyond transcription are less well characterized. This study reveals a novel transcription-independent role of calcineurin during the temperature stress response in the human fungal pathogen Cryptococcus neoformans. The diffusely cytoplasmic calcineurin catalytic subunit Cna1 relocates to endoplasmic reticulum (ER)-associated puncta and the mother-bud neck when cells are subjected to 37°C. More than 50% of Cna1 puncta contain the P-body constituent decapping enzyme Dcp1 and the stress granule constituent poly(A)-binding protein Pub1. These results support a model in which calcineurin orchestrates thermal stress responses by associating with sites of mRNA processing. © 2011, American Society for Microbiology. All Rights Reserved.

Authors
Kozubowski, L; Aboobakar, EF; Cardenas, ME; Heitman, J
MLA Citation
Kozubowski, L, Aboobakar, EF, Cardenas, ME, and Heitman, J. "Calcineurin colocalizes with P-bodies and stress granules during thermal stress in cryptococcus neoformans." Eukaryotic Cell 10.11 (2011): 1396-1402.
PMID
21724937
Source
scival
Published In
Eukaryotic cell
Volume
10
Issue
11
Publish Date
2011
Start Page
1396
End Page
1402
DOI
10.1128/EC.05087-11

Exploiting and subverting tor signaling in the pathogenesis of fungi, parasites, and viruses

Authors
Shertz, CA; Cardenas, ME
MLA Citation
Shertz, CA, and Cardenas, ME. "Exploiting and subverting tor signaling in the pathogenesis of fungi, parasites, and viruses." PLoS Pathogens 7.9 (2011).
PMID
21980290
Source
scival
Published In
PLoS pathogens
Volume
7
Issue
9
Publish Date
2011
DOI
10.1371/journal.ppat.1002269

Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki.Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions.The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Authors
Shertz, CA; Bastidas, RJ; Li, W; Heitman, J; Cardenas, ME
MLA Citation
Shertz, CA, Bastidas, RJ, Li, W, Heitman, J, and Cardenas, ME. "Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom." BMC genomics 11 (September 23, 2010): 510-.
Website
http://hdl.handle.net/10161/4347
PMID
20863387
Source
epmc
Published In
BMC Genomics
Volume
11
Publish Date
2010
Start Page
510
DOI
10.1186/1471-2164-11-510

TORC1 signaling in the budding yeast endomembrane system and control of cell-cell adhesion in pathogenic fungi

The rapamycin-sensitive TORC1 protein kinase is the central component of a conserved signal transduction cascade controlling cell growth in response to nutrients and growth factors. Groundbreaking studies are uncovering novel roles for the endomembrane vesicular trafficking system as a platform for TORC1 signaling. TORC1 components, regulators, and major effectors have been localized to late endosomes and the vacuolar periphery in yeast. Mutations in the class C Vps, Ego/Gse, and PAS protein complexes, involved in vesicular trafficking and protein sorting, in combination with mutation of the nonessential Tor1 kinase severely compromise or abolish cell growth. Class C Vps complex function sustains amino acid homeostasis for efficient TORC1 signaling. The Ego/Gse complex (EGOC) GTPase activity responds to amino acids to regulate permease sorting and activate TORC1 signaling. The emerging view is that amino acids are sensed in intimate association with the endomembrane system, which facilitates molecular interactions to enable TORC1 activation and signaling. Novel roles for TORC1 are also surfacing throughout the fungal kingdom. TORC1 regulates filamentous growth in fungi. In the human pathogen Candida albicans, TORC1 controls transcriptional programs including those involved in ribosome biogenesis and nutritional control. Remarkably, in C. albicans TORC1 governs expression of adhesins, which promote tissue adherence, biofilm formation, and virulence. These studies reveal TORC1 pathway wiring plasticity as pathogenic yeasts adapted to their host niche environments. A preeminent role for TORC1 signaling in fungal virulence has attracted interest in the use of rapamycin and its analogs in antifungal therapy. © 2010 Elsevier Inc.

Authors
Bastidas, RJ; Cardenas, ME
MLA Citation
Bastidas, RJ, and Cardenas, ME. "TORC1 signaling in the budding yeast endomembrane system and control of cell-cell adhesion in pathogenic fungi." Enzymes 27.C (July 30, 2010): 199-227.
Source
scopus
Published In
Enzymes
Volume
27
Issue
C
Publish Date
2010
Start Page
199
End Page
227
DOI
10.1016/S1874-6047(10)27011-7

On the roles of calcineurin in fungal growth and pathogenesis

Calcineurin is a calcium-activated phosphatase that controls morphogenesis and stress responses in eukaryotes. Fungal pathogens have adopted the calcineurin pathway to survive and effectively propagate within the host. The difficulty in treating fungal infections stems from similarities between pathogen and host eukaryotic cells. Using calcineurin inhibitors such as cyclosporin A or tacrolimus (FK506) in combination with antifungal drugs, including azoles or echinocandins, renders these drugs fungicidal, even towards drug-resistant species or strains, making calcineurin a promising drug target. This article summarizes the current understanding of the calcineurin pathway and its roles in governing the growth and virulence of pathogenic fungi, and compares and contrasts the roles of calcineurin in fungal pathogens that infect humans (Candida albicans and Cryptococcus neoformans) or plants (Magnaporthe oryzae and Ustilago maydis). Further investigation of calcineurin biology will advance opportunities to develop novel antifungal therapeutic approaches and provide insight into the evolution of virulence. © 2010 Springer Science+Business Media, LLC.

Authors
Chen, Y-L; Kozubowski, L; Cardenas, ME; Heitman, J
MLA Citation
Chen, Y-L, Kozubowski, L, Cardenas, ME, and Heitman, J. "On the roles of calcineurin in fungal growth and pathogenesis." Current Fungal Infection Reports 4.4 (2010): 244-255.
Source
scival
Published In
Current Fungal Infection Reports
Volume
4
Issue
4
Publish Date
2010
Start Page
244
End Page
255
DOI
10.1007/s12281-010-0027-5

The protein kinase Tor1 regulates adhesin gene expression in Candida albicans.

Eukaryotic cell growth is coordinated in response to nutrient availability, growth factors, and environmental stimuli, enabling cell-cell interactions that promote survival. The rapamycin-sensitive Tor1 protein kinase, which is conserved from yeasts to humans, participates in a signaling pathway central to cellular nutrient responses. To gain insight into Tor-mediated processes in human fungal pathogens, we have characterized Tor signaling in Candida albicans. Global transcriptional profiling revealed evolutionarily conserved roles for Tor1 in regulating the expression of genes involved in nitrogen starvation responses and ribosome biogenesis. Interestingly, we found that in C. albicans Tor1 plays a novel role in regulating the expression of several cell wall and hyphal specific genes, including adhesins and their transcriptional repressors Nrg1 and Tup1. In accord with this transcriptional profile, rapamycin induced extensive cellular aggregation in an adhesin-dependent fashion. Moreover, adhesin gene induction and cellular aggregation of rapamycin-treated cells were strongly dependent on the transactivators Bcr1 and Efg1. These findings support models in which Tor1 negatively controls cellular adhesion by governing the activities of Bcr1 and Efg1. Taken together, these results provide evidence that Tor1-mediated cellular adhesion might be broadly conserved among eukaryotic organisms.

Authors
Bastidas, RJ; Heitman, J; Cardenas, ME
MLA Citation
Bastidas, RJ, Heitman, J, and Cardenas, ME. "The protein kinase Tor1 regulates adhesin gene expression in Candida albicans." PLoS pathogens 5.2 (February 6, 2009): e1000294-.
PMID
19197361
Source
epmc
Published In
PLoS pathogens
Volume
5
Issue
2
Publish Date
2009
Start Page
e1000294
DOI
10.1371/journal.ppat.1000294

Human protein phosphatase PP6 regulatory subunits provide Sit4-dependent and rapamycin-sensitive sap function in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G1 to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G1 gene expression and DNA synthesis, and partially abrogate the G1 cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2α phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans. © 2009 Morales-Johansson et al.

Authors
Morales-Johansson, H; Puria, R; Brautigan, DL; Cardenas, ME
MLA Citation
Morales-Johansson, H, Puria, R, Brautigan, DL, and Cardenas, ME. "Human protein phosphatase PP6 regulatory subunits provide Sit4-dependent and rapamycin-sensitive sap function in Saccharomyces cerevisiae." PLoS ONE 4.7 (2009).
PMID
19621075
Source
scival
Published In
PloS one
Volume
4
Issue
7
Publish Date
2009
DOI
10.1371/journal.pone.0006331

Signaling cascades as drug targets in model and pathogenic fungi.

Microbes evolved to produce natural products that inhibit growth of competing soil microorganisms. In many cases these compounds act on fungi, which are eukaryotes with conserved gene sequences closely related to metazoans, including humans. The calcineurin inhibitors cyclosporin A and FK-506, the Tor inhibitor rapamycin, and the Hsp90 inhibitor geldanamycin, all act via targets conserved from yeast to humans. This allows the use of genetically tractable fungi as models to elucidate how these drugs and their targets function in yeast and human cells. These inhibitors also enable studies aimed at harnessing their intrinsic antimicrobial activities to develop novel antifungal therapies. Extensive studies have revealed a globally conserved role for the Tor protein in regulating growth and proliferation in response to nutrients, and targeting its essential functions results in robust antifungal action. Similarly, a conserved and essential role for calcineurin in fungal virulence has been established and could be targeted by inhibitors for therapeutic uses in a variety of clinical settings. Finally, the discovery that inhibitors of calcineurin or Hsp90 result in dramatic synergism with either azoles or glucan synthase inhibitors (candins) provides another therapeutic vantage point. Taken together, these fungal targets and their inhibitors provide a robust platform from which to develop novel antimicrobial therapies.

Authors
Bastidas, RJ; Reedy, JL; Morales-Johansson, H; Heitman, J; Cardenas, ME
MLA Citation
Bastidas, RJ, Reedy, JL, Morales-Johansson, H, Heitman, J, and Cardenas, ME. "Signaling cascades as drug targets in model and pathogenic fungi." Current opinion in investigational drugs (London, England : 2000) 9.8 (August 2008): 856-864. (Review)
PMID
18666033
Source
epmc
Published In
Current Opinion in Investigational Drugs
Volume
9
Issue
8
Publish Date
2008
Start Page
856
End Page
864

Nutritional control via Tor signaling in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae senses and responds to nutrients by adapting its growth rate and undergoing morphogenic transitions to ensure survival. The Tor pathway is a major integrator of nutrient-derived signals that in coordination with other signaling pathways orchestrates cell growth. Recent advances have identified novel Tor kinase substrates and established the protein trafficking membranous network and the nucleus as platforms for Tor signaling. These and other recent findings delineate distinct signaling branches emanating from membrane-associated Tor complexes to control cell growth.

Authors
Rohde, JR; Bastidas, R; Puria, R; Cardenas, ME
MLA Citation
Rohde, JR, Bastidas, R, Puria, R, and Cardenas, ME. "Nutritional control via Tor signaling in Saccharomyces cerevisiae." Current opinion in microbiology 11.2 (April 8, 2008): 153-160. (Review)
PMID
18396450
Source
epmc
Published In
Current Opinion in Microbiology
Volume
11
Issue
2
Publish Date
2008
Start Page
153
End Page
160
DOI
10.1016/j.mib.2008.02.013

A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in saccharomyces cerevisiae

The ammonium permease Mep2 is required for the induction of pseudohyphal growth, a process in Saccharomyces cerevisiae that occurs in response to nutrient limitation. Mep2 has both a transport and a regulatory function, supporting models in which Mep2 acts as a sensor of ammonium availability. Potentially similar ammonium permease-dependent regulatory cascades operate in other fungi, and they may also function in animals via the homologous Rh proteins; however, little is known about the molecular mechanisms that mediate ammonium sensing. We show that Mep2 is localized to the cell surface during pseudohyphal growth, and it is required for both filamentous and invasive growth. Analysis of site-directed Mep2 mutants in residues lining the ammonia-conducting channel reveal separation of function alleles (transport and signaling defective; transport-proficient/signaling defective), indicating transport is necessary but not sufficient to sense ammonia. Furthermore, Mep2 overexpression enhances differentiation under normally repressive conditions and induces a transcriptional profile that is consistent with activation of the mitogen-activated protein (MAP) kinase pathway. This finding is supported by epistasis analysis establishing that the known role of the MAP kinase pathway in pseudohyphal growth is linked to Mep2 function. Together, these data strengthen the model that Mep2-like proteins are nutrient sensing transceptors that govern cellular differentiation. © 2008 by The American Society for Cell Biology.

Authors
Rutherford, JC; Chua, G; Hughes, T; Cardenas, ME; Heitman, J
MLA Citation
Rutherford, JC, Chua, G, Hughes, T, Cardenas, ME, and Heitman, J. "A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in saccharomyces cerevisiae." Molecular Biology of the Cell 19.7 (2008): 3028-3039.
PMID
18434596
Source
scival
Published In
Molecular Biology of the Cell
Volume
19
Issue
7
Publish Date
2008
Start Page
3028
End Page
3039
DOI
10.1091/mbc.E08-01-0033

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has developed specialized mechanisms that enable growth on suboptimal nitrogen sources. Exposure of yeast cells to poor nitrogen sources or treatment with the Tor kinase inhibitor rapamycin elicits activation of Gln3 and transcription of nitrogen catabolite-repressed (NCR) genes whose products function in scavenging and metabolizing nitrogen. Here, we show that mutations in class C and D Vps components, which mediate Golgi-to-endosome vesicle transport, impair nuclear translocation of Gln3, NCR gene activation, and growth in poor nitrogen sources. In nutrient-replete conditions, a significant fraction of Gln3 is peripherally associated with light membranes and partially colocalizes with Vps10-containing foci. These results reveal a role for Golgi-to-endosome vesicular trafficking in TORC1-controlled nuclear translocation of Gln3 and support a model in which Tor-mediated signaling in response to nutrient cues occurs in these compartments. These findings have important implications for nutrient sensing and growth control via mTor pathways in metazoans. © 2008 by The National Academy of Sciences of the USA.

Authors
Puria, R; Zurita-Martinez, SA; Cardenas, ME
MLA Citation
Puria, R, Zurita-Martinez, SA, and Cardenas, ME. "Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae." Proceedings of the National Academy of Sciences of the United States of America 105.20 (2008): 7194-7199.
PMID
18443284
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
105
Issue
20
Publish Date
2008
Start Page
7194
End Page
7199
DOI
10.1073/pnas.0801087105

Yeast as a model to study the immunosuppressive and chemotherapeutic drug rapamycin

Rapamycin is a natural product of a soil bacterium and has potent immunosuppressive, and antiproliferative actions. First identified in 1975 as an antifungal drug, rapamycin languished in obscurity after it was found to cause bone marrow suppression [112]. Interest in rapamycin was later rekindled when it was discovered to be structurally related to the potent T-cell inhibitor FK506 [65]. Subsequent studies, conducted first in yeast and then in mammalian cells, revealed the molecular basis of therapeutic action. Rapamycin diffuses into the cell and binds to a small cellular protein, FKBP12, forming an FKBP12-rapamycin protein-drug complex that is the active intracellular agent. This complex then binds to and inhibits the Tor kinases, which function in nutrient sensing pathways that control cell growth and differentiation (Figure 1) (reviewed in [101]). The Tor kinases and FKBP12 are conserved from yeast to worms, flies, plants, and humans. Rapamycin has received FDA approval as an immunosuppressant, and more recently as an antiproliferative agent to inhibit restenosis following cardiac stenting [84]. Recent studies indicate that rapamycin and its analogs will find additional clinical applications as chemotherapeutic agents, topical immunosuppressive therapies in dermatology, and novel antifungal agents [33, 45, 83, 88, 116]. We review here the use of yeast as a model to elucidate the molecular basis of therapeutics for this exciting natural product. The history of the budding yeast Saccharomyces cerevisiae as a model to identify the targets and molecular basis of therapeutic action of rapamycin and other immunosuppressive drugs began with the discovery of rapamycin (sirolimus), cyclosporine A (CsA), and FK506 (tracrolimus) themselves. These immunosuppressive drugs were identified in three independent screens conducted at three different pharmaceutical companies. CsA was discovered at Sandoz Pharmaceuticals in a screen of soil samples for inhibitors of a mixed lymphocyte response (MLR) assay. CsA is produced by a fungus, Tolypocladium inflatum, which was isolated from a soil sample from northern Norway [13]. CsA received FDA approval in 1983 as an immunosuppressant to prevent and treat graft rejection in organ transplant recipients, revolutionized organ transplant therapy, and became the gold standard for immunosuppressive therapies. FK506 was subsequently discovered at Fujisawa Pharmaceuticals in 1987 in a soil sample taken from the Tsukuba region of northern Japan and found to be a macrolide produced by the soil bacterium S. tsukubaensis [65]. FK506 (tacrolimus) received FDA approval in 1994 and has gone on to have considerable impact in transplant medicine. Rapamycin was discovered from a screen for novel natural products in 1975 at Wyeth-Ayerst Pharmaceuticals [112, 125]. In this case, the screen was for antifungal activity, and the rapamycin producing bacterium, S. hygroscopicus, was discovered in an isolate from the beaches of Easter Island (Rapa nui). Rapamycin remains one of the most potent anti-Candida drugs ever discovered [5], however the early finding that it caused bone marrow suppression halted development as an antimicrobial agent. It was only following the discovery of FK506 in 1987, and the appreciation that FK506 and rapamycin are structurally related, that rapamycin was resurrected from the shelf and studies began again in earnest to understand its molecular targets and possible therapeutic applications. Studies in yeast that began in the late 1980s defined the molecular targets of rapamycin, contributed to elucidate the mechanism of action of FK506 and CsA, and fueled comparable studies in mammalian T-cells that led to considerable insights into the molecular basis of therapeutic action (reviewed in [20]). Early studies from Merck demonstrated that FK506 and rapamycin inhibit T-cell proliferation by blocking different signaling pathways [38, 39]. FK506, like CsA, blocked the T-cell antigen response pathway necessary for signaling cascades to drive expression of hundreds of genes required for T-cell activation. In contrast, rapamycin had no effect on the T-cell antigen response pathway, but instead blocked T-cell proliferation in response to interleukin-2 (IL-2). FK506 and rapamycin were found to act as reciprocal antagonists [38, 39], suggesting that the two exerted their actions via a common target. Concurrently, biochemical studies led to the identification of a small abundant cellular binding protein, FK506 binding protein of 12 kDa (FKBP12), which is bound with high affinity to either FK506 or to rapamycin [49, 117]. However, given that FKBP12 is an abundant, ubiquitous protein expressed in all cells in the human body, a general view was that its drug-binding activity might not be involved in a very specific action in lymphocytes. Studies in yeast resolved this dilemma, and established unequivocally the central role of the FKBP12 protein in rapamycin action. Around this time, an FKBP12 homolog that shared 54% amino acid sequence identity with the human FKBP12 protein was identified in the yeast S. cerevisiae [52, 132]. Subsequently the crystal structures were solved for both the yeast and the human protein and found to be essentially superimposable [104, 123]. The gene encoding the yeast FKBP12 protein was cloned and disrupted. Whereas wild-type yeast cells are exquisitely sensitive to growth inhibition by rapamycin, with a minimum inhibitory concentration (MIC) of 25 ng/ml, yeast cells lacking FKBP12 were completely viable and only slightly reduced in growth rate [52, 67, 132]. Thus inhibition of an essential function of FKBP12 could not explain the potent toxic action of FKBP12. Taken together, these findings support a model in which both the FKBP12 protein and its ligand rapamycin are both required to exert a toxic effect in yeast. These findings also established unequivocally that FKBP12 plays a central role in the action of rapamycin, and the next challenge then was to identify the molecular target of the FKBP12-rapamycin complex. The targets of the FKBP12-rapamycin complex, the products of the TOR1 and TOR2 genes (target of rapamycin), were discovered in a genetic screen in yeast searching for rapamycin resistant mutants [51]. Mutations in three different genes were identified. First, mutations in the FPR1 gene encoding FKBP12 were found to be recessive, and resulted from amino acid substitutions that based on structural studies of the FKBP12-rapamycin complex were predicted to be critical for rapamycin binding. Importantly, mutations in two other genes identified were genetically distinct from FPR1, mapped to two different genomic locations and conferred dominant, or semidominant, drug resistance in genetic crosses. Based on an unusual genetic behavior between alleles of these three genes, known as nonallelic noncomplementation, it was proposed that the three might form a physical complex. The TOR1 and TOR2 genes were subsequently cloned by the Hall and Livi laboratories, revealing that they encode extremely large proteins, ∼280 kDa, that Tor1 and Tor2 are homologs of each other, and that both share a C-terminal domain with homology to lipid and protein kinases [18, 54, 71]. Further studies demonstrated that FKBP12-rapamycin forms a physical complex with the yeast Tor1 and Tor2 proteins [22, 76, 118]. Later, work from five different groups converged to identify the mammalian Tor homolog (mTor) via its ability to bind the FKBP12-rapamycin complex [15, 27, 105, 106]. In addition, it was demonstrated that yeast Tor-mTor hybrid genes are capable of providing Tor function in yeast cells [2]. Tor homologs were later identified in other organisms; similar to S. cerevisiae two Tor proteins have been characterized in S. pombe, and a single Tor homolog has been identified in C. albicans, C. neoformans, D. melanogaster, A. thaliana, and H. sapiens [15, 32, 51, 71, 82, 92, 105, 131, 136]. © 2007 Springer.

Authors
Rohde, JR; Zurita-Martinez, SA; Cardenas, ME
MLA Citation
Rohde, JR, Zurita-Martinez, SA, and Cardenas, ME. "Yeast as a model to study the immunosuppressive and chemotherapeutic drug rapamycin." (December 1, 2007): 347-374. (Chapter)
Source
scopus
Publish Date
2007
Start Page
347
End Page
374
DOI
10.1007/978-1-4020-5963-6_13

Sensing the environment: Lessons from fungi

All living organisms use numerous signal-transduction systems to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we review recent progress in our understanding of how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental cues.

Authors
Bahn, Y-S; Xue, C; Idnurm, A; Rutherford, JC; Heitman, J; Cardenas, ME
MLA Citation
Bahn, Y-S, Xue, C, Idnurm, A, Rutherford, JC, Heitman, J, and Cardenas, ME. "Sensing the environment: Lessons from fungi." Nature Reviews Microbiology 5.1 (2007): 57-69.
PMID
17170747
Source
scival
Published In
Nature Reviews Microbiology
Volume
5
Issue
1
Publish Date
2007
Start Page
57
End Page
69
DOI
10.1038/nrmicro1578

Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae

The Tor kinases regulate responses to nutrients and control cell growth. Unlike most organisms that only contain one Tor protein, Saccharomyces cerevisiae expresses two, Tor1 and Tor2, which are thought to share all of the rapamycin-sensitive functions attributable to Tor signaling. Here we conducted a genetic screen that defined the global TOR1 synthetic fitness or lethal interaction gene network. This screen identified mutations in distinctive functional categories that impaired vacuolar function, including components of the EGO/Gse and PAS complexes that reduce fitness. In addition, tor1 is lethal in combination with mutations in class C Vps complex components. We find that Tor1 does not regulate the known function of the class C Vps complex in protein sorting. Instead class C vps mutants fail to recover from rapamycin-induced growth arrest or to survive nitrogen starvation and have low levels of amino acids. Remarkably, addition of glutamate or glutamine restores viability to a tor1 pep3 mutant strain. We conclude that Tor1 is more effective than Tor2 at providing rapamycin-sensitive Tor signaling under conditions of amino acid limitation, and that an intact class C Vps complex is required to mediate intracellular amino acid homeostasis for efficient Tor signaling. Copyright © 2007 by the Genetics Society of America.

Authors
Zurita-Martinez, SA; Puria, R; Pan, X; Boeke, JD; Cardenas, ME
MLA Citation
Zurita-Martinez, SA, Puria, R, Pan, X, Boeke, JD, and Cardenas, ME. "Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae." Genetics 176.4 (2007): 2139-2150.
PMID
17565946
Source
scival
Published In
Genetics
Volume
176
Issue
4
Publish Date
2007
Start Page
2139
End Page
2150
DOI
10.1534/genetics.107.072835

Tor and cyclic AMP-protein kinase A: Two parallel pathways regulating expression of genes required for cell growth

In the budding yeast Saccharomyces cerevisiae, the Tor and cyclic AMP-protein kinase A (cAMP-PKA) signaling cascades respond to nutrients and regulate coordinately the expression of genes required for cell growth, including ribosomal protein (RP) and stress-responsive (STRE) genes. The inhibition of Tor signaling by rapamycin results in repression of the RP genes and induction of the STRE genes. Mutations that hyperactivate PKA signaling confer resistance to rapamycin and suppress the repression of RP genes imposed by rapamycin. By contrast, partial inactivation of PKA confers rapamycin hypersensitivity but only modestly affects RP gene expression. Complete inactivation of PKA impairs RP gene expression and concomitantly enhances STRE gene expression; remarkably, this altered transcriptional pattern is still sensitive to rapamycin and thus subject to Tor control. These findings illustrate how the Tor and cAMP-PKA signaling pathways respond to nutrient signals to govern gene expression required for cell growth via two parallel routes, and they have broad implication for our understanding of analogous regulatory networks in normal and neoplastic mammalian cells.

Authors
Zurita-Martinez, SA; Cardenas, ME
MLA Citation
Zurita-Martinez, SA, and Cardenas, ME. "Tor and cyclic AMP-protein kinase A: Two parallel pathways regulating expression of genes required for cell growth." Eukaryotic Cell 4.1 (2005): 63-71.
PMID
15643061
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
1
Publish Date
2005
Start Page
63
End Page
71
DOI
10.1128/EC.4.1.63-71.2005

TOR controls transcriptional and translational programs via Sap-Sit4 protein phosphatase signaling effectors

The Tor kinases are the targets of the immunosuppressive drug rapamycin and couple nutrient availability to cell growth. In the budding yeast Saccharomyces cerevisiae, the PP2A-related phosphatase Sit4 together with its regulatory subunit Tap42 mediates several Tor signaling events. Sit4 interacts with other potential regulatory proteins known as the Saps. Deletion of the SAP or SIT4 genes confers increased sensitivity to rapamycin and defects in expression of subsets of Tor-regulated genes. Sap155, Sap185, or Sap190 can restore these responses. Strains lacking Sap185 and Sap190 are hypersensitive to rapamycin, and this sensitivity is Gcn2 dependent and correlated with a defect in translation, constitutive eukaryotic initiation factor 2α hyperphosphorylation, induction of GCN4 translation, and hypersensitivity to amino acid starvation. We conclude that Tor signals via Sap-Sit4 complexes to control both transcriptional and translational programs that couple cell growth to amino acid availability.

Authors
Rohde, JR; Campbell, S; Zurita-Martinez, SA; Cutler, NS; Ashe, M; Cardenas, ME
MLA Citation
Rohde, JR, Campbell, S, Zurita-Martinez, SA, Cutler, NS, Ashe, M, and Cardenas, ME. "TOR controls transcriptional and translational programs via Sap-Sit4 protein phosphatase signaling effectors." Molecular and Cellular Biology 24.19 (2004): 8332-8341.
PMID
15367655
Source
scival
Published In
Molecular and Cellular Biology
Volume
24
Issue
19
Publish Date
2004
Start Page
8332
End Page
8341
DOI
10.1128/MCB.24.19.8332-8341.2004

Nutrient signaling through TOR kinases controls gene expression and cellular differentiation in fungi

The TOR kinases were first identified in Saccharomyces cerevisiae as the targets of the immunosuppressive drug rapamycin. Subsequent studies employing rapamycin as a tool in yeast have given us insight into the structure and function of the TOR kinases, as well as the biological role of the TOR signaling program in transmitting nutrient signals to promote cell growth. One of the major advances from this area has been in defining an unexpected role for TOR signaling in the regulation of transcription. The identification of target genes subject to regulation by TOR has provided a platform for the dissection of the signaling events downstream of the TOR kinases. Studies aimed at understanding TOR-regulated transcription have begun to shed light on how TOR signaling cooperates with other signaling programs. In addition, the TOR pathway regulates the developmental program of pseudohyphal differentiation in concert with highly conserved MAP kinase and PKA signaling programs. Remarkably, rapamycin also blocks filamentation in a number of important human and plant pathogens and the mechanism of rapamycin action is conserved in Candida albicans and Cryptococcus neoformans. The antimicrobial properties of less immunosuppressive analogs of rapamycin hold promise for the development of an effective anti-fungal therapy.

Authors
Rohde, JR; Cardenas, ME
MLA Citation
Rohde, JR, and Cardenas, ME. "Nutrient signaling through TOR kinases controls gene expression and cellular differentiation in fungi." Current Topics in Microbiology and Immunology 279 (2003): 53-72.
PMID
14560951
Source
scival
Published In
Current topics in microbiology and immunology
Volume
279
Publish Date
2003
Start Page
53
End Page
72

The Tor pathway regulates gene expression by linking nutrient sensing to histone acetylation

The Tor pathway mediates cell growth in response to nutrient availability, in part by inducing ribosomal protein (RP) gene expression via an unknown mechanism. Expression of RP genes coincides with recruitment of the Esa1 histone acetylase to RP gene promoters. We show that inhibition of Tor with rapamycin releases Esa1 from RP gene promoters and leads to histone H4 deacetylation without affecting promoter occupancy by Rap1 and Abf1. Genetic and biochemical evidence identifies Rpd3 as the major histone deacetylase responsible for reversing histone H4 acetylation at RP gene promoters in response to Tor inhibition by rapamycin or nutrient limitation. Our results illustrate that the Tor pathway links nutrient sensing with histone acetylation to control RP gene expression and cell growth.

Authors
Rohde, JR; Cardenas, ME
MLA Citation
Rohde, JR, and Cardenas, ME. "The Tor pathway regulates gene expression by linking nutrient sensing to histone acetylation." Molecular and Cellular Biology 23.2 (2003): 629-635.
PMID
12509460
Source
scival
Published In
Molecular and Cellular Biology
Volume
23
Issue
2
Publish Date
2003
Start Page
629
End Page
635
DOI
10.1128/MCB.23.2.629-635.2003

Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATalpha and MATa cells. Virulence has been linked to the mating type, and MATalpha cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20alpha, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20alpha, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20alpha pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20alpha gene, the MATalpha locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.

Authors
Wang, P; Nichols, CB; Lengeler, KB; Cardenas, ME; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Wang, P, Nichols, CB, Lengeler, KB, Cardenas, ME, Cox, GM, Perfect, JR, and Heitman, J. "Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans." Eukaryot Cell 1.2 (April 2002): 257-272.
PMID
12455960
Source
pubmed
Published In
Eukaryotic cell
Volume
1
Issue
2
Publish Date
2002
Start Page
257
End Page
272

Calcineurin is essential for survival during membrane stress in Candida albicans.

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.

Authors
Cruz, MC; Goldstein, AL; Blankenship, JR; Del Poeta, M; Davis, D; Cardenas, ME; Perfect, JR; McCusker, JH; Heitman, J
MLA Citation
Cruz, MC, Goldstein, AL, Blankenship, JR, Del Poeta, M, Davis, D, Cardenas, ME, Perfect, JR, McCusker, JH, and Heitman, J. "Calcineurin is essential for survival during membrane stress in Candida albicans." EMBO J 21.4 (February 15, 2002): 546-559.
PMID
11847103
Source
pubmed
Published In
EMBO Journal
Volume
21
Issue
4
Publish Date
2002
Start Page
546
End Page
559

The TOR signal transduction cascade controls cellular differentiation in response to nutrients.

Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.

Authors
Cutler, NS; Pan, X; Heitman, J; Cardenas, ME
MLA Citation
Cutler, NS, Pan, X, Heitman, J, and Cardenas, ME. "The TOR signal transduction cascade controls cellular differentiation in response to nutrients." Mol Biol Cell 12.12 (December 2001): 4103-4113.
PMID
11739804
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
12
Issue
12
Publish Date
2001
Start Page
4103
End Page
4113

Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR.

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans. Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.

Authors
Cruz, MC; Goldstein, AL; Blankenship, J; Del Poeta, M; Perfect, JR; McCusker, JH; Bennani, YL; Cardenas, ME; Heitman, J
MLA Citation
Cruz, MC, Goldstein, AL, Blankenship, J, Del Poeta, M, Perfect, JR, McCusker, JH, Bennani, YL, Cardenas, ME, and Heitman, J. "Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR." Antimicrob Agents Chemother 45.11 (November 2001): 3162-3170.
PMID
11600372
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
45
Issue
11
Publish Date
2001
Start Page
3162
End Page
3170
DOI
10.1128/AAC.45.11.3162-3170.2001

Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans.

Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39 degrees C and attenuated for virulence, whereas cpa2 mutants are viable at 39 degrees C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.

Authors
Wang, P; Cardenas, ME; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Wang, P, Cardenas, ME, Cox, GM, Perfect, JR, and Heitman, J. "Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans." EMBO Rep 2.6 (June 2001): 511-518.
PMID
11415984
Source
pubmed
Published In
EMBO Reports
Volume
2
Issue
6
Publish Date
2001
Start Page
511
End Page
518
DOI
10.1093/embo-reports/kve109

The TOR kinases link nutrient sensing to cell growth.

Rapamycin is an immunosuppressive natural product that inhibits the proliferation of T-cells in response to nutrients and growth factors. Rapamycin binds to the peptidyl-prolyl isomerase FKBP12 and forms protein-drug complexes that inhibit signal transduction by the TOR kinases. The FKBP12 and TOR proteins are conserved from fungi to humans, and in both organisms the TOR signaling pathway plays a role in nutrient sensing. In response to nitrogen sources or amino acids, TOR regulates both transcription and translation, enabling cells to appropriately respond to growth-promoting signals. Rapamycin is having a profound impact on clinical medicine and was approved as an immunosuppressant for transplant recipients in 1999. Ongoing clinical studies address new clinical applications for rapamycin as an antiproliferative drug for chemotherapy and invasive cardiology.

Authors
Rohde, J; Heitman, J; Cardenas, ME
MLA Citation
Rohde, J, Heitman, J, and Cardenas, ME. "The TOR kinases link nutrient sensing to cell growth." J Biol Chem 276.13 (March 30, 2001): 9583-9586. (Review)
PMID
11266435
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
13
Publish Date
2001
Start Page
9583
End Page
9586
DOI
10.1074/jbc.R000034200

Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans.

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

Authors
Fox, DS; Cruz, MC; Sia, RA; Ke, H; Cox, GM; Cardenas, ME; Heitman, J
MLA Citation
Fox, DS, Cruz, MC, Sia, RA, Ke, H, Cox, GM, Cardenas, ME, and Heitman, J. "Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans." Mol Microbiol 39.4 (February 2001): 835-849.
PMID
11251806
Source
pubmed
Published In
Molecular Microbiology
Volume
39
Issue
4
Publish Date
2001
Start Page
835
End Page
849

The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

The Ess1/Pin1 peptidyl-prolyl isomerase (PPIase) is thought to control mitosis by binding to cell cycle regulatory proteins and altering their activity. Here we isolate temperature-sensitive ess1 mutants and identify six multicopy suppressors that rescue their mitotic-lethal phenotype. None are cell cycle regulators. Instead, five encode proteins involved in transcription that bind DNA, modify chromatin structure or are regulatory subunits of RNA polymerase II. A sixth suppressor, cyclophilin A, is a member of a distinct family of PPIases that are targets of immuno suppressive drugs. We show that the expression of some but not all genes is decreased in ess1 mutants, and that Ess1 interacts with the C-terminal domain (CTD) of RNA polymerase II in vitro and in vivo. The results forge a strong link between PPIases and the transcription machinery and suggest a new model for how Ess1/Pin1 controls mitosis. In this model, Ess1 binds and isomerizes the CTD of RNA polymerase II, thus altering its interaction with proteins required for transcription of essential cell cycle genes.

Authors
Wu, X; Wilcox, CB; Devasahayam, G; Hackett, RL; Arévalo-Rodríguez, M; Cardenas, ME; Heitman, J; Hanes, SD
MLA Citation
Wu, X, Wilcox, CB, Devasahayam, G, Hackett, RL, Arévalo-Rodríguez, M, Cardenas, ME, Heitman, J, and Hanes, SD. "The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery." EMBO J 19.14 (July 17, 2000): 3727-3738.
PMID
10899126
Source
pubmed
Published In
EMBO Journal
Volume
19
Issue
14
Publish Date
2000
Start Page
3727
End Page
3738
DOI
10.1093/emboj/19.14.3727

Cyclophilin A and Ess1 interact with and regulate silencing by the Sin3-Rpd3 histone deacetylase.

Three families of prolyl isomerases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. All 12 cyclophilins and FKBPs are dispensable for growth in yeast, whereas the one parvulin homolog, Ess1, is essential. We report here that cyclophilin A becomes essential when Ess1 function is compromised. We also show that overexpression of cyclophilin A suppresses ess1 conditional and null mutations, and that cyclophilin A enzymatic activity is required for suppression. These results indicate that cyclophilin A and Ess1 function in parallel pathways and act on common targets by a mechanism that requires prolyl isomerization. Using genetic and biochemical approaches, we found that one of these targets is the Sin3-Rpd3 histone deacetylase complex, and that cyclophilin A increases and Ess1 decreases disruption of gene silencing by this complex. We show that conditions that favor acetylation over deacetylation suppress ess1 mutations. Our findings support a model in which Ess1 and cyclophilin A modulate the activity of the Sin3-Rpd3 complex, and excess histone deacetylation causes mitotic arrest in ess1 mutants.

Authors
Arévalo-Rodríguez, M; Cardenas, ME; Wu, X; Hanes, SD; Heitman, J
MLA Citation
Arévalo-Rodríguez, M, Cardenas, ME, Wu, X, Hanes, SD, and Heitman, J. "Cyclophilin A and Ess1 interact with and regulate silencing by the Sin3-Rpd3 histone deacetylase." EMBO J 19.14 (July 17, 2000): 3739-3749.
PMID
10899127
Source
pubmed
Published In
EMBO Journal
Volume
19
Issue
14
Publish Date
2000
Start Page
3739
End Page
3749
DOI
10.1093/emboj/19.14.3739

Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system. Existing therapies include amphotericin B, fluconazole, and flucytosine, which are limited by toxic side effects and the emergence of drug resistance. We recently demonstrated that the protein phosphatase calcineurin is required for growth at 37 degrees C and virulence of C. neoformans. Because calcineurin is the target of potent inhibitors in widespread clinical use, cyclosporine and FK506 (tacrolimus), it is an attractive drug target for novel antifungal agents. Here we have explored the synergistic potential of combining the calcineurin inhibitor FK506 or its nonimmunosuppressive analog, L-685,818, with other antifungal agents and examined the molecular basis of FK506 action by using genetically engineered fungal strains that lack the FK506 target proteins FKBP12 and calcineurin. We demonstrate that FK506 exhibits marked synergistic activity with the H(+)ATPase inhibitor bafilomycin A(1) via a novel action distinct from calcineurin loss of function. FK506 also exhibits synergistic activity with the pneumocandin MK-0991/caspofungin acetate (formerly L-743,873), which targets the essential beta-1,3 glucan synthase, and in this case, FK506 action is mediated via FKBP12-dependent inhibition of calcineurin. Finally, we demonstrate that FK506 and fluconazole have synergistic activity that is independent of both FKBP12 and calcineurin and may involve the known ability of FK506 to inhibit multidrug resistance pumps, which are known to export azoles from fungal cells. In summary, our studies illustrate the potential for synergistic activity of a variety of different drug combinations and the power of molecular genetics to define the mechanisms of drug action, as well as identify a novel action of FK506 that could have profound implications for therapeutic or toxic effects in other organisms, including humans.

Authors
Del Poeta, M; Cruz, MC; Cardenas, ME; Perfect, JR; Heitman, J
MLA Citation
Del Poeta, M, Cruz, MC, Cardenas, ME, Perfect, JR, and Heitman, J. "Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans." Antimicrob Agents Chemother 44.3 (March 2000): 739-746.
PMID
10681348
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
44
Issue
3
Publish Date
2000
Start Page
739
End Page
746

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.

Authors
Lorenz, MC; Pan, X; Harashima, T; Cardenas, ME; Xue, Y; Hirsch, JP; Heitman, J
MLA Citation
Lorenz, MC, Pan, X, Harashima, T, Cardenas, ME, Xue, Y, Hirsch, JP, and Heitman, J. "The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae." Genetics 154.2 (February 2000): 609-622.
PMID
10655215
Source
pubmed
Published In
Genetics
Volume
154
Issue
2
Publish Date
2000
Start Page
609
End Page
622

Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptococcus neoformans via cyclophilin-dependent inhibition of calcineurin.

Cyclosporine (CsA) is an immunosuppressive and antimicrobial drug which, in complex with cyclophilin A, inhibits the protein phosphatase calcineurin. We recently found that Cryptococcus neoformans growth is resistant to CsA at 24 degrees C but sensitive at 37 degrees C and that calcineurin is required for growth at 37 degrees C and pathogenicity. Here CsA analogs were screened for toxicity against C. neoformans in vitro. In most cases, antifungal activity was correlated with cyclophilin A binding in vitro and inhibition of the mixed-lymphocyte reaction and interleukin 2 production in cell culture. Two unusual nonimmunosuppressive CsA derivatives, (gamma-OH) MeLeu(4)-Cs (211-810) and D-Sar (alpha-SMe)(3) Val(2)-DH-Cs (209-825), which are also toxic to C. neoformans were identified. These CsA analogs inhibit C. neoformans via fungal cyclophilin A and calcineurin homologs. Our findings identify calcineurin as a novel antifungal drug target and suggest nonimmunosuppressive CsA analogs warrant investigation as antifungal agents.

Authors
Cruz, MC; Del Poeta, M; Wang, P; Wenger, R; Zenke, G; Quesniaux, VF; Movva, NR; Perfect, JR; Cardenas, ME; Heitman, J
MLA Citation
Cruz, MC, Del Poeta, M, Wang, P, Wenger, R, Zenke, G, Quesniaux, VF, Movva, NR, Perfect, JR, Cardenas, ME, and Heitman, J. "Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptococcus neoformans via cyclophilin-dependent inhibition of calcineurin." Antimicrob Agents Chemother 44.1 (January 2000): 143-149.
PMID
10602736
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
44
Issue
1
Publish Date
2000
Start Page
143
End Page
149

The TOR signaling cascade regulates gene expression in response to nutrients.

Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to man. The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. We have analyzed gene expression in yeast cells exposed to rapamycin using arrays representing the whole yeast genome. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression. gln3 mutations were found to confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants. We find that Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. In summary, our results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability.

Authors
Cardenas, ME; Cutler, NS; Lorenz, MC; Di Como, CJ; Heitman, J
MLA Citation
Cardenas, ME, Cutler, NS, Lorenz, MC, Di Como, CJ, and Heitman, J. "The TOR signaling cascade regulates gene expression in response to nutrients." Genes Dev 13.24 (December 15, 1999): 3271-3279.
PMID
10617575
Source
pubmed
Published In
Genes & development
Volume
13
Issue
24
Publish Date
1999
Start Page
3271
End Page
3279

Antifungal activities of antineoplastic agents: Saccharomyces cerevisiae as a model system to study drug action.

Recent evolutionary studies reveal that microorganisms including yeasts and fungi are more closely related to mammals than was previously appreciated. Possibly as a consequence, many natural-product toxins that have antimicrobial activity are also toxic to mammalian cells. While this makes it difficult to discover antifungal agents without toxic side effects, it also has enabled detailed studies of drug action in simple genetic model systems. We review here studies on the antifungal actions of antineoplasmic agents. Topics covered include the mechanisms of action of inhibitors of topoisomerases I and II; the immunosuppressants rapamycin, cyclosporin A, and FK506; the phosphatidylinositol 3-kinase inhibitor wortmannin; the angiogenesis inhibitors fumagillin and ovalicin; the HSP90 inhibitor geldanamycin; and agents that inhibit sphingolipid metabolism. In general, these natural products inhibit target proteins conserved from microorganisms to humans. These studies highlight the potential of microorganisms as screening tools to elucidate the mechanisms of action of novel pharmacological agents with unique effects against specific mammalian cell types, including neoplastic cells. In addition, this analysis suggests that antineoplastic agents and derivatives might find novel indications in the treatment of fungal infections, for which few agents are presently available, toxicity remains a serious concern, and drug resistance is emerging.

Authors
Cardenas, ME; Cruz, MC; Del Poeta, M; Chung, N; Perfect, JR; Heitman, J
MLA Citation
Cardenas, ME, Cruz, MC, Del Poeta, M, Chung, N, Perfect, JR, and Heitman, J. "Antifungal activities of antineoplastic agents: Saccharomyces cerevisiae as a model system to study drug action." Clin Microbiol Rev 12.4 (October 1999): 583-611. (Review)
PMID
10515904
Source
pubmed
Published In
Clinical microbiology reviews
Volume
12
Issue
4
Publish Date
1999
Start Page
583
End Page
611

TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals.

Rapamycin is a natural product with potent antifungal and immunosuppressive activities. Rapamycin binds to the FKBP12 prolyl isomerase, and the resulting protein-drug complex inhibits the TOR kinase homologs. Both the FKBP12 and the TOR proteins are highly conserved from yeast to man, and genetic and biochemical studies reveal that these proteins are the targets of rapamycin in vivo. Treatment of yeast or mammalian cells with rapamycin inhibits translational initiation of a subset of mRNAs and dramatically represses ribosomal mRNA and tRNA transcription. Furthermore, rapamycin exposure blocks cell cycle progression in the early G1 phase of the cell cycle, driving cells into a G0 state and, ultimately, triggering autophagy. Recent findings reveal that the upstream factors regulating the TOR signaling cascade are involved in detecting amino acids, nutrients, or growth factors. These findings indicate that the TOR proteins function in a signal transduction pathway that coordinates nutritional and mitogenic signals to control protein biosynthesis and degradation.

Authors
Cutler, NS; Heitman, J; Cardenas, ME
MLA Citation
Cutler, NS, Heitman, J, and Cardenas, ME. "TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals." Mol Cell Endocrinol 155.1-2 (September 10, 1999): 135-142. (Review)
PMID
10580846
Source
pubmed
Published In
Molecular and Cellular Endocrinology
Volume
155
Issue
1-2
Publish Date
1999
Start Page
135
End Page
142

Secretion of FK506/FK520 and rapamycin by Streptomyces inhibits the growth of competing Saccharomyces cerevisiae and Cryptococcus neoformans.

FK506 and rapamycin are immunosuppressants that inhibit signalling cascades required for T-cell activation, yet both are natural products of Streptomyces that live in the soil. FK506 and rapamycin also have potent antimicrobial activity against yeast and pathogenic fungi, suggesting a natural role in inhibiting growth of competing micro-organisms. The immunosuppressive and antimicrobial activities of FK506 and rapamycin are mediated by binding to the FKBP12 prolyl isomerase and the resulting FKBP12/FK506 and FKBP12/rapamycin complexes inhibit conserved protein targets, either the phosphatase calcineurin or the TOR (target of rapamycin) kinases, respectively. Streptomyces sp., 'Streptomyces hygroscopicus subsp. ascomyceticus' and Streptomyces hygroscopicus, which produce FK506, FK520 (also known as ascomycin, a C21 ethyl derivative of FK506) and rapamycin, respectively, produced toxins that inhibited the growth of competing cells of the yeast Saccharomyces cerevisiae and the pathogenic fungus Cryptococcus neoformans. Yeast and fungal mutants lacking FKBP12 or expressing dominant drug-resistant calcineurin or TOR mutants were resistant to FK506 and rapamycin, and to the toxins produced by Streptomyces. Streptomyces strains with mutations in the FK506 or rapamycin biosynthetic enzymes were impaired in toxin production. Finally, the toxins secreted by 'S. hygroscopicus subsp. ascomyceticus' and S. hygroscopicus promoted formation of FKBP12/calcineurin and FKBP12/TOR complexes in a two-hybrid assay and mutations that rendered calcineurin or TOR drug-resistant prevented interaction. These observations support the hypothesis that Streptomyces evolved to secrete FK506, FK520 and rapamycin as toxins to inhibit the growth of competing yeast and fungi.

Authors
Arndt, C; Cruz, MC; Cardenas, ME; Heitman, J
MLA Citation
Arndt, C, Cruz, MC, Cardenas, ME, and Heitman, J. "Secretion of FK506/FK520 and rapamycin by Streptomyces inhibits the growth of competing Saccharomyces cerevisiae and Cryptococcus neoformans." Microbiology 145 ( Pt 8) (August 1999): 1989-2000.
PMID
10463165
Source
pubmed
Published In
Microbiology (Reading, England)
Volume
145 ( Pt 8)
Publish Date
1999
Start Page
1989
End Page
2000
DOI
10.1099/13500872-145-8-1989

Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast.

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Authors
Alarcon, CM; Heitman, J; Cardenas, ME
MLA Citation
Alarcon, CM, Heitman, J, and Cardenas, ME. "Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast." Mol Biol Cell 10.8 (August 1999): 2531-2546.
PMID
10436010
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
10
Issue
8
Publish Date
1999
Start Page
2531
End Page
2546

Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents.

Authors
Cruz, MC; Cavallo, LM; Görlach, JM; Cox, G; Perfect, JR; Cardenas, ME; Heitman, J
MLA Citation
Cruz, MC, Cavallo, LM, Görlach, JM, Cox, G, Perfect, JR, Cardenas, ME, and Heitman, J. "Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans." Mol Cell Biol 19.6 (June 1999): 4101-4112.
PMID
10330150
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
6
Publish Date
1999
Start Page
4101
End Page
4112

CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations and interacts with Hsp90.

Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast.

Authors
Dolinski, KJ; Cardenas, ME; Heitman, J
MLA Citation
Dolinski, KJ, Cardenas, ME, and Heitman, J. "CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations and interacts with Hsp90." Mol Cell Biol 18.12 (December 1998): 7344-7352.
PMID
9819421
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
18
Issue
12
Publish Date
1998
Start Page
7344
End Page
7352

Signal-transduction cascades as targets for therapeutic intervention by natural products.

Many bacteria and fungi produce natural products that are toxic to other microorganisms and have a variety of physiological effects in animals. Recent studies have revealed that, in several cases, the targets of these agents are components of conserved signal-transduction cascades. This article looks at the mechanisms of action of five natural products--the immunosuppressants cyclosporin A, FK506 and rapamycin, and the antiproliferative agents wortmannin and geldanamycin. These mechanisms reveal the importance of signal-transduction cascades as targets for therapeutic intervention and the enormous utility of studies of natural-product action in simple model genetic systems.

Authors
Cardenas, ME; Sanfridson, A; Cutler, NS; Heitman, J
MLA Citation
Cardenas, ME, Sanfridson, A, Cutler, NS, and Heitman, J. "Signal-transduction cascades as targets for therapeutic intervention by natural products." Trends Biotechnol 16.10 (October 1998): 427-433. (Review)
PMID
9807840
Source
pubmed
Published In
Trends in Biotechnology
Volume
16
Issue
10
Publish Date
1998
Start Page
427
End Page
433

Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells.

The role of the mammalian target of rapamycin (mTOR) was investigated in insulin responsive cell lines. mTOR was expressed at high levels in insulin responsive cell types and in 3T3-L1 cells mTOR expression levels increased dramatically as cells differentiated from fibroblasts into insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically localized to microsomal membranes. Protein kinase activity directed towards mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was due to an autokinase activity present in mTOR. The mTOR autokinase and the protein kinase activity of the p110 alpha isoform of PI 3-kinase shared several notable similarities; (a) both were maximally active in the presence of Mn2+ but also showed significant activity in the presence of Mg2+ (b) neither were inhibited by the presence of non-ionic detergent and (c) both were inhibited by wortmannin and LY294002, known inhibitors of the PI 3-kinase lipid kinase activity. These data taken together indicate the autokinase activity lay in the PI 3-kinase homology domain. In summary mTOR is a membrane anchored protein kinase that is active in conditions encountered in vivo and the fact it is highly expressed in insulin responsive cell types is consistent with a role in insulin signalling.

Authors
Withers, DJ; Ouwens, DM; Nave, BT; van der Zon, GC; Alarcon, CM; Cardenas, ME; Heitman, J; Maassen, JA; Shepherd, PR
MLA Citation
Withers, DJ, Ouwens, DM, Nave, BT, van der Zon, GC, Alarcon, CM, Cardenas, ME, Heitman, J, Maassen, JA, and Shepherd, PR. "Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells." Biochem Biophys Res Commun 241.3 (December 29, 1997): 704-709.
PMID
9434772
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
241
Issue
3
Publish Date
1997
Start Page
704
End Page
709
DOI
10.1006/bbrc.1997.7878

All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae.

The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding. We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae. Five cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.

Authors
Dolinski, K; Muir, S; Cardenas, M; Heitman, J
MLA Citation
Dolinski, K, Muir, S, Cardenas, M, and Heitman, J. "All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae." Proc Natl Acad Sci U S A 94.24 (November 25, 1997): 13093-13098.
PMID
9371805
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
24
Publish Date
1997
Start Page
13093
End Page
13098

Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae.

Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

Authors
Dolinski, K; Scholz, C; Muir, RS; Rospert, S; Schmid, FX; Cardenas, ME; Heitman, J
MLA Citation
Dolinski, K, Scholz, C, Muir, RS, Rospert, S, Schmid, FX, Cardenas, ME, and Heitman, J. "Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae." Mol Biol Cell 8.11 (November 1997): 2267-2280.
PMID
9362068
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
8
Issue
11
Publish Date
1997
Start Page
2267
End Page
2280

STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae.

Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate.

Authors
Cutler, NS; Heitman, J; Cardenas, ME
MLA Citation
Cutler, NS, Heitman, J, and Cardenas, ME. "STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae." J Biol Chem 272.44 (October 31, 1997): 27671-27677.
PMID
9346907
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
44
Publish Date
1997
Start Page
27671
End Page
27677

Calcineurin mutants render T lymphocytes resistant to cyclosporin A.

The immunosuppressants cyclosporin A (CsA) and FK506 have been widely used to prevent and treat graft rejection after human organ and tissue transplantations. CsA and FK506 associate with intracellular binding proteins (i.e., CsA with cyclophilin A and FK506 with FKBP12) to form protein/drug complexes that suppress the immune system by preventing activation of T cells in response to antigen presentation. The common target of CsA and FK506 is calcineurin, a Ca2+/calmodulin-regulated, serine/threonine-specific protein phosphatase that regulates the nuclear import of a transcription factor, NF-AT, required for expression of T cell activation genes. In previous studies, we identified calcineurin mutations that block binding by the cyclophilin A/CsA or FKBP12/FK506 complexes and thereby render yeast cells resistant to the antifungal effects of CsA or FK506. In this report, we demonstrate that the corresponding mutations in murine calcineurin render the T cell receptor signal transduction cascade CsA resistant in human Jurkat T cells. Our findings support the recently determined calcineurin X-ray crystal structure, provide evidence that calcineurin is the only CsA-sensitive component limiting signaling from the T cell receptor to the nucleus, and suggest a means to render cells and tissues resistant to the toxic side effects of CsA and FK506.

Authors
Zhu, D; Cardenas, ME; Heitman, J
MLA Citation
Zhu, D, Cardenas, ME, and Heitman, J. "Calcineurin mutants render T lymphocytes resistant to cyclosporin A." Mol Pharmacol 50.3 (September 1996): 506-511.
PMID
8794888
Source
pubmed
Published In
Molecular pharmacology
Volume
50
Issue
3
Publish Date
1996
Start Page
506
End Page
511

Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast.

In complex with the prolyl isomerase FKBP12, the natural product rapamycin blocks signal transduction in organisms as diverse as yeast and man. The yeast targets of FKBP12-rapamycin, TOR1 and TOR2, are large proteins with homology to lipid and protein kinases. A mammalian FKBP12-rapamycin binding protein, RAFT1, shares 39% and 43% identity with TOR1 and TOR2 proteins, respectively but has not been linked to rapamycin action in vivo. We find that when expressed in yeast, neither wild-type nor mutant RAFT1 complemented tor mutations or conferred rapamycin resistance. In contrast, TOR1-RAFT1 and TOR1-RAFT1 hybrid proteins containing the carboxy-terminal RAFT1 kinase domain complemented tor2 and tor1 mutant strains, respectively. Moreover, TOR2-RAFT1 and TOR1-RAFT1 hybrid proteins mutated at the position corresponding to rapamycin-resistant TOR mutants (S20351) conferred rapamycin resistance. Like the TOR2 protein, the TOR2-RAFT1 proteins were stably expressed, localized to the vacuolar surface, and associated with a phosphatidylinositol-4 kinase activity. These findings directly link the mammalian TOR homolog RAFT1 to rapamycin action in vivo and indicate that the TOR/RAFT1 kinase domain has been functionally conserved from yeast to man.

Authors
Alarcon, CM; Cardenas, ME; Heitman, J
MLA Citation
Alarcon, CM, Cardenas, ME, and Heitman, J. "Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast." Genes Dev 10.3 (February 1, 1996): 279-288.
PMID
8595879
Source
pubmed
Published In
Genes & development
Volume
10
Issue
3
Publish Date
1996
Start Page
279
End Page
288

FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity.

In complex with the immunophilin FKBP12, the natural product rapamycin inhibits signal transduction events required for G1 to S phase cell cycle progression in yeast and mammalian cells. Genetic studies in yeast first implicated the TOR1 and TOR2 proteins as targets of the FKBP12-rapamycin complex. We report here that the TOR2 protein is membrane associated and localized to the surface of the yeast vacuole. Immunoprecipitated TOR2 protein contains readily detectable phosphatidylinositol-4 (PI-4) kinase activity attributable to either a TOR2 intrinsic activity or to a PI-4 kinase tightly associated with TOR2. Importantly, we find that rapamycin stimulates FKBP12 binding to wild-type TOR2 but not to a rapamycin-resistant TOR2-1 mutant protein. Surprisingly, FKBP12-rapamycin binding does not markedly inhibit the PI kinase activity associated with TOR2, but does cause a delocalization of TOR2 from the vacuolar surface, which may deprive the TOR2-associated PI-4 kinase activity of its in vivo substrate. Several additional findings indicate that vacuolar localization is important for TOR2 function and, conversely, that TOR2 modulates vacuolar morphology and segregation. These studies demonstrate that TOR2 is an essential, highly conserved component of a signal transduction pathway regulating cell cycle progression conserved from yeast to man.

Authors
Cardenas, ME; Heitman, J
MLA Citation
Cardenas, ME, and Heitman, J. "FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity." EMBO J 14.23 (December 1, 1995): 5892-5907.
PMID
8846782
Source
pubmed
Published In
EMBO Journal
Volume
14
Issue
23
Publish Date
1995
Start Page
5892
End Page
5907

Molecular mechanisms of immunosuppression by cyclosporine, FK506, and rapamycin.

The immunosuppressant cyclosporine A revolutionized treatment of graft rejection. Two newer agents, FK506 and rapamycin, show great clinical potential. These drugs suppress the immune system by forming protein-drug complexes that interact with and inhibit key components of the signal transduction pathways required for T-cell activation. The target of the cyclophilin A-cyclosporine A and FKBP12-FK506 complexes is calcineurin, a protein phosphatase required for signaling via the T-cell receptor. Cyclosporine A and FK506 nephrotoxicity may reflect renal-specific functions of calcineurin. The target of the FKBP12-rapamycin complex is TOR, a lipid and protein kinase homolog that is likely to be required for T-cell proliferation in response to interleukin-2. The identification of cyclosporine A, FK506, and rapamycin targets reveals much concerning T-cell signaling and provides the means to design novel immunosuppressants with reduced toxicity.

Authors
Cardenas, ME; Zhu, D; Heitman, J
MLA Citation
Cardenas, ME, Zhu, D, and Heitman, J. "Molecular mechanisms of immunosuppression by cyclosporine, FK506, and rapamycin." Curr Opin Nephrol Hypertens 4.6 (November 1995): 472-477. (Review)
PMID
8591053
Source
pubmed
Published In
Current Opinion in Nephrology and Hypertension
Volume
4
Issue
6
Publish Date
1995
Start Page
472
End Page
477

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

Authors
Hemenway, CS; Dolinski, K; Cardenas, ME; Hiller, MA; Jones, EW; Heitman, J
MLA Citation
Hemenway, CS, Dolinski, K, Cardenas, ME, Hiller, MA, Jones, EW, and Heitman, J. "vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly." Genetics 141.3 (November 1995): 833-844.
PMID
8582630
Source
pubmed
Published In
Genetics
Volume
141
Issue
3
Publish Date
1995
Start Page
833
End Page
844

Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae.

Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast. Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus. We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate [3H]myristate when expressed in yeast. Both wild-type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations. In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity. In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A. Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506. This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex.

Authors
Zhu, D; Cardenas, ME; Heitman, J
MLA Citation
Zhu, D, Cardenas, ME, and Heitman, J. "Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae." J Biol Chem 270.42 (October 20, 1995): 24831-24838.
PMID
7559604
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
42
Publish Date
1995
Start Page
24831
End Page
24838

Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae.

In complex with the peptidyl-prolyl isomerase cyclophilin A, the immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, which regulates signal transduction. We isolated and characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive. Three mutations (G70S, H90Y, and G102A) alter single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophilin A. By Western analysis, all three mutant proteins are expressed in yeast. In vitro, two purified mutant cyclophilins (G70S, G102A) retain prolyl isomerase activity and have moderately reduced affinity for CsA and calcineurin but, when bound to CsA, do bind and inhibit calcineurin phosphatase activity. In contrast, the purified H90Y mutant cyclophilin is dramatically decreased in prolyl isomerase activity, CsA affinity, and calcineurin binding and inhibition. These studies identify conserved cyclophilin A residues that participate in CsA binding and catalysis.

Authors
Cardenas, ME; Lim, E; Heitman, J
MLA Citation
Cardenas, ME, Lim, E, and Heitman, J. "Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae." J Biol Chem 270.36 (September 8, 1995): 20997-21002.
PMID
7673124
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
36
Publish Date
1995
Start Page
20997
End Page
21002

Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.

Authors
Cardenas, ME; Muir, RS; Breuder, T; Heitman, J
MLA Citation
Cardenas, ME, Muir, RS, Breuder, T, and Heitman, J. "Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A." EMBO J 14.12 (June 15, 1995): 2772-2783.
PMID
7540976
Source
pubmed
Published In
EMBO Journal
Volume
14
Issue
12
Publish Date
1995
Start Page
2772
End Page
2783

Role of calcium in T-lymphocyte activation.

Authors
Cardenas, ME; Heitman, J
MLA Citation
Cardenas, ME, and Heitman, J. "Role of calcium in T-lymphocyte activation." Adv Second Messenger Phosphoprotein Res 30 (1995): 281-298. (Review)
PMID
7695994
Source
pubmed
Published In
Advances in Second Messenger and Phosphoprotein Research
Volume
30
Publish Date
1995
Start Page
281
End Page
298

Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands.

The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.

Authors
Cardenas, ME; Hemenway, C; Muir, RS; Ye, R; Fiorentino, D; Heitman, J
MLA Citation
Cardenas, ME, Hemenway, C, Muir, RS, Ye, R, Fiorentino, D, and Heitman, J. "Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands." EMBO J 13.24 (December 15, 1994): 5944-5957.
PMID
7529175
Source
pubmed
Published In
EMBO Journal
Volume
13
Issue
24
Publish Date
1994
Start Page
5944
End Page
5957

Antifungal effects of cyclosporine and FK 506 are mediated via immunophilin-dependent calcineurin inhibition.

Authors
Heitman, J; Cardenas, ME; Breuder, T; Hemenway, C; Muir, RS; Lim, E; Goetz, L; Zhu, D; Lorenz, M; Dolinski, K
MLA Citation
Heitman, J, Cardenas, ME, Breuder, T, Hemenway, C, Muir, RS, Lim, E, Goetz, L, Zhu, D, Lorenz, M, and Dolinski, K. "Antifungal effects of cyclosporine and FK 506 are mediated via immunophilin-dependent calcineurin inhibition." Transplant Proc 26.5 (October 1994): 2833-2834.
PMID
7524220
Source
pubmed
Published In
Transplantation Proceedings
Volume
26
Issue
5
Publish Date
1994
Start Page
2833
End Page
2834

Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains.

The immunophilin-immunosuppressant complexes cyclophilin-cyclosporin A (CsA) and FKBP12-FK506 inhibit the phosphatase calcineurin to block T-cell activation. Although cyclophilin A, FKBP12, and calcineurin are highly conserved from yeast to man, none had previously been shown to be essential for viability. We find that CsA-sensitive yeast strains are FK506 hypersensitive and demonstrate that calcineurin is required for viability in these strains. Mutants lacking cyclophilin A or FKBP12 are resistant to CsA or FK506, respectively. Thus, both the immunosuppressive and the antifungal actions of CsA and FK506 result from calcineurin inhibition by immunophilin-drug complexes. In yeast strains in which calcineurin is not essential, calcineurin inhibition or mutation of calcineurin confers hypersensitivity to LiCl or NaCl, suggesting that calcineurin regulates cation transport.

Authors
Breuder, T; Hemenway, CS; Movva, NR; Cardenas, ME; Heitman, J
MLA Citation
Breuder, T, Hemenway, CS, Movva, NR, Cardenas, ME, and Heitman, J. "Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains." Proc Natl Acad Sci U S A 91.12 (June 7, 1994): 5372-5376.
PMID
7515500
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
91
Issue
12
Publish Date
1994
Start Page
5372
End Page
5376

Yeast as model T cells

The immunosuppressants cyclosporin A, FK-506, and rapamycin prevent T-cell activation by inhibiting intermediate signal transduction steps. Studies have focused on their mechanisms of action, with the aim of both designing novel immunosuppressants and understanding signal transduction. Biochemical studies first identified the primary drug targets, the immunophilins cyclophilin and FKBP. Genetic studies in yeast demonstrated that the active agents in vivo are toxic protein-drug complexes. The target of cyclophilin-CsA and FKBP12-FK-506, the calcium- and calmodulin-regulated phosphatase calcineurin, regulates nuclear import of a T-cell-specific transcription factor during response to antigen. In yeast, calcineurin is required for recovery from pheromone-induced cell cycle arrest. The challenges ahead are to understand the normal cellular roles of the immunophilins, to biochemically define the direct target of the FKBP12-rapamycin complex, and to translate recent advances into the design of novel immunosuppressants. © 1994 ESCOM Science Publishers B.V.

Authors
Cardenas, ME; Lorenz, M; Hemenway, C; Heitman, J
MLA Citation
Cardenas, ME, Lorenz, M, Hemenway, C, and Heitman, J. "Yeast as model T cells." Perspectives in Drug Discovery and Design 2.1 (1994): 103-126.
Source
scival
Published In
Perspectives in Drug Discovery and Design
Volume
2
Issue
1
Publish Date
1994
Start Page
103
End Page
126
DOI
10.1007/BF02171739

The regulation of DNA topoisomerase II by casein kinase II

DNA topoisomerase II is an essential nuclear enzyme required for the proper condensation and segregation of chromosomes during mitotic and meiotic cell division. The enzyme exists in the cell as a phosphoprotein, and it is most highly phosphorylated in G2 and M-phases of the cell cycle. We have shown that topoisomerase II is the target of casein kinase II (CKII) in yeast by comparison of in vivo and in vitro phosphotryptic peptide maps. Limited proteolysis and probing with domain specific antibodies show that with the exception of a weakly modified residue between aa 660 and aa 1250, all residues modified by CKII are in the last 200 amino acids of yeast topoisomerase II. This C-terminal domain is the least conserved region of the enzyme and truncation of the enzyme shows that it is nonessential for activity in vitro. However, the fully dephosphorylated full-size protein is nearly inactive in decatenation assays, and activity can be restored by phosphorylation by CKII. To reconcile these observations, we propose that the Cterminal region is a negative regulatory domain, counteracted by phosphorylation within the domain itself. To test this hypothesis we have mutagenised 12 potential CKII phosphoacceptor sites in the C-terminus of topoisomerase II and introduced the mutant genes into a yeast strain which has a temperature sensitive top2 gene. The growth of the transformed strains is monitored at nonpermissive temperature to determine whether C-terminal phosphorylation is important for mitotic growth. In addition, we have purified the mutant enzymes to homogeneity for in vitro assays. © 1994.

Authors
Alghisi, GC; Roberts, E; Cardenas, ME; Gasser, SM
MLA Citation
Alghisi, GC, Roberts, E, Cardenas, ME, and Gasser, SM. "The regulation of DNA topoisomerase II by casein kinase II." Cellular and Molecular Biology Research 40.5-6 (1994): 563-571.
PMID
7735331
Source
scival
Published In
Cellular and Molecular Biology Research
Volume
40
Issue
5-6
Publish Date
1994
Start Page
563
End Page
571

Identification of Immunosuppressive Drug Targets in Yeast

Cells transfer information from the cell surface to the nucleus via signal transduction pathways. Although much is known about the two ends of such pathways, membrane receptors and nuclear transcription factors, the intermediate steps have remained elusive. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin inhibit conserved elements of signal transduction cascades. The primary drug targets, the immunophilins cyclophilin and FKBP, and the target of the cyclophilin-CsA and FKBP- FK506 complexes, the phosphatase calcineurin, are highly conserved from yeast to human. Studies on immunosuppressant mechanisms of action are providing insights into signal transduction in both yeast and T lymphocytes. © 1993 Academic Press. All rights reserved.

Authors
Heitman, J; Koller, A; Cardenas, ME; Hall, MN
MLA Citation
Heitman, J, Koller, A, Cardenas, ME, and Hall, MN. "Identification of Immunosuppressive Drug Targets in Yeast." Methods 5.2 (1993): 176-187.
Source
scival
Published In
Methods
Volume
5
Issue
2
Publish Date
1993
Start Page
176
End Page
187
DOI
10.1006/meth.1993.1022

Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme

Mitotic division in yeast requires the activity of topoisomerase II, a DNA topology modifying enzyme that is able to disentangle sister chromatids after DNA replication. Previous work has shown that topoisomerase II is a phosphoprotein in intact yeast cells. We show here that when dephosphorylated in vitro, topoisomerase II is unable to cleave or decatenate kinetoplast DNA. An efficient kinase activity that modifies topoisomerase II on seven major sites was found to copurify with the enzyme purified from yeast. Characterization of this kinase, analysis of phosphotryptic peptides, and studies with a yeast mutant deficient in casein kinase II, indicate that the copurifying kinase is casein kinase II (CKII). Topoisomerase II itself has no self-phosphorylating activity. Modification of topoisomerase II by the copurifying kinase is sufficient to restore decatenation activity after dephosphorylation by alkaline phosphatase. The CKII target sites have been mapped to multiple serine and threonine residues on 4 tryptic fragments within the C-terminal 350 amino acids of yeast topoisomerase II. These results are consistent with a model in which the C-terminal domain of topoisomerase II is a negative regulatory domain that is neutralized by phosphorylation.

Authors
Cardenas, ME; Walter, R; Hanna, D; Gasser, SM
MLA Citation
Cardenas, ME, Walter, R, Hanna, D, and Gasser, SM. "Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme." Journal of Cell Science 104.2 (1993): 533-543.
PMID
8389377
Source
scival
Published In
Journal of Cell Science
Volume
104
Issue
2
Publish Date
1993
Start Page
533
End Page
543

Regulation of topoisomerase II by phosphorylation: A role for casein kinase II

Authors
Cardenas, ME; Gasser, SM
MLA Citation
Cardenas, ME, and Gasser, SM. "Regulation of topoisomerase II by phosphorylation: A role for casein kinase II." Journal of Cell Science 104.2 (1993): 219-225.
PMID
8389373
Source
scival
Published In
Journal of Cell Science
Volume
104
Issue
2
Publish Date
1993
Start Page
219
End Page
225

A Xenopus egg factor with DNA-binding properties characteristic of terminus-specific telomeric proteins

We have identified a Xenupus laevis protein factor that specifically recognizes vertebrate telomeric repeats at DNA ends. This factor, called Xenopus telomere end factor (XTEF), is detected predominantly in extracts of Xenopus eggs and ovaries, which are estimated to contain sufficient XTEF to bind ∼3 × 107 DNA ends. In contrast, XTEF is much less abundant (∼90 per cell) in extracts of somatic cell nuclei. Mobility retardation analysis of the XTEF activity in egg extracts indicates that this factor binds the vertebrate telomeric repeat sequence (TTAGGG)2 when present in a single-stranded 3′ overhang. Single-stranded 3′ extensions of (TTTGGG)2, (AAAGGG)2, (TTACCC)2, or a nonrepetitive sequence fail to bind XTEF efficiently, whereas changes in the double-stranded sequence 5′ to the TTAGGG repeat tail are tolerated. TTAGGG repeats are not recognized at internal positions, at a 5′ protruding end, or in double-stranded DNA. In addition, the factor does not bind RNA with single-stranded UUAGGG repeats at a 3′ end. XTEF-DNA complexes form and are stable in high salt. The DNA-binding properties of XTEF resemble the characteristics of a class of terminus-specific telomere proteins identified previously in hypotrichous ciliates.

Authors
Cardenas, ME; Bianchi, A; Lange, TD
MLA Citation
Cardenas, ME, Bianchi, A, and Lange, TD. "A Xenopus egg factor with DNA-binding properties characteristic of terminus-specific telomeric proteins." Genes and Development 7.5 (1993): 883-894.
PMID
7684008
Source
scival
Published In
Genes and Development
Volume
7
Issue
5
Publish Date
1993
Start Page
883
End Page
894

Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast

Topoisomerase II (topoII) and RAP1 (Repressor Activator Protein 1) are two abundant nuclear proteins with proposed structural roles in the higherorder organization of chromosomes. Both proteins cofractionate as components of nuclear scaffolds from vegetatively growing yeast cells, and both proteins are present as components of pachytene chromosomes, cofractionating with an insoluble subfraction of meiotic nuclei. Immunolocalization using antibodies specific for topoII shows staining of an axial core of the yeast meiotic chromosome, extending the length of the synaptonemal complex. RAP1, on the other hand, is located at the ends of the paired bivalent chromosomes, consistent with its ability to bind telomeric sequences in vitro. In interphase nuclei, again in contrast to antitopoII, anti-RAP1 gives a distinctly punctate staining that is located primarily at the nuclear periphery. Approximately 16 brightly staining foci can be identified in a diploid nucleus stained with anti-RAP1 antibodies, suggesting that telomeres are grouped together, perhaps through interaction with the nuclear envelope.

Authors
Klein, F; Laroche, T; Cardenas, ME; Hofmann, JF-X; Schweizer, D; Gasser, SM
MLA Citation
Klein, F, Laroche, T, Cardenas, ME, Hofmann, JF-X, Schweizer, D, and Gasser, SM. "Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast." Journal of Cell Biology 117.5 (1992): 935-948.
PMID
1315786
Source
scival
Published In
The Journal of Cell Biology
Volume
117
Issue
5
Publish Date
1992
Start Page
935
End Page
948
DOI
10.1083/jcb.117.5.935

Topoisomerase II: its functions and phosphorylation

The gene encoding topoisomerase II in yeast is unique and essential, required for both mitotic and meiotic proliferation. The use of temperature-sensitive mutants in topoisomerase II have demonstrated roles in the relaxation of tortional stress, reduction of recombination rates, and in the separation of sister chromatids after replication. In vertebrate cells, topoisomerase II was shown to be the most abundant component of the metaphase chromosomal scaffold, and has been shown to play a role in chromosome condensation in vitro. The cell cycle control of chromosome condensation may well require phosphorylation of topoisomerase II, since the enzyme is more highly phosphorylated in metaphase than in G1. Recent studies have identified casein kinase II as the major enzyme phosphorylating topoisomerase II in intact yeast cells. The target sites of CKII are exclusively in the C-terminal 400 amino acids of topoisomerase II, the region that is most divergent among the eukaryotic type II enzymes and which is absent in the bacterial gyrase homologues. © 1992 Kluwer Academic Publishers.

Authors
Gasser, SM; Walter, R; Dang, Q; Cardenas, ME
MLA Citation
Gasser, SM, Walter, R, Dang, Q, and Cardenas, ME. "Topoisomerase II: its functions and phosphorylation." Antonie van Leeuwenhoek 62.1-2 (1992): 15-24.
PMID
1332607
Source
scival
Published In
Antonie van Leeuwenhoek
Volume
62
Issue
1-2
Publish Date
1992
Start Page
15
End Page
24
DOI
10.1007/BF00584459

Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.

Authors
Cardenas, ME; Dang, Q; Glover, CVC; Gasser, SM
MLA Citation
Cardenas, ME, Dang, Q, Glover, CVC, and Gasser, SM. "Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo." EMBO Journal 11.5 (1992): 1785-1796.
PMID
1316274
Source
scival
Published In
EMBO Journal
Volume
11
Issue
5
Publish Date
1992
Start Page
1785
End Page
1796

The composition and morphology of yeast nuclear scaffolds

The yeast nuclear scaffold has been shown to bind with high affinity to genomic sequences that permit autonomous DNA replication of plasmids (ARS elements). We describe here conditions for the isolation of a histone-free nuclear substructure, the nuclear scaffold, from Saccharomyces cerevisiae. We examine the protein composition of this structure, and the conditions under which topoisomerase II, the nuclear factor RAP-I and RNA polymerase II cofractionate with the scaffold. We find that exposure of nuclei to a combined metal and heat treatment (0.5mM Cu2+, 37°C) prior to detergent extraction is required for effective stabilization of these proteins with the scaffold. Electron microscopy of the residual nuclei extracted with non-ionic detergents shows the absence of a typical peripheral lamina structure.

Authors
Cardenas, ME; Laroche, T; Gasser, SM
MLA Citation
Cardenas, ME, Laroche, T, and Gasser, SM. "The composition and morphology of yeast nuclear scaffolds." Journal of Cell Science 96.3 (1990): 439-450.
PMID
22096810
Source
scival
Published In
Journal of cell science
Volume
96
Issue
3
Publish Date
1990
Start Page
439
End Page
450

Studies on scaffold attachment sites and their relation to genome function

Authors
Gasser, SM; Amati, BB; Cardenas, ME; Hofmann, JF-X
MLA Citation
Gasser, SM, Amati, BB, Cardenas, ME, and Hofmann, JF-X. "Studies on scaffold attachment sites and their relation to genome function." International Review of Cytology 119 (1989): 57-96.
PMID
2695485
Source
scival
Published In
International review of cytology
Volume
119
Publish Date
1989
Start Page
57
End Page
96

Endogenous polymers of ADP-ribose are associated with the nuclear matrix.

The metabolism of nuclear polymers of ADP-ribose has been implicated in several chromatin-associated processes. However, the distribution of endogenous ADP-ribose polymers in the nucleus or within different fractions of chromatin has not been studied. Using a procedure which allowed the radiolabeling and detection of endogenous polymers of ADP-ribose, we have analyzed the nuclear distribution of these polymers in untreated cells and in cells subjected to hyperthermia, N-methyl-N'-nitro-N-nitrosoguanidine, or both. When isolated nuclei from cells subjected to any of these conditions were digested with micrococcal nuclease such that 80% of the DNA was released, 90% of the total poly(ADP-ribose) remained with the micrococcal nuclease resistant chromatin fraction. When nuclear matrix fractions were prepared by exhaustive DNase I digestion in combination with three different salt extraction procedures (2 M NaCl, 300 mM (NH4)2SO4 or 25 mM lithium diiodosalicylate), the matrices contained less than 1% of the total nuclear DNA but 50 to 70% of the total poly(ADP-ribose). These data suggest that the nuclear matrix may be a major site of poly(ADP-ribose) metabolism.

Authors
Cardenas-Corona, ME; Jacobson, EL; Jacobson, MK
MLA Citation
Cardenas-Corona, ME, Jacobson, EL, and Jacobson, MK. "Endogenous polymers of ADP-ribose are associated with the nuclear matrix." J Biol Chem 262.31 (November 5, 1987): 14863-14866.
PMID
3667608
Source
pubmed
Published In
The Journal of biological chemistry
Volume
262
Issue
31
Publish Date
1987
Start Page
14863
End Page
14866

Studies of endogenous mono(ADP-ribosylation)

Authors
Jacobson, MK; Payne, DM; Cardenas, ME
MLA Citation
Jacobson, MK, Payne, DM, and Cardenas, ME. "Studies of endogenous mono(ADP-ribosylation)." Federation Proceedings 44.3 (1985): No.-1587.
Source
scival
Published In
Federation Proceedings
Volume
44
Issue
3
Publish Date
1985
Start Page
No.
End Page
1587

Glutamine metabolism during aerial mycelium growth of Neurospora crassa

Authors
Cardenas, ME; Hansberg, W
MLA Citation
Cardenas, ME, and Hansberg, W. "Glutamine metabolism during aerial mycelium growth of Neurospora crassa." Journal of General Microbiology 130.7 (1984): 1733-1741.
PMID
22096812
Source
scival
Published In
Journal of General Microbiology
Volume
130
Issue
7
Publish Date
1984
Start Page
1733
End Page
1741

Glutamine requirement for aerial mycelium growth in Neurospora crassa

Authors
Cardenas, ME; Hansberg, W
MLA Citation
Cardenas, ME, and Hansberg, W. "Glutamine requirement for aerial mycelium growth in Neurospora crassa." Journal of General Microbiology 130.7 (1984): 1723-1732.
PMID
22096811
Source
scival
Published In
Journal of General Microbiology
Volume
130
Issue
7
Publish Date
1984
Start Page
1723
End Page
1732
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