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Nicchitta, Christopher Vincent

Overview:

  Our laboratory studies the cellular architecture and regulation of protein synthesis, with the goal of understanding how cells regulate the subcellular organization and temporal dynamics of protein synthesis. We focus on mRNA localization - the process by which cells control where and when a protein is synthesized by localizing its mRNA to a discrete location(s) in the cell. Such regulation is critical for many aspects of cell dynamics, cell signaling and cell division. Of the diverse mRNA localization phenomena that have been identified to date, the most prominent is mRNA localization to the endoplasmic reticulum (ER). mRNA localization to the ER operates on an unusually large scale (essentially the entire mRNA transcriptome is partially represented on the ER, with those mRNAs encoding secretory and membrane proteins being highly ER-enriched), and continuously– all newly exported mRNAs undergo selection for translation in the cytosol and/or the ER compartments.

We use a broad array of experimental approaches - biochemistry, cell biology, genomics, and computational biology - and are focusing on several related themes. First, we are working to identify the mRNA-encoded signals used to target mRNAs to the ER as well as the cellular factors that recognize these signals. One mechanism, in which a signal in nascent secretory and membrane proteins directs mRNA recruitment to the ER, has been previously described. It is clear though that there are multiple pathways that direct mRNAs to the ER, including pathways that direct cytosolic and nucleoplasmic protein-encoding mRNAs to the ER. We are also investigating how, once localized, mRNAs are anchored to the ER membrane. In a recent study, we reported that the cohort of mRNAs encoding organelle resident proteins(e.g., nuclear envelope, ER, Golgi, lysosomes, peroxisomes) are localized tothe ER and directly anchored to components of the ER membrane. We are very interested in understanding the cis-encoded anchoring signals and the integral membrane proteins that function in mRNA anchoring to biological membrane, and lastly, how direct mRNA anchoring influences mRNA translation and mRNA stability.


In parallel efforts, we discovered that mRNA translation is under distinct regulatory control in the cytosol and ER compartments, with translation being 3-5 fold more efficient on the ER. These differences are substantial and suggest that mRNA localization to the ER may represent an important post-transcriptional gene expression mechanism. To gain insight into the mechanisms and factors responsible for the compartmental regulation of mRNA translation we are using traditional biochemical approaches (pulse-labeling, cell fractionation, immunoprecipitation, proteomics) as well as genomic approaches (ribosome footprinting, deep sequencing).

Positions:

Professor of Cell Biology

Cell Biology
School of Medicine

Associate Professor of Pathology

Pathology
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Director, of Graduate Studies

Graduate School
Graduate School

Associate Dean of Research Training

School of Medicine
School of Medicine

Education:

Ph.D. 1987

Ph.D. — University of Pennsylvania

News:

Grants:

Organization and Function of Cellular Structure

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

RNA localization in neural stem cells during cortical development

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
July 01, 2016
End Date
June 30, 2019

Eukaryotic Initiation Factor 2a Signaling in Dystonia Pathogenesis and Treatment

Administered By
Neurology, Movement Disorders
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
July 01, 2016
End Date
June 30, 2018

Dissecting the role of the exon junction complex in embryonic corticogenesis

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
April 01, 2016
End Date
March 31, 2018

Mechanisms of Translational Compartmentalization in Eukaryotic Cells

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2017
End Date
November 30, 2017

Training Program in Developmental and Stem Cell Biology

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2001
End Date
October 31, 2017

mRNA Localization in Organelle Biogenesis

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2012
End Date
July 31, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

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

Regulation of mRNA Partitioning to the Endoplasmic Reticulum

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2007
End Date
October 31, 2012

Role Of Surfactant In Innate and Adaptive Immunity

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
July 01, 2001
End Date
September 30, 2011

Rapidly Degraded Polypeptides in Protein Quality Control

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2008
End Date
August 31, 2009

Mechanism of Chaperone-Mediated Tumor Rejection

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2004
End Date
February 28, 2009

The Molecular Mechanism of GRP94 Function

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1997
End Date
April 30, 2007

Novel mechanisms governing cellular mRNA localization

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 02, 2004
End Date
February 28, 2007

Structure and Regulation of hsp90 Chaperones

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
May 01, 2005
End Date
October 31, 2005

Regulation of GRP94: Targeting Novel Cancer Therapeutics

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2002
End Date
June 30, 2005

GRP94-Mediated Immune Responses Evoked by Cell Death

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2001
End Date
January 31, 2004

The Molecular Mechanism Of Translocation

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1994
End Date
April 30, 2003

Intracellular Trafficking of Exogenous Chaperones

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1999
End Date
June 30, 2002

The Role Of Lumenal Proteins In Protein Translocation

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1994
End Date
April 30, 1999

The Role Of Lumenal Proteins And Protein Translocation

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1997
End Date
April 30, 1998

The Role Of Lumenal Proteins And Protein Transiocation

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1995
End Date
April 30, 1998
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Publications:

Flavivirus RNA transactions from viral entry to genome replication.

Upon release of the ∼11 kb single-stranded positive polarity dengue virus genomic RNA (gRNA) into the cytoplasm of an infected cell, it serves as the template for translation of the viral polyprotein, which is cleaved into three structural and seven non-structural proteins. The structural organization of the viral replication complex and RNA is not known but it is increasingly becoming evident that the viral gRNA and replication intermediates adopt unique structural features and localize to discrete regions in the infected cell. Both structure and location play multiple roles ranging from evasion of innate immune response to recruitment of viral and host proteins for translation and replication of the message. This review visits the various transactions that the viral gRNA undergoes between entry and RNA synthesis with the view that some of these events may be targeted by antiviral compounds. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research.

Authors
Garcia-Blanco, MA; Vasudevan, SG; Bradrick, SS; Nicchitta, C
MLA Citation
Garcia-Blanco, MA, Vasudevan, SG, Bradrick, SS, and Nicchitta, C. "Flavivirus RNA transactions from viral entry to genome replication." Antiviral research 134 (October 2016): 244-249.
PMID
27666184
Source
epmc
Published In
Antiviral Research
Volume
134
Publish Date
2016
Start Page
244
End Page
249
DOI
10.1016/j.antiviral.2016.09.010

Complementary Roles of GADD34- and CReP-Containing Eukaryotic Initiation Factor 2α Phosphatases during the Unfolded Protein Response.

Phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation in stressed cells. While phosphorylated eIF2α (P-eIF2α) attenuates global protein synthesis, mRNAs encoding stress proteins are more efficiently translated. Two eIF2α phosphatases, containing GADD34 and CReP, catalyze P-eIF2α dephosphorylation. The current view of GADD34, whose transcription is stress induced, is that it functions in a feedback loop to resolve cell stress. In contrast, CReP, which is constitutively expressed, controls basal P-eIF2α levels in unstressed cells. Our studies show that GADD34 drives substantial changes in mRNA translation in unstressed cells, particularly targeting the secretome. Following activation of the unfolded protein response (UPR), rapid translation of GADD34 mRNA occurs and GADD34 is essential for UPR progression. In the absence of GADD34, eIF2α phosphorylation is persistently enhanced and the UPR translational program is significantly attenuated. This "stalled" UPR is relieved by the subsequent activation of compensatory mechanisms that include AKT-mediated suppression of PKR-like kinase (PERK) and increased expression of CReP mRNA, partially restoring protein synthesis. Our studies highlight the coordinate regulation of UPR by the GADD34- and CReP-containing eIF2α phosphatases to control cell viability.

Authors
Reid, DW; Tay, ASL; Sundaram, JR; Lee, ICJ; Chen, Q; George, SE; Nicchitta, CV; Shenolikar, S
MLA Citation
Reid, DW, Tay, ASL, Sundaram, JR, Lee, ICJ, Chen, Q, George, SE, Nicchitta, CV, and Shenolikar, S. "Complementary Roles of GADD34- and CReP-Containing Eukaryotic Initiation Factor 2α Phosphatases during the Unfolded Protein Response." Molecular and cellular biology 36.13 (July 2016): 1868-1880.
PMID
27161320
Source
epmc
Published In
Molecular and Cellular Biology
Volume
36
Issue
13
Publish Date
2016
Start Page
1868
End Page
1880
DOI
10.1128/mcb.00190-16

Simple and inexpensive ribosome profiling analysis of mRNA translation.

The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.

Authors
Reid, DW; Shenolikar, S; Nicchitta, CV
MLA Citation
Reid, DW, Shenolikar, S, and Nicchitta, CV. "Simple and inexpensive ribosome profiling analysis of mRNA translation." Methods (San Diego, Calif.) 91 (December 2015): 69-74.
PMID
26164698
Source
epmc
Published In
Methods
Volume
91
Publish Date
2015
Start Page
69
End Page
74
DOI
10.1016/j.ymeth.2015.07.003

LOCAL TRANSLATION. Comment on "Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling".

Jan et al. (Research Articles, 7 November 2014, p. 716) propose that ribosomes translating secretome messenger RNAs (mRNAs) traffic from the cytosol to the endoplasmic reticulum (ER) upon emergence of the signal peptide and return to the cytosol after termination. An accounting of controls demonstrates that mRNAs initiate translation on ER-bound ribosomes and that ribosomes are retained on the ER through many cycles of translation.

Authors
Reid, DW; Nicchitta, CV
MLA Citation
Reid, DW, and Nicchitta, CV. "LOCAL TRANSLATION. Comment on "Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling"." Science (New York, N.Y.) 348.6240 (June 11, 2015): 1217-.
PMID
26068841
Source
epmc
Published In
Science
Volume
348
Issue
6240
Publish Date
2015
Start Page
1217
DOI
10.1126/science.aaa7257

Diversity and selectivity in mRNA translation on the endoplasmic reticulum.

Pioneering electron microscopy studies defined two primary populations of ribosomes in eukaryotic cells: one freely dispersed through the cytoplasm and the other bound to the surface of the endoplasmic reticulum (ER). Subsequent investigations revealed a specialized function for each population, with secretory and integral membrane protein-encoding mRNAs translated on ER-bound ribosomes, and cytosolic protein synthesis was widely attributed to free ribosomes. Recent findings have challenged this view, and transcriptome-scale studies of mRNA distribution and translation have now demonstrated that ER-bound ribosomes also function in the translation of a large fraction of mRNAs that encode cytosolic proteins. These studies suggest a far more expansive role for the ER in transcriptome expression, where membrane and secretory protein synthesis represents one element of a multifaceted and dynamic contribution to post-transcriptional gene expression.

Authors
Reid, DW; Nicchitta, CV
MLA Citation
Reid, DW, and Nicchitta, CV. "Diversity and selectivity in mRNA translation on the endoplasmic reticulum." Nature reviews. Molecular cell biology 16.4 (April 2015): 221-231. (Review)
PMID
25735911
Source
epmc
Published In
Nature Reviews Molecular Cell Biology
Volume
16
Issue
4
Publish Date
2015
Start Page
221
End Page
231
DOI
10.1038/nrm3958

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Authors
LaMonte, G; Walzer, KA; Lacsina, J; Nicchitta, C; Chi, J-T
MLA Citation
LaMonte, G, Walzer, KA, Lacsina, J, Nicchitta, C, and Chi, J-T. "Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum." Journal of Visualized Experiments 106 (2015).
Source
crossref
Published In
Journal of Visualized Experiments
Issue
106
Publish Date
2015
DOI
10.3791/53214

De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum.

The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER.

Authors
Jagannathan, S; Reid, DW; Cox, AH; Nicchitta, CV
MLA Citation
Jagannathan, S, Reid, DW, Cox, AH, and Nicchitta, CV. "De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum." RNA (New York, N.Y.) 20.10 (October 2014): 1489-1498.
PMID
25142066
Source
epmc
Published In
RNA (New York, N.Y.)
Volume
20
Issue
10
Publish Date
2014
Start Page
1489
End Page
1498
DOI
10.1261/rna.045526.114

A novel ribosomopathy caused by dysfunction of RPL10 disrupts neurodevelopment and causes X-linked microcephaly in humans.

Neurodevelopmental defects in humans represent a clinically heterogeneous group of disorders. Here, we report the genetic and functional dissection of a multigenerational pedigree with an X-linked syndromic disorder hallmarked by microcephaly, growth retardation, and seizures. Using an X-linked intellectual disability (XLID) next-generation sequencing diagnostic panel, we identified a novel missense mutation in the gene encoding 60S ribosomal protein L10 (RPL10), a locus associated previously with autism spectrum disorders (ASD); the p.K78E change segregated with disease under an X-linked recessive paradigm while, consistent with causality, carrier females exhibited skewed X inactivation. To examine the functional consequences of the p.K78E change, we modeled RPL10 dysfunction in zebrafish. We show that endogenous rpl10 expression is augmented in anterior structures, and that suppression decreases head size in developing morphant embryos, concomitant with reduced bulk translation and increased apoptosis in the brain. Subsequently, using in vivo complementation, we demonstrate that p.K78E is a loss-of-function variant. Together, our findings suggest that a mutation within the conserved N-terminal end of RPL10, a protein in close proximity to the peptidyl transferase active site of the 60S ribosomal subunit, causes severe defects in brain formation and function.

Authors
Brooks, SS; Wall, AL; Golzio, C; Reid, DW; Kondyles, A; Willer, JR; Botti, C; Nicchitta, CV; Katsanis, N; Davis, EE
MLA Citation
Brooks, SS, Wall, AL, Golzio, C, Reid, DW, Kondyles, A, Willer, JR, Botti, C, Nicchitta, CV, Katsanis, N, and Davis, EE. "A novel ribosomopathy caused by dysfunction of RPL10 disrupts neurodevelopment and causes X-linked microcephaly in humans." Genetics 198.2 (October 2014): 723-733.
PMID
25316788
Source
epmc
Published In
Genetics
Volume
198
Issue
2
Publish Date
2014
Start Page
723
End Page
733
DOI
10.1534/genetics.114.168211

Multifunctional roles for the protein translocation machinery in RNA anchoring to the endoplasmic reticulum.

Signal sequence-encoding mRNAs undergo translation-dependent localization to the endoplasmic reticulum (ER) and at the ER are anchored via translation on Sec61-bound ribosomes. Recent investigations into the composition and membrane association characteristics of ER-associated mRNAs have, however, revealed both ribosome-dependent (indirect) and ribosome-independent (direct) modes of mRNA association with the ER. These findings raise important questions regarding our understanding of how mRNAs are selected, localized, and anchored to the ER. Using semi-intact tissue culture cells, we performed a polysome solubilization screen and identified conditions that distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. To gain insight into the molecular basis of direct mRNA anchoring to the ER, we performed RNA-protein UV photocross-linking studies in rough microsomes and demonstrate that numerous ER integral membrane proteins display RNA binding activity. Quantitative proteomic analyses of HeLa cytosolic and ER-bound polysome fractions identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins, whereas translocon accessory proteins, such as ribophorin I, were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocross-linked ER protein-RNA adducts identified Sec61α,β and ribophorin I as ER-poly(A) mRNA-binding proteins, suggesting unexpected roles for the protein translocation and modification machinery in mRNA anchoring to the ER. In summary, we propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable an mRNA transcriptome-wide function for the ER in protein synthesis.

Authors
Jagannathan, S; Hsu, JC-C; Reid, DW; Chen, Q; Thompson, WJ; Moseley, AM; Nicchitta, CV
MLA Citation
Jagannathan, S, Hsu, JC-C, Reid, DW, Chen, Q, Thompson, WJ, Moseley, AM, and Nicchitta, CV. "Multifunctional roles for the protein translocation machinery in RNA anchoring to the endoplasmic reticulum." The Journal of biological chemistry 289.37 (September 2014): 25907-25924.
PMID
25063809
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
37
Publish Date
2014
Start Page
25907
End Page
25924
DOI
10.1074/jbc.m114.580688

The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum.

The unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress.

Authors
Reid, DW; Chen, Q; Tay, AS-L; Shenolikar, S; Nicchitta, CV
MLA Citation
Reid, DW, Chen, Q, Tay, AS-L, Shenolikar, S, and Nicchitta, CV. "The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum." Cell 158.6 (September 2014): 1362-1374.
PMID
25215492
Source
epmc
Published In
Cell
Volume
158
Issue
6
Publish Date
2014
Start Page
1362
End Page
1374
DOI
10.1016/j.cell.2014.08.012

Rare codons regulate KRas oncogenesis.

Oncogenic mutations in the small Ras GTPases KRas, HRas, and NRas render the proteins constitutively GTP bound and active, a state that promotes cancer. Ras proteins share ~85% amino acid identity, are activated by and signal through the same proteins, and can exhibit functional redundancy. Nevertheless, manipulating expression or activation of each isoform yields different cellular responses and tumorigenic phenotypes, even when different ras genes are expressed from the same locus. We now report a novel regulatory mechanism hardwired into the very sequence of RAS genes that underlies how such similar proteins impact tumorigenesis differently. Specifically, despite their high sequence similarity, KRAS is poorly translated compared to HRAS due to enrichment in genomically underrepresented or rare codons. Converting rare to common codons increases KRas expression and tumorigenicity to mirror that of HRas. Furthermore, in a genome-wide survey, similar gene pairs with opposing codon bias were identified that not only manifest dichotomous protein expression but also are enriched in key signaling protein classes and pathways. Thus, synonymous nucleotide differences affecting codon usage account for differences between HRas and KRas expression and function and may represent a broader regulation strategy in cell signaling.

Authors
Lampson, BL; Pershing, NLK; Prinz, JA; Lacsina, JR; Marzluff, WF; Nicchitta, CV; MacAlpine, DM; Counter, CM
MLA Citation
Lampson, BL, Pershing, NLK, Prinz, JA, Lacsina, JR, Marzluff, WF, Nicchitta, CV, MacAlpine, DM, and Counter, CM. "Rare codons regulate KRas oncogenesis." Curr Biol 23.1 (January 7, 2013): 70-75.
PMID
23246410
Source
pubmed
Published In
Current Biology
Volume
23
Issue
1
Publish Date
2013
Start Page
70
End Page
75
DOI
10.1016/j.cub.2012.11.031

Rare codons regulate KRas oncogenesis

Oncogenic mutations in the small Ras GTPases KRas, HRas, and NRas render the proteins constitutively GTP bound and active, a state that promotes cancer [1]. Ras proteins share ∼85% amino acid identity [2], are activated by [3] and signal through [4] the same proteins, and can exhibit functional redundancy [5, 6]. Nevertheless, manipulating expression or activation of each isoform yields different cellular responses [7-10] and tumorigenic phenotypes [11-13], even when different ras genes are expressed from the same locus [6]. We now report a novel regulatory mechanism hardwired into the very sequence of RAS genes that underlies how such similar proteins impact tumorigenesis differently. Specifically, despite their high sequence similarity, KRAS is poorly translated compared to HRAS due to enrichment in genomically underrepresented or rare codons. Converting rare to common codons increases KRas expression and tumorigenicity to mirror that of HRas. Furthermore, in a genome-wide survey, similar gene pairs with opposing codon bias were identified that not only manifest dichotomous protein expression but also are enriched in key signaling protein classes and pathways. Thus, synonymous nucleotide differences affecting codon usage account for differences between HRas and KRas expression and function and may represent a broader regulation strategy in cell signaling. © 2013 Elsevier Ltd.

Authors
Lampson, BL; Pershing, NLK; Prinz, JA; Lacsina, JR; Marzluff, WF; Nicchitta, CV; MacAlpine, DM; Counter, CM
MLA Citation
Lampson, BL, Pershing, NLK, Prinz, JA, Lacsina, JR, Marzluff, WF, Nicchitta, CV, MacAlpine, DM, and Counter, CM. "Rare codons regulate KRas oncogenesis." Current Biology 23.1 (2013): 70-75.
Source
scival
Published In
Current Biology
Volume
23
Issue
1
Publish Date
2013
Start Page
70
End Page
75
DOI
10.1016/j.cub.2012.11.031

Induction of the unfolded protein response drives enhanced metabolism and chemoresistance in glioma cells.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics.

Authors
Epple, LM; Dodd, RD; Merz, AL; Dechkovskaia, AM; Herring, M; Winston, BA; Lencioni, AM; Russell, RL; Madsen, H; Nega, M; Dusto, NL; White, J; Bigner, DD; Nicchitta, CV; Serkova, NJ; Graner, MW
MLA Citation
Epple, LM, Dodd, RD, Merz, AL, Dechkovskaia, AM, Herring, M, Winston, BA, Lencioni, AM, Russell, RL, Madsen, H, Nega, M, Dusto, NL, White, J, Bigner, DD, Nicchitta, CV, Serkova, NJ, and Graner, MW. "Induction of the unfolded protein response drives enhanced metabolism and chemoresistance in glioma cells. (Published online)" PLoS One 8.8 (2013): e73267-.
PMID
24039668
Source
pubmed
Published In
PloS one
Volume
8
Issue
8
Publish Date
2013
Start Page
e73267
DOI
10.1371/journal.pone.0073267

Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis.

OBJECTIVE: The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll-like receptor (TLR)-mediated macrophage activation in RA. METHODS: RA synovial fluid was used to activate macrophages and HEK-TLR-2 and HEK-TLR-4 cells. Neutralizing antibodies to TLR-2, TLR-4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin-8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti-gp96 antiserum or control serum. RESULTS: RA synovial fluid induced the activation of macrophages and HEK-TLR-2 and HEK-TLR-4 cells. RA synovial fluid-induced macrophage and HEK-TLR-2 activation was suppressed by neutralizing anti-gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum-induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination. CONCLUSION: These observations support the notion that gp96 plays a role as an endogenous TLR-2 ligand in RA and identify the TLR-2 pathway as a therapeutic target.

Authors
Huang, Q-Q; Koessler, RE; Birkett, R; Dorfleutner, A; Perlman, H; Haines, GK; Stehlik, C; Nicchitta, CV; Pope, RM
MLA Citation
Huang, Q-Q, Koessler, RE, Birkett, R, Dorfleutner, A, Perlman, H, Haines, GK, Stehlik, C, Nicchitta, CV, and Pope, RM. "Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis." Arthritis Rheum 64.11 (November 2012): 3638-3648.
PMID
22777994
Source
pubmed
Published In
Arthritis and Rheumatism
Volume
64
Issue
11
Publish Date
2012
Start Page
3638
End Page
3648
DOI
10.1002/art.34610

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance.

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Authors
LaMonte, G; Philip, N; Reardon, J; Lacsina, JR; Majoros, W; Chapman, L; Thornburg, CD; Telen, MJ; Ohler, U; Nicchitta, CV; Haystead, T; Chi, J-T
MLA Citation
LaMonte, G, Philip, N, Reardon, J, Lacsina, JR, Majoros, W, Chapman, L, Thornburg, CD, Telen, MJ, Ohler, U, Nicchitta, CV, Haystead, T, and Chi, J-T. "Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance." Cell Host Microbe 12.2 (August 16, 2012): 187-199.
PMID
22901539
Source
pubmed
Published In
Cell Host and Microbe
Volume
12
Issue
2
Publish Date
2012
Start Page
187
End Page
199
DOI
10.1016/j.chom.2012.06.007

Development of a Grp94 inhibitor.

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.

Authors
Duerfeldt, AS; Peterson, LB; Maynard, JC; Ng, CL; Eletto, D; Ostrovsky, O; Shinogle, HE; Moore, DS; Argon, Y; Nicchitta, CV; Blagg, BSJ
MLA Citation
Duerfeldt, AS, Peterson, LB, Maynard, JC, Ng, CL, Eletto, D, Ostrovsky, O, Shinogle, HE, Moore, DS, Argon, Y, Nicchitta, CV, and Blagg, BSJ. "Development of a Grp94 inhibitor." J Am Chem Soc 134.23 (June 13, 2012): 9796-9804.
PMID
22642269
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
134
Issue
23
Publish Date
2012
Start Page
9796
End Page
9804
DOI
10.1021/ja303477g

The enduring enigma of nuclear translation.

Although the physical separation of transcription in the nucleus and translation in the cytoplasm has presided as a fundamental tenet of cell biology for decades, it has not done so without recurring challenges and contentious debate. In this issue, David et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201112145) rekindle the controversy by providing convincing experimental evidence for nuclear translation.

Authors
Reid, DW; Nicchitta, CV
MLA Citation
Reid, DW, and Nicchitta, CV. "The enduring enigma of nuclear translation." J Cell Biol 197.1 (April 2, 2012): 7-9.
PMID
22472436
Source
pubmed
Published In
The Journal of Cell Biology
Volume
197
Issue
1
Publish Date
2012
Start Page
7
End Page
9
DOI
10.1083/jcb.201202140

Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling.

In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a more substantial role for the ER cellular protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression.

Authors
Reid, DW; Nicchitta, CV
MLA Citation
Reid, DW, and Nicchitta, CV. "Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling." J Biol Chem 287.8 (February 17, 2012): 5518-5527.
PMID
22199352
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
8
Publish Date
2012
Start Page
5518
End Page
5527
DOI
10.1074/jbc.M111.312280

Premature translational termination products are rapidly degraded substrates for MHC class I presentation.

Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.

Authors
Lacsina, JR; Marks, OA; Liu, X; Reid, DW; Jagannathan, S; Nicchitta, CV
MLA Citation
Lacsina, JR, Marks, OA, Liu, X, Reid, DW, Jagannathan, S, and Nicchitta, CV. "Premature translational termination products are rapidly degraded substrates for MHC class I presentation." PLoS One 7.12 (2012): e51968-.
PMID
23251665
Source
pubmed
Published In
PloS one
Volume
7
Issue
12
Publish Date
2012
Start Page
e51968
DOI
10.1371/journal.pone.0051968

Polysome profiling of the malaria parasite Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in P. falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.

Authors
Lacsina, JR; LaMonte, G; Nicchitta, CV; Chi, J-T
MLA Citation
Lacsina, JR, LaMonte, G, Nicchitta, CV, and Chi, J-T. "Polysome profiling of the malaria parasite Plasmodium falciparum." Mol Biochem Parasitol 179.1 (September 2011): 42-46.
PMID
21605599
Source
pubmed
Published In
Molecular and Biochemical Parasitology
Volume
179
Issue
1
Publish Date
2011
Start Page
42
End Page
46
DOI
10.1016/j.molbiopara.2011.05.003

Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells.

The mRNA transcriptome is currently thought to be partitioned between the cytosol and endoplasmic reticulum (ER) compartments by binary selection; mRNAs encoding cytosolic/nucleoplasmic proteins are translated on free ribosomes, and mRNAs encoding topogenic signal-bearing proteins are translated on ER-bound ribosomes, with ER localization being conferred by the signal-recognition particle pathway. In subgenomic and genomic analyses of subcellular mRNA partitioning, we report an overlapping subcellular distribution of cytosolic/nucleoplasmic and topogenic signal-encoding mRNAs, with mRNAs of both cohorts displaying noncanonical subcellular partitioning patterns. Unexpectedly, the topogenic signal-encoding mRNA transcriptome was observed to partition in a hierarchical, cohort-specific manner. mRNAs encoding resident proteins of the endomembrane system were clustered at high ER-enrichment values, whereas mRNAs encoding secretory pathway cargo were broadly represented on free and ER-bound ribosomes. Two distinct modes of mRNA association with the ER were identified. mRNAs encoding endomembrane-resident proteins were bound via direct, ribosome-independent interactions, whereas mRNAs encoding secretory cargo displayed predominantly ribosome-dependent modes of ER association. These data indicate that mRNAs are partitioned between the cytosol and ER compartments via a hierarchical system of intrinsic and encoded topogenic signals and identify mRNA cohort-restricted modes of mRNA association with the ER.

Authors
Chen, Q; Jagannathan, S; Reid, DW; Zheng, T; Nicchitta, CV
MLA Citation
Chen, Q, Jagannathan, S, Reid, DW, Zheng, T, and Nicchitta, CV. "Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells." Mol Biol Cell 22.14 (July 15, 2011): 2646-2658.
PMID
21613539
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
22
Issue
14
Publish Date
2011
Start Page
2646
End Page
2658
DOI
10.1091/mbc.E11-03-0239

Out with the old, in with the new? Comparing methods for measuring protein degradation.

Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.

Authors
Yewdell, JW; Lacsina, JR; Rechsteiner, MC; Nicchitta, CV
MLA Citation
Yewdell, JW, Lacsina, JR, Rechsteiner, MC, and Nicchitta, CV. "Out with the old, in with the new? Comparing methods for measuring protein degradation." Cell Biol Int 35.5 (May 2011): 457-462. (Review)
PMID
21476986
Source
pubmed
Published In
Cell Biology International
Volume
35
Issue
5
Publish Date
2011
Start Page
457
End Page
462
DOI
10.1042/CBI20110055

Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.

The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise approximately 30% of the transcriptome, the localization of mRNAs to the ER represents an extraordinarily prominent, ubiquitous, and yet poorly understood RNA localization phenomenon.The partitioning of mRNAs to the ER is generally thought to be achieved by the signal recognition particle (SRP) pathway. In this pathway, mRNA localization to the ER is determined by the translation product - translation yields an N-terminal signal sequence or a topogenic signal that is recognized by the SRP and the resulting mRNA-ribosome-SRP complex is then recruited to the ER membrane. Recent studies have demonstrated that mRNAs can be localized to the ER via a signal sequence and/or translation-independent pathway(s) and that discrete sets of cytosolic protein-encoding mRNAs are enriched on the ER membrane, though they lack an encoded signal sequence. These key findings reopen investigations into the mechanism(s) that govern mRNA localization to the ER. In this contribution, we describe two independent methods that can be utilized to study this important and poorly understood aspect of eukaryotic cell biology. These methods comprise two independent means of fractionating tissue culture cells to yield free/cytosolic polyribosomes and ER membrane-bound polyribosomes. Detailed methods for the fractionation and characterization of the two polyribosome pools are provided.

Authors
Jagannathan, S; Nwosu, C; Nicchitta, CV
MLA Citation
Jagannathan, S, Nwosu, C, and Nicchitta, CV. "Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation." Methods Mol Biol 714 (2011): 301-321.
PMID
21431749
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
714
Publish Date
2011
Start Page
301
End Page
321
DOI
10.1007/978-1-61779-005-8_19

Re-examination of CD91 function in GRP94 (glycoprotein 96) surface binding, uptake, and peptide cross-presentation.

GRP94 (gp96)-peptide complexes can be internalized by APCs and their associated peptides cross-presented to yield activation of CD8(+) T cells. Investigations into the identity (or identities) of GRP94 surface receptors have yielded conflicting results, particularly with respect to CD91 (LRP1), which has been proposed to be essential for GRP94 recognition and uptake. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in mouse embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNA interference or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of receptor-associated protein, an established CD91 ligand. Surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed after treatment of MEF cell lines with heparin, sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentation of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to the CD91 ligands receptor-associated protein or activated α(2)-macroglobulin and occurred primarily via a fluid-phase, rather than receptor-mediated, uptake pathway. These data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis, and peptide cross-presentation and identify a role for heparin sulfate proteoglycans in GRP94 surface binding.

Authors
Jockheck-Clark, AR; Bowers, EV; Totonchy, MB; Neubauer, J; Pizzo, SV; Nicchitta, CV
MLA Citation
Jockheck-Clark, AR, Bowers, EV, Totonchy, MB, Neubauer, J, Pizzo, SV, and Nicchitta, CV. "Re-examination of CD91 function in GRP94 (glycoprotein 96) surface binding, uptake, and peptide cross-presentation." J Immunol 185.11 (December 1, 2010): 6819-6830.
PMID
21048103
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
185
Issue
11
Publish Date
2010
Start Page
6819
End Page
6830
DOI
10.4049/jimmunol.1000448

Gp93, the Drosophila GRP94 ortholog, is required for gut epithelial homeostasis and nutrient assimilation-coupled growth control.

GRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. This dichotomy suggests that GRP94 is required for the functional expression of secretory and/or membrane proteins that enable the integration of cells into tissues. To explore this hypothesis, we have identified the Drosophila ortholog of GRP94, Gp93, and report that Gp93 is an essential gene in Drosophila. Loss of zygotic Gp93 expression is late larval-lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. Gp93 mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of Gp93-expression are profound; Gp93 mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of Gp93 expression resemble those reported for mutations in labial, an endodermal homeotic gene required for copper cell specification, and alpha-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding lab protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton to confer epithelial membrane specialization.

Authors
Maynard, JC; Pham, T; Zheng, T; Jockheck-Clark, A; Rankin, HB; Newgard, CB; Spana, EP; Nicchitta, CV
MLA Citation
Maynard, JC, Pham, T, Zheng, T, Jockheck-Clark, A, Rankin, HB, Newgard, CB, Spana, EP, and Nicchitta, CV. "Gp93, the Drosophila GRP94 ortholog, is required for gut epithelial homeostasis and nutrient assimilation-coupled growth control." Dev Biol 339.2 (March 15, 2010): 295-306.
PMID
20044986
Source
pubmed
Published In
Developmental Biology
Volume
339
Issue
2
Publish Date
2010
Start Page
295
End Page
306
DOI
10.1016/j.ydbio.2009.12.023

Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 independent (Journal of Immunology (2009) 183, (4205-4210))

Authors
Lev, A; Dimberu, P; Das, SR; Maynard, JC; Nicchitta, CV; Bennink, JR; Yewdell, JW
MLA Citation
Lev, A, Dimberu, P, Das, SR, Maynard, JC, Nicchitta, CV, Bennink, JR, and Yewdell, JW. "Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 independent (Journal of Immunology (2009) 183, (4205-4210))." Journal of Immunology 185.1 (2010): 770--.
Source
scival
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
185
Issue
1
Publish Date
2010
Start Page
770-
DOI
10.4049/jimmunol.1090046

Glioblastoma proto-oncogene SEC61gamma is required for tumor cell survival and response to endoplasmic reticulum stress.

Glioblastoma multiforme is the most prevalent type of adult brain tumor and one of the deadliest tumors known to mankind. The genetic understanding of glioblastoma multiforme is, however, limited, and the molecular mechanisms that facilitate glioblastoma multiforme cell survival and growth within the tumor microenvironment are largely unknown. We applied digital karyotyping and single nucleotide polymorphism arrays to screen for copy-number changes in glioblastoma multiforme samples and found that the most frequently amplified region is at chromosome 7p11.2. The high resolution of digital karyotyping and single nucleotide polymorphism arrays permits the precise delineation of amplicon boundaries and has enabled identification of the minimal region of amplification at chromosome 7p11.2, which contains two genes, EGFR and SEC61gamma. SEC61gamma encodes a subunit of a heterotrimeric protein channel located in the endoplasmic reticulum (ER). In addition to its high frequency of gene amplification in glioblastoma multiforme, SEC61gamma is also remarkably overexpressed in 77% of glioblastoma multiforme but not in lower-grade gliomas. The small interfering RNA-mediated knockdown of SEC61gamma expression in tumor cells led to growth suppression and apoptosis. Furthermore, we showed that pharmacologic ER stress agents induce SEC61gamma expression in glioblastoma multiforme cells. Together, these results indicate that aberrant expression of SEC61gamma serves significant roles in glioblastoma multiforme cell survival likely via a mechanism that is involved in the cytoprotective ER stress-adaptive response to the tumor microenvironment.

Authors
Lu, Z; Zhou, L; Killela, P; Rasheed, AB; Di, C; Poe, WE; McLendon, RE; Bigner, DD; Nicchitta, C; Yan, H
MLA Citation
Lu, Z, Zhou, L, Killela, P, Rasheed, AB, Di, C, Poe, WE, McLendon, RE, Bigner, DD, Nicchitta, C, and Yan, H. "Glioblastoma proto-oncogene SEC61gamma is required for tumor cell survival and response to endoplasmic reticulum stress." Cancer Res 69.23 (December 1, 2009): 9105-9111.
PMID
19920201
Source
pubmed
Published In
Cancer Research
Volume
69
Issue
23
Publish Date
2009
Start Page
9105
End Page
9111
DOI
10.1158/0008-5472.CAN-09-2775

Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 independent.

Cross-priming, the activation of naive CD8+ T cells by dendritic cells presenting Ags synthesized by other cells, is believed to play an important role in the generation of antiviral and antitumor responses. The molecular mechanism(s) underlying cross-priming remain poorly defined and highly controversial. GRP94 (gp96), an abundant endoplasmic reticulum chaperone with innate immune-activating capacity, has been widely reported to play a major role in cross-priming. In this study, we show that cells whose expression of GRP94 is silenced via transient or stable transfection with GRP94-directed small interfering RNAs demonstrate no reduction in their abilities to generate class I peptide complexes in cultured cells or to prime antiviral CD8+ T cell responses in vivo. In demonstrating the dispensability of GRP94, our finding points to the importance of alternative mechanisms for generation of class I peptide complexes from endogenous and exogenous Ags and immunogens.

Authors
Lev, A; Dimberu, P; Das, SR; Maynard, JC; Nicchitta, CV; Bennink, JR; Yewdell, JW
MLA Citation
Lev, A, Dimberu, P, Das, SR, Maynard, JC, Nicchitta, CV, Bennink, JR, and Yewdell, JW. "Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 independent." J Immunol 183.7 (October 1, 2009): 4205-4210.
PMID
19752220
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
183
Issue
7
Publish Date
2009
Start Page
4205
End Page
4210
DOI
10.4049/jimmunol.0901828

Heat shock protein 96 is elevated in rheumatoid arthritis and activates macrophages primarily via TLR2 signaling.

Macrophages are important mediators of chronic inflammation and are prominent in the synovial lining and sublining of patients with rheumatoid arthritis (RA). Recently, we demonstrated increased TLR2 and TLR4 expression and increased response to microbial TLR2 and TLR4 ligands in macrophages from the joints of RA. The current study characterized the expression of the 96-kDa heat shock glycoprotein (gp96) in the joints of RA and its role as an endogenous TLR ligand to promote innate immunity in RA. gp96 was increased in RA compared with osteoarthritis and arthritis-free control synovial tissues. The expression of gp96 strongly correlated with inflammation and synovial lining thickness. gp96 was increased in synovial fluid from the joints of RA compared with disease controls. Recombinant gp96 was a potent activator of macrophages and the activation was mediated primarily through TLR2 signaling. The cellular response to gp96 was significantly stronger with RA synovial macrophages compared with peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNF-alpha, and IL-8, but not TLR4, was significantly induced by gp96, and the induction was significantly greater in purified RA synovial macrophages. The expression of TLR2, but not TLR4, on synovial fluid macrophages strongly correlated with the level of gp96 in the synovial fluid. The present study documents the potential role of gp96 as an endogenous TLR2 ligand in RA and provides insight into the mechanism by which gp96 promotes the chronic inflammation of RA, identifying gp96 as a potential new therapeutic target.

Authors
Huang, Q-Q; Sobkoviak, R; Jockheck-Clark, AR; Shi, B; Mandelin, AM; Tak, PP; Haines, GK; Nicchitta, CV; Pope, RM
MLA Citation
Huang, Q-Q, Sobkoviak, R, Jockheck-Clark, AR, Shi, B, Mandelin, AM, Tak, PP, Haines, GK, Nicchitta, CV, and Pope, RM. "Heat shock protein 96 is elevated in rheumatoid arthritis and activates macrophages primarily via TLR2 signaling." J Immunol 182.8 (April 15, 2009): 4965-4973.
PMID
19342676
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
182
Issue
8
Publish Date
2009
Start Page
4965
End Page
4973
DOI
10.4049/jimmunol.0801563

Cell biology: How to combat stress.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Cell biology: How to combat stress." Nature 457.7230 (February 5, 2009): 668-669.
PMID
19194438
Source
pubmed
Published In
Nature
Volume
457
Issue
7230
Publish Date
2009
Start Page
668
End Page
669
DOI
10.1038/457668a

Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 independent (Journal of Immunology (2009) 183, (4205-4210))

Authors
Lev, A; Dimberu, P; Das, SR; Maynard, JC; Nicchitta, CV; Bennink, JR; Yewdell, JW
MLA Citation
Lev, A, Dimberu, P, Das, SR, Maynard, JC, Nicchitta, CV, Bennink, JR, and Yewdell, JW. "Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 independent (Journal of Immunology (2009) 183, (4205-4210))." Journal of Immunology 183.7 (2009).
Source
scival
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
183
Issue
7
Publish Date
2009
DOI
10.4049/jimmunol.1090046

Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo.

CD8(+) T cells (T(CD8+)) differentiate into effector cells following recognition of specific peptide-major histocompatibility complex (MHC) class I complexes (pMHC-I) on the surface of professional APCs (pAPCs), such as dendritic cells. Antigenic pMHC-I can be generated from two spatially distinct sources. The direct presentation pathway involves generation of peptide from protein substrate synthesized within the cell that is presenting the pMHC-I. Alternatively, the cross presentation pathway involves presentation of antigen that is not synthesized within the presenting cell, but is derived from exogenous proteins synthesized within other donor cells. The mechanisms by which cross presentation of exogenous antigens occur in vivo remain controversial. The C-type lectin scavenger receptor A (SR-A) has been implicated in a number of potential cross presentation pathways, including the presentation of peptide bound to heat shock proteins, such as glycoprotein 96 (gp96), and the transfer of pMHC-I from a donor cell to the pAPC. We demonstrate here that initiation of T(CD8+) responses is normal in mice lacking SR-A, and that the redundancy of ligand binding exhibited by the SR family is likely to be an important mechanism that ensures cross presentation in vivo. These observations emphasize the requirement to target multiple receptors and antigen-processing pathways during the rational design of vaccines aimed at eliciting protective T(CD8+).

Authors
Tewalt, EF; Maynard, JC; Walters, JJ; Schell, AM; Berwin, BL; Nicchitta, CV; Norbury, CC
MLA Citation
Tewalt, EF, Maynard, JC, Walters, JJ, Schell, AM, Berwin, BL, Nicchitta, CV, and Norbury, CC. "Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo." Immunology 125.4 (December 2008): 480-491.
PMID
18489571
Source
pubmed
Published In
Immunology
Volume
125
Issue
4
Publish Date
2008
Start Page
480
End Page
491
DOI
10.1111/j.1365-2567.2008.02861.x

The exception that reinforces the rule: crosspriming by cytosolic peptides that escape degradation.

The nature of crosspriming immunogens for CD8(+) T cell responses is highly controversial. By using a panel of T cell receptor-like antibodies specific for viral peptides bound to mouse D(b) major histocompatibility complex class I molecules, we show that an exceptional peptide (PA(224-233)) expressed as a viral minigene product formed a sizeable cytosolic pool continuously presented for hours after protein synthesis was inhibited. PA(224-233) pool formation required active cytosolic heat-shock protein 90 but not ER g96 and uniquely enabled crosspriming by this peptide. These findings demonstrate that exceptional class I binding oligopeptides that escape proteolytic degradation are potent crosspriming agents. Thus, the feeble immunogenicity of natural proteasome products in crosspriming can be attributed to their evanescence in donor cells and not an absolute inability of cytosolic oligopeptides to be transferred to and presented by professional antigen-presenting cells.

Authors
Lev, A; Takeda, K; Zanker, D; Maynard, JC; Dimberu, P; Waffarn, E; Gibbs, J; Netzer, N; Princiotta, MF; Neckers, L; Picard, D; Nicchitta, CV; Chen, W; Reiter, Y; Bennink, JR; Yewdell, JW
MLA Citation
Lev, A, Takeda, K, Zanker, D, Maynard, JC, Dimberu, P, Waffarn, E, Gibbs, J, Netzer, N, Princiotta, MF, Neckers, L, Picard, D, Nicchitta, CV, Chen, W, Reiter, Y, Bennink, JR, and Yewdell, JW. "The exception that reinforces the rule: crosspriming by cytosolic peptides that escape degradation." Immunity 28.6 (June 2008): 787-798.
PMID
18549799
Source
pubmed
Published In
Immunity
Volume
28
Issue
6
Publish Date
2008
Start Page
787
End Page
798
DOI
10.1016/j.immuni.2008.04.015

Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum.

The process of mRNA localization typically utilizes cis-targeting elements and trans-recognition factors to direct the compartmental organization of translationally suppressed mRNAs. mRNA localization to the endoplasmic reticulum (ER), in contrast, occurs via a co-translational, signal sequence/signal recognition particle (SRP)-dependent mechanism. We have utilized cell fractionation/cDNA microarray analysis, shRNA-mediated suppression of SRP expression, and mRNA reporter construct studies to define the role of the SRP pathway in ER-directed mRNA localization. Cell fractionation studies of mRNA partitioning between the cytosol and ER demonstrated the expected enrichment of cytosolic/nucleoplasmic protein-encoding mRNAs and secretory/integral membrane protein-encoding mRNAs in the cytosol and ER fractions, respectively, and identified a subpopulation of cytosolic/nucleoplasmic protein-encoding mRNAs in the membrane-bound mRNA pool. The latter finding suggests a signal sequence-independent pathway of ER-directed mRNA localization. Extending from these findings, mRNA partitioning was examined in stable SRP54 shRNA knockdown HeLa cell lines. shRNA-directed reductions in SRP did not globally alter mRNA partitioning patterns, although defects in membrane protein processing were observed, further suggesting the existence of multiple pathways for mRNA localization to the ER. ER localization of GRP94-encoding mRNA was observed when translation was disabled by mutation of the start codon/insertion of a 5'UTR stem-loop structure or upon deletion of the encoded signal sequence. Combined, these data indicate that the mRNA localization to the ER can be conferred independent of the signal sequence/SRP pathway and suggest that mRNA localization to the ER may utilize cis-encoded targeting information.

Authors
Pyhtila, B; Zheng, T; Lager, PJ; Keene, JD; Reedy, MC; Nicchitta, CV
MLA Citation
Pyhtila, B, Zheng, T, Lager, PJ, Keene, JD, Reedy, MC, and Nicchitta, CV. "Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum." RNA 14.3 (March 2008): 445-453.
PMID
18192611
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
14
Issue
3
Publish Date
2008
Start Page
445
End Page
453
DOI
10.1261/rna.721108

Divergent regulation of protein synthesis in the cytosol and endoplasmic reticulum compartments of mammalian cells.

In eukaryotic cells, mRNAs encoding signal sequence-bearing proteins undergo translation-dependent trafficking to the endoplasmic reticulum (ER), thereby restricting secretory and integral membrane protein synthesis to the ER compartment. However, recent studies demonstrating that mRNAs encoding cytosolic/nucleoplasmic proteins are represented on ER-bound polyribosomes suggest a global role for the ER in cellular protein synthesis. Here, we examined the steady-state protein synthesis rates and compartmental distribution of newly synthesized proteins in the cytosol and ER compartments. We report that ER protein synthesis rates exceed cytosolic protein synthesis rates by 2.5- to 4-fold; yet, completed proteins accumulate to similar levels in the two compartments. These data suggest that a significant fraction of cytosolic proteins undergo synthesis on ER-bound ribosomes. The compartmental differences in steady-state protein synthesis rates correlated with a divergent regulation of the tRNA aminoacylation/deacylation cycle. In the cytosol, two pathways were observed to compete for aminoacyl-tRNAs-protein synthesis and aminoacyl-tRNA hydrolysis-whereas on the ER tRNA deacylation is tightly coupled to protein synthesis. These findings identify a role for the ER in global protein synthesis, and they suggest models where compartmentalization of the tRNA acylation/deacylation cycle contributes to the regulation of global protein synthesis rates.

Authors
Stephens, SB; Nicchitta, CV
MLA Citation
Stephens, SB, and Nicchitta, CV. "Divergent regulation of protein synthesis in the cytosol and endoplasmic reticulum compartments of mammalian cells." Mol Biol Cell 19.2 (February 2008): 623-632.
PMID
18077556
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
19
Issue
2
Publish Date
2008
Start Page
623
End Page
632
DOI
10.1091/mbc.E07-07-0677

Analysis of mRNA partitioning between the cytosol and endoplasmic reticulum compartments of mammalian cells.

All eukaryotic cells display a dramatic partitioning of mRNAs between the cytosol and endoplasmic reticulum (ER) compartments-mRNAs encoding secretory and integral membrane proteins are highly enriched on ER-bound ribosomes and mRNAs encoding cytoplasmic/nucleoplasmic proteins are enriched on cytosolic ribosomes. In current views, this partitioning phenomenon occurs through positive selection-mRNAs encoding signal sequence-bearing proteins are directed into the signal recognition particle pathway early in translation and trafficked as mRNA/ribosome/nascent polypeptide chain complexes to the ER. In the absence of an encoded signal sequence, mRNAs undergo continued translation on cytosolic ribosomes. Recent genome-wide analyses of mRNA partitioning between the cytosol and the ER compartments have identified subsets of mRNAs that are non-canonically partitioned to the ER-although lacking an encoded signal sequence, they are translated on ER-bound ribosomes. These findings suggest that multiple, and as yet unidentified, pathways exist for directing mRNA partitioning in the cell. In this contribution, we briefly review the literature describing the subcellular partitioning patterns of mRNAs and present a detailed methodology for studying this fundamental, yet poorly understood process.

Authors
Stephens, SB; Dodd, RD; Lerner, RS; Pyhtila, BM; Nicchitta, CV
MLA Citation
Stephens, SB, Dodd, RD, Lerner, RS, Pyhtila, BM, and Nicchitta, CV. "Analysis of mRNA partitioning between the cytosol and endoplasmic reticulum compartments of mammalian cells." Methods Mol Biol 419 (2008): 197-214.
PMID
18369985
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
419
Publish Date
2008
Start Page
197
End Page
214
DOI
10.1007/978-1-59745-033-1_14

In vitro and tissue culture methods for analysis of translation initiation on the endoplasmic reticulum.

For mRNAs encoding secretory and integral membrane proteins, translation initiation is thought to begin a process of mRNA localization where mRNA/ribosome/nascent chain complexes (RNCs) are trafficked from the cytosol compartment to the endoplasmic reticulum (ER). At the ER membrane, RNCs bind to a protein-conducting channel via the large ribosomal subunit and protein translocation ensues through coupling of the ribosomal nascent protein exit site with the protein-conducting channel. At the termination of translation, ribosomal subunits are thought to dissociate from the ER to return to a common cytoplasmic pool and participate in additional cycles of initiation, translation, targeting, termination, and ER membrane release. Experimental evidence has demonstrated that ER-membrane ribosomes are capable of de novo initiation, that mRNA partitioning to the ER membrane does not, per se, require translation of an encoded signal sequence, and that ribosomal subunit dissociation from the ER membrane is not obligatorily coupled to protein synthesis termination. These findings suggest that the cycle of protein synthesis-initiation, elongation, and termination-can occur on the two-dimensional plane of the ER membrane and challenge current views on the subcellular restriction of translation initiation to the cytosol, the role of the ribosome cycle in partitioning mRNA between the cytosol and ER, and the in vivo basis for termination-induced ribosomal subunit dissociation. In the following chapter, we provide detailed experimental methods to study protein synthesis initiation on the ER membrane.

Authors
Stephens, SB; Nicchitta, CV
MLA Citation
Stephens, SB, and Nicchitta, CV. "In vitro and tissue culture methods for analysis of translation initiation on the endoplasmic reticulum." Methods Enzymol 431 (2007): 47-60. (Review)
PMID
17923230
Source
pubmed
Published In
Methods in Enzymology
Volume
431
Publish Date
2007
Start Page
47
End Page
60
DOI
10.1016/S0076-6879(07)31004-5

Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses.

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (> or =50 microg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses.

Authors
Warger, T; Hilf, N; Rechtsteiner, G; Haselmayer, P; Carrick, DM; Jonuleit, H; von Landenberg, P; Rammensee, H-G; Nicchitta, CV; Radsak, MP; Schild, H
MLA Citation
Warger, T, Hilf, N, Rechtsteiner, G, Haselmayer, P, Carrick, DM, Jonuleit, H, von Landenberg, P, Rammensee, H-G, Nicchitta, CV, Radsak, MP, and Schild, H. "Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses." J Biol Chem 281.32 (August 11, 2006): 22545-22553.
PMID
16754684
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
32
Publish Date
2006
Start Page
22545
End Page
22553
DOI
10.1074/jbc.M502900200

The DRiP hypothesis decennial: support, controversy, refinement and extension.

In 1996, to explain the rapid presentation of viral proteins to CD8+ T cells, it was proposed that peptides presented by MHC class I molecules derive from defective ribosomal products (DRiPs), presumed to be polypeptides arising from in-frame translation that fail to achieve native structure owing to inevitable imperfections in transcription, translation, post-translational modifications or protein folding. Here, we consider findings that address the DRiP hypothesis, and extend the hypothesis by proposing that cells possess specialized machinery, possibly in the form of "immunoribosomes", to couple protein synthesis to antigen presentation.

Authors
Yewdell, JW; Nicchitta, CV
MLA Citation
Yewdell, JW, and Nicchitta, CV. "The DRiP hypothesis decennial: support, controversy, refinement and extension." Trends Immunol 27.8 (August 2006): 368-373.
PMID
16815756
Source
pubmed
Published In
Trends in Immunology
Volume
27
Issue
8
Publish Date
2006
Start Page
368
End Page
373
DOI
10.1016/j.it.2006.06.008

Synthesis of Hsp90 dimerization modulators.

The synthesis and evaluation of several chemical modulators of heat shock protein 90 (Hsp90) dimerization is presented. These agents may represent useful tools to study the importance of N-terminal dimerization and also to determine subunit interface(s) in Hsp90.

Authors
Chiosis, G; Aguirre, J; Nicchitta, CV
MLA Citation
Chiosis, G, Aguirre, J, and Nicchitta, CV. "Synthesis of Hsp90 dimerization modulators." Bioorg Med Chem Lett 16.13 (July 1, 2006): 3529-3532.
PMID
16621545
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
16
Issue
13
Publish Date
2006
Start Page
3529
End Page
3532
DOI
10.1016/j.bmcl.2006.03.092

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94.

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.

Authors
Chu, F; Maynard, JC; Chiosis, G; Nicchitta, CV; Burlingame, AL
MLA Citation
Chu, F, Maynard, JC, Chiosis, G, Nicchitta, CV, and Burlingame, AL. "Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94." Protein Sci 15.6 (June 2006): 1260-1269.
PMID
16731965
Source
pubmed
Published In
Protein Science
Volume
15
Issue
6
Publish Date
2006
Start Page
1260
End Page
1269
DOI
10.1110/ps.052065106

mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation.

Eukaryotic cells utilize a cycle of ribosome trafficking on the endoplasmic reticulum (ER) to partition mRNAs between the cytosol and ER compartments. In this process, ribosomes engaged in the synthesis of signal sequence-bearing proteins are trafficked to the endoplasmic reticulum via the signal-recognition particle pathway and are released from the ER upon translation termination. Though the processes governing ribosome trafficking to the ER are well understood, little is known regarding the complementary ribosome release process. In this study, Coxsackie B virus (CBV) infection was used to inactivate the initiation stage of protein synthesis, thereby limiting translation to the elongation and termination stages. Ribosome partitioning between the cytosol and ER compartments was examined to determine the role of termination in ribosome release from the ER. CBV infection resulted in efficient cleavage of eIF4G and PABP, coincident with polyribosome breakdown in the cytosol and ER compartments. Termination resulted in the continued association of ribosomes with the ER compartment, rather than the expected process of ribosome release. Analyses of ribosome/mRNA loading patterns in the cytosol and ER revealed that CBV infection was accompanied by a suppression of mRNA translation in the cytosol and the sustained, although reduced, translation in the ER compartment. Direct biosynthetic labeling experiments demonstrated that protein synthesis on the ER was enhanced relative to the cytosol following CBV infection. In total, these data demonstrate that ribosome and mRNA release from the ER is regulated independent of translation termination and identify the ER as a privileged site for protein synthesis.

Authors
Lerner, RS; Nicchitta, CV
MLA Citation
Lerner, RS, and Nicchitta, CV. "mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation." RNA 12.5 (May 2006): 775-789.
PMID
16540694
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
12
Issue
5
Publish Date
2006
Start Page
775
End Page
789
DOI
10.1261/rna.2318906

Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model.

mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER) -- bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on cytosolic ribosomes. The partitioning of mRNAs to the ER occurs by positive selection; cytosolic ribosomes engaged in the translation of signal-sequence-bearing proteins are engaged by the signal-recognition particle (SRP) pathway and subsequently trafficked to the ER. Studies have demonstrated that, in addition to the SRP pathway, mRNAs encoding cytosolic proteins can also be partitioned to the ER, suggesting that RNA partitioning in the eukaryotic cell is a complex process requiring the activity of multiple RNA-partitioning pathways. In this review, key findings on this topic are discussed, and the template-partitioning model, describing a hypothetical mechanism for RNA partitioning in the eukaryotic cell, is proposed.

Authors
Nicchitta, CV; Lerner, RS; Stephens, SB; Dodd, RD; Pyhtila, B
MLA Citation
Nicchitta, CV, Lerner, RS, Stephens, SB, Dodd, RD, and Pyhtila, B. "Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model." Biochem Cell Biol 83.6 (December 2005): 687-695. (Review)
PMID
16333319
Source
pubmed
Published In
Biochemistry and Cell Biology
Volume
83
Issue
6
Publish Date
2005
Start Page
687
End Page
695
DOI
10.1139/o05-147

Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation.

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.

Authors
Stephens, SB; Dodd, RD; Brewer, JW; Lager, PJ; Keene, JD; Nicchitta, CV
MLA Citation
Stephens, SB, Dodd, RD, Brewer, JW, Lager, PJ, Keene, JD, and Nicchitta, CV. "Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation." Mol Biol Cell 16.12 (December 2005): 5819-5831.
PMID
16221886
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
16
Issue
12
Publish Date
2005
Start Page
5819
End Page
5831
DOI
10.1091/mbc.E05-07-0685

Enhancement of cancer radiation therapy by use of adenovirus-mediated secretable glucose-regulated protein 94/gp96 expression.

Tumor-derived glucose-regulated protein 94 (GRP94/gp96) has shown great promise as a tumor vaccine. However, current protein-based approaches require the availability of large quantities of tumor tissue, which are often not possible. In addition, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of a combined GRP94/gp96-based genetic immunotherapy and radiation therapy strategy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding a modified, secretable form of GRP94 gene (AdsGRP94) was constructed and evaluated in various antitumor experiments. Lethally irradiated, virus-infected cells were used as vaccines. Adenoviral vectors were also injected directly into tumors in conjunction with tumor irradiation. Vaccination with lethally irradiated, AdsGRP94-infected 4T1 cells completely prevented subsequent tumor growth from challenge inoculations of as many as 10(7) cells per mouse. In established tumor models, vaccinations alone had minimal effect on local and metastatic tumor growth. However, when vaccination was combined with radiation therapy and i.t. AdsGRP94 injections, local tumor growth and pulmonary metastasis were markedly inhibited. In some cases, complete tumor regression was observed. In these cases, the mice were resistant to subsequent tumor challenge and remain tumor free up to 10 months after initial therapy. Our results indicate that combined AdsGRP94-based immunotherapy and radiation therapy may be a potentially effective strategy for cancer treatment.

Authors
Liu, S; Wang, H; Yang, Z; Kon, T; Zhu, J; Cao, Y; Li, F; Kirkpatrick, J; Nicchitta, CV; Li, C-Y
MLA Citation
Liu, S, Wang, H, Yang, Z, Kon, T, Zhu, J, Cao, Y, Li, F, Kirkpatrick, J, Nicchitta, CV, and Li, C-Y. "Enhancement of cancer radiation therapy by use of adenovirus-mediated secretable glucose-regulated protein 94/gp96 expression." Cancer Res 65.20 (October 15, 2005): 9126-9131.
PMID
16230366
Source
pubmed
Published In
Cancer Research
Volume
65
Issue
20
Publish Date
2005
Start Page
9126
End Page
9131
DOI
10.1158/0008-5472.CAN-05-0945

HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor.

The molecular chaperone heat shock protein 90 (Hsp90) and its accessory cochaperones function by facilitating the structural maturation and complex assembly of client proteins, including steroid hormone receptors and selected kinases. By promoting the activity and stability of these signaling proteins, Hsp90 has emerged as a critical modulator in cell signaling. Here, we present evidence that Hsp90 chaperone activity is regulated by reversible acetylation and controlled by the deacetylase HDAC6. We show that HDAC6 functions as an Hsp90 deacetylase. Inactivation of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from an essential cochaperone, p23, and a loss of chaperone activity. In HDAC6-deficient cells, Hsp90-dependent maturation of the glucocorticoid receptor (GR) is compromised, resulting in GR defective in ligand binding, nuclear translocation, and transcriptional activation. Our results identify Hsp90 as a target of HDAC6 and suggest reversible acetylation as a unique mechanism that regulates Hsp90 chaperone complex activity.

Authors
Kovacs, JJ; Murphy, PJM; Gaillard, S; Zhao, X; Wu, J-T; Nicchitta, CV; Yoshida, M; Toft, DO; Pratt, WB; Yao, T-P
MLA Citation
Kovacs, JJ, Murphy, PJM, Gaillard, S, Zhao, X, Wu, J-T, Nicchitta, CV, Yoshida, M, Toft, DO, Pratt, WB, and Yao, T-P. "HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor." Mol Cell 18.5 (May 27, 2005): 601-607.
PMID
15916966
Source
pubmed
Published In
Molecular Cell
Volume
18
Issue
5
Publish Date
2005
Start Page
601
End Page
607
DOI
10.1016/j.molcel.2005.04.021

Catheter-mediated subselective intracoronary gene delivery to the rabbit heart: introduction of a novel method.

BACKGROUND: Recent studies suggest that gene therapy using replication-deficient adenoviruses will benefit treatment of cardiovascular diseases including heart failure. A persistent hurdle is the effective and reproducible delivery of a transgene to the myocardium with minimal iatrogenic morbidity. In this study, we sought to design a relatively non-invasive percutaneous gene delivery system that would maximize cardiac transgene expression and minimize mortality after intracoronary adenovirus injection. METHODS: Adult rabbits received a left circumflex coronary artery (LCx) infusion of 5x10(11) total viral particles of an adenovirus containing the marker transgene beta-galactosidase (Adeno-betaGal) via either a continuous infusion method utilizing an oxygenated, normothermic, physiologic pH Krebs solution driven by a Langendorff apparatus (n=12) or a timed bolus and set concentration at a constant infusion rate to the LCx (n=12). Six rabbits underwent global transgene delivery via an invasive method involving intraventricular delivery and aortic root cross-clamping. The efficacy of transgene expression via these three distinct delivery methods was determined in the left ventricle at 5 days by histological staining and colorimetric quantification assay. RESULTS: While the open-chest, aortic cross-clamping method provides the highest level of gene expression throughout the heart, the morbidity of this procedure is clinically prohibitive. Percutaneous LCx delivery of Adeno-betaGal using the Langendorff apparatus was associated with the lowest morbidity and mortality while still supporting significant myocardial gene expression. CONCLUSIONS: Percutaneous delivery of an adenovirus solution using a continuous infusion of oxygenated Krebs solution via a Langendorff apparatus appears to be a gene delivery modality offering the best compromise of gene expression and clinical utility to maximize any potential therapeutic outcome.

Authors
Parsa, CJ; Reed, RC; Walton, GB; Pascal, LS; Thompson, RB; Petrofski, JA; Emani, SM; Folgar, F; Riel, RU; Nicchitta, CV; Koch, WJ
MLA Citation
Parsa, CJ, Reed, RC, Walton, GB, Pascal, LS, Thompson, RB, Petrofski, JA, Emani, SM, Folgar, F, Riel, RU, Nicchitta, CV, and Koch, WJ. "Catheter-mediated subselective intracoronary gene delivery to the rabbit heart: introduction of a novel method." J Gene Med 7.5 (May 2005): 595-603.
PMID
15651066
Source
pubmed
Published In
The Journal of Gene Medicine
Volume
7
Issue
5
Publish Date
2005
Start Page
595
End Page
603
DOI
10.1002/jgm.704

Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10-29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Tie, J-K; Nicchitta, C; Heijne, GV; Stafford, DW
MLA Citation
Tie, J-K, Nicchitta, C, Heijne, GV, and Stafford, DW. "Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation." Journal of Biological Chemistry 280.16 (2005): 16410-16416.
PMID
15716279
Source
scival
Published In
Journal of Biological Chemistry
Volume
280
Issue
16
Publish Date
2005
Start Page
16410
End Page
16416
DOI
10.1074/jbc.M500765200

Adenosine nucleotides and the regulation of GRP94-client protein interactions.

The molecular chaperone heat shock protein 90 (Hsp90) serves essential roles in the regulation of signaling protein function, trafficking, and turnover. Hsp90 function is intimately linked to intrinsic ATP binding and hydrolysis activities, the latter of which is under the regulatory control of accessory factors. Glucose-regulated protein of 94 kDa (GRP94), the endoplasmic reticulum Hsp90, is highly homologous to cytosolic Hsp90. However, neither accessory factors nor adenosine nucleotides have been clearly implicated in the regulation of GRP94-client protein interactions. In the current study, the structural and regulatory consequences of adenosine nucleotide binding to GRP94 were investigated. We report that apo-GRP94 undergoes a time- and temperature-dependent tertiary conformational change that exposes a site(s) of protein-protein interaction; ATP, ADP, and radicicol markedly suppress this conformational change. In concert with these findings, ATP and ADP act identically to suppress GRP94 homooligomerization, as well as both local and global conformational activity. To identify a role(s) for ATP or ADP in the regulation of GRP94-client protein interactions, immunoglobulin (Ig) heavy chain folding intermediates containing bound GRP94 and immunoglobulin binding protein (BiP) were isolated from myeloma cells, and the effects of adenosine nucleotides on chaperone-Ig heavy chain interactions were examined. Whereas ATP elicited efficient release of BiP from both wild-type and mutant Ig heavy chain intermediates, GRP94 remained in stable association with Ig heavy chains in the presence of ATP or ADP. On the basis of these data, we propose that structural maturation of the client protein substrate, rather than ATP binding or hydrolysis, serves as the primary signal for dissociation of GRP94-client protein complexes.

Authors
Rosser, MFN; Trotta, BM; Marshall, MR; Berwin, B; Nicchitta, CV
MLA Citation
Rosser, MFN, Trotta, BM, Marshall, MR, Berwin, B, and Nicchitta, CV. "Adenosine nucleotides and the regulation of GRP94-client protein interactions." Biochemistry 43.27 (July 13, 2004): 8835-8845.
PMID
15236592
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
27
Publish Date
2004
Start Page
8835
End Page
8845
DOI
10.1021/bi049539q

Glucose-regulated protein 94/glycoprotein 96 elicits bystander activation of CD4+ T cell Th1 cytokine production in vivo.

Glucose-regulated protein 94 (GRP94/gp96), the endoplasmic reticulum heat shock protein 90 paralog, elicits both innate and adaptive immune responses. Regarding the former, GRP94/gp96 stimulates APC cytokine expression and dendritic cell maturation. The adaptive component of GRP94/gp96 function reflects a proposed peptide-binding activity and, consequently, a role for native GRP94/gp96-peptide complexes in cross-presentation. It is by this mechanism that tumor-derived GRP94/gp96 is thought to suppress tumor growth and metastasis. Recent data have demonstrated that GRP94/gp96-elicited innate immune responses can be sufficient to suppress tumor growth and metastasis. However, the immunological processes activated in response to tumor Ag-negative sources of GRP94/gp96 are currently unknown. We have examined the in vivo immunological response to nontumor sources of GRP94/gp96 and report that administration of syngeneic GRP94/gp96- or GRP94/gp96-N-terminal domain-secreting KBALB fibroblasts to BALB/c mice stimulates CD11b(+) and CD11c(+) APC function and promotes bystander activation of CD4(+) T cell Th1 cytokine production. Only modest activation of CD8(+) T cell or NK cell cytolytic function was observed. The GRP94/gp96-dependent induction of CD4(+) T cell cytokine production was markedly inhibited by carrageenan, indicating an essential role for APC in this response. These results identify the bystander activation of CD4(+) T lymphocytes as a previously unappreciated immunological consequence of GRP94/gp96 administration and demonstrate that GRP94/gp96-elicited alterations in the in vivo cytokine environment influence the development of CD4(+) T cell effector functions, independently of its proposed function as a peptide chaperone.

Authors
Baker-LePain, JC; Sarzotti, M; Nicchitta, CV
MLA Citation
Baker-LePain, JC, Sarzotti, M, and Nicchitta, CV. "Glucose-regulated protein 94/glycoprotein 96 elicits bystander activation of CD4+ T cell Th1 cytokine production in vivo." J Immunol 172.7 (April 1, 2004): 4195-4203.
PMID
15034032
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
172
Issue
7
Publish Date
2004
Start Page
4195
End Page
4203

The messenger and the message: gp96 (GRP94)-peptide interactions in cellular immunity.

Vaccination of mice with tumor-derived stress proteins, such as Hsp70 and gp96 (GRP94), can elicit antitumor immune responses, yielding a marked suppression of tumor growth and metastasis. The molecular basis for this response is proposed to reflect a peptide-binding function for these proteins. In this view, stress proteins bind the antigenic peptide repertoire of their parent cell, and when provided to the immune system, tumor-derived stress protein-peptide complexes are processed by antigen-presenting cells (APCs) to yield the subsequent activation of tumor-directed cytotoxic T lymphocyte activity. This model predicts that stress proteins, whose primary intracellular function concerns the proper folding and assembly of nascent polypeptides, intersect with the cellular pathways responsible for the generation, processing, or assembly (or all) of peptide antigens onto nascent major histocompatability class I molecules. Recent insights into the pathways for peptide generation now allow this hypothesis to be critically examined, which is the subject of this review.

Authors
Nicchitta, CV; Carrick, DM; Baker-Lepain, JC
MLA Citation
Nicchitta, CV, Carrick, DM, and Baker-Lepain, JC. "The messenger and the message: gp96 (GRP94)-peptide interactions in cellular immunity." Cell Stress Chaperones 9.4 (2004): 325-331. (Review)
PMID
15633290
Source
pubmed
Published In
Cell Stress and Chaperones
Volume
9
Issue
4
Publish Date
2004
Start Page
325
End Page
331

Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation.

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 A of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.

Authors
Soldano, KL; Jivan, A; Nicchitta, CV; Gewirth, DT
MLA Citation
Soldano, KL, Jivan, A, Nicchitta, CV, and Gewirth, DT. "Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation." J Biol Chem 278.48 (November 28, 2003): 48330-48338.
PMID
12970348
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
48
Publish Date
2003
Start Page
48330
End Page
48338
DOI
10.1074/jbc.M308661200

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells.

gp96 (GRP94) elicits antigen-presenting cell (APC) activation and can direct peptides into the cross- presentation pathways of APC. These responses arise through interactions of gp96 with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC. Previously, CD91, the alpha2-macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class-A (SR-A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re-presentation are inhibited by the SR-A inhibitory ligand fucoidin, although fucoidin was without effect on alpha2-macroglobulin binding or uptake. Ectopic expression of SR-A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR-A-/- mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR-A in the regulation of cellular responses to heat shock proteins.

Authors
Berwin, B; Hart, JP; Rice, S; Gass, C; Pizzo, SV; Post, SR; Nicchitta, CV
MLA Citation
Berwin, B, Hart, JP, Rice, S, Gass, C, Pizzo, SV, Post, SR, and Nicchitta, CV. "Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells." EMBO J 22.22 (November 17, 2003): 6127-6136.
PMID
14609958
Source
pubmed
Published In
EMBO Journal
Volume
22
Issue
22
Publish Date
2003
Start Page
6127
End Page
6136
DOI
10.1093/emboj/cdg572

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes.

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.

Authors
Lerner, RS; Seiser, RM; Zheng, T; Lager, PJ; Reedy, MC; Keene, JD; Nicchitta, CV
MLA Citation
Lerner, RS, Seiser, RM, Zheng, T, Lager, PJ, Reedy, MC, Keene, JD, and Nicchitta, CV. "Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes." RNA 9.9 (September 2003): 1123-1137.
PMID
12923260
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
9
Issue
9
Publish Date
2003
Start Page
1123
End Page
1137

Come forth CD1d: Hsp110 in the regulation of intestinal epithelial CD1d expression.

CD1d, a nonclassical MHC class I-like molecule, is prominently expressed on intestinal epithelial cells and is thought to function in the regulation of intestinal intraepithelial lymphocyte activity. Hsp110, an abundant heat shock protein present in essentially all mammalian tissues, has now been shown to upregulate CD1d expression in colonic tissue culture cell lines. Might this abundant chaperone serve an autocrine function in the regulation of CD1d expression?

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Come forth CD1d: Hsp110 in the regulation of intestinal epithelial CD1d expression." J Clin Invest 112.5 (September 2003): 646-648.
PMID
12952910
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
112
Issue
5
Publish Date
2003
Start Page
646
End Page
648
DOI
10.1172/JCI19641

GRP94/gp96 elicits ERK activation in murine macrophages. A role for endotoxin contamination in NF-kappa B activation and nitric oxide production.

Vaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-kappa B signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-kappa B signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 microg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses.

Authors
Reed, RC; Berwin, B; Baker, JP; Nicchitta, CV
MLA Citation
Reed, RC, Berwin, B, Baker, JP, and Nicchitta, CV. "GRP94/gp96 elicits ERK activation in murine macrophages. A role for endotoxin contamination in NF-kappa B activation and nitric oxide production." J Biol Chem 278.34 (August 22, 2003): 31853-31860.
PMID
12805368
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
34
Publish Date
2003
Start Page
31853
End Page
31860
DOI
10.1074/jbc.M305480200

ISO: a critical evaluation of the role of peptides in heat shock/chaperone protein-mediated tumor rejection.

Authors
Baker-LePain, JC; Reed, RC; Nicchitta, CV
MLA Citation
Baker-LePain, JC, Reed, RC, and Nicchitta, CV. "ISO: a critical evaluation of the role of peptides in heat shock/chaperone protein-mediated tumor rejection." Curr Opin Immunol 15.1 (February 2003): 89-94. (Review)
PMID
12495739
Source
pubmed
Published In
Current Opinion in Immunology
Volume
15
Issue
1
Publish Date
2003
Start Page
89
End Page
94

GRP94 (gp96) and GRP94 N-terminal geldanamycin binding domain elicit tissue nonrestricted tumor suppression.

In chemical carcinogenesis models, GRP94 (gp96) elicits tumor-specific protective immunity. The tumor specificity of this response is thought to reflect immune responses to GRP94-bound peptide antigens, the cohort of which uniquely identifies the GRP94 tissue of origin. In this study, we examined the apparent tissue restriction of GRP94-elicited protective immunity in a 4T1 mammary carcinoma model. We report that the vaccination of BALB/c mice with irradiated fibroblasts expressing a secretory form of GRP94 markedly suppressed 4T1 tumor growth and metastasis. In addition, vaccination with irradiated cells secreting the GRP94 NH(2)-terminal geldanamycin-binding domain (NTD), a region lacking canonical peptide-binding motifs, yielded a similar suppression of tumor growth and metastatic progression. Conditioned media from cultures of GRP94 or GRP94 NTD-secreting fibroblasts elicited the up-regulation of major histocompatibility complex class II and CD86 in dendritic cell cultures, consistent with a natural adjuvant function for GRP94 and the GRP94 NTD. Based on these findings, we propose that GRP94-elicited tumor suppression can occur independent of the GRP94 tissue of origin and suggest a primary role for GRP4 natural adjuvant function in antitumor immune responses.

Authors
Baker-LePain, JC; Sarzotti, M; Fields, TA; Li, C-Y; Nicchitta, CV
MLA Citation
Baker-LePain, JC, Sarzotti, M, Fields, TA, Li, C-Y, and Nicchitta, CV. "GRP94 (gp96) and GRP94 N-terminal geldanamycin binding domain elicit tissue nonrestricted tumor suppression." J Exp Med 196.11 (December 2, 2002): 1447-1459.
PMID
12461080
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
196
Issue
11
Publish Date
2002
Start Page
1447
End Page
1459

A platform for compartmentalized protein synthesis: protein translation and translocation in the ER.

Recent advances in the study of protein translocation across the membrane of the endoplasmic reticulum include insights into the mechanism of signal-sequence function. Biochemical and genetic studies have provided further evidence that lumenal proteins perform direct roles in secretory protein translocation and in the regulation of protein-conducting-channel permeability during membrane protein integration. A hypothesis identifying the endoplasmic reticulum as a site of mRNA localization and compartmentalized protein synthesis has been suggested.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "A platform for compartmentalized protein synthesis: protein translation and translocation in the ER." Curr Opin Cell Biol 14.4 (August 2002): 412-416. (Review)
PMID
12383790
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
14
Issue
4
Publish Date
2002
Start Page
412
End Page
416

GRP94-associated enzymatic activities. Resolution by chromatographic fractionation.

GRP94 (gp96), which performs established functions as a molecular chaperone and immune system modulator, has been reported to display a number of intrinsic enzymatic activities, including ATP hydrolysis, protein phosphorylation, and aminopeptidase. In observing that GRP94 co-purified with bacterial beta-galactosidase through multiple chromatographic steps, we have examined the hypothesis that the reported enzymatic activities of GRP94 may reflect co-purification of contaminant enzymes, rather than intrinsic catalytic functions. In subjecting GRP94 to increasingly stringent chromatographic purification, we report that a GRP94 carboxyl-terminal directed protein kinase activity could be separated from GRP94 by heparin affinity chromatography. Analysis of the kinase substrate specificity indicates that this kinase is distinct from casein kinase II, which is known to co-purify with GRP94. Electrophoretically pure GRP94 displayed low, but significant levels of aminopeptidase activity. Further purification of GRP94 by anion exchange and heparin affinity chromatography yielded resolution of GRP94 from the aminopeptidase activity. Furthermore, exhaustive trypsinolysis of GRP94 preparations displaying aminopeptidase activity yielded complete proteolysis of GRP94 but did not affect aminopeptidase activity. These results are discussed with respect to current models for GRP94 function and the role of such co-purifying (poly)peptides in the generation of GRP94-dependent cellular immune responses.

Authors
Reed, RC; Zheng, T; Nicchitta, CV
MLA Citation
Reed, RC, Zheng, T, and Nicchitta, CV. "GRP94-associated enzymatic activities. Resolution by chromatographic fractionation." J Biol Chem 277.28 (July 12, 2002): 25082-25089.
PMID
11983709
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
28
Publish Date
2002
Start Page
25082
End Page
25089
DOI
10.1074/jbc.M203195200

Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis.

In current views, translation-coupled ribosome binding to the endoplasmic reticulum (ER) membrane is transient, with association occurring via the signal recognition particle pathway and dissociation occurring upon the termination of protein synthesis. Recent studies indicate, however, that ribosomal subunits remain membrane-bound following the termination of protein synthesis. To define the mechanism of post-termination ribosome association with the ER membrane, membrane-bound ribosomes were detergent-solubilized from tissue culture cells at different stages of the protein synthesis cycle, and the composition of the ribosome-associated membrane protein fraction was determined. We report that ribosomes reside in stable association with the Sec61alpha-translocon following the termination stage of protein synthesis. Additionally, in vitro experiments revealed that solubilized, gradient-purified ribosome-translocon complexes were able to initiate the translation of secretory and cytosolic proteins and were functional in assays of signal sequence recognition. Using this experimental system, synthesis of signal sequence-bearing polypeptides yielded a tight ribosome-translocon junction; synthesis of nascent polypeptides lacking a signal sequence resulted in a disruption of this junction. On the basis of these data, we propose that in situ, ribosomes reside in association with the translocon throughout the cycle of protein synthesis, with membrane release occurring upon translation of proteins lacking topogenic signals.

Authors
Potter, MD; Nicchitta, CV
MLA Citation
Potter, MD, and Nicchitta, CV. "Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis." J Biol Chem 277.26 (June 28, 2002): 23314-23320.
PMID
11964406
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
26
Publish Date
2002
Start Page
23314
End Page
23320
DOI
10.1074/jbc.M202559200

Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides.

GRP94(gp96) elicits CD8(+) T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in alpha(2)-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated alpha(2)-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.

Authors
Berwin, B; Hart, JP; Pizzo, SV; Nicchitta, CV
MLA Citation
Berwin, B, Hart, JP, Pizzo, SV, and Nicchitta, CV. "Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides." J Immunol 168.9 (May 1, 2002): 4282-4286.
PMID
11970968
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
168
Issue
9
Publish Date
2002
Start Page
4282
End Page
4286

Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules.

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the Kb-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.

Authors
Berwin, B; Rosser, MFN; Brinker, KG; Nicchitta, CV
MLA Citation
Berwin, B, Rosser, MFN, Brinker, KG, and Nicchitta, CV. "Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules." Traffic 3.5 (May 2002): 358-366.
PMID
11967129
Source
pubmed
Published In
Traffic
Volume
3
Issue
5
Publish Date
2002
Start Page
358
End Page
366

Probing protein-protein interactions with chemical cross-linking and differential nano-LC-MS

Protein-protein interaction was investigated using chemical cross-linking and differential nano-LC-MS. The tryptic digestion mixtures were analyzed by nano-LC-MS. GGH-ecotin D137Y was used as a model protein and cross-linked by primary amine reactive cross-linker, disuccinimidyl suberate (DSS). A retrospective analysis of the wild-type ecotin X-ray crystal structure shows that the cross-linked residues are all on the dimeric interface.

Authors
Chu, F; Baker, P; Mahrus, S; Craik, CS; Rosser, M; Nicchitta, CV; Burlingame, AL
MLA Citation
Chu, F, Baker, P, Mahrus, S, Craik, CS, Rosser, M, Nicchitta, CV, and Burlingame, AL. "Probing protein-protein interactions with chemical cross-linking and differential nano-LC-MS." Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics (2002): 289-290.
Source
scival
Published In
Proceedings 50th ASMS Conference on Mass Spectrometry and Allied Topics
Publish Date
2002
Start Page
289
End Page
290

Signal sequence function in the mammalian endoplasmic reticulum: A biological perspective

The discovery of the signal peptide, an amino-terminal protein sequence that specifies targeting of newly synthesized polypeptides to the endoplasmic reticulum (ER), stands as one of the most significant in cell biology. The signal peptide performs a targeting function in the cell and serves as a paradigm for the processes by which proteins are targeted to other organelles of the eukaryotic cell, such as the nucleus, the mitochondria, and the peroxisome. Central to signal sequence function is its composite secondary structure, a conserved tripartite motif consisting of a positively charged amino terminus, a central hydrophobic core, and a carboxy-terminal polar domain. Of the three domains, it is the central hydrophobic core, a continuous stretch of 7-15 hydrophobic amino acids, that is functionally dominant. By virtue of its mean hydrophobicity, the central hydrophobic domain disposes the signal sequence to direct interactions with the lipid bilayer. Nonetheless, the predominant view in the cell biology community is that signal sequences function through direct protein-protein interactions to specify both targeting to the ER and regulation of the protein translocation machinery. This review focuses on signal sequence function, with critical emphasis on discerning lipid-dependent versus protein-dependent interactions with and within the ER membrane. © 2002.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Signal sequence function in the mammalian endoplasmic reticulum: A biological perspective." Current Topics in Membranes 52 (2002): 483-499.
Source
scival
Published In
Current Topis in Membranes
Volume
52
Publish Date
2002
Start Page
483
End Page
499

To find the road traveled to tumor immunity: the trafficking itineraries of molecular chaperones in antigen-presenting cells.

Molecular chaperones, both endoplasmic reticulum and cytosol derived, have been identified as tumor rejection antigens; in animal models, they can elicit prophylactic and therapeutic immune responses against their tumor of origin. Chaperone immunogenic activity derives from three principal characteristics: they bind an array of immunogenic (poly)peptides, they can be efficiently internalized by professional antigen-presenting cells, and once internalized, they traffic to a subcellular compartment(s) where peptide release can occur. Within the antigen-presenting cell, chaperone-derived peptides can be assembled onto major histocompatibility class I molecules for presentation at the antigen-presenting cell surface, thereby yielding the requisite and specific CD8+ T-cell responses that contribute to the process of tumor rejection. Though it is clear that chaperones, in particular GRP94 (gp96), calreticulin and Hsp70, can elicit cellular immune responses, the subcellular basis of chaperone processing by antigen-presenting cells remains mysterious. In this review, we discuss recent reports describing the identification of a chaperone internalization receptor and the physiological release of chaperones from necrotic cells, and we present views on the trafficking pathways within antigen-presenting cells that may function to deliver the chaperone-associated peptides to subcellular organelles for their subsequent exchange onto major histocompatibility complex molecules.

Authors
Berwin, B; Nicchitta, CV
MLA Citation
Berwin, B, and Nicchitta, CV. "To find the road traveled to tumor immunity: the trafficking itineraries of molecular chaperones in antigen-presenting cells." Traffic 2.10 (October 2001): 690-697. (Review)
PMID
11576445
Source
pubmed
Published In
Traffic
Volume
2
Issue
10
Publish Date
2001
Start Page
690
End Page
697

Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96.

Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

Authors
Berwin, B; Reed, RC; Nicchitta, CV
MLA Citation
Berwin, B, Reed, RC, and Nicchitta, CV. "Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96." J Biol Chem 276.24 (June 15, 2001): 21083-21088.
PMID
11279246
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
24
Publish Date
2001
Start Page
21083
End Page
21088
DOI
10.1074/jbc.M101836200

Ribosome exchange revisited: a mechanism for translation-coupled ribosome detachment from the ER membrane.

In current models, ribosome release from the endoplasmic reticulum (ER) is coupled to the termination of protein translation. Thus, coincident with termination, membrane-bound ribosomes dissociate into their component subunits and are released into the cytosol. Here, we review past and current data and propose that the affinity of the ribosome for the ER membrane is decreased during translation, with ribosome release occurring when a membrane-bound ribosome is engaged in the synthesis of a protein lacking a signal sequence. Our model emphasizes a role for the conformation of the large ribosomal subunit in the regulation of membrane affinity and provides a mechanism for translation-coupled ribosome release.

Authors
Potter, MD; Seiser, RM; Nicchitta, CV
MLA Citation
Potter, MD, Seiser, RM, and Nicchitta, CV. "Ribosome exchange revisited: a mechanism for translation-coupled ribosome detachment from the ER membrane." Trends Cell Biol 11.3 (March 2001): 112-115. (Review)
PMID
11306271
Source
pubmed
Published In
Trends in Cell Biology
Volume
11
Issue
3
Publish Date
2001
Start Page
112
End Page
115

Sensitivity of Mature ErbB2 to Geldanamycin Is Conferred by Its Kinase Domain and Is Mediated by the Chaperone Protein Hsp90

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.

Authors
Xu, W; Mimnaugh, E; Rosser, MFN; Nicchitta, C; Marcu, M; Yarden, Y; Neckers, L
MLA Citation
Xu, W, Mimnaugh, E, Rosser, MFN, Nicchitta, C, Marcu, M, Yarden, Y, and Neckers, L. "Sensitivity of Mature ErbB2 to Geldanamycin Is Conferred by Its Kinase Domain and Is Mediated by the Chaperone Protein Hsp90." Journal of Biological Chemistry 276.5 (2001): 3702-3708.
PMID
11071886
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
5
Publish Date
2001
Start Page
3702
End Page
3708
DOI
10.1074/jbc.M006864200

The fate of membrane-bound ribosomes following the termination of protein synthesis.

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.

Authors
Seiser, RM; Nicchitta, CV
MLA Citation
Seiser, RM, and Nicchitta, CV. "The fate of membrane-bound ribosomes following the termination of protein synthesis." J Biol Chem 275.43 (October 27, 2000): 33820-33827.
PMID
10931837
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
43
Publish Date
2000
Start Page
33820
End Page
33827
DOI
10.1074/jbc.M004462200

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane.

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.

Authors
Potter, MD; Nicchitta, CV
MLA Citation
Potter, MD, and Nicchitta, CV. "Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane." J Biol Chem 275.43 (October 27, 2000): 33828-33835.
PMID
10924518
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
43
Publish Date
2000
Start Page
33828
End Page
33835
DOI
10.1074/jbc.M005294200

Chaperone-mediated cross-priming: a hitchhiker's guide to vesicle transport (review).

The resident endoplasmic reticulum (ER) chaperone proteins GRP94 (gp96) and calreticulin can activate the immune system to slow or stop the progression of tumors by escorting tumor-derived peptides into the endogenous antigen presentation pathway of antigen presenting cells (APC). Although the phenomenology of cross-priming is well worked out, the mechanism(s) remains unclear. Continuing insights into cellular protein trafficking pathways suggest several means by which chaperones could travel from the extracellular space into the endosome, lysosome or ER of APC. In particular, proteins that cycle between two or more compartments and those that undergo and mediate retrograde flow offer models of how exogenous chaperones might travel in the APC. New insights into how non-chaperone proteins access the APC antigen presentation pathway also suggest several ways this process could occur.

Authors
Reed, RC; Nicchitta, CV
MLA Citation
Reed, RC, and Nicchitta, CV. "Chaperone-mediated cross-priming: a hitchhiker's guide to vesicle transport (review)." Int J Mol Med 6.3 (September 2000): 259-264. (Review)
PMID
10934286
Source
pubmed
Published In
International journal of molecular medicine
Volume
6
Issue
3
Publish Date
2000
Start Page
259
End Page
264

A topological study of the human gamma-glutamyl carboxylase.

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Authors
Tie, J; Wu, SM; Jin, D; Nicchitta, CV; Stafford, DW
MLA Citation
Tie, J, Wu, SM, Jin, D, Nicchitta, CV, and Stafford, DW. "A topological study of the human gamma-glutamyl carboxylase." Blood 96.3 (August 1, 2000): 973-978.
PMID
10910912
Source
pubmed
Published In
Blood
Volume
96
Issue
3
Publish Date
2000
Start Page
973
End Page
978

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding.

X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a K(d) of 200 nm. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In [(3)H]NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NECA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.

Authors
Rosser, MF; Nicchitta, CV
MLA Citation
Rosser, MF, and Nicchitta, CV. "Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding." J Biol Chem 275.30 (July 28, 2000): 22798-22805.
PMID
10816561
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
30
Publish Date
2000
Start Page
22798
End Page
22805
DOI
10.1074/jbc.M001477200

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity.

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket that also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. Although this domain is essential for Hsp90 function, the molecular basis for adenosine nucleotide-dependent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, remains to be established. We report that bis-ANS (1,1'-bis(4-anilino-5-napthalenesulfonic acid), an environment sensitive fluorophore known to interact with nucleotide-binding domains, binds to the adenosine nucleotide-binding domain of GRP94 and thereby activates its molecular chaperone and peptide binding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activates GRP94 function. bis-ANS activation of GRP94 function was efficiently blocked by radicicol, an established inhibitory ligand for the adenosine nucleotide binding pocket. Confirmation of the N-terminal nucleotide binding pocket as the bis-ANS-binding site was obtained following covalent incorporation of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labeled limit digestion products. These data identify a ligand dependent regulation of GRP94 function and suggest a model whereby GRP94 function is regulated through a ligand-dependent conversion of GRP94 from an inactive to an active conformation.

Authors
Wassenberg, JJ; Reed, RC; Nicchitta, CV
MLA Citation
Wassenberg, JJ, Reed, RC, and Nicchitta, CV. "Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity." J Biol Chem 275.30 (July 28, 2000): 22806-22814.
PMID
10816560
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
30
Publish Date
2000
Start Page
22806
End Page
22814
DOI
10.1074/jbc.M001476200

Role of chaperones in antigen processing.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Role of chaperones in antigen processing." Immunol Invest 29.2 (May 2000): 101-104.
PMID
10854175
Source
pubmed
Published In
Immunological Investigations (Informa)
Volume
29
Issue
2
Publish Date
2000
Start Page
101
End Page
104

Ribosome-independent regulation of translocon composition and Sec61alpha conformation.

In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61alpha, ribophorin I, and TRAPalpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61alpha, ribophorin I, or TRAPalpha were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61alpha was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61alpha to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61alpha, indicating that a protease-resistant conformation of Sec61alpha can be conferred in a ribosome-independent manner.

Authors
Potter, MD; Nicchitta, CV
MLA Citation
Potter, MD, and Nicchitta, CV. "Ribosome-independent regulation of translocon composition and Sec61alpha conformation." J Biol Chem 275.3 (January 21, 2000): 2037-2045.
PMID
10636907
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
3
Publish Date
2000
Start Page
2037
End Page
2045

The immunological properties of endoplasmic reticulum chaperones: a conflict of interest?

ER chaperones are abundant and highly conserved proteins that display both peptide binding and chaperone activity. Of the family of chaperones present in the mammalian ER, GRP94 and calreticulin are apparently unique in their ability to elicit CD8+ T-cell responses against components of their bound-peptide pools. The ability of GRP94 and calreticulin to elicit CD8+ T-cell responses indicates that both proteins bind peptides suitable for assembly on to MHC class-I molecules. The capacity to function as molecular chaperones and as peptide-binding proteins capable of transferring, directly or indirectly, peptides on to class-I molecules, indicates that GRP94 and calreticulin participate in the regulation of both peptide and polypeptide traffic in the ER. Perspectives on the regulation of and interplay between the peptide binding and chaperone activity of GRP94 and calreticulin are discussed.

Authors
Nicchitta, CV; Reed, RC
MLA Citation
Nicchitta, CV, and Reed, RC. "The immunological properties of endoplasmic reticulum chaperones: a conflict of interest?." Essays Biochem 36 (2000): 15-25. (Review)
PMID
12471899
Source
pubmed
Published In
Essays in biochemistry
Volume
36
Publish Date
2000
Start Page
15
End Page
25

Structural determinants for signal sequence function in the mammalian endoplasmic reticulum.

Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.

Authors
Zheng, T; Nicchitta, CV
MLA Citation
Zheng, T, and Nicchitta, CV. "Structural determinants for signal sequence function in the mammalian endoplasmic reticulum." J Biol Chem 274.51 (December 17, 1999): 36623-36630.
PMID
10593964
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
51
Publish Date
1999
Start Page
36623
End Page
36630

Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages.

Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elicits cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell-derived peptides. Elicitation of a CTL response requires that GRP94-associated peptides be transferred onto major histocompatability complex (MHC) class I molecules, a process that is postulated to accompany GRP94 internalization by antigen presenting cells, such as macrophages (Mphi) and dendritic cells (DC). In studies of GRP94 uptake in elicited Mphi, we report that Mphi display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis. GRP94 internalized by this pathway co-localized predominately with transferrin-positive early endosomes. At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed. When GRP94 was present in the medium, and thus accessible to both receptor-mediated and fluid phase internalization pathways, internalization was modestly inhibited in the presence of yeast mannan, a competitive inhibitor of mannose/fucose receptor activity, and substantially inhibited by dimethylamiloride, an inhibitor of macropinocytosis. GRP94 internalized via macropinocytosis did not display prominent co-staining with the lysosomal marker LAMP-2. These data identify multiple pathways of GRP94 internalization and indicate that receptor-dependent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety. Most significantly, these data demonstrate the existence of cell surface receptor(s), apparently unique to antigen presenting cells, that function in the binding and internalization of the ER chaperone GRP94.

Authors
Wassenberg, JJ; Dezfulian, C; Nicchitta, CV
MLA Citation
Wassenberg, JJ, Dezfulian, C, and Nicchitta, CV. "Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages." J Cell Sci 112 ( Pt 13) (July 1999): 2167-2175.
PMID
10362546
Source
pubmed
Published In
Journal of cell science
Volume
112 ( Pt 13)
Publish Date
1999
Start Page
2167
End Page
2175

Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.

Authors
Nair, S; Wearsch, PA; Mitchell, DA; Wassenberg, JJ; Gilboa, E; Nicchitta, CV
MLA Citation
Nair, S, Wearsch, PA, Mitchell, DA, Wassenberg, JJ, Gilboa, E, and Nicchitta, CV. "Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides." J Immunol 162.11 (June 1, 1999): 6426-6432.
PMID
10352256
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
162
Issue
11
Publish Date
1999
Start Page
6426
End Page
6432

Structural transitions accompanying the activation of peptide binding to the endoplasmic reticulum Hsp90 chaperone GRP94.

GRP94, the endoplasmic reticulum Hsp90 paralog, binds a diverse array of peptides, a subset of which are suitable for assembly onto nascent MHC class I molecules. At present, the mechanism, site, and regulation of peptide binding to GRP94 are unknown. Using VSV8, the immunodominant peptide epitope of the vesicular stomatitis virus, and native, purified GRP94, we have investigated GRP94-peptide complex formation. The formation of stable GRP94-VSV8 complexes was slow; competition studies demonstrated that peptide binding to GRP94 was specific. VSV8 binding to GRP94 was stimulated 2-fold or 4-fold, respectively, following chemical denaturation/renaturation or transient heat shock. The activation of GRP94-peptide binding occurred coincident with a stable, tertiary conformational change, as identified by tryptophan fluorescence and proteolysis studies. Analysis of GRP94 secondary structure by circular dichroism spectroscopy indicated an identical alpha-helical content for the native, chemically denatured/renatured, and heat-shocked forms of GRP94. Through use of the environment-sensitive fluorophores acrylodan and Nile Red, it was observed that the activation of peptide binding was accompanied by enhanced peptide and solvent accessibility to a hydrophobic binding site(s). Peptide binding to native or activated GRP94 was identical in the presence or absence of ATP or ADP. These results are discussed with respect to a model in which peptide binding to GRP94 occurs within a hydrophobic binding pocket whose accessibility is conformationally regulated in an adenine nucleotide-independent manner.

Authors
Wearsch, PA; Voglino, L; Nicchitta, CV
MLA Citation
Wearsch, PA, Voglino, L, and Nicchitta, CV. "Structural transitions accompanying the activation of peptide binding to the endoplasmic reticulum Hsp90 chaperone GRP94." Biochemistry 37.16 (April 21, 1998): 5709-5719.
PMID
9548957
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
16
Publish Date
1998
Start Page
5709
End Page
5719
DOI
10.1021/bi9801006

Biochemical, cell biological and immunological issues surrounding the endoplasmic reticulum chaperone GRP94/gp96.

The past year has born witness to compelling demonstrations of the utility of peptide complexes with glucose regulated protein 94 (GRP94, also known as gp96) in cancer immunotherapy. Insights into the structural basis of peptide binding to GRP94 have been obtained and the role of the transporter for antigen presentation in defining the GRP94-bound peptide composition has been determined.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Biochemical, cell biological and immunological issues surrounding the endoplasmic reticulum chaperone GRP94/gp96." Curr Opin Immunol 10.1 (February 1998): 103-109. (Review)
PMID
9523119
Source
pubmed
Published In
Current Opinion in Immunology
Volume
10
Issue
1
Publish Date
1998
Start Page
103
End Page
109

Protein translocation across the mammalian endoplasmic reticulum: A tale of tunnels and ratchets?

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Protein translocation across the mammalian endoplasmic reticulum: A tale of tunnels and ratchets?." Chemtracts 11.6 (1998): 408-422.
Source
scival
Published In
Chemtracts
Volume
11
Issue
6
Publish Date
1998
Start Page
408
End Page
422

Regulation of the ribosome-membrane junction at early stages of presecretory protein translocation in the mammalian endoplasmic reticulum.

A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome-membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation. In a study of secretory protein precursors, formation of a protease-resistant ribosome-membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome-membrane junction by the nascent chain is presented.

Authors
Nicchitta, CV; Zheng, T
MLA Citation
Nicchitta, CV, and Zheng, T. "Regulation of the ribosome-membrane junction at early stages of presecretory protein translocation in the mammalian endoplasmic reticulum." J Cell Biol 139.7 (December 29, 1997): 1697-1708.
PMID
9412465
Source
pubmed
Published In
The Journal of Cell Biology
Volume
139
Issue
7
Publish Date
1997
Start Page
1697
End Page
1708

Structure and folding of nascent polypeptide chains during protein translocation in the endoplasmic reticulum.

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo and in vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.

Authors
Haynes, RL; Zheng, T; Nicchitta, CV
MLA Citation
Haynes, RL, Zheng, T, and Nicchitta, CV. "Structure and folding of nascent polypeptide chains during protein translocation in the endoplasmic reticulum." J Biol Chem 272.27 (July 4, 1997): 17126-17133.
PMID
9202031
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
27
Publish Date
1997
Start Page
17126
End Page
17133

Identification of a novel stage of ribosome/nascent chain association with the endoplasmic reticulum membrane.

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.

Authors
Murphy, EC; Zheng, T; Nicchitta, CV
MLA Citation
Murphy, EC, Zheng, T, and Nicchitta, CV. "Identification of a novel stage of ribosome/nascent chain association with the endoplasmic reticulum membrane." J Cell Biol 136.6 (March 24, 1997): 1213-1226.
PMID
9087438
Source
pubmed
Published In
The Journal of Cell Biology
Volume
136
Issue
6
Publish Date
1997
Start Page
1213
End Page
1226

Interaction of endoplasmic reticulum chaperone GRP94 with peptide substrates is adenine nucleotide-independent.

GRP94, the endoplasmic reticulum paralog of hsp90, has recently been identified as a peptide and adenine nucleotide-binding protein. To determine if adenine nucleotides directly contribute to the regulation of GRP94 peptide binding activity, an in vitro peptide binding assay was developed. Using purified GRP94, we observed specific, saturable, temperature-sensitive binding of the peptide VSV8, a known in vivo ligand. ATP was without effect on VSV8 binding to GRP94, whether present during or subsequent to peptide binding. To evaluate the interaction of GRP94 with adenine nucleotides, the ATP binding and hydrolysis activities were directly assayed. Only negligible binding of ATP to GRP94 was observed. In addition, analysis of the GRP94 adenine nucleotide content indicated that GRP94 did not copurify with bound adenine nucleotides. GRP94 preparations exhibited low ATPase and apparent autophosphorylation activities. Further purification, combined with inhibitor studies, indicated that both activities were the result of trace contamination (<0.1%) with casein kinase II. On the basis of these data, we propose that the peptide binding activity of GRP94 is adenine nucleotide-independent and that ATP binding and hydrolysis are not inherent properties of GRP94.

Authors
Wearsch, PA; Nicchitta, CV
MLA Citation
Wearsch, PA, and Nicchitta, CV. "Interaction of endoplasmic reticulum chaperone GRP94 with peptide substrates is adenine nucleotide-independent." J Biol Chem 272.8 (February 21, 1997): 5152-5156.
PMID
9030582
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
8
Publish Date
1997
Start Page
5152
End Page
5156

Membrane insertion, glycosylation, and oligomerization of inositol trisphosphate receptors in a cell-free translation system.

In order to study the membrane topology, processing, and oligomerization of inositol trisphosphate receptor (IP3R) isoforms, we have utilized RNA templates encoding putative transmembrane domains to program a cell-free translation system of rabbit reticulocyte lysates supplemented with canine pancreas microsomes. In the absence of microsomes, translation of the RNA templates encoding all the putative transmembrane domains present in the C-terminal segment of the type I (1TM) and type III (3TM) IP3R isoforms resulted in a 62- and 59-kDa polypeptide, respectively. In both cases, an additional band approximately 3 kDa larger was observed upon the addition of microsomes. Both bands in the translation doublet were integrated into microsomal membranes and were full-length translation products, as shown by sedimentation through a sucrose cushion and immunoprecipitation with C-terminal isoform-specific antibodies. With both isoforms, N-glycopeptidase F digestion indicates that the upper band in the doublet corresponds to a glycosylated translation product. A 17-kDa protected fragment was observed after proteinase-K digestion of 1TM translated in the presence of microsomes. The pattern and size of protected fragments was consistent with the current six-transmembrane domain model of IP3R topology. Cotranslation of both 1TM and 3TM RNA templates in the presence of microsomes followed by immunoprecipitation with isoform specific antibodies revealed coimmunoprecipitation of translation products. This was not observed when the isoforms were translated separately and then mixed, suggesting that heteroligomerization occurs cotranslationally. A construct encoding only the first putative transmembrane domain of the type I isoform was found to be sufficient for integration into membranes but was unable to oligomerize with either 1TM or 3TM. Cotranslation experiments using additional constructs indicate that the major structural determinant for homoligomerization lies between putative transmembrane domain 5 and the C terminus. A second oligomerization domain involved in stabilization of heteroligomers is present within the first four transmembrane domains.

Authors
Joseph, SK; Boehning, D; Pierson, S; Nicchitta, CV
MLA Citation
Joseph, SK, Boehning, D, Pierson, S, and Nicchitta, CV. "Membrane insertion, glycosylation, and oligomerization of inositol trisphosphate receptors in a cell-free translation system." J Biol Chem 272.3 (January 17, 1997): 1579-1588.
PMID
8999831
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
3
Publish Date
1997
Start Page
1579
End Page
1588

Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain.

GRP94 is an abundant, resident glycoprotein of the mammalian endoplasmic reticulum lumen and member of the hsp90 family of molecular chaperones. To identify the structure/function relationships which define the molecular basis of GRP94 activity, we have performed a structural analysis of native GRP94 and identified a discrete domain, representing amino acids 676-719, which regulates dimerization and displays autonomous oligomerization activity. Velocity sedimentation and gel filtration chromatography were used to identify native GRP94 as a dimer with an extended, rod-like shape. Limited proteolysis resulted in the loss of approximately 16 kDa from the C-terminus and disassembly into monomers, implicating the C-terminus as the site of assembly. An assembly function for the C-terminal domain was established by analysis of the quaternary structure of C-terminal constructs synthesized either in vitro or through recombinant expression. In vitro translation was used to demonstrate that a C-terminal 20 kDa domain was both necessary and sufficient for dimerization. Structural studies of recombinant fusion protein constructs yielded identification of a 44 amino acid domain that displayed autonomous dimerization activity and conferred a highly elongated structure, characteristic of native GRP94, to the fusion protein. These data, combined with molecular dimensions obtained from rotary shadowing electron microscopy, provide a structural model of GRP94 and identify the molecular basis of GRP94 self-assembly.

Authors
Wearsch, PA; Nicchitta, CV
MLA Citation
Wearsch, PA, and Nicchitta, CV. "Endoplasmic reticulum chaperone GRP94 subunit assembly is regulated through a defined oligomerization domain." Biochemistry 35.51 (December 24, 1996): 16760-16769.
PMID
8988013
Source
pubmed
Published In
Biochemistry
Volume
35
Issue
51
Publish Date
1996
Start Page
16760
End Page
16769
DOI
10.1021/bi962068q

Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C).

Enterotoxigenic Escherichia coli elaborate a peptide called heat-stable enterotoxin (ST), which binds to and activates the intestinal ST receptor (STaR). STaR, also known as guanylyl cyclase C (GC-C), is a member of the transmembrane guanylyl cyclase receptor family. The mRNA for STaR encodes an approximately 120 kDa protein with the N-terminal ligand binding domain on the cell surface. Ligand affinity cross-linking studies have previously demonstrated several species of potential ST binding proteins, ranging in size from approximately 50 to 160 kDa. Although these smaller forms of STaR (50-80 kDa) have been proposed to act in vivo as toxin binding proteins, their biogenesis and localization have not previously been examined. Using pulse labeling in vivo and synchronized translation in vitro, we demonstrate that these smaller forms represent incomplete translational products and are not formed through limited proteolysis of the full-length receptor, as had previously been believed. We determined, using fluorescence confocal microscopy and surface labeling, that only approximately 25% of cellular receptors are expressed at the surface, while the remaining population is retained within the endoplasmic reticulum. Only full-length receptor is found at the surface of the cell, indicating this to be the biologically active form of STaR responsible for interacting with the heat-stable enterotoxin and other luminal intestinal peptides. The large intracellular receptor population, and potential for function before translocation to the cell surface, may impact on how pharmacologic modulators of this clinically important receptor are designed.

Authors
Rudner, XL; Nicchitta, C; Almenoff, JS
MLA Citation
Rudner, XL, Nicchitta, C, and Almenoff, JS. "Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C)." Biochemistry 35.33 (August 20, 1996): 10680-10686.
PMID
8718857
Source
pubmed
Published In
Biochemistry
Volume
35
Issue
33
Publish Date
1996
Start Page
10680
End Page
10686
DOI
10.1021/bi9607721

Purification and partial molecular characterization of GRP94, an ER resident chaperone.

GRP94 is a resident glycoprotein of the endoplasmic reticulum (ER) and a member of the hsp90 family of molecular chaperones. Current experimental evidence indicates that GRP94 functions in an as yet undefined manner in protein folding and assembly in the ER. We report a rapid, high-yield GRP94 purification procedure that yields milligram quantities of homogeneous protein suitable for structural and biochemical analyses. Beginning with a rough microsome fraction derived from porcine pancreas, GRP94 was isolated by partial detergent extraction, anion exchange, and gel filtration chromatography. With this procedure, approximately 3 mg of homogeneous GRP94 can be prepared from a 25-g pancreas. Heterogeneity in the migration of purified GRP94 on native and denaturing PAGE was observed and demonstrated to reflect variability in the N-linked glycosylation state of the protein. Analysis by native and two-dimensional nonreducing/reducing gels indicated that the protein exists as a dimer of noncovalently associated subunits. The membrane localization of GRP94 in isolated pancreatic microsomes was assessed by alkali and detergent extraction. By comparison with the resident ER integral membrane protein TRAPalpha, GRP94 exists as a soluble, lumenal protein.

Authors
Wearsch, PA; Nicchitta, CV
MLA Citation
Wearsch, PA, and Nicchitta, CV. "Purification and partial molecular characterization of GRP94, an ER resident chaperone." Protein Expr Purif 7.1 (February 1996): 114-121.
PMID
9172775
Source
pubmed
Published In
Protein Expression and Purification
Volume
7
Issue
1
Publish Date
1996
Start Page
114
End Page
121
DOI
10.1006/prep.1996.0015

Protein translocation in the endoplasmic reticulum: The search for a unified molecular mechanism

The development of procedures for the reconstitution of protein translocation in the endoplasmic reticulum (ER) has allowed substantial new insights into the molecular mechanism of translocation. With these techniques, investigators are beginning to address the functional role of individual membrane and lumenal protein components in translocation. Efforts are now focused on the functional analysis of the translocation channel, the regulation of ribosome binding and the role of molecular chaperones in vectorial transport across the lipid bilayer. Current models of translocation suggest a system of remarkable simplicity; the molecular mechanism of translocation, however, remains elusive. ©1996 Academic Press Ltd.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Protein translocation in the endoplasmic reticulum: The search for a unified molecular mechanism." Seminars in Cell and Developmental Biology 7.4 (1996): 497-503.
Source
scival
Published In
Seminars in Cell and Developmental Biology
Volume
7
Issue
4
Publish Date
1996
Start Page
497
End Page
503
DOI
10.1006/scdb.1996.0062

Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.

Authors
Nicchitta, CV; Murphy, EC; Haynes, R; Shelness, GS
MLA Citation
Nicchitta, CV, Murphy, EC, Haynes, R, and Shelness, GS. "Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane." J Cell Biol 129.4 (May 1995): 957-970.
PMID
7744967
Source
pubmed
Published In
The Journal of Cell Biology
Volume
129
Issue
4
Publish Date
1995
Start Page
957
End Page
970

Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

Authors
Yu, H; Nicchitta, CV; Kumar, J; Becker, M; Toyoshima, I; Sheetz, MP
MLA Citation
Yu, H, Nicchitta, CV, Kumar, J, Becker, M, Toyoshima, I, and Sheetz, MP. "Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis." Mol Biol Cell 6.2 (February 1995): 171-183.
PMID
7787244
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
6
Issue
2
Publish Date
1995
Start Page
171
End Page
183

Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation.

The role of the lumenal contents (reticuloplasm) of the endoplasmic reticulum (ER) in protein translocation was determined by in vitro analysis. Depletion of the reticuloplasm from mammalian rough microsomes revealed two distinct stages of the translocation reaction. The first stage, translocation up to and including signal peptide cleavage, was insensitive to the loss of the reticuloplasm, whereas the second stage, net transfer of the nascent chain into the ER lumen, was reticuloplasm dependent. In reticuloplasm-depleted membranes, signal-cleaved and glycosylated translocation intermediates were observed to transit free from the translocation channel to the cis, or cytoplasmic, side of the membrane. This translocation defect was complemented by reconstitution of lumenal proteins into depleted membranes. We propose that lumenal proteins are necessary for unidirectional protein translocation in mammalian ER.

Authors
Nicchitta, CV; Blobel, G
MLA Citation
Nicchitta, CV, and Blobel, G. "Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation." Cell 73.5 (June 4, 1993): 989-998.
PMID
8500184
Source
pubmed
Published In
Cell
Volume
73
Issue
5
Publish Date
1993
Start Page
989
End Page
998

Membrane topology and biogenesis of eukaryotic signal peptidase.

The signal peptidase complex (SPC) is a hetero-oligomeric membrane protein containing subunits of 12, 18, 21, 22/23, and 25 kDa. The 18- and 21-kDa subunits are mammalian homologs of SEC11 protein, which is necessary for signal peptide processing and cell viability in the yeast Saccharomyces cerevisiae. The functional and/or structural contributions of the 12-, 22/23-, and 25-kDa subunits to SPC activity have not yet been elucidated. To explore the structure of SPC subunits and their relationships to signal peptide processing and protein translocation, we have examined their endoplasmic reticulum (ER) membrane topology and biogenesis. Signal peptidase activity and SPC subunits are resistant to protease treatment in intact and detergent-solubilized membranes. Heat-denatured SPC subunits and SPC subunits translated in vitro are, however, protease sensitive, suggesting that the assembly of the oligomeric complex confers protease resistance. To define the membrane topology of SPC subunits, both wild-type subunits and subunit fusion proteins containing additional sites for N-linked glycosylation were assembled into microsomal membranes in vitro. Despite the presence of multiple hydrophobic domains, each subunit is anchored to the ER membrane by a single amino-terminal transmembrane domain in an Ncytoplasmic Cexoplasmic (type II) orientation. This topology places the bulk of the protein mass in the ER lumen and positions a putative serine-containing active site domain in SPC 18 and 21 at the same relative distance from the membrane as the analogous region in Escherichia coli leader peptidase. These studies have also revealed that, in spite of the temporal and perhaps physical association of the SPC with the process of protein translocation, SPC subunits integrate into the ER membrane by a signal recognition particle-dependent pathway and, hence, rely on the existence of a preformed translocation apparatus for their own membrane assembly.

Authors
Shelness, GS; Lin, L; Nicchitta, CV
MLA Citation
Shelness, GS, Lin, L, and Nicchitta, CV. "Membrane topology and biogenesis of eukaryotic signal peptidase." J Biol Chem 268.7 (March 5, 1993): 5201-5208.
PMID
8444896
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
7
Publish Date
1993
Start Page
5201
End Page
5208

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation.

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

Authors
Migliaccio, G; Nicchitta, CV; Blobel, G
MLA Citation
Migliaccio, G, Nicchitta, CV, and Blobel, G. "The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation." J Cell Biol 117.1 (April 1992): 15-25.
PMID
1313437
Source
pubmed
Published In
The Journal of Cell Biology
Volume
117
Issue
1
Publish Date
1992
Start Page
15
End Page
25

Biochemical fractionation and assembly of the membrane components that mediate nascent chain targeting and translocation.

Fractionation of a microsomal detergent extract with ammonium sulfate allows separation of the signal recognition particle receptor (SR alpha), which is required for targeting of the nascent chain, from other microsomal proteins, such as signal peptidase, whose activity is displayed during subsequent translocation. The reconstituted SR alpha-enriched fraction is functional in assays of precursor targeting and elongation arrest release but lacks translocation activity. This defect can be complemented by addition, prior to reconstitution, of a separate protein subfraction. In addition, protein components necessary for translocation can be reversibly depleted from the complementary fraction, under conditions where precursor targeting is retained, by sulfhydryl-directed chromatography. Thus, precursor binding and translocation can be biochemically uncoupled, indicating that they are sequential reactions mediated by distinct components.

Authors
Nicchitta, CV; Migliaccio, G; Blobel, G
MLA Citation
Nicchitta, CV, Migliaccio, G, and Blobel, G. "Biochemical fractionation and assembly of the membrane components that mediate nascent chain targeting and translocation." Cell 65.4 (May 17, 1991): 587-598.
PMID
1851670
Source
pubmed
Published In
Cell
Volume
65
Issue
4
Publish Date
1991
Start Page
587
End Page
598

Reconstitution of secretory protein translocation from detergent-solubilized rough microsomes.

Authors
Nicchitta, C; Migliaccio, G; Blobel, G
MLA Citation
Nicchitta, C, Migliaccio, G, and Blobel, G. "Reconstitution of secretory protein translocation from detergent-solubilized rough microsomes." Methods in cell biology 34 (1991): 263-285.
PMID
1943804
Source
scival
Published In
Methods in cell biology
Volume
34
Publish Date
1991
Start Page
263
End Page
285

Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins.

Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane. The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent. The protein composition of the reconstituted vesicles differed markedly from that of native vesicles. Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation.

Authors
Watanabe, M; Nicchitta, CV; Blobel, G
MLA Citation
Watanabe, M, Nicchitta, CV, and Blobel, G. "Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins." Proc Natl Acad Sci U S A 87.5 (March 1990): 1960-1964.
PMID
2408048
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
5
Publish Date
1990
Start Page
1960
End Page
1964

Assembly of translocation-competent proteoliposomes from detergent-solubilized rough microsomes.

Canine pancreas rough microsomes were solubilized in a high salt buffer containing sodium cholate, a detergent extract prepared by high speed centrifugation, and vesicles were reconstituted from the extract by a detergent dialysis procedure. The reconstituted vesicles, lacking resident lumenal proteins, translocated in vitro-synthesized preprolactin in a cotranslational, SRP-dependent manner. The translocated precursor was processed to mature prolactin and protected from digestion by exogenous protease. Vesicles were also reconstituted from detergent extracts depleted of glycoproteins by chromatography on concanavalin-Sepharose. The depleted vesicles, containing a full complement of SRP receptor but deficient in the glycoprotein subunit of signal peptidase, the ribophorins, and other glycoprotein components, were functional in the targeting and binding of nascent preprolactin but exhibited a greatly diminished capacity for translocation.

Authors
Nicchitta, CV; Blobel, G
MLA Citation
Nicchitta, CV, and Blobel, G. "Assembly of translocation-competent proteoliposomes from detergent-solubilized rough microsomes." Cell 60.2 (January 26, 1990): 259-269.
PMID
2297788
Source
pubmed
Published In
Cell
Volume
60
Issue
2
Publish Date
1990
Start Page
259
End Page
269

Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation.

We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.

Authors
Nicchitta, CV; Blobel, G
MLA Citation
Nicchitta, CV, and Blobel, G. "Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation." J Cell Biol 108.3 (March 1989): 789-795.
PMID
2537834
Source
pubmed
Published In
The Journal of Cell Biology
Volume
108
Issue
3
Publish Date
1989
Start Page
789
End Page
795

Characteristics of GTP-mediated microsomal Ca2+ release.

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.

Authors
Joseph, SK; Rice, HL; Nicchitta, CV
MLA Citation
Joseph, SK, Rice, HL, and Nicchitta, CV. "Characteristics of GTP-mediated microsomal Ca2+ release." Biochim Biophys Acta 945.2 (November 22, 1988): 185-194.
PMID
3056523
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
945
Issue
2
Publish Date
1988
Start Page
185
End Page
194

GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability.

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5'-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5'-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.

Authors
Nicchitta, CV; Joseph, SK; Williamson, JR
MLA Citation
Nicchitta, CV, Joseph, SK, and Williamson, JR. "GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability." Biochem J 248.3 (December 15, 1987): 741-747.
PMID
2829838
Source
pubmed
Published In
The Biochemical journal
Volume
248
Issue
3
Publish Date
1987
Start Page
741
End Page
747

Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP-dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high-affinity (K(m) = 0.9 μM) GTPase. The effect of PEG reflect an increase in the V(max) of this activity; no effects on K(m) were observed. The concentration dependence for PEG-dependent stimulation of the high-affinity GTPase exactly mimicked that for GTP-dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP-dependent Ca2+ release, the microsomal PEG-stimulated GTPase was competitively inhibited by the GTP analog GTPγS (K(i) = 60 nM). It is proposed that the PEG-stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+ fluxes.

Authors
Nicchitta, CV; Joseph, SK; Williamson, JR
MLA Citation
Nicchitta, CV, Joseph, SK, and Williamson, JR. "Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release." Journal of Pharmacology and Experimental Therapeutics 240.1 (1987): 243-248.
Source
scival
Published In
Journal of Pharmacology and Experimental Therapeutics
Volume
240
Issue
1
Publish Date
1987
Start Page
243
End Page
248

Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release.

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP-dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high-affinity (Km = 0.9 microM) GTPase. The effects of PEG reflect an increase in the Vmax of this activity; no effects on Km were observed. The concentration dependence for PEG-dependent stimulation of the high-affinity GTPase exactly mimicked that for GTP-dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP-dependent Ca2+ release, the microsomal PEG-stimulated GTPase was competitively inhibited by the GTP analog GTP gamma S (Ki = 60 nM). It is proposed that the PEG-stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+ fluxes.

Authors
Nicchitta, CV; Joseph, SK; Williamson, JR
MLA Citation
Nicchitta, CV, Joseph, SK, and Williamson, JR. "Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release." FEBS Lett 209.2 (December 15, 1986): 243-248.
PMID
3025017
Source
pubmed
Published In
FEBS Letters
Volume
209
Issue
2
Publish Date
1986
Start Page
243
End Page
248

Cyclosporine augments receptor-mediated cellular Ca2+ fluxes in isolated hepatocytes.

The immunosuppressive agent, cyclosporine, has been found to augment receptor-stimulated calcium fluxes in isolated hepatocytes. After treatment of Quin 2-loaded hepatocytes with cyclosporine, both the amplitude and duration of the vasopressin-induced rise in the cytosolic free Ca2+ are increased. These effects are dependent upon the concentration and time of exposure of the cells to cyclosporine. Cyclosporine increases both 45Ca2+ influx across the plasma membrane and the cellular calcium content. The total cellular magnesium, sodium, and potassium contents are not affected by cyclosporine. However, cyclosporine treatment, per se, has no apparent effect on the cytosolic free Ca2+ concentration as assayed by Quin 2 fluorescence. The increase in total cell calcium is associated with progressive increases in the calcium content of the endoplasmic reticular and mitochondrial calcium pools. The vasopressin-induced net efflux of Ca2+ from hepatocytes was 2-fold greater after treatment with 10 micrograms/ml cyclosporine for 10 min, but the lag time prior to the onset of Ca2+ efflux was not affected. These results are interpreted on the basis of cyclosporine having a primary effect on increasing the permeability of the plasma membrane to Ca2+, thereby leading to an increase of the calcium content of the hormone-sensitive intracellular calcium pool.

Authors
Nicchitta, CV; Kamoun, M; Williamson, JR
MLA Citation
Nicchitta, CV, Kamoun, M, and Williamson, JR. "Cyclosporine augments receptor-mediated cellular Ca2+ fluxes in isolated hepatocytes." J Biol Chem 260.25 (November 5, 1985): 13613-13618.
PMID
3877052
Source
pubmed
Published In
The Journal of biological chemistry
Volume
260
Issue
25
Publish Date
1985
Start Page
13613
End Page
13618

Spermine. A regulator of mitochondrial calcium cycling.

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.

Authors
Nicchitta, CV; Williamson, JR
MLA Citation
Nicchitta, CV, and Williamson, JR. "Spermine. A regulator of mitochondrial calcium cycling." J Biol Chem 259.21 (November 10, 1984): 12978-12983.
PMID
6238031
Source
pubmed
Published In
The Journal of biological chemistry
Volume
259
Issue
21
Publish Date
1984
Start Page
12978
End Page
12983

High-performance liquid chromatographic separation and quantification of alanopine and strombine in crude tissue extracts.

A method for the separation and quantification of the levels of alanopine and strombine in neutralized, perchloric acid extracts of tissues of marine invertebrates is presented. The method is based on high-performance liquid chromatographic (HPLC) separation, postcolumn derivatization using o-phthaldialdehyde and sodium hypochlorite, and subsequent fluorometric detection. Isocratic separation results in the rapid elution of alanopine and strombine, with elution times of 4.7 and 5.4 min, respectively. The sensitivity of this method is in the range 50-250 pmol. However, the fluorometric detection approach provides the capability for even greater sensitivity.

Authors
Fiore, GB; Nicchitta, CV; Ellington, WR
MLA Citation
Fiore, GB, Nicchitta, CV, and Ellington, WR. "High-performance liquid chromatographic separation and quantification of alanopine and strombine in crude tissue extracts." Anal Biochem 139.2 (June 1984): 413-417.
PMID
6206747
Source
pubmed
Published In
Analytical Biochemistry
Volume
139
Issue
2
Publish Date
1984
Start Page
413
End Page
417

Effects of spermine on Ca2+-cycling in rat liver mitochondria

Authors
Nicchitta, CV; Williamson, JR
MLA Citation
Nicchitta, CV, and Williamson, JR. "Effects of spermine on Ca2+-cycling in rat liver mitochondria." Federation Proceedings 43.6 (1984): No.-925.
Source
scival
Published In
Federation Proceedings
Volume
43
Issue
6
Publish Date
1984
Start Page
No.
End Page
925

PARTIAL-PURIFICATION AND CHARACTERIZATION OF A STROMBINE DEHYDROGENASE FROM THE ADDUCTOR MUSCLE OF THE MUSSEL MODIOLUS-SQUAMOSUS

Authors
NICCHITTA, CV; ELLINGTON, WR
MLA Citation
NICCHITTA, CV, and ELLINGTON, WR. "PARTIAL-PURIFICATION AND CHARACTERIZATION OF A STROMBINE DEHYDROGENASE FROM THE ADDUCTOR MUSCLE OF THE MUSSEL MODIOLUS-SQUAMOSUS." COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 77.1 (1984): 233-236.
Source
wos-lite
Published In
Comparative Biochemistry and Physiology -- Part B: Biochemistry and
Volume
77
Issue
1
Publish Date
1984
Start Page
233
End Page
236
DOI
10.1016/0305-0491(84)90249-9

Re-evaluating the role of heat-shock protein-peptide interactions in tumour immunity.

Early investigations into the immune surveillance of chemically-induced sarcomas led to two important concepts in tumour immunobiology: one, tumour rejection can be elicited by immune recognition of tumour antigens; and two, tumours express unique sets of antigens, which are known as tumour-specific antigens. The pioneering studies of Srivastava and colleagues led to the proposal that heat-shock proteins (HSPs) function as ubiquitous tumour-specific antigens, with the specificity residing in a population of bound peptides that identify the tissue of origin of the HSP. However, recent findings, including new data on the cell biology of peptide generation and trafficking, have called into question the specificity of tumour rejection that is induced by HSPs.

Authors
Nicchitta, CV
MLA Citation
Nicchitta, CV. "Re-evaluating the role of heat-shock protein-peptide interactions in tumour immunity." Nat Rev Immunol 3.5: 427-432. (Review)
PMID
12766764
Source
pubmed
Published In
Nature Reviews Immunology
Volume
3
Issue
5
Start Page
427
End Page
432
DOI
10.1038/nri1089
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