1987 — 2000 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Biology of Mammalian Anion Transport
Physiological evidence supports the evidence of a wide variety of anion transporters which play essential roles in pH and ionic homeostasis in all animal cells. We have identified a family of genes related to the well characterized anion transporter of the erythrocyte, band 3. Preliminary data show that transcripts of these genes are detectable in all mammalian cells examined. The research proposed herein is concerned with the cloning of these genes and the physiological characterization of their products. We will clone cDNAs encoding these putative anion transporters from tissue-specific cell lines and determine their complete nucleotide sequences, from which the amino acid sequences will be deduced. Antibodies against defined regions of the polypeptides encoded by these genes will be raised in rabbits, and will be used to determine their tissue and cell-type specificity as well as to help define functionally important domains. Expression of the clones in Xenopus oocytes or transfected mammalian cell lines will allow us to determine their ion selectivity, pH dependence and pharmacological properties. These studies will permit, for the first time, the assignment of previously described anion transport activities to specific proteins and the elucidation of their roles in the tissues where they are normally expressed. Finally, we will continue to apply new molecular biological approaches to the identification and cloning of a broad class of genes which are likely to mediate the transport of ions across cellular membranes.
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0.958 |
1989 — 1994 |
Kopito, Ron |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Presidential Young Investigator Award
This Presidential Young Investigator award is to support research into the molecular biology of membrane proteins in the department of Biological Sciences at Stanford University. The specific class of membrane proteins under investigation are the anion transporters, which catalyze the exchange of chloride and bicarbonate across the cell membrane and are also a site of primary attachment of the cytoskeleton of the cell to the membrane. The ultimate aim is to determine the structure- function relationships among several related anion transporters and to determine their physiological function, for instance, do they regulate intracellular pH. This research has fundamental importance to our understanding of the role of the plasma membrane and its component parts on cell function. The relationship of membrane structure and function will be investigated using molecular biologic techniques.
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1 |
1991 — 1998 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular and Cellular Biology of the Cftr |
0.958 |
1997 — 2000 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Function of Nucleotide Binding Domains in Cftr
DESCRIPTION: Cystic fibrosis is the most common lethal hereditary disease in the US and is caused by mutations in the gene encoding the CFTR chloride channel. The studies proposed herein are designed to further understanding of the molecular basis of CFTR Cl-channel function and the mechanism by which mutations that cause CF lead to channel dysfunction. CFTR activity is controlled by direct interaction of ATP with the nucleotide binding folds (NBFs) on the CFTR polypeptide, but the mechanism by which this interaction leads to channel gating is not understood. The principal aim of this research is therefore to elucidate the role of nucleotide binding and hydrolysis in gating CFTR. Five specific aims are proposed. They are: (1) to define the roles of NBF1 and NBF2; (2) to define the roles of endogenous sulfhydryls in controlling the activities of the NBFs, and the coupling thereof to channel gating; (3) to obtain biochemically pure, native, reconstituted CFTR suitable for biochemical investigations; (4) to determine the affinity and rate constants for ATP binding to and hydrolysis by purified native CFTR and to correlate ATP binding and hydrolysis kinetics with biophysical properties controlling channel gating and (5) to determine the mechanism by which CF-causing mutations, particularly those mapping to NBFs and R domain affect CFTR gating.
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0.958 |
1999 — 2003 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Folding and Degradation of Membrane Proteins in the Er.
Quality control pathways monitor the folding and assembly of newly synthesized secretory and membrane proteins, retaining some abnormal proteins in the endoplasmic reticulum and diverting others for degradation by cytoplasmic proteasomes. The operation of these quality control pathways underlies the cellular basis of human genetic diseases like cystic fibrosis that arise from mutations which alter the normal folding and assembly of integral membrane proteins. How the decision to degrade a misfolded or unassembled membrane protein is made, and the nature of the cellular machinery which recognizes and dislocates these proteins to cytoplasmic proteasomes for degradation is not known. Elucidating these processes is the long-term objective of the proposed research. The specific aims constitute a comprehensive approach toward these goals by addressing four questions: (1) What is the molecular basis for CFTR misfolding? (2) How do cis-degradation signals determine the fate of integral membrane proteins? (3) What is the nature of the membrane apparatus through which integral membrane proteins are dislocated from the ER? (4) What is the role of cytoplasmic factors in dislocation of integral membrane proteins from the endoplasmic reticulum?
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0.958 |
2002 — 2019 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Protein Aggregation and Inclusion Body Formation
DESCRIPTION (provided by applicant): Accumulation of protein aggregates and ubiquitin (Ub) into cytoplasmic inclusion bodies (IB) is the single most definitive diagnostic neuropathological marker of neurodegenerative disease. Despite this universal diagnostic significance, the cell biological mechanisms underlying IB formation and, indeed, whether formation of these structures reflects a pathogenic or protective process, remains an unresolved mystery. The long-term objective of this research project is to elucidate the molecular mechanisms that underlie IB formation and Ub deposition and to develop an integrated understanding of how mammalian cells respond to the chronic expression of pathogenic, aggregation-prone proteins. The research supported by this project during the previous funding period exploited single-cell analysis of a cellular model of Huntington's disease (HD) to show that, although mature IB contain both aggregated huntingtin (htt) and Ub, the two proteins are recruited to IB with vastly different kinetics. These observations, together with emerging data from genetic models of neurodegenerative disease, suggest a model in which chronic expression of a folding-defective aggregation-prone protein like htt, burdens the cell's proteostasis capacity (ie, the cell's ability to maintain the correct dynamic equilibrium between protein folding and degradation) leading to a progressive diversion of normal proteins from productive folding to the ubiquitin proteasome system, ultimately overwhelming the cell's capacity to degrade proteins. The research in this proposal aims to rigorously testand refine this emerging model using state-of-the-art proteomic and bioimaging technologies. These studies will provide a comprehensive understanding of the dynamic interaction between protein aggregation and proteostasis and will illuminate one of the longest-standing controversies in neurodegenerative disease biology.
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0.958 |
2004 — 2005 |
Kopito, Ron R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A High Throughput Assay For Pharmacological Chaperones
DESCRIPTION (provided by applicant): Many inherited diseases, including cystic fibrosis, diabetes, and familial hypercholesterolemia are caused by mutations that impair the folding and intracellular trafficking of ion channels, transporters and receptors that are normally expressed at the plasma membrane. There is compelling evidence demonstrating that the mutant phenotype of many of these mutants can be suppressed by treatment with pharmacological chaperones, small high affinity ligands that bind to and stabilize the native 3-dimensional structure of their respective targets. To develop pharmacological chaperones with therapeutic potential, it is necessary to identify small high affinity ligands that can stabilize the native state for proteins without known high affinity ligands. The exploratory project described here is aimed at the development of a general, robust cell based assay that can be used in highthroughput screening platforms to identify novel pharmacological chaperones based on their ability to increase the efficiency of protein folding. This homogenous assay exploits a highly sensitive enzymatic complementation between a small peptide (S-peptide) and RNAseA and the use of a fluorogenic substrate. It is proposed to demonstrate the feasibility of this assay with two model substrate, the G-protein coupled V2 vasopressin receptor, mutations in which are linked to nephrogenic diabetes insipidus and the cystic fibrosis transmembrane conductance regulator.
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0.958 |
2005 — 2006 |
Kopito, Ron R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Ubiquitin Deficiency in Aging and Neurodegeneration
DESCRIPTION (provided by applicant): The ubiquitin proteasome system (UPS) is the principal mechanism for degrading unwanted proteins in eukaryotic cells. The UPS contributes an important role in protecting cells against stress resulting from environmental damage to proteins and against the potentially deleterious consequences of the production of error-containing polypeptides. A decline in activity of specific elements of the UPS has been reported to occur during normal aging and in the pathogenesis of many late-onset neurodegenerative diseases. Despite tantalizing genetic evidence linking neurodegenerative diseases like Parkinson's to specific lesions in components of the UPS, the role of this proteolytic system in maintaining protein homeostasis and resisting stress in intact animals has never been studied. The proposed exploratory project aims to create mouse lines that are defective in the ability to induce expression of ubiquitin, a key step in the UPS, in response to stress. Mice harboring deletions of the two polyubiquitin genes, UbB and UbC will be bred and intercrossed to produce lines containing progressively severe ubiquitin deficiencies. These ubiquitin-deficient mice will be closely monitored for life-span, as well as developmental, neurological, behavioral and growth phenotypes. The effect of ubiquitin deficiency on the sensitivity to environmental neurotoxins will be evaluated. Finally these mice will be backcrossed with C57BL/6 mice to generate congenic lines suitable for breeding with established and emerging models of aging and neurodegeneration.
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0.958 |
2006 — 2021 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Ubiquitin Proteasome System in Er Quality Control
DESCRIPTION (provided by applicant): Folding and assembly of proteins synthesized in the endoplasmic reticulum is closely monitored by a quality control apparatus that diverts folding-defective products to the cytosol to be degraded by the ubiquitin-proteasome system by a process known as endoplasmic reticulum-associated degradation (ERAD). The long-term goal of this project is to elucidate the mechanisms by which ERAD recognizes and destroys its targets. In the previous funding period we successfully implemented a large scale proteomic analysis of the mammalian ERAD system that allowed us to create the first comprehensive course-grained map of the mammalian ERAD interactome in mammals. We used these data to identify new ERAD components and protein complexes and to propose a novel hypothesis in which mannose trimming of core N-glycans orchestrates the ordered recognition and delivery of folding defective proteins. The studies proposed in the present application seek to test this hypothesis, to refine the ERAD interaction network and discover new ERAD components not based on orthology to fungi. To this end we will exploit emerging technologies such as gene editing, ultrahigh density shRNA and genetic interaction mapping.
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0.958 |
2007 — 2011 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Cellular Mechanisms of Protein Aggregation and Inclusion Body Formation
DESCRIPTION (provided by applicant): Deposition of aggregates of misfolded protein into intracellular inclusion bodies (IB) is a prominent cytopathological feature of nearly every known neurodegenerative disease. A confluence of recent data has challenged the widespread belief that IB are pathogenic. Studies from conditional animal models of several neurodegenerative diseases including tauopathy, Huntington's disease and spinocerebellar ataxia type 7 reveals that neurons are endowed with the capacity to recover from the toxicity of misfolded mutant proteins, and are able to clear intracellular IB. Together, these findings support a cytoprotective role for IB formation. We have demonstrated that dynein-dependent transport of polyubiquitinated misfolded or aggregated proteins are delivered to cytoplasmic IB via dynein-dependent transport on microtubule tracks. Such dynein-dependent IB are called aggresomes (AG). We previously hypothesized that AG formation can contribute to clearance of toxic misfolded or aggregated proteins by facilitating their degradation in lysosomes by autophagy, and recent studies strongly support a role for autophagy as a cytoprotective mechanism in human disease and animal models thereof. The studies proposed here are focused on elucidating the mechanisms by which potential proteotoxins are recognized and transported to AG for autophagic degradation. To this end, three specific aims are proposed. The first aim will use novel synthetic substrates to assess the roles of polyubiquitination and aggregation as cis-acting signals for targeting to AG in vivo and in cell-free extracts. The second aim will exploit a novel cell-free AG formation assay to purify and identify trans-acting factors that couple AG substrates to cytoplasmic dynein. The third aim will define the structural and functional relationship between AG formation and autophagic degradation of misfolded or aggregated proteins using a novel assay and genetic screen in yeast. PUBLIC HEALTH REVELANCE: The formation of toxic protein aggregates underlies the pathogenesis of most neurodegenerative disorders. Cells are known to possess mechanisms to counteract the formation of and to eliminate these toxic protein species, but the mechanisms by which this occurs is not understood in sufficient detail to develop viable therapeutic strategies. The research in this proposal will use biochemical and genetic means to understand the mechanism by which cells can protect themselves against toxic aggregated proteins.
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0.958 |
2007 — 2009 |
Kopito, Ron R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Ubiquitin Proteasome System in Quality Control
[unreadable] DESCRIPTION (provided by applicant): Production of folding-defective proteins underlies the pathogenesis of many heritable disorders in man, yet the unique features of misfolded proteins and the cellular machinery that recognizes them remain largely unknown. The long-term goal of this project is to identify the cellular factors that couple the recognition of folding-defective proteins in the endoplasmic reticulum to their degradation by a process known as ER-associated degradation (ERAD). Despite considerable progress made in linking the cytoplasmic ubiquitin-proteasome system (UPS) to ERAD, fundamental questions remain unanswered. Foremost amongst these is the nature of the cis and trans signals that identify a protein as an ERAD substrate and the molecular events that divert proteins from the biosynthetic folding pathway to ERAD. Previous work from my lab and many others has firmly established a central role for the UPS in the targeting of misfolded ER proteins for degradation. The immediate goal of this project is thus to identify the cellular machinery by which malfolded proteins in the ER are recognized. We will use a combination of functional genomics and traditional cell biology to identify E3 Ub ligases and other critical elements of the UPS that collaborate in the recognition of ERAD substrates. To this end, three specific aims are proposed. In the first aim we plan to screen libraries of small hairpin interfering RNA (shRNA) directed against all probable UPS components in the human genome. Hits from this screen will be validated by a battery of assays and the interaction of the newly identified components with substrate and other ERAD machinery will be studied in detail. Preliminary data are included identifying Hrd1 as an E3 ligase for degradation of unassembled AMPA-type glutamate receptor subunits, thereby validating the efficacy of our functional genomics screen. Thus, the second aim is to elucidate the cis- and trans- acting factors that promoting Hrd1- dependent degradation of GluR1 subunits. The third aim complements the first by performing a comprehensive biochemical and microarray investigation of how cellular stress response pathways respond to topologically and structurally distinct classes of ERAD substrate. It is anticipated that this analysis will provide new understanding of how cells respond to physiologically relevant protein stress, and will facilitate the identification of new components of that direct specific classes of substrate to degradation by ERAD. [unreadable] [unreadable] [unreadable]
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0.958 |