1985 — 1988 |
Horwich, Arthur L |
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. |
Expression of Cdna For Human Ornithine Transcarbamylase
Ornithine transcarbamylase (OTC) is a mitochondrial enzyme of the urea cycle, encoded on the X chromosome, whose inherited deficiency leads to severe, often lethal, ammonia intoxication in affected humans. The proposed studies are directed 1) to understanding the system of mitochondrial compartmentation, whereby mitochondrial protein precursors, encoded in the nucleus and synthesized on cytoplasmic polyribosomes, are posttranslationally recognized by mitochondria, translocated across one or both membranes, and proteolytically processed to their active forms; and 2) to developing gene therapy for hepatic enzyme deficiencies. In both areas of investigation the experimental approaches taken will involve manipulation of the cloned biologically active cDNA sequence encoding human OTC. The studies of mitochondrial import are aimed at: definition of the sequences present in the cytoplasmically-synthesized precursor of OTC that are required for its import; study of putative cytoplasmic factors and outer membrane receptor molecules involved with recognition of the OTC precursor by mitochondria; and clarification of whether one or two NH2-terminal proteolytic processing steps are required to produce the mature OTC subunit. The coding sequence of the NH2-terminal leader portion, programmed for expression in eukaryotic cells, will be joined with that of a cytoplasmic enzyme to determine whether the leader alone is sufficient to direct mitochondrial import. The coding sequence of the entire OTC precursor, programmed for expression, will be subjected to deletion and point mutational alteration, and the effects upon import measured. The precursor coding sequence will also be programmed for high level expression in both eukaryotic and prokaryotic cells, enabling isolation of the precursor for use as a substrate in binding and proteolysis studies. Gene replacement experiments will aim to provide sufficient additional hepatic OTC activity, expressed from an introduced sequence, to permit correction of deficient nitrogen catabolism in OTC-deficient mice. The OTC cDNA sequence will be programmed for expression in a cloned retroviral genome that will be packaged into retroviral particles. The particles will be used to infect OTC-deficient mouse embryos and the liver of these animals analyzed for presence of the introduced sequence, and for enzymatically active human OTC.
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1 |
1988 — 1989 |
Horwich, Arthur L |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Genetics and Medical Sciences |
1 |
1988 — 1992 |
Horwich, Arthur L |
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. |
Targeting of Human Otc to the Mitochondrial Matrix
The proposed studies are directed to understanding the system of mitochondrial compartmentation, whereby mitochondrial protein precursors, encoded in the nucleus and synthesized on cytoplasmic polyribosomes, are posttranslationally recognized via their NH2- terminal leader peptides, translocated across one or both membranes, and proteolytically processed to their active forms. We will continue to use as a model for analysis of this system the human mitochondrial matrix enzyme ornithine transcarbamylase (OTC), which catalyzes the second step of the urea cycle in mammals. The studies proposed here are aimed at: identification and characterization of components of the mitochondria of both Saccharomyces cerevisiae and mammalian cells that are involved with specific recognition, import to the matrix compartment, and proteolytic processing of the OTC precursor; determination of whether such components are shared by other proteins destined for mitochondria; and analysis of the higher-order structure of the OTC leader peptide. In both yeast and mammalian cells, genetic approaches will be taken to isolating genes encoding import components: suppressing mutations will be isolated in both Saccharomyces and HeLa cells that permit mutant OTC precursors to reach the mitochondria; mutations that block import/processing of the wild-type OTC precursor and result in a conditional growth-deficient state will also be isolated in Saccharomyces. Biochemical approaches will be taken to studying components involved with import. The wild-type OTC precursor will be overproduced in E. coli by mutagenizing a plasmid programming production of a fusion protein joining the wild-type OTC leader peptide with galactokinase, and assaying for high- level expression of enzyme activity by a colony color assay. The overproduced precursor will be purified and used in a variety of studies, including mitochondrial binding studies designed to examine kinetics of recognition by the organelles and competition for recognition with other precursors; crosslinking reactions designed to identify specific components that interact with the OTC precursor; and structural studies designed to analyze the higher-order structure of the leader peptide. In vitro synthesized precursors with either reactive amino acid side chains or enzymatically active mature portions will also be used as affinity- labeling reagents, to identify additional interacting mitochondrial components.
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1 |
1990 — 1992 |
Horwich, Arthur L |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Genetics--Medical Genetics |
1 |
1993 — 1996 |
Horwich, Arthur L |
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 Groel in the Bacterial Cytoplasm
The proposed studies are aimed at defining the function of the homooligomeric double ring 14mer complex, groEL, in folding and assembly of newly-translated proteins in the bacterial cytoplasm. During the current project period we have identified an apparently global role for a homologue in the mitochondrial matrix, hsp60 complex, in mediating folding and assembly of proteins newly-imported into the organelles. We now wish to assess whether groEL could have a similar general role for assisting newly-translated proteins to reach their native forms. such a possibility is suggested by observations: that groEL is essential for cell viability at all temperatures; that bacteriophage assembly is impaired in several nonlethal groEL mutants; and that groEL and its cooperating component groES can mediate refolding in vitro of several polypeptides diluted from denaturant. We will assess the role of groEL in vivo by producing temperature-sensitive or cold-sensitive lethal mutants. Our approach will involve direct homologous insertion of in vitro-mutagenized groEL sequences into the E. coli chromosome. Mutagenesis approaches will include directed codon changes, doped oligonucleotide mutagenesis, and alanine scanning mutagenesis. After shift to nonpermissive temperature, we will compare wild-type and conditional lethal mutant cells for: fractionation behavior of groEL complexes; rat of protein translation and pattern of translation products; half-life and solubility of the newly-translated proteins; translation and acquisition of enzyme activity of several proteins specifically induced at nonpermissive temperature, including beta- galactosidase and galactokinase. groEL complexes from both conditional lethal mutants and slow-growing nonlethal mutants will be overproduced and purified, to allow functional studies in vitro to distinguish specific defects, enabling ultimately, once a groEL crystal structure is determined, structure-function understandings. We will also examine where polypeptides are bound at groEL, whether at its outer surface or within the cavity of the rings, by examination i the scanning transmission electron microscope of undecagold-conjugated dihydrofolate reductase bound to groEL.
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1 |
1993 — 1996 |
Horwich, Arthur L |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Genetics: Medical Genetics |
1 |
1997 — 2000 |
Horwich, Arthur L |
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. |
Groel-Mediated Protein Folding
DESCRIPTION: This proposal, which is a continuation of an earlier funded proposal, is aimed at further defining the role of GroEL and its cochaperonin, GroES, in protein folding in vivo. Unfolded polypeptides form ternary complexes with GroEL/GroEs and bind either trans, i.e., on the opposite side from GroES, or bind cis, i.e., on the same side as GroES within a central chamber formed from GroEL and GroES. Folding may proceed differently at the trans and cis sides. A major goal (Aim 1) is to determine precisely the perecentage and identity of the E. coli proteins which are substrates of GroEL. These studies will exploit rapid onset temperature-sensitive mutants of GroEL to be constructed in the P.I.'s laboratory, to address a variety of questions: Are large proteins (>60 kDa) which should be incapable of binding cis because of their size also substrates of GroEL? if so, do their misfolded forms bind productively in trans, i.e, are they unfolded and released by GroEL for a subsequent round of folding in solution? Are small proteins (<10 kDa), which because of their size potentially can fold more rapidly than they can bind GroEL, also substrates of GroEL/GroES? If so, the P.I. will use such proteins for mechanistic studies by deuterium exchange and NMR spectroscopy to elucidate cis-mediated folding. A second goal (Aim 2) is to observe directly the conformation changes associated with substrate binding to GroEL. Protease and tryptophan fluorescence studies are proposed to characterize conformational changes which accompany the binding of metastable rubisco and mitochondrial malate dehydrogenase (mMDH) forms to GroEL. Stopped-flow pyrene fluorescence studies of pyrene-labeled rubisco and dihydrofolate reductase (DHFR) are also proposed. The latter studies would permit quantitative comparisons of unfolding kinetics (monitored by fluorescence changes) with binding kinetics (monitored by fluorescence anisotropy measurements) and help establish whether "native-like" intermediates need to first unfold in order to bind GroEL or whether they can bind GroEL and then unfold. A third goal (Aim 3) is to determine the fate of substrate molecules starting in a cis ternary complex with GroEL/GroES. Can polypeptides, for example, leave the cis side in a non-native state, or are they released only in the native state with release of non-native forms occuring only from the trans side (as is hypothesized by some current models)? A "cis-only" chaperonin complex constructed in the P.I.'s laboratory, in conjunction with GroEL-mutant "traps" that are capable of binding but not releasing non-native polypeptides, will be used to test these hypotheses.
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1 |
2001 — 2006 |
Horwich, Arthur L |
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. |
Mechanism of Action of the Hsp100 Chaperone Clpa
DESCRIPTION (applicant's description): The objective of the proposed studies is to provide a mechanistic understanding of ATP-dependent unfolding and proteolysis mediated by HSP100 chaperone ring structures in association with cylindrical proteases, resembling the action of 19S "cap" assemblies in directing proteins for degradation by the eukaryotic 20S proteasome cylinder. In particular, we are studying the mechanism by which the bacterial HSP100 chaperone, CIpA, a hexameric ring with two ATP binding domains in each of its 84 kDa subunits, mediates ATP dependent unfolding of protein substrates and commits them to translocation into and degradation by the coaxially bound cognate protease, CIpP, a stacked double ring tetradecamer of identical 23 kDa serine protease subunits. Translocation of several model substrates will be studied using fluorescence dynamics measurements, assessing whether the unfolded proteins are directionally translocated into ClpP. The hypothesis that proteolysis does not commence until substrate translocation is completed will be tested using fusion proteins. The site of substrate binding on ClpA will be determined by EM analysis of gold-tagged substrate proteins bound to CIpAP complexes. The dynamic ATP-directed movements of ClpA in directing unfolding and translocation will also be studied, using rapid-freezing and cryoEM examination of CIpAP complexes in various nucleotide states. Structure-function analyses will be carried out, employing random and site-directed mutants of CIpA, both in vivo and in vitro, to study function, and crystallographic studies of various forms of CIpA to obtain high-resolution structural information.
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1 |
2004 — 2007 |
Horwich, Arthur L |
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. |
Chaperonin-Mediated Protein Folding
[unreadable] DESCRIPTION (provided by applicant): This proposal seeks to support an ongoing Iongterm project concerning the mechanism of action of the essential megadalton-sized double ring molecular machines known as chaperonins, which mediate ATPdependent protein folding to the native state in a variety of cellular compartments. Here, we propose studies of the bacterial chaperonin, GroEL, and its cooperating co-chaperonin, GroES, both in vivo and in vitro. One study in vivo will aim to resolve the physiological action of GroEL, not well-understood due to lack of a tight conditional mutant, by identifying a chemical compound that binds to the GroEL ATP binding pocket and produces immediate-onset loss of function in vivo. We will examine whether the protein translation machinery is halted by such arrest of the GroEL folding machine, and will inspect for misfolding/aggregation both of nascent polypeptides, if they are produced, and of pre-existent proteins. This will allow us to observe the immediate physiologic consequences of disruption of the chaperonin system and should identify its authentic and essential substrates. In a second area, mechanistic studies will be carried out in vitro, following on our earlier studies of the machine itself, directing analysis to the polypeptide substrate. We will analyze the global topology of substrate proteins while bound to GroEL, using oxidative crosslinking between native and engineered cysteines in the substrates themselves, addressing whether the topology is unique and potentially native-like, random, or a limited ensemble of states. We will also address whether bound polypeptides occupy a characteristic topology relative to GroEL, with a particular portion bound inside the central cavity of a GroEL ring while the remainder is localized outside in the bulk solution, using external labeling of the exposed portion of substrate protein and single particle imaging with cryoEM. We will also address actions of nucleotide/GroES binding on GroEL-substrate binary complexes. One study will use cryoEM to examine complexes formed by addition of ADP/GroES to GroEL-rhodanese, in which apical domain movement is severely retarded, potentially providing a structure of a GroEL-GroES collision complex. In a second study, the question of whether ATP/GroES binding to GroEL-substrate complexes produces an active, forced, unfolding of substrate protein prior to its release into the central cavity will be studied using tritium and hydrogen/deuterium exchange experiments. [unreadable] [unreadable]
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1 |
2010 |
Horwich, Arthur L |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Progressive Aggregation Despite Chaperone Association @ University of Washington
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.
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0.97 |