Our research program has attempted to unravel the complex regulatory processes which contribute to the regulation of cellular protein levels. As an example of this general problem, our focus has been, and will continue to be, on the key enzyme in the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme-A reductase.
Our Specific Aims for the proposed grant period are sharply focused on the degradation of HMG-CoA reductase which is a major mechanism regulating the level of reductase in the cell. This work promises fundamental insights into a novel problem in cell biochemistry, protein degradation in the endoplasmic reticulum, for which we have an excellent system for study. We will extend and refine our analysis of mutants in the membrane domain of HMG-CoA reductase in order to more precisely define the subregions or residues essential for degradation. For these experiments, we will continue to use as a model, the fusion protein, HMGal, which contains the membrane domain of HMG-CoA reductase fused to beta-galactosidase and responds to regulatory signals like native reductase. We will initiate an analysis of the biochemical components involved in the regulated degradation of reductase with particular attention to the protease(s). We plan to purify ALLN sensitive, ER protease(s). We have shown previously that the protease inhibitor peptide ALLN inhibits reductase degradation in vivo. We now have ALLN resistant cells with accelerated reductase degradation and elevated protease levels and these cells will be used as starting material for purification. We will characterize mutants of CHO cells which are altered in their ability to degrade reductase. We have successfully used a selection based on the fusion protein, HMhyg, which consists of the membrane domain of reductase fused to hygromycin phosphotransferase which confers hygromycin resistance upon cells in the absence of mevalonate but not in the presence. We have isolated two mutants which exhibit unregulated, rapid degradation of HMhyg, HMGal and HMG-CoA reductase. The genetic characterization of these mutants will lead to a biochemical understanding of the degradation process. We will also exploit a newly developed in vitro system for studying the degradation of reductase which uses permeabilized cells and exhibits physiologically relevant in vitro degradation. This system will be critical in directly defining the cellular location and biochemical components involved in the process.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL026502-13
Application #
2216008
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1980-12-01
Project End
1998-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
13
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Moriyama, T; Wada, M; Urade, R et al. (2001) 3-hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum. Arch Biochem Biophys 386:205-12
Cheng, H H; Xu, L; Kumagai, H et al. (1999) Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase. J Biol Chem 274:17171-8
Moriyama, T; Sather, S K; McGee, T P et al. (1998) Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease. J Biol Chem 273:22037-43
Meigs, T E; Sherwood, S W; Simoni, R D (1995) Farnesyl acetate, a derivative of an isoprenoid of the mevalonate pathway, inhibits DNA replication in hamster and human cells. Exp Cell Res 219:461-70
Kumagai, H; Chun, K T; Simoni, R D (1995) Molecular dissection of the role of the membrane domain in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. J Biol Chem 270:19107-13
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Sherwood, S W; Kung, A L; Roitelman, J et al. (1993) In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal. Proc Natl Acad Sci U S A 90:3353-7
Inoue, S; Sharma, R C; Schimke, R T et al. (1993) Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase. J Biol Chem 268:5894-8
Roitelman, J; Olender, E H; Bar-Nun, S et al. (1992) Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum. J Cell Biol 117:959-73
Inoue, S; Simoni, R D (1992) 3-Hydroxy-3-methylglutaryl-coenzyme A reductase and T cell receptor alpha subunit are differentially degraded in the endoplasmic reticulum. J Biol Chem 267:9080-6

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