Nearly all aspects of cell growth and metabolism rely upon precise control of protein levels. This balance is not achieved solely through the regulation of protein synthesis, but depends as well upon the rates of protein degradation. Metabolic shifts, environmental stress, and cellular differentiation all are accompanied bby selective proteolysis as the cell adapts to its new condition. To accomplish this, the machinery for intracellular proteolysis must possess both specificity and great flexibility. In eukaryotes, one pathway of intracellular proteolysis involves a 76 amino acid polypeptide, ubiquitin (Ub), which is attached covalently to proteins prior to their degradation. Two questions concerning this system will be addressed: what features of the target protein make it a substrate for ubiquitination, and what features of the resultant conjugate are recognized for subsequent proteolysis. A series of native and modified forms of cytochrome c's and bovine pancreatic trypsin inhibitor will be used as substrates to examine: 1) selectivity among a well-defined set of target proteins that represent folded, misfolded and unfolded conformations; 2) characteristics of ubiquitination sites -- sequence, tertiary structure, and flexibility; 3) involvement of the substrate N-terminus in target recognition; 4) the apparent cooperativity or processivity of ubiquitination. The structures of free Ub and various Ub-amide and Ub-protein conjugates will be compared by one- and two-dimensional 1H nuclear magnetic resonance spectroscopy. Ubiquitinated degradation intermediates of apo-cytochrome c will be sought in yeast. Enzymes of the yeast Ub pathway will be purified and an in vitro ubiquitination system established. Ub conjugation to various mutant apo-cytochrome c's in vitro will be compared with their in vivo turnover rates.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037666-05
Application #
3293166
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-12-01
Project End
1992-09-29
Budget Start
1990-12-01
Budget End
1992-09-29
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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