Recent findings indicate that formation of isoaspartate (isoAsp) via deamidation of labile Asn-X sequences or direct isomerization at certain Asp-X sequences is a major source of spontaneous protein damage at physiological pH and temperature and that this process may have important consequences for protein function and turnover. This proposal seeks to understand the role of protein sequence and structure on formation of isoaspartate and to explore the effects of isomerization on protein function. Four experimental approaches are proposed. First, we will determine the sites of isoAsp formation in synapsin 1, tissue plasminogen activator, triosephosphate isomerase and the major intrinsic protein of the eye lens. All four of these proteins appear to generate isoAsp at significant rates at physiological pH and temperature. Second, we will synthesize a series homologous Asn- and Asp-containing pentapeptides in which the amino acids neighboring the Asn or Asp are varied in a systematic way. Substitutions will be designed to fill in several critical gaps in our knowledge of how neighboring amino acids influence the propensity of Asn or Asp to isomerize. Knowledge of the sites of isoAsp formation in the above proteins and synthetic peptides will be used to strengthen or refute our current paradigm, which emphasizes the nature of the amino acid linked to the Asn/Asp-carboxyl, together with regional flexibility, to predict sites of isoAsp formation.
Our third aim i s to determine to what extent isoAsp effects several functional attributes of synapsin 1, including its ability to be phosphorylated by the cAMP- and type II calcium/calmodulin-dependent protein kinases, its ability to bind to small synaptic vesicles, and its ability to bind F-actin. Finally, we will investigate the possibility that protein sequences which are prone to forming isoaspartate may also be prone to crosslinking. These studies should provide important new information on basic mechanisms which govern the stability and integrity of proteins in vitro and in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS029421-02
Application #
3416236
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1991-05-01
Project End
1994-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697