The overall goal of this project is to understand the role of protein carboxyl methylation in neuronal function. There are two current hypotheses of methylation function in eucaryotes: (1) that it mediates some aspect of stimulus-response coupling via the reversible modification of protein function, and (2) that it facilitates the repair or degradation of defective proteins which contain abnormal forms of aspartic acid. In testing the regulation hypothesis, we propose to measure the ability of certain neurotransmitters and depolarizing agents to stimulate transient protein methylation (as indicated by the evolution of radiolabeled methanol), to search in purified subfractions of brain for specific protein which serve as preferential substrates in an in vitro transient methylation assay, and, to determine if the in vitro transient methylation reaction is regulated by the second messengers cAMP, cGMP, Ca++ or phosphatidyl inositol. Our strategy in these experiments is derived from recent indications that carboxyl methylation may serve as an initial activation step in a more complex protein modification reaction than hitherto assumed. An alternative role for carboxyl methylation in the repair or degradatiom of damaged proteins is suggested by the recently discovered selectivity of the methyltransferase enzyme for proteins containing abnormal forms of aspartate, particularly L-isoaspartyl and D-aspartyl residues. We propose to determine if the methyl accepting substrates we have located in synaptic membranes and myelin are enriched in these atypical forms of aspartate, and, to determine if the levels of these abnormal proteins and/or the methyltransferase enzyme changes significantly with age in the human or in association with Alzheimer's disease. Establishing a firm role for carboxyl methylation should provide important new insight on fundamental mechanisms of cellular regulation and/or aging processes in the mammalian nervous system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Modified Research Career Development Award (K04)
Project #
5K04NS001082-02
Application #
3074923
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1986-03-01
Project End
1991-02-28
Budget Start
1987-03-01
Budget End
1988-02-29
Support Year
2
Fiscal Year
1987
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
Najbauer, J; Johnson, B A; Aswad, D W (1992) Analysis of stable protein methylation in cultured cells. Arch Biochem Biophys 293:85-92
Johnson, B A; Aswad, D W (1991) Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins. Anal Biochem 192:384-91
Johnson, B A; Shirokawa, J M; Geddes, J W et al. (1991) Protein L-isoaspartyl methyltransferase in postmortem brains of aged humans. Neurobiol Aging 12:19-24
Najbauer, J; Johnson, B A; Aswad, D W (1991) Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells. Anal Biochem 197:412-20
Najbauer, J; Aswad, D W (1990) Diversity of methyl acceptor proteins in rat pheochromocytoma (PC12) cells revealed after treatment with adenosine dialdehyde. J Biol Chem 265:12717-21
Johnson, B A; Aswad, D W (1990) Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage. Biochemistry 29:4373-80
Johnson, B A; Shirokawa, J M; Aswad, D W (1989) Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase. Arch Biochem Biophys 268:276-86
Johnson, B A; Shirokawa, J M; Hancock, W S et al. (1989) Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone. J Biol Chem 264:14262-71
Henzel, W J; Stults, J T; Hsu, C A et al. (1989) The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites. J Biol Chem 264:15905-11
Aswad, D W; Johnson, B A; Langmack, E L et al. (1988) Modification of isoaspartyl peptides and proteins by protein carboxyl methyltransferase from bovine brain. Adv Exp Med Biol 231:247-59