COTRANSLATIONAL PROCESSING AND PROTEIN TURNOVER

共翻译加工和蛋白质周转

• 批准号: 2138822
• 负责人: RALPH A BRADSHAW
• 金额: $ 22.91万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138822
• 关键词: acetyl coA acetyltransferase; acetylation; aminohydrolases; aminopeptidase; animal genetic material tag; animal tissue; arginine; carbon nitrogen ligase; cell free system; enzyme mechanism; enzyme structure; enzyme substrate; genetic library; hypoxanthine phosphoribosyltransferase; immunoprecipitation; methionine; molecular cloning; nucleic acid sequence; polymerase chain reaction; polysomes; posttranslational modifications; protein degradation; protein purification; protein reconstitution; protein sequence; site directed mutagenesis; tissue /cell culture; ubiquitin

The co-translational processing of eukaryotic proteins generally produces four main classes of proteins: those with and without initiator Met and those with and without N-alpha-acetylation. Methionine aminopeptidase (MAP) and N-alpha-acetyltransferase (NAT), enzymes associated with the ribosomes, apparently affect these modifications. Importantly, the structures generated apparently dictate the long-term stability of many intracellular proteins in eukaryotes and can direct the turnover of these proteins via the ubiquitin-based degradation system. Two main lines of experimentation are proposed to further clarify these relationships. In the first, porcine liver NAT and MAP, which act co-translationally, and protein N- terminal, asparagine deamidase, which acts post-translationally, will be examined with respect to structure, properties and specificity. Full-length cDNA sequences will be obtained as will precipitating antibodies directed against each of the 3 enzymes. The combined effect of these agents, either in sub-groups or en bloc, is to provide (presumably well regulated) access of selected intracellular proteins to the degradation machinery. The second part of the proposal deals with the function of these enzymes in protein degradation using rabbit reticulocyte lysate. Lysate will be used to express transcripts (both native and altered) to generate appropriate proteins and to induce degradation by the ubiquitin-dependent pathway. Participants in the modification and turnover pathways will be manipulated (or neutralized) with antisera and/or inhibitors, to determine their role in the process. In the whole cell experiments, transcripts will be generated in situ from appropriately tailored plasmids and the degradation of the resulting proteins monitored. Two proteins (asparagine synthetase and hypoxanthine phosphoribosyl transferase) will be used as the principal substrates in these studies. Appropriate nucleic acid and immunological reagents are either in-hand or will be generated.

真核蛋白的共翻译加工一般产生