COTRANSLATIONAL PROCESSING AND PROTEIN TURNOVER

共翻译加工和蛋白质周转

• 批准号: 2905287
• 负责人: RALPH A BRADSHAW
• 金额: $ 22.57万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2905287
• 关键词: active sites; acyltransferase; aminohydrolases; aminopeptidase; cell growth regulation; chimeric proteins; confocal scanning microscopy; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; fungal genetics; glycosylation; immunoaffinity chromatography; immunoprecipitation; isozymes; matrix assisted laser desorption ionization; methionine; molecular cloning; posttranslational modifications; protein degradation; protein localization; protein sequence; site directed mutagenesis; ubiquitin

DESCRIPTION: (Adapted from the application) N-terminal processing of proteins in eukaryotes occurs both co- and post-translationally and the resulting modifications are involved in translocation, regulation of activity and turnover. The initial reactions involve the removal of the initiator methionine and the addition of N-alpha acetyl groups by methionine aminopeptidases (Met AP) and N-alpha-acetyl transferases (NAT), respectively. Each exists in multiple isoforms that appear to differ in specificity, regulation of cellular location. Using recombinant material, the role of the two types of yeast MetAPs will be determined and the proteins characterized with respect to structural and functional properties, including metal content, substrate specificity and catalytic organization. Site-directed mutagenesis and chemical inhibitors will be extensively used. Cellular localization will also be determined with recombinant fusion proteins and immunological reagents. Recombinant human isoforms will also be prepared and examined for putative glycosylations and the role of the type II enzyme in cell cycle regulation. Similar studies will be carried out with the NATs, with a focus on the subforms specific for N-terminal Met (produced by penultimate Asp, Glu and Asn-the "DEN" subset). In parallel to the NATs that modify the Gly, Ala, Ser, and Thr (GAST) group, the M-NATs exist as a pair of proteins that may functions as a heterodimer complex. This hypothesis will be tested as a part of the structural characterizations. Two enzymes putatively involved in downstream processing of DEN proteins that will also be investigated. In the first case, yeast will be screened (and enzyme isolated if detected) for an acyl amino acid hydrolase, specific for acetyl Met groups, that could destabilize DEN proteins for degradation by the N-end Rule. In the second, human protein N-terminal asparagine amidohydrolase, which converts N-terminal Asn residues to Asp (the yeast form also acts on Gln), will be isolated and further characterized. Finally, yeast null strains, deficient in several of these (and other) co-/post- translationally active enzymes, will be used to confirm their putative physiological roles and to identify proteins degraded in an N-terminal specific manner.

描述：（根据应用程序进行了改编）N末端处理 真核生物中的蛋白质在跨化和后均发生，以及 由此产生的修改涉及易位，调节 活动和营业额。初始反应涉及去除 引发剂蛋氨酸和通过 蛋氨酸氨基肽酶（MET AP）和N-α-乙酰基转移酶（NAT），，， 分别。每种都存在于多种同工型中，似乎有所不同 特异性，细胞位置的调节。使用重组材料， 将确定两种类型的酵母Metaps的作用，并 相对于结构和功能的蛋白质 特性，包括金属含量，底物特异性和催化性 组织。定点诱变和化学抑制剂将是 广泛使用。细胞定位也将通过 重组融合蛋白和免疫学试剂。重组人 同工型还将进行准备并检查假定的糖基化 以及II型酶在细胞周期调节中的作用。相似的 研究将与NAT一起进行，重点 特定于N端MET（由倒数第二ASP，GLU和ASN-THE-THE-THE-THE-TEM “ den”子集）。与修改Gly，Ala，Ser和 thr（gast）组，M-NAT作为一对蛋白质存在 充当异二聚体复合物。该假设将被检验为 结构特征的一部分。涉及两种酶 在下游加工的DEN蛋白中，也将进行研究。 在第一种情况下，将筛选酵母（如果 检测到的）对于乙酰氨基酸水解酶，特异于乙酰基Met 群体，这可能会破坏den蛋白的稳定性，从而通过n端降解 规则。在第二个，人类蛋白N末端天冬酰胺氨基水解酶， 将N末端ASN残基转换为ASP（酵母形式也作用于 GLN），将被隔离并进一步表征。最后，酵母效机 菌株，缺乏这些（和其他）共同/后的菌株 翻译活性酶将用于确认其推定 生理角色并确定在N末端降解的蛋白质 具体方式。