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：(改编自应用程序)N端处理 真核生物中的蛋白质以共翻译和翻译后的方式存在，并且 由此产生的修饰涉及转位、调节 活动和营业额。最初的反应包括移除 引发剂蛋氨酸和N-α乙酰基的加成 蛋氨酸氨基肽酶(Met AP)和N-α-乙酰基转移酶(NAT)， 分别进行了分析。每种异构体都有多种不同的异构体 特异性，细胞位置的调节。利用重组材料， 这两种类型的酵母MetAP的作用将被确定，并且 具有结构和功能特征的蛋白质 性能，包括金属含量、底物专一性和催化 组织。定点突变和化学抑制剂将是 被广泛使用。蜂窝定位也将通过 重组融合蛋白和免疫试剂。重组人 还将准备和检查可能的糖基化的异构体。 以及II型酶在细胞周期调节中的作用。类似 将与NAT一起进行研究，重点是子表单 N-末端蛋氨酸(由倒数第二个天冬氨酸、谷氨酸和天冬氨酸产生-The “DEN”子集)。与修改Gly、Ala、Ser和 THR(GAST)组，M-NAT以一对蛋白质的形式存在，可能 起到杂二聚体的作用。这一假设将作为一种 结构特征的一部分。推测涉及的两种酶 在DEN蛋白的下游加工中，也将进行调查。 在第一种情况下，酵母将被筛选(并分离酶，如果 检测到)乙酰蛋氨酸特有的酰基氨基酸水解酶 基团，这可能会破坏DEN蛋白的稳定性，使其被N-端降解 规则。第二种是人蛋白N末端天冬酰胺氨基水解酶， 它将N-末端的天冬氨酸残基转化为天冬氨酸(酵母形式也作用于 Gln)，将对其进行分离和进一步鉴定。最后，酵母为空 菌株，缺乏这些(和其他)co-/post- 翻译活性的酶，将被用来确认其推测的 生理作用和鉴定N-末端降解的蛋白质 具体的方式。