COTRANSLATIONAL PROCESSING AND PROTEIN TURNOVER

共翻译加工和蛋白质周转

• 批准号: 2701065
• 负责人: RALPH A BRADSHAW
• 金额: $ 22.96万
• 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/2701065
• 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-末端处理 真核生物中的蛋白质既发生在协同作用下，也发生在协同作用后， 由此产生的修饰涉及易位，调节 活动和营业额。最初的反应包括去除 引发剂甲硫氨酸和N-α乙酰基的加成， 甲硫氨酸氨基肽酶（Met AP）和N-α-乙酰基转移酶（NAT）， 分别每一种都存在于多个亚型中，这些亚型在 特异性，细胞定位的调节。使用重组材料， 两种类型的酵母MetAP的作用将被确定， 蛋白质的结构和功能特征 性质，包括金属含量、底物特异性和催化性 organization.定点诱变和化学抑制剂将被 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 还将制备同种型并检查推定的糖基化 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，重点是子形式 特异于N-末端Met（由倒数第二个Asp、Glu和Asn产生）， “DEN”子集）。与修饰Gly、Ala、Ser和 Thr（GAST）组，M-NAT作为一对蛋白质存在， 作为异二聚体复合物起作用。这一假设将作为一个 部分结构特征。涉及两种酶 在DEN蛋白的下游加工中也将被研究。 在第一种情况下，将筛选酵母（如果需要，则分离酶）。 检测）的酰基氨基酸水解酶，特异于乙酰Met 基团，它们可能会使DEN蛋白不稳定，从而通过N端降解 统治在第二，人蛋白N-末端天冬酰胺水解酶， 其将N-末端Asn残基转化为Asp（酵母形式也作用于 Gln），将被分离并进一步表征。最后，酵母空 这些（和其他）共同/后- 将用于确认其推定的 生理作用，并鉴定N-末端降解的蛋白质 具体方式。