COTRANSLATIONAL PROCESSING AND PROTEIN TURNOVER

共翻译加工和蛋白质周转

• 批准号: 2659954
• 负责人: RALPH A BRADSHAW
• 金额: $ 10.4万
• 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 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2659954
• 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通常产生 四种主要类型的蛋白质：具有和不具有起始剂Met的蛋白质， 那些有和没有N-α-乙酰化。 硫氨酸氨基肽酶 (MAP)和N-α-乙酰基转移酶（NAT），与 核糖体，显然影响这些修改。 重要的是 所产生的结构显然决定了许多国家的长期稳定， 真核生物中的细胞内蛋白质，并可以指导这些 蛋白质通过基于泛素的降解系统。 两条主线 提出实验以进一步阐明这些关系。 在 第一，猪肝NAT和MAP，它们协同作用， 蛋白质N-末端，天冬酰胺脱酰胺酶，其作用于胰后， 将在结构、性质和特异性方面进行检查。 将获得全长cDNA序列， 针对3种酶中每一种的抗体。 的共同作用 这些试剂，无论是在亚组或整体，是提供（大概 良好调节）选定的细胞内蛋白质进入细胞内， 降解机制 建议的第二部分涉及 这些酶在使用兔网织红细胞的蛋白质降解中的功能 裂解物。 裂解物将用于表达转录物（天然的和非天然的）。 改变）以产生适当的蛋白质并诱导降解， 泛素依赖途径。 参与修改和周转的人员 途径将用抗血清和/或 抑制剂，以确定其在该过程中的作用。 在整个细胞 实验中，转录本将在原位从适当的 定制的质粒，并监测所得蛋白质的降解。 两种蛋白质（天冬酰胺合成酶和次黄嘌呤磷酸核糖基 转移酶）将用作这些研究中的主要底物。 适当的核酸和免疫试剂或者在手边，或者 将被生成。