COTRANSLATIONAL PROCESSING AND PROTEIN TURNOVER

真核生物蛋白质的共翻译加工通常产生 四类主要蛋白质：有无引发剂Met和 那些有和没有N-α-乙酰化的。甲硫氨酸氨基肽酶 (MAP)和N-α-乙酰转移酶(NAT)，与 核糖体显然会影响这些修饰。重要的是， 所产生的结构显然决定了许多 真核生物中的细胞内蛋白质，并可以指导这些蛋白质的周转 蛋白质通过基于泛素的降解系统。两条主线 为了进一步阐明这些关系，建议进行实验。在……里面 第一个是猪肝NAT和MAP，它们共同翻译，以及 蛋白质N端，天冬酰胺脱酰胺酶，翻译后起作用， 将从结构、性质和特异性方面进行检查。 将获得全长cdna序列，并将进行沉淀 针对这三种酶中每一种的抗体。的综合效应 这些代理，无论是分组还是整体，将提供(假定 调控良好的)选定的胞内蛋白进入 降解机器。建议的第二部分涉及 这些酶在兔网织红细胞降解蛋白质中的作用 裂解液。裂解物将用于表达转录本(包括原生和 改变)以产生适当的蛋白质，并通过 泛素依赖途径。参与修改和更替的人员 通路将被抗血清和/或 抑制物，以确定它们在这一过程中的作用。在整个细胞中 实验，成绩单将由适当的现场生成 量身定制的质粒和由此产生的蛋白质的降解情况受到监测。 两种蛋白质(天冬酰胺合成酶和次黄嘌呤磷酸核糖 在这些研究中，将使用转移酶)作为主要底物。 合适的核酸和免疫试剂要么手头有，要么 将会被生成。

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.