ATP--UBIQUITIN-DEPENDENT PROTEOLYSIS

越来越多的证据表明，ATP偶联的共价 连接低分子量多肽泛素，以 真核生物中的各种靶蛋白是基本的。 对细胞调节的重要性。该计划的长远目标 建议的研究是为了了解这一新奇的邮局的功能 细胞调节中的翻译修饰，重点是其 在依赖能量的蛋白质降解中的作用。这些研究将 测试泛素在蛋白质周转中的作用模型 建议观察到的短路降解的特异性- 活的和异常的蛋白质是由 降解性和可变性交替命运的共轭池 拆卸，其中泛素部分是非生产性的 乳沟。此分区的基础被假定为依赖于 对靶蛋白结构不稳定程度的研究 泛素的共价结合；因此，降解剂 特异性主要存在于目标的特征中 蛋白质，而不是在泛素连接的步骤。模型 预测短寿命和长寿命的蛋白质都应该是 发现是结合的，但两个泛素池是连接的 靶蛋白在不同的降解过程中的分配应该不同 和拆卸。此外，该模型预测， 泛素化应导致普遍增强的蛋白水解物 目标蛋白的敏感性。两个独立的， 将采取免费的方法。免疫化学 将使用各种方法来定量和分离脉冲标记的游离 和结合泛素库来确定蛋白质的范围 培养人肺内靶蛋白的半衰期 成纤维细胞。由于泛素周转率很高， 这些方法将用于校正标签合并到 分离的结合物的泛素部分。其他研究将 鉴定和表征血清的作用机制 显示的诱导游离态和结合态净值增加的因子 成纤维细胞内的泛素池。酶催化作用 然后从兔肝中提纯泛素结合物 根据它们的功能和酶的特性 机制。这些纯的结合酶将被用于 体外模型研究以测试靶标的泛素化 蛋白质诱导普遍增强的蛋白水解性，因为 它的作用机制。

Accumulating evidence indicates that the ATP-coupled covalent ligation of the low molecular weight polypeptide, ubiquitin, to various target proteins within eukaryotes is of fundamental importance to cellular regulation. The long-term objective of the proposed research is to understand the function of this novel post- translational modification in cell regulation, with emphasis on its role in energy-dependent protein degradation. The studies will test a model for the role of ubiquitin in protein turnover which proposes that the observed specificity of degradation of short- lived and abnormal proteins results from the partitioning of the conjugate pool between alternate fates of degradation and disassembly, in which the ubiquitin moiety is non-productively cleaved. The basis for this partitioning is hypothesized to depend on the degree of target protein structural instability induced by the convalent conjugation of ubiquitin; therefore, the degradative specificity would principally reside in features of the target protein and not in the step of ubiquitin ligation. The model predicts that both short-lived and long-lived proteins should be found conjugated, but that the two pools of ubiquitin-ligated target proteins should partition differently between degradation and disassembly. In addition, the model predicts that ubiquitination should lead to a generally enhanced proteolytic susceptibility of the target protein. Two independent, complimentary approaches will be taken. Immunochemical methods will be used to quantitate and isolate pulse-labeled free and conjugated ubiquitin pools to determine the range of protein half lives for target proteins within cultured human lung fibroblasts. Because of the significant rate of ubiquitin turnover, these methods will be used to correct for label incorporation into the ubiquitin moiety of the isolated conjugates. Other studies will identify and characterize the mechanism of action for a serum factor shown to induce net increases in both free and conjugated ubiquitin pools within the fibroblasts. The enzymes catalyzing ubiquitin conjugation will be purified from rabbit liver then characterized in terms of their function and enzymatic mechanism. These pure conjugating enzymes will then be used for in vitro model studies to test whether ubiquitination of target proteins induces a general enhanced proteolytic susceptibility as its mechanism of action.