ATP--UBIQUITIN-DEPENDENT PROTEOLYSIS

越来越多的证据表明， 将低分子量多肽泛素连接到 真核生物中的各种靶蛋白是重要的 对细胞调控的重要性。 的长期目标 这项研究的目的是了解这种新的后- 翻译修饰在细胞调控中的作用，重点是其 在能量依赖性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的模型， 建议，观察到的降解的特异性短- 活的和不正常的蛋白质是由蛋白质的分配引起的。 交替降解结局之间的结合物合并液， 拆卸，其中泛素部分是非生产性的 劈开的 假设这种划分的基础取决于 诱导的靶蛋白结构不稳定程度 泛蛋白的共价共轭;因此，降解 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中。 模型 预测短寿命和长寿命的蛋白质都应该是 发现结合，但两个池的泛素连接， 靶蛋白在降解之间分配应该不同 和拆卸。 此外，该模型预测， 泛素化会导致蛋白水解的普遍增强， 靶蛋白的敏感性。 两个独立的， 将采取互补办法。 免疫化学 方法将用于定量和分离脉冲标记的游离 和缀合的泛素池以确定蛋白质的范围 靶蛋白在培养人肺中的半衰期 成纤维细胞 由于泛素周转的显著速率， 这些方法将用于校正标记掺入 分离的缀合物的泛素部分。 其他研究将 确定和表征血清的作用机制 因子显示诱导游离和共轭 成纤维细胞内的泛素池。 酶催化 将从兔肝中纯化泛素缀合物， 其特征在于其功能和酶 机制 然后，这些纯的缀合酶将用于 体外模型研究，以测试靶蛋白的泛素化是否 蛋白质诱导一般增强的蛋白水解敏感性， 其作用机制。

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.