CONTROL OF CELLULAR PROTEOLYSIS

通过细胞质泛素依赖途径降解的蛋白质有 第一次与泛素共价连接。这些泛素-蛋白质结合物 然后被蛋白质的降解成分识别和降解 路径。这项拟议研究的主要目的之一是确定 该蛋白水解组分并对该蛋白水解物进行了表征 以泛素-钙调蛋白为底物。 为了确定泛素-蛋白质结合物如何被识别和 被泛素依赖途径的蛋白分解成分降解， 它需要一种具有已知结构的衬底，对于该衬底， 蛋白水解物可以被表征。网柄网柄菌钙调素形式 与泛素，特别是与赖氨酸115的一对一的偶联物。全 到目前为止已报道的其他底物蛋白与泛素在 多个位点，而这些蛋白质中的结合位点还有待于 已定义。使用泛素-钙调蛋白作为底物，我们已经初步 在网织红细胞中发现一种可降解的ATP依赖的蛋白酶 钙调素只以结合形式存在。建议的研究包括 该酶的连续纯化及对其切割特性的研究 泛素化的钙调蛋白。含有异肽的合成肽 连接，以及与泛素结合的小钙调蛋白片段， 将作为潜在的底物或抑制剂进行测试。这些结果， 结合共轭钙调素的裂解位点的作图， 将被用来检验这一假设，即等肽链接区 作为蛋白水解酶的识别位点。 从大多数来源分离的钙调蛋白含有一个三甲基赖氨酸 第115位，不能与泛素结合。我们目前的情况 假设是这种赖氨酸的翻译后甲基化 通过将该蛋白质排除在降解之外，使其稳定 泛素依赖途径。这一假设将通过测量来检验 不同形态显微注射钙调素的降解率 正常和泛素依赖条件下的卵母细胞 路径已处于非活动状态。我们还将测试乙酰化是否 钙调素的N-末端通过使用细菌发挥相同的功能 表达的鸡钙调蛋白含有一个未阻断的N-末端。

Proteins to be degraded via the cytoplasmic ubiquitin-dependent pathway are first covalently linked with ubiquitin. These ubiquitin-protein conjugates are then recognized and degraded by the proteolytic component of the pathway. One of the major thrusts of this proposed research is to identify this proteolytic component and to characterize the proteolytic reaction using ubiquitin-calmodulin as a substrate. In order to determine how an ubiquitin-protein conjugate is recognized and degraded by the proteolytic component of the ubiquitin dependent pathway, it requires a substrate having a known structure, and for which the proteolytic products can be characterized. Dictyostelium calmodulin forms a one-to-one conjugate with ubiquitin and specifically at lysine 115. All other substrate proteins reported to date are conjugated with ubiquitin at multiple sites, and the conjugation sites in these proteins have yet to be defined. Using ubiquitin-calmodulin as a substrate, we have tentatively identified an ATP-dependent protease in reticulocytes which degrades calmodulin only in the conjugated form. The proposed studies include the continued purification of this protease and to characterize the cleavage of ubiquitinated calmodulin. Synthetic peptides containing the isopeptide linkage, as well as small calmodulin fragments conjugated to ubiquitin, will be tested as potential substrates or as inhibitors. These results, together with the mapping of the cleavage sites of conjugated calmodulin, will be used to test the hypothesis that the isopeptide linkage region serves as the recognition site for the protease. Calmodulin isolated from most sources contains a trimethyllysine at position 115 and cannot be conjugated with ubiquitin. Our current hypothesis is that the post-translational methylation of this lysine stabilizes this protein by excluding it from degradation via the ubiquitin-dependent pathway. This hypothesis will be tested by measuring the degradative rates of various forms of microinjected calmodulin in oocytes under normal and under conditions in which the ubiquitin-dependent pathway has been rendered inactive. We will also test whether acetylation of the N-terminus of calmodulin serves the same function by using bacterial expressed chicken calmodulin which contains an unblocked N-terminus.