CONTROL OF CELLULAR PROTEOLYSIS

通过细胞质泛素依赖性途径降解的蛋白质是 首先与泛素共价连接。 这些泛素-蛋白质结合物 然后被蛋白质的蛋白水解成分识别并降解 通路 这项拟议研究的主要目标之一是确定 该蛋白水解组分并表征蛋白水解反应 使用泛素-钙调蛋白作为底物。 为了确定泛素-蛋白质结合物如何被识别， 被泛素依赖性途径的蛋白水解组分降解， 它需要具有已知结构的衬底， 可以表征蛋白水解产物。 网柄藻钙调素型 与泛素的一对一缀合物，特别是在赖氨酸115处。 所有 迄今为止报道的其它底物蛋白质与泛素在 多个位点，这些蛋白质中的结合位点还没有被发现。 定义了 以泛素-钙调素为底物，我们初步 鉴定了网织红细胞中的ATP依赖性蛋白酶， 钙调素仅以共轭形式存在。 建议的研究包括 继续纯化该蛋白酶，并表征 泛素化钙调蛋白 含有异肽的合成肽 连接，以及与泛素缀合的小钙调蛋白片段， 将作为潜在底物或抑制剂进行测试。 这些结果， 与缀合的钙调蛋白的切割位点的作图一起， 将用于检验异肽连接区 作为蛋白酶的识别位点。 从大多数来源分离的钙调素含有三甲基赖氨酸， 位置115并且不能与遍在蛋白缀合。 我们目前 一种假说是，这种赖氨酸的翻译后甲基化 通过排除该蛋白质经由酶的降解来稳定该蛋白质。 泛素依赖途径。 这一假设将通过测量 微注射不同形式钙调素的降解率 卵母细胞在正常和条件下，其中泛素依赖的 已使通路失活。 我们还将测试乙酰化是否 钙调蛋白N端的一个突变体也具有同样的功能， 表达的鸡钙调蛋白，其含有未封闭的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.