Regulation of Proteasome Activity by Ubp6 and Hul5

描述(申请人提供)：蛋白酶体被认为是细胞蛋白质降解机制的中心组成部分，但对其调节机制知之甚少。在这项提案中提出的初步数据中，我们报告了钙调蛋白是蛋白酶体的主要调节因子的证据。蛋白酶体似乎有三个不同的钙调蛋白结合位点，分别位于Rpn11亚基和蛋白酶体相关蛋白Hul5和Ubp6上。值得注意的是，这三种钙调蛋白的“受体”都以酶促方式作用于蛋白酶体结合的泛素链。我们已经确定了这些蛋白质中钙调素结合部位的特定氨基酸替换。这些突变都不是蛋白酶体功能的亚型。然而，当突变进入典型的蛋白酶体低构型rpn4，而rpn4不能调节其蛋白酶体水平来补偿功能缺陷时，它们会导致蛋白酶体功能的显著恢复。因此，钙调蛋白显然直接与蛋白酶体结合以抑制其活性。值得注意的是，蛋白酶体中的钙调蛋白结合位点从酵母到人类都是严格保守的，这表明这里描述的调节机制是普遍存在的。我们基于生物化学和遗传学的结合，对钙调蛋白对蛋白酶体的调节进行了详细的分析。我们将调查钙调蛋白改变蛋白酶体活性的哪些方面，主要候选蛋白包括Rpn11、Hul5和Ubp6的酶活性。我们还将确定钙是否参与了这一调控，以及钙调素下游的调节因子，如钙调素依赖的激酶和钙调神经磷酸酶是否参与了该途径。这些研究将阐明一条令人惊讶的新信号通路，该通路连接了真核生物的两个主要调控系统。基于蛋白酶体上钙调素结合位点的多样性，以及蛋白酶体中每个钙调素受体蛋白的关键作用，这似乎是一个高度复杂的调控途径。 公共卫生相关性：蛋白酶体对人类健康非常重要；蛋白酶体抑制剂已被证明在临床上对几种癌症非常有效，最近，蛋白酶体与许多其他疾病有关，了解越来越详细。这项提案描述了一种似乎是通过中央细胞调节因子钙调蛋白来调节蛋白酶体的主要新途径的发现。对这一新途径的更好理解可能会为各种疾病机制提供新的线索。

DESCRIPTION (provided by applicant): The proteasome is thought to be a central component of cellular protein degradation machinery, but little is known about the mechanisms by which it is regulated. In preliminary data presented in this proposal, we report evidence that calmodulin is a major regulator of the proteasome. Proteasomes appear to have three distinct binding sites for calmodulin, located on the subunit Rpn11 and on the proteasome-associated proteins Hul5 and Ubp6. Remarkably, all three of these "receptors" for calmodulin act enzymatically on proteasome-bound ubiquitin chains. We have characterized specific amino acid substitutions in the calmodulin-binding sites each of these proteins. None of these mutations is detectably hypomorphic for proteasome function. However, when the mutations are crossed into the canonical proteasome hypomorph rpn4, which cannot adjust its proteasome levels to compensate for functional defects, they result in a major recovery of proteasome function. Thus, calmodulin apparently binds directly to the proteasome to inhibit its activity. Remarkably, the calmodulin binding sites in the proteasome are strictly conserved from yeast to humans, suggesting that the regulatory mechanism described here is universal. We propose a detailed analysis of proteasome regulation by calmodulin, based on a combination of biochemistry and genetics. We will investigate what aspect of proteasome activity is altered by calmodulin, prominent candidates including the enzymatic activities of Rpn11, Hul5, and Ubp6. We will also determine whether calcium is involved in the regulation, and whether regulators that function downstream of calmodulin, such as calmodulin-dependent kinases and calcineurin, are involved in the pathway. The studies will elucidate a surprising new signaling pathway that couples two major regulatory systems of eukaryotes. Based on the multiplicity of calmodulin binding sites on the proteasome, and the key roles of each of the proteasome's calmodulin receptor proteins, this already appears to be a highly intricate regulatory pathway. PUBLIC HEALTH RELEVANCE: The proteasome is very significant for human health; proteasome inhibitors have proven to be quite effective in clinical use against several cancers and more recently, proteasomes have been linked to many other diseases, with increasingly detailed understanding. This proposal describes the discovery of what appears to be a major new pathway for regulating the proteasome via a central cellular regulator, calmodulin. A better understanding of this new pathway may potentially shed new light on a variety of disease mechanisms.