Regulating Proteolysis to Dissect Apoptosis

描述(由申请人提供)：这项提案的长期目标是了解特定的caspase切割事件在驱动细胞凋亡中的作用。细胞凋亡是一种无私的过程，目的是去除受感染的、DNA受损的或癌前细胞。最后一步是由一类细胞内的半胱氨酸蛋白酶驱动的，称为半胱氨酸酶，它通过对人类细胞中多达1000种蛋白质的特定(通常是单一的)切割来解构细胞。蛋白质分解的目标教会了我们许多关于维持体内平衡的细胞通路和网络，以及驱动这一过程的凋亡机制。我们的假设是，许多凋亡靶点形成了功能网络或支柱，当它们单独被切割时，就可以触发细胞凋亡。不幸的是，鉴于如此多的靶点同时被caspase切割，单个蛋白分解事件的重要性无法评估。为了系统地解决这个问题，我们开发了一个技术平台，使我们能够开始剖析切割单个靶点在推动细胞凋亡中的重要性。这些包括开发一种部位特异性的蛋白酶(狙击手)，它被一个小分子(雷帕霉素)激活，并切割包含其特定识别序列的单一靶标，这是人类蛋白质组中没有的。第二种是翻译后基因替换载体，它使我们能够引入感兴趣的靶基因，用狙击点取代caspase位点，同时将shRNA表达到内源性caspase靶点。这使得内源性caspase敏感等位基因可以快速有效地替换为特定的狙击敏感等位基因。第三种技术允许我们使用我们实验室建立的蛋白质组学方法来跟踪蛋白质降解的详细事件，该方法用于在蛋白质降解过程中标记新产生的N-末端。我们将把这些技术应用于三个由caspase蛋白分解触发的微型网络，它们被认为是细胞凋亡的关键驱动因素和标志，包括：DNA损伤的激活和DNA修复的抑制，caspase蛋白分解后集中在细胞核中的信号酶，以及26S蛋白酶体中在凋亡过程中被切割并使清除激活的caspase的蛋白酶体失效的亚基。具体目标#1：确定caspase激活的DNA酶(CAD)和邻近的修复酶在细胞凋亡中的位置特异性蛋白水解酶激活的生化和细胞后果。特定目标#2：确定ABL激酶和CDC25A磷酸酶位点特异性蛋白分解的生化和细胞后果。具体目标#3：确定26S蛋白酶体的19S调节颗粒的caspase样裂解的生化和细胞后果。这些实验应该会极大地提高我们对特定的蛋白分解事件如何引发、敏化和驱动细胞凋亡的理解。通过小分子调控的、定点选择性的蛋白分解来点燃信号事件，为剖析复杂的蛋白水解酶信号通路建立了新的范式。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand the role of specific caspase cleavage events in driving apoptosis. Apoptosis is an altruistic process for removing infected, DNA damaged, or precancerous cells. The final steps are driven by a class of intracellular cysteine proteases, known as caspases, that deconstruct the cell by specific (and usually single) cleavage of up to 1000 proteins in human cells. The targets of proteolysis teach us much about cellular pathways and networks that maintain homeostasis as well as the apoptotic machinery that drives the process. Our hypothesis is that many targets of apoptosis form functional webs or struts that when cleaved alone can trigger apoptosis. Unfortunately, given that so many targets are cleaved simultaneously by caspases, the importance of individual proteolytic events can not be assessed. To systematically attack this problem we have developed a platform of technologies that allows us to begin to dissect the importance of cutting individual targets in driving apoptosis. These include the development of a site-specific protease (SNIPer) which is activated by a small molecule (rapamycin) and cleaves single targets containing its specific recognition sequence that is not found in the human proteome. The second is a post- translational gene replacement vector, which enables us to introduce the target gene of interest with a SNIPer site replacing a caspase site and simultaneously expressing an shRNA into the endogenous caspase target. This allows rapid and effective replacement of the endogenous caspase sensitive allele, with a specific SNIPer sensitive allele. A third technology permits us to follow the detailed events of proteolysis using a proteomic method established in our lab for tagging newly created N-termini during proteolysis. We will apply these technologies on three mini-networks that are triggered by caspase proteolysis and are thought to be critical drivers and hallmarks of apoptosis including: activation of DNA damage and inhibition of DNA repair, signaling enzymes that concentrate in the nucleus following caspase proteolysis, and subunits in the 26S proteasome that are cleaved during apoptosis and disable the proteasome which clears activated caspases. Specific Aim #1: Determine the biochemical and cellular consequences for site-specific proteolytic activation of the caspase activated DNase (CAD) and neighboring repair enzymes in apoptosis. Specific Aim#2: Determine the biochemical and cellular consequences of site-specific proteolysis of Abl kinase and CDC25A phosphatase. Specific Aim#3: Determine the biochemical and cellular consequences of caspase-like cleavages of the 19S regulatory particle of the 26S proteasome. These experiments should greatly enhance our understanding of how specific proteolytic events can spark, sensitize, and drive apoptosis. The ignition of signaling events via small molecule regulated, site-selective proteolysis sets a new paradigm for dissecting complex protease signaling pathways.