Regulating Proteolysis to Dissect Apoptosis

描述（由申请人提供）：本提案的长期目标是了解特定胱天蛋白酶裂解事件在驱动细胞凋亡中的作用。细胞凋亡是一个利他的过程，用于清除感染的，DNA损伤的或癌前细胞。最后的步骤是由一类细胞内半胱氨酸蛋白酶驱动的，称为半胱天冬酶，它通过特异性（通常是单一）切割人类细胞中多达1000种蛋白质来解构细胞。蛋白质水解的靶点告诉我们很多关于维持稳态的细胞通路和网络以及驱动该过程的凋亡机制的信息。我们的假设是，许多细胞凋亡的目标形成功能网或支柱，当单独切割时可以触发细胞凋亡。不幸的是，鉴于这么多的目标被半胱天冬酶同时切割，单个蛋白水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驱动细胞凋亡中的重要性。这些包括开发一种位点特异性蛋白酶（SNIPer），它被小分子（雷帕霉素）激活，并切割含有其在人类蛋白质组中未发现的特异性识别序列的单一靶标。第二种是翻译后基因置换载体，它使我们能够用SNIPer位点置换半胱天冬酶位点，同时将shRNA表达到内源性半胱天冬酶靶标中，从而引入感兴趣的靶基因。这允许用特异性SNIPer敏感等位基因快速有效地替换内源性胱天蛋白酶敏感等位基因。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跟踪蛋白质水解的详细事件，用于在蛋白质水解过程中标记新创建的N-末端。我们将这些技术应用于三个由caspase蛋白水解触发的小型网络，这些网络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蛋白水解后集中在细胞核中的信号酶，以及26 S蛋白酶体中的亚基，这些亚基在细胞凋亡过程中被切割并使清除激活的caspase的蛋白酶体失活。具体目标1：确定凋亡中caspase激活的DNA酶（CAD）和邻近修复酶的位点特异性蛋白水解激活的生化和细胞后果。具体目标#2：确定Abl激酶和CDC 25 A磷酸酶的位点特异性蛋白水解的生物化学和细胞后果。具体目标#3：确定26 S蛋白酶体的19 S调节颗粒的半胱天冬酶样切割的生物化学和细胞后果。这些实验应该大大提高我们的理解，如何特定的蛋白水解事件可以火花，敏化，并驱动细胞凋亡。通过小分子调节的，位点选择性的蛋白质水解的信号事件的点火设置了一个新的范例解剖复杂的蛋白酶信号通路。

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.