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caspase切割事件在驱动细胞凋亡中的作用。细胞凋亡是一种利他的过程，用于清除受感染的、DNA受损的或癌前细胞。最后的步骤是由一类细胞内半胱氨酸蛋白酶驱动的，这种酶被称为半胱氨酸酶，它通过对人体细胞中多达1000种蛋白质的特异性（通常是单次）切割来解构细胞。蛋白质水解的目标告诉我们很多关于维持体内平衡的细胞途径和网络，以及驱动这一过程的凋亡机制。我们的假设是，许多细胞凋亡的靶点形成了功能性的网或支，当它们被单独劈开时可以触发细胞凋亡。不幸的是，考虑到如此多的靶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，使我们能够开始分析切割单个靶点在驱动细胞凋亡中的重要性。其中包括一种位点特异性蛋白酶（SNIPer）的开发，它被一种小分子（雷帕霉素）激活，并切割包含其在人类蛋白质组中未发现的特定识别序列的单个靶标。第二种是翻译后基因替代载体，它使我们能够用SNIPer位点替代caspase位点引入感兴趣的靶基因，同时在内源性caspase靶标中表达shRNA。这允许快速和有效地替换内源性半胱天冬酶敏感等位基因，与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跟踪蛋白质水解的详细事件，用于标记蛋白质水解过程中新创建的n端。我们将把这些技术应用于三个由半胱天冬酶蛋白水解引发的微型网络，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，半胱天冬酶蛋白水解后集中在细胞核中的信号酶，以及26S蛋白酶体中的亚基，这些亚基在细胞凋亡过程中被切割，并使清除活化半胱天冬酶的蛋白酶体失效。特定目标#1：确定凋亡中caspase活化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。特定目标#2：确定Abl激酶和CDC25A磷酸酶位点特异性蛋白水解的生化和细胞后果。特定目标#3：确定26S蛋白酶体的19S调节颗粒的caspase样切割的生化和细胞后果。这些实验将极大地增强我们对特定蛋白水解事件如何引发、致敏和驱动细胞凋亡的理解。通过小分子调控、位点选择性蛋白水解引发的信号事件为解剖复杂蛋白酶信号通路提供了新的范例。

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.