Chemically Interrogating Clostridium difficile Glucosylating Toxin Activation

6.项目摘要/摘要 6.A.职业发展计划 在这项提案中，我描述了我的计划，以阐明自我蛋白分解调节细菌的机制。 毒素功能。到目前为止，在我的博士后工作中，我发现了一种独特的蛋白酶激活机制 并描述了半胱氨酸蛋白酶结构域(CPD)在自动处理新发现的 细菌毒素。我已经确定了这个新的蛋白水解酶家族的第一个化学抑制剂，并开始 将这些抑制剂发展成化学探针，以研究蛋白酶底物的专一性和功能。 在提案的指导阶段，我将继续学习如何合成小分子抑制剂和 斯坦福大学Matthew Bogyo博士实验室中监测和扰乱CPD激活的探测器。这 阶段将取决于博乔实验室内研究的深度和多样性以及出色的研究 斯坦福大学的环境。我将用一门表面等离子激元课程来补充我的机构环境。 共鸣和参观未来合作者的实验室(俄克拉荷马州立大学的吉米·巴拉德博士)在那里我 将学习如何培养艰难梭菌并提纯其糖化毒素。 在斯坦福大学，我将继续利用学院内优秀的职业发展资源 通过参加旨在帮助博士后研究员过渡到学术职位的讲座，医学。这就做 继续咨询我的导师博乔博士和一个由约翰博士组成的非正式指导委员会 Boothroyd和K.Christopher Garcia博士在斯坦福大学帮助我成功地驾驭学术工作 市场，申请资金，成立我自己的研究小组。 一旦我在一家研究机构获得了独立的职位，我就会应用探针和抑制剂 在指导阶段开发，以研究自身蛋白分解在调节毒素功能中的作用 在细胞和中毒的动物模型中。从长远来看，我将把这种新的方法应用于对蛋白酶进行成像 以及我在细菌遗传学和生物化学方面的背景，在 不同的系统。 6.b.研究计划 大多数分泌的细菌毒素是以非活性前体的形式产生的，这些前体在 遇到了真核细胞。虽然真核蛋白水解酶通常会激活这些毒素，但一组精选的 毒素被内部的半胱氨酸蛋白酶结构域(CPD)自动蛋白分解激活。细胞毒作用 艰难梭菌糖化毒素的一部分由CPD激活，CPD本身在结合 真核特异性小分子六氢肌醇(InsP6)。尽管自动处理对于 毒素激活，InsP6如何激活CPD，以及CPD在细胞中何时何地分解这些毒素，是 未知。这项提案概述了使用小分子来解决以下这些重要问题的计划 CPD介导的艰难梭菌糖基化毒素的激活。初步鉴定的小分子抑制剂 研究将发展成探针，允许在体外、宿主细胞内和 动物。这些探头将允许通过以下方式实时监控毒素激活的时间和位置 共价标记激活的CPD。与结构方法相结合，这些探头还将有助于分析 CPD底物的特异性和功能，这将反过来为针对抑制艰难梭菌的策略提供信息 葡萄糖化毒素功能。

6. Project Summary/Abstract 6.A. Career Development Plan In this proposal, I describe my plans to elucidate the mechanism by which autoproteolysis regulates bacterial toxin function. In my postdoctoral work thus far, I have discovered a unique mechanism of protease activation and delineated a role for a cysteine protease domain (CPD) in autoprocessing a newly recognized family of bacterial toxins. I have identified the first chemical inhibitors of this novel protease family and have begun developing these inhibitors into chemical probes to study protease substrate specificity and function. In the mentored phase of the proposal, I will continue to learn how to synthesize small molecule inhibitors and probes to monitor and perturb CPD activation in the lab of Dr. Matthew Bogyo at Stanford University. This phase will rely on the depth and diversity of research within the Bogyo lab and the excellent research environment at Stanford. I will supplement my institutional environment with a course in surface plasmon resonance and a visit to a future collaborator's lab (Dr. Jimmy Ballard at Oklahoma State University) where I will learn how to culture Clostridium difficile and purify its glucosylating toxins. At Stanford, I will continue to capitalize on the excellent career development resources within the School of Medicine by attending lectures directed at helping postdoctoral fellows transition into academic positions. I will continue to consult with my advisor, Dr. Bogyo, and an informal mentoring committee, consisting of Dr. John Boothroyd and Dr. K. Christopher Garcia at Stanford, to help me successfully navigate the academic job market, apply for funding, and set-up my own research group. Once I acquire an independent position at a research institution, I will apply the probes and inhibitors developed during the mentored phase to study the function of autoproteolysis in regulating toxin function within cells and in animal models of intoxication. In the long-term, I will apply this new method for imaging protease function, along with my background in bacterial genetics and biochemistry, to study bacterial proteases in diverse systems. 6.B. Research Plan Most secreted bacterial toxins are produced as inactive precursors that become proteolytically activated upon encountering a eukaryotic cell. Whereas eukaryotic proteases typically activate these toxins, a select group of toxins are autoproteolytically activated by an internal cysteine protease domain (CPD). The cytotoxic function of Clostridium difficile glucosylating toxins is activated by the CPD, which itself is activated upon binding the eukaryotic-specific small molecule inositol hexakisphosphate (InsP6). Although autoprocessing is essential for toxin activation, how InsP6 activates the CPD, and when and where in the cell the CPD cleaves these toxins, is unknown. This proposal outlines plans to use small molecules to address these important questions regarding CPD-mediated activation of C. difficile glucosylating toxins. Small molecule inhibitors identified in preliminary studies will be developed into probes that allow CPD activation to be visualized in vitro, within host cells, and in animals. These probes will permit the timing and location of toxin activation to be monitored in real-time by covalently tagging activated CPD. Combined with structural methods, the probes will also facilitate analyses of CPD substrate specificity and function, which will in turn inform strategies directed at inhibiting C. difficile glucosylating toxin function.