Activity-based protein profiling of the human rhomboid proteases for inhibitor discovery and enzyme characterization

水解酶是利用水裂解共价键的蛋白质，是最大的一类酶 在人体内，并在许多生理途径中发挥重要作用， 神经系统信号尽管这类酶的流行和重要性， 许多水解酶的特性仍然很差。在这些研究较少的成员中，菱形膜内 蛋白酶（RIP），其在生物制品的疏水环境内执行水解化学。 膜，是特别感兴趣的。神经退行性疾病中这些酶的突变和失调 疾病和多种类型的癌症促使人们对它们的发展有更深入的了解。 生理作用和研究其作为治疗靶点的潜力;然而， 目前缺乏这些研究。基于活性的蛋白质谱（ABPP）技术，其中活性 定点化学探针用于检测酶活性，促进了许多酶的表征， 丝氨酸水解酶作为酶超家族的成员，RIP代表了有希望的研究候选者 通过ABPP方法，然后可以利用该方法来发现选择性抑制剂并表征活性物质， 这些酶的蛋白形式。该提案旨在（1）开发优化的ABPP测定法以监测活性 复杂生物样品中五种人菱形蛋白酶的每一种，（2）产生并筛选文库 作为这些酶的潜在抑制剂的N-磺酰化杂环，和（3）表征蛋白水解 ER定位的RIP RHBDL 4的加工以及加工对酶活性的影响。这些研究 将有助于研究人类RIP的生理作用及其作为治疗药物的潜力 目标的此外，拟议的项目将提供一个机会，以培训奥伯林本科研究人员在 当代化学生物学方法，包括合成化学，生物化学和生物化学技术， 分子生物学奥伯林的研究生将学习理论和先进的实验室技能，而不是其他 涵盖在本科课堂设置，同时发展他们的能力，分析和提出科学 数据总的来说，这些活动将丰富奥伯林学院的研究环境，并帮助准备 跨学科分子科学的下一代研究人员。

Hydrolases, proteins that cleave covalent bonds using water, represent one of the largest classes of enzymes in the human body and play essential roles in numerous physiological pathways ranging from digestion to nervous system signaling. Despite the prevalence and importance of this enzyme class, the biological functions of many hydrolases remain poorly characterized. Among these less studied members, rhomboid intramembrane proteases (RIPs), which perform hydrolysis chemistry within the hydrophobic environment of biological membranes, are of particular interest. Mutation and dysregulation of these enzymes in neurodegenerative diseases and multiple types of cancer motivates the development of an enhanced understanding of their physiological roles and an investigation of their potential to serve as therapeutic targets; however, methods to conduct these studies are currently lacking. Activity-based protein profiling (ABPP) technology, in which active site-directed chemical probes are used to detect enzyme activity, has facilitated the characterization of many serine hydrolases. As members of this superfamily of enzymes, RIPs represent promising candidates for study by ABPP methods, which can then be leveraged to discover selective inhibitors and to characterize the active proteoforms of these enzymes. This proposal aims to (1) develop optimized ABPP assays to monitor the activity of each of the five human rhomboid proteases in complex biological samples, (2) generate and screen a library of N-sulfonylated heterocycles as potential inhibitors for these enzymes, and (3) characterize proteolytic processing of the ER-localized RIP RHBDL4 and the impact of processing on enzymatic activity. These studies will empower efforts to investigate the physiological roles of the human RIPs and their potential as therapeutic targets. Further, the proposed project will provide an opportunity to train Oberlin undergraduate researchers in contemporary chemical biology methods that incorporate techniques from synthetic chemistry, biochemistry, and molecular biology. Research students at Oberlin will learn theory and advanced laboratory skills not otherwise covered in an undergraduate classroom setting while developing their ability to analyze and present scientific data. Collectively, these activities will enrich the research environment at Oberlin College and help prepare the next generation of researchers in interdisciplinary molecular science.