Structure and Regulation of Ectodomain Sheddases in Development and Disease

项目摘要/摘要 胞外结构域脱落是生物学中使细胞能够改变膜蛋白的基本过程。 拓扑，影响细胞黏附和信号转导通路。因此，在体内发挥作用的酶 胞外区脱落对人类健康至关重要，在疾病发病机制中也处于失衡状态。尽管 这些酶对人类健康的重要性，以及控制酶原的分子和原子细节 成熟、激活、底物选择性、大分子抑制和亚细胞区划 仅限于几个例子。我的研究计划的长期目标是研究机械细节 ，揭示了支持胞外结构域脱落的普遍概念 酶的功能和将酶定位到生物利基中的独特性质。最初，我们 正在优先研究去整合素和金属蛋白酶家族，特别是ADAM17，以推出我的 研究计划担任新的首席研究员。 ADAMS是一种复杂的多域1型跨膜酶，在发育过程中具有重要功能， 受精和免疫。ADAM17被认为可以处理80多种不同的蛋白质底物，并具有必不可少的 在免疫、皮肤形态发生和心脏瓣膜形成的发育中的作用。所有亚当斯最初都是 翻译为酶原，受控制酶潜伏期的原结构域结合。ADAM17酶原 成熟和通过细胞的运输依赖于与不活跃的菱形体家族的相互作用 蛋白质、IRHOM1和-2。IRhoms恰如其分地被定位为ADAM17活动的重要调节者。尽管 ADAM17在发展和动态平衡中的作用得到了很好的表征，重大缺陷仍然存在 了解调节酶活性的分子事件，如酶原成熟、酶 激活并与调节蛋白结合。为了解决这些知识差距，我的研究计划将 将新的创新方法引入该领域，并利用我在结构和细胞生物学方面的经验 描述调控ADAM17活性的原子细节。具体地说，我们将确定原子细节 并揭示导致酶特异性和抑制作用的特性。此外, 我们建议使用X射线结晶学和电子显微镜来揭示ADAM17的机制细节 辅助蛋白IRHOM1和2的激活和识别。最后，我们将使用一个无偏倚的蛋白质组 质谱法特异性评价ADAM17-iRhom复合体在酶激活过程中的作用 除鉴定调节ADAM17的亚细胞室和活性调节蛋白外 活动。在未来五年，我们的目标是制定一个统一的ADAM17监管模式，并构建 基金会，以扩大我们的努力，包括额外的膜系留蛋白水解酶和家族功能 外域脱落。

Project Summary/Abstract Ectodomain shedding is a fundamental process in biology that enables a cell to change the membrane protein topology, affecting cell adhesion and signal transduction pathways. Accordingly, the enzymes that function in ectodomain shedding are vital to human health and dysregulated in disease pathogenesis. Despite the importance of these enzymes in human health, the molecular and atomic details that govern zymogen maturation, activation, substrate selectivity, macromolecular inhibition and subcellular compartmentalization are limited to a few examples. The long-term goal of my research program is to study the mechanistic details of membrane tethered enzymes, revealing the universal concepts in ectodomain shedding that underpin enzyme function and the unique properties that position an enzyme into a biological niche. Initially, we are prioritizing the study of the a disintegrin and metalloproteinase family, specifically ADAM17, to launch my research program as a new principal investigator. ADAMs are complex multidomain type1 transmembrane enzyme that have essential functions in development, fertilization and immunity. ADAM17 is credited to process over 80 different protein substrates and have essential functions in immunity, skin morphogenesis and development of heart valve formation. All ADAMs are initially translated as a zymogen, bound by a prodomain that functions to control enzyme latency. ADAM17 zymogen maturation and transport through the cell is dependent on an interaction with a family of inactive rhomboid proteins, iRhom1 and -2. Appropriately, iRhoms are positioned as vital regulators of ADAM17 activity. Despite ADAM17s well characterized roles in development and homeostasis, significant shortcomings persist in understanding the molecular events that regulate enzyme activity, such as zymogen maturation, enzyme activation and association with regulatory proteins. To address these knowledge gaps, my research program will bring new innovative approaches to the field and leverage my experience in structural and cell biology to characterize the atomic details that regulate ADAM17 activity. Specifically, we will determine the atomic details of the ADAM prodomain and uncover the properties that lead to enzyme specificity and inhibition. In addition, we propose to use X-ray crystallography and electron microscopy to reveal the mechanistic details into ADAM17 activation and recognition by the accessory proteins, iRhom1 and 2. Finally, we will use an unbiased proteomic mass spectroscopy approach to specifically evaluate the ADAM17-iRhom complex during enzyme activation in addition to identifying the subcellular compartment and activity modulating proteins in regulating ADAM17 activity. In the next five years, we aim to develop a unified model for ADAM17 regulation and construct the foundation to expand our efforts to include additional membrane tethered proteases and families that function in ectodomain shedding.