Structure and Regulation of Ectodomain Sheddases in Development and Disease

项目总结/摘要 胞外域脱落是生物学中的一个基本过程，它使细胞能够改变膜蛋白 拓扑结构，影响细胞粘附和信号转导途径。因此，在细胞中起作用的酶 胞外域脱落对人类健康至关重要，并且在疾病发病机制中失调。尽管 这些酶在人类健康中的重要性，控制酶原的分子和原子细节 成熟、活化、底物选择性、大分子抑制和亚细胞区室化是 仅限于几个例子。我研究计划的长期目标是研究 膜系酶，揭示了细胞外结构域脱落的普遍概念， 酶的功能和独特的性质，使酶进入生物生态位。最初我们 正在优先研究解整合素和金属蛋白酶家族，特别是ADAM 17， 作为一名新的首席研究员。 亚当斯是复杂的多结构域1型跨膜酶，在发育中具有重要功能， 受精和免疫。ADAM 17被认为可以处理80多种不同的蛋白质底物，并具有重要的生物学功能。 在免疫、皮肤形态发生和心脏瓣膜形成的发育中起作用。所有亚当斯最初都是 翻译为酶原，由一个功能控制酶潜伏期的前结构域结合。ADAM 17酶原 成熟和通过细胞的运输依赖于与非活性菱形蛋白家族的相互作用。 iRhom 1和iRhom 2。iRhoms被恰当地定位为ADAM 17活性的重要调节剂。尽管 ADAM 17在发育和体内平衡中的作用得到了很好的表征，但仍然存在显著的缺点， 了解调节酶活性的分子事件，如酶原成熟，酶 激活和与调节蛋白的结合。为了解决这些知识差距，我的研究计划将 为该领域带来新的创新方法，并利用我在结构和细胞生物学方面的经验， 描述了调节ADAM 17活性的原子细节。具体来说，我们将确定原子细节 并揭示导致酶特异性和抑制的特性。此外，本发明还提供了一种方法， 我们建议使用X射线晶体学和电子显微镜来揭示ADAM 17的机制细节 辅助蛋白iRhom 1和2的激活和识别。最后，我们将使用一个公正的蛋白质组学 质谱法，以具体评估在酶活化过程中的ADAM 17-iRhom复合物， 除了鉴定亚细胞区室和调节ADAM 17活性的蛋白质外， 活动在未来五年内，我们的目标是开发一个统一的ADAM 17调控模型，并构建一个 基金会，以扩大我们的努力，包括额外的膜拴系蛋白酶和家庭的功能，在 胞外域脱落

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.