CAREER: Biochemical and Structural Characterization of Intramembrane Proteases

从细菌到人类，基本的分子信号控制着细胞行为的多个方面。最近，一种被称为调节性膜内蛋白分解(RIP)的通用信号传递范式被发现。在RIP中，膜蛋白在其膜内区域被切割，从而产生水溶性信号。这些信号重新定位在细胞内或细胞外介质中，并作为细胞新陈代谢和发育的调节器。膜内切割酶构成了一类新的完整的膜蛋白。这项研究的目的是通过关注一个普遍存在的称为菱形体的膜内蛋白水解酶家族来揭示RIP的分子机制。这项工作将分为三个方面：(I)调节菱形体膜内酶活性的因素的生化研究；(Ii)识别区分好的和不好的底物的特征，并确定切割位点的准确位置；(Iii)阐明菱形的三维结构，以便从结构角度解释功能数据。为了实现这些目标，生化和生物物理方法将与结构技术相结合，如高分辨率电子低温显微镜、核磁共振和X射线结晶学。广泛影响：纽约城市学院有相当大比例的少数族裔和新移民学生代表不足。概述的多学科研究将吸引和培训来自生物、化学和物理一般领域的研究生和本科生，并推动他们详细了解生物过程。这些学生将获得膜蛋白研究的经验，并接受现代结构生物学的一般原理和应用方面的培训，特别是电子冷冻显微镜。在纽约结构生物学中心的领导下，每年将开发和提供一门关于大分子组装的电子冷冻显微镜的研究生课程，并向纽约市的研究生社区开放。

From bacteria to humans, essential molecular signals control multiple aspects of cellular behavior. Recently, a universal signaling paradigm, termed regulated intramembrane proteolysis, (RIP) has been discovered. In RIP, membrane proteins are cleaved in their intramembrane regions resulting in water-soluble signals. These signals relocate within the cell or to the extracellular media and act as modulators of cell metabolism and development. The intramembrane cutting enzymes constitute a novel class of integral membrane proteins. The goal of this research is to unravel the molecular mechanisms underlying RIP by focusing on a ubiquitous intramembrane protease family called Rhomboids. The work will be divided in three fronts: (I) biochemical study of the factors that regulate Rhomboid intramembrane protease activity; (II) identification of the features that distinguish good substrates from bad ones and determine the precise location of the cleavage site; and (III) elucidation of the three-dimensional structure of Rhomboid to allow interpretation of functional data in structural terms. To achieve these goals, biochemical and biophysical approaches will be used in combination with structural techniques such as high-resolution electron-cryo microscopy, nuclear magnetic resonance and X-ray crystallography.Broader impact: The City College of New York has a substantial proportion of underrepresented minority and new immigrant students. The outlined multidisciplinary research will draw and train graduate and undergraduate students from the general areas of biology, chemistry and physics with a drive to understand biological processes in detail. These students will gain experience in membrane protein research and be trained in the principles and applications of modern structural biology in general, and of electron cryo-microscopy in particular. A graduate course in electron cryo-microscopy of macromolecular assemblies will be developed and offered every year, under the leadership of the New York Structural Biology Center, and open to the New York City graduate student community.