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，一个普遍的信号模式，被称为调节膜内蛋白水解，（RIP）已被发现。在RIP中，膜蛋白在其膜内区域被切割，导致水溶性信号。这些信号在细胞内或细胞外介质中重新定位，并作为细胞代谢和发育的调节剂。膜内切割酶是一类新的膜蛋白。这项研究的目标是通过关注一个普遍存在的称为Rhomboids的膜内蛋白酶家族来解开RIP的分子机制。这项工作将分为三个方面：（I）调节Rhomboid膜内蛋白酶活性的因素的生物化学研究;（II）识别区分好底物和坏底物的特征，并确定切割位点的精确位置;和（III）阐明Rhomboid的三维结构，以便在结构方面解释功能数据。为了实现这些目标，将结合使用生物化学和生物物理学方法以及结构技术，如高分辨率电子低温显微镜、核磁共振和X射线晶体学。更广泛的影响：纽约城市学院有相当大比例的代表性不足的少数民族和新移民学生。概述的多学科研究将吸引和培养来自生物学，化学和物理学一般领域的研究生和本科生，以详细了解生物过程。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原理和应用的培训，特别是电子冷冻显微镜。在纽约结构生物学中心的领导下，每年将开发和提供大分子组装体电子冷冻显微镜研究生课程，并向纽约市研究生社区开放。

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.