Collaborative Research: Mechanism of Ste24, a Novel Integral Membrane Zinc Metalloprotease that Promotes Catalysis Within an Intramembrane Chamber

凭借这一奖项，化学部门的生命过程化学项目正在资助普渡大学的克莉丝汀·赫里西纳博士和明尼苏达大学的马克·迪斯特法诺博士来定义酵母菌S.酿酒酵母整合膜蛋白Ste 24的功能。Ste 24是一类新型膜蛋白酶的创始成员，这些酶催化蛋白质的水解;这些反应切割原始蛋白质。 Ste 24的独特之处在于它在两个不同的位点一个接一个地切割蛋白质（而不是像大多数蛋白酶那样仅在一个位点切割蛋白质），并且它只切割附着于特定脂质的蛋白质。 Ste 24不仅在反应性上而且在结构上都是独特的;例如，与迄今为止描述的其他膜内蛋白酶相比，它具有大的、充满水的、膜内桶形反应室，其在两端都被封端。拟议的研究检查底物如何进入和退出Ste 24的腔室，它是如何被识别的，以及Ste 24如何介导蛋白水解。这项工作为研究生创造了机会，既了解Ste 24蛋白酶的结构和生化功能，也精通使用膜蛋白的方法和工具，这些研究是新兴科学前沿的核心。此外，本研究项目还结合了一项拓展计划，通过参与实践研究，让来自经济困难背景的高中生及其教师了解膜蛋白生物化学和分子生物学的现代问题和技术。本研究项目旨在使用一系列新的化学探针和生物化学方法，从分子水平上了解Ste 24的作用机制。Ste 24结合和介导膜内室内a-因子底物前体蛋白水解的方式使用结构引导的诱变、酶测定、合成肽底物和抑制剂、光亲和底物类似物探针、时间分辨荧光光谱和计算建模来确定。此外，该研究的重点是使用定点诱变和生物化学测定来鉴定导致腔室内部的四个大门户中的哪一个用于a因子从酶的进入和退出。这些研究提供了对Ste 24的作用机制和底物特异性的更好理解，并揭示了这个保守的蛋白酶家族所特有的新的基本性质。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Christine Hrycyna from Purdue University and Mark Distefano from the University of Minnesota to define how the yeast S. cerevisiae integral membrane protein Ste24 functions. Ste24 is the founding member of a novel class of membrane proteases, enzymes that catalyze the hydrolysis of proteins; these reactions cleave the original proteins. What is unique to Ste24 is that it cleaves proteins at two different sites one after the other (rather than cleaving the proteins at only one site as the majority proteases do) and it only cleaves proteins that are attached to specific lipids. Ste24 is unique not only in reactivity but also in structure; for example, in contrast to the other of intramembrane proteases described to date, it has a large, water-filled, intramembrane barrel-shaped reaction chamber that is capped at both ends. The proposed studies examine how a substrate enters and exits the chamber of Ste24, how it is being recognized, and how Ste24 mediates the proteolysis. This work creates opportunities for graduate students to both understand the structure and biochemical function of the Ste24 protease, as well as become versed in the use of methods and tools for working with membrane proteins, studies of which are central to an emerging frontier of science. Furthermore, the research program is integrated with an outreach program that exposes high school students from economically-disadvantaged backgrounds and their teachers to modern questions and techniques in membrane protein biochemistry and molecular biology via participation in hands-on research.This research project aims to understand the mechanism of action of Ste24 at the molecular level using an array of novel chemical probes and biochemical methods. The manner in which Ste24 binds and mediates the proteolysis of a-factor substrate precursors inside the intramembrane chamber is determined using structure-guided mutagenesis, enzymatic assays, synthetic peptide substrates and inhibitors, photoaffinity substrate-analog probes, time-resolved fluorescence spectroscopy, and computational modeling. Furthermore, the research focuses on the identification of which of the four large portals that lead to the interior of the chamber are used for the entry and exit of a-factor from the enzyme using site-directed mutagenesis and biochemical assays. These studies provide a better understanding of the mechanism of action and substrate specificity of Ste24 and reveal new fundamental properties unique to this conserved family of proteases.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.