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

合作研究：Ste24 的机制，一种新型整体膜锌金属蛋白酶，可促进膜内室内的催化作用

• 批准号: 1905156
• 负责人: Christine Hrycyna
• 金额: $ 35.7万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1905156&HistoricalAwards=false
• 关键词: Collaborative; Research; Mechanism; Ste24; Novel

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.

有了这个奖项，化学部门的化学过程计划是资助DRS。来自普渡大学的Christine Hrycyna和明尼苏达大学的Mark Distefano定义了酵母S.酿酒酵母的整体膜蛋白Ste24的功能。 Ste24是一类新型膜蛋白酶的创始成员，该酶是催化蛋白质水解的酶。这些反应裂解了原始蛋白质。 Ste24所独有的是，它在两个不同的位点上裂解蛋白质（而不是像大多数蛋白酶一样在一个位点上裂解蛋白质），并且只能切割附着在特定脂质上的蛋白质。 Ste24不仅在反应性上，而且在结构上都是独一无二的。例如，与迄今为止描述的另一个膜内蛋白酶相比，它具有大型，水填充的膜内枪管内反应室，两端都被盖住。拟议的研究检查了底物如何进入和退出Ste24腔室，如何被识别以及Ste24如何介导蛋白水解。这项工作为研究生提供了了解Ste24蛋白酶的结构和生化功能的机会，并精通使用方法和工具与膜蛋白一起使用，研究对于新兴的科学领域至关重要。此外，该研究计划与一项外展计划集成在一起，该计划使高中生从经济偏离的背景及其老师及其老师揭示了膜蛋白质生物化学和分子生物学的现代问题和技术，通过参与动手研究的研究。本研究项目旨在了解STE24在Modecar comport a drogners afio and a array boio boio andaray boray botaray saray saray and array saray sears a array and array searn of array searn a dray and ar are a raray lode and bo are bo are ar a rare and array consectiqual s。 Ste24使用结构引导的诱变，酶促测定，合成肽底物和抑制剂，PhotoAbrate-Analog-Analog-Analog-Analog-Analog探针，时间分辨出荧光荧光模型和计算模型，使用结构引导的诱变，合成肽底物和抑制作用来确定STE24结合和介导A因子底物前体的蛋白水解。此外，该研究的重点是使用位于定位的诱变和生化试验的四个大型门户中的哪个大型门户中的哪个识别导致腔室内部的四个大门口中的哪个。这些研究可以更好地了解Ste24的作用机理和底物特异性，并揭示了这种保守的蛋白酶家族独有的新基本特性。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛的影响审查标准通过评估来获得支持的。

项目成果

Photoswitchable Isoprenoid Lipids Enable Optical Control of Peptide Lipidation.

• DOI: 10.1021/acschembio.2c00645
• 发表时间: 2022-10-21
• 期刊: ACS CHEMICAL BIOLOGY
• 影响因子: 4
• 作者: Morstein, Johannes;Bader, Taysir;Cardillo, Ariana L.;Schackmann, Julian;Ashok, Sudhat;Hougland, James L.;Hrycyna, Christine A.;Trauner, Dirk H.;Distefano, Mark D.
• 通讯作者: Distefano, Mark D.