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.

获得该奖项后，化学部的生命过程化学计划资助普渡大学的Christine Hrycyna博士和明尼苏达大学的Mark Distefano博士，以确定酵母酿酒酵母整合膜蛋白Ste24的功能。Ste24是一类新的膜蛋白水解酶的创始成员，这种酶催化蛋白质的水解；这些反应裂解原始蛋白质。Ste24的独特之处在于，它在两个不同的位置一个接一个地切割蛋白质(而不是像大多数蛋白酶那样只在一个位置切割蛋白质)，而且它只切割附着在特定脂类上的蛋白质。STE24不仅在反应活性上是独一无二的，而且在结构上也是独一无二的；例如，与迄今描述的另一种膜内蛋白酶相比，它有一个巨大的、充满水的膜内桶形反应室，两端都有盖子。拟议的研究考察了底物如何进入和离开Ste24的小室，它是如何被识别的，以及Ste24是如何介导蛋白质分解的。这项工作为研究生创造了了解Ste24蛋白酶的结构和生化功能的机会，并精通使用处理膜蛋白的方法和工具，对膜蛋白的研究是一个新兴的科学前沿的中心。此外，该研究计划与一个推广计划相结合，通过参与动手研究，让来自经济困难背景的高中生和他们的老师接触膜蛋白生物化学和分子生物学的现代问题和技术。本研究项目旨在利用一系列新的化学探针和生化方法在分子水平上了解Ste24的作用机制。通过结构导向突变、酶分析、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模拟，确定了Ste24结合和介导膜内α-因子底物前体蛋白降解的方式。此外，研究的重点是利用定点突变和生化分析来鉴定通向小室内部的四个大入口中的哪一个用于从酶中进出α-因子。这些研究提供了对STE24作用机制和底物专一性的更好的理解，并揭示了这一保守的蛋白质家族独有的新的基本性质。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.