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

基本信息

批准号：
1905204
负责人：
Mark Distefano
金额：
$ 34.2万
依托单位：
University of Minnesota-Twin Cities
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 2023-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1905204&HistoricalAwards=false
关键词：
Collaborative Research Mechanism Ste24 Novel

项目摘要

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.

通过该奖项，化学部门的生命过程化学项目正在资助博士。普渡大学的 Christine Hrycyna 和明尼苏达大学的 Mark Distefano 定义了酿酒酵母整合膜蛋白 Ste24 的功能。 Ste24 是一类新型膜蛋白酶（催化蛋白质水解的酶）的创始成员；这些反应会裂解原始蛋白质。 Ste24 的独特之处在于，它在两个不同位点依次裂解蛋白质（而不是像大多数蛋白酶那样仅在一个位点裂解蛋白质），并且仅裂解附着在特定脂质上的蛋白质。 Ste24不仅在反应活性上而且在结构上都是独一无二的；例如，与迄今为止描述的其他膜内蛋白酶相比，它具有一个两端加盖的大型、充满水的膜内桶形反应室。拟议的研究考察了底物如何进入和离开 Ste24 的腔室、如何被识别以及 Ste24 如何介导蛋白水解。这项工作为研究生创造了机会，让他们了解 Ste24 蛋白酶的结构和生化功能，并熟练使用膜蛋白的方法和工具，膜蛋白的研究是新兴科学前沿的核心。此外，该研究项目还与一项外展项目相结合，该项目通过参与实践研究，让来自经济困难背景的高中生及其老师了解膜蛋白生物化学和分子生物学的现代问题和技术。该研究项目旨在了解使用一系列新型化学探针和生化方法在分子水平上研究 Ste24 的作用机制。 Ste24 结合并介导膜内 a 因子底物前体蛋白水解的方式是通过结构引导诱变、酶分析、合成肽底物和抑制剂、光亲和底物模拟探针、时间分辨荧光光谱和计算建模。此外，该研究的重点是使用定点诱变和生化测定来识别通向室内部的四个大门户中的哪一个用于a因子进入和退出酶。这些研究使人们更好地了解 Ste24 的作用机制和底物特异性，并揭示了这个保守的蛋白酶家族独特的新基本特性。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力优点和技术进行评估，被认为值得支持。更广泛的影响审查标准。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Methoxy-Substituted Nitrodibenzofuran-Based Protecting Group with an Improved Two-Photon Action Cross-Section for Thiol Protection in Solid Phase Peptide Synthesis

具有改进的双光子作用截面的甲氧基取代的硝基二苯并呋喃保护基，用于固相肽合成中的硫醇保护

DOI：
10.1021/acs.joc.9b02751
发表时间：
2019-12
期刊：
The Journal of Organic Chemistry
影响因子：
0
作者：
Bader, Taysir K.;Xu, Feng;Hodny, Michael H.;Blank, David A.;Distefano, Mark D.
通讯作者：
Distefano, Mark D.

Chapter Eight - Synthesis and NMR Characterization of the Prenylated Peptide, a-Factor

第八章 - 异戊二烯化肽 a 因子的合成和 NMR 表征