CAREER: Elucidating the role of ATP in Cytosolic Iron Sulfur Cluster Biogenesis

职业：阐明 ATP 在胞质铁硫簇生物发生中的作用

• 批准号: 1555295
• 负责人: Deborah Perlstein
• 金额: $ 77.76万
• 依托单位: Trustees of Boston University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1555295&HistoricalAwards=false
• 关键词: CAREER; Elucidating; role; ATP; Cytosolic

With this CAREER award from the Chemistry of Life Processes Program, Professor Deborah Perlstein at Boston University is investigating how clusters of iron and sulfur are assembled and transferred to their appropriate proteins in the cytosol of eukaryotic cells. Cluster biosynthesis is essential for cell growth and division since proteins requiring iron sulfur clusters play central roles in DNA replication and repair as well as many other important cellular functions. Despite its unquestionable importance, scientists understand remarkably little about the molecular details of how these iron sulfur clusters are synthesized and ultimately transferred into the appropriate proteins/enzymes. Professor Perlstein's lab is using a combination of genetic, biochemical, and biophysical approaches to shed light on how assembly of a cluster on a protein scaffold takes place as well as on the subsequent steps required to ultimately insert the cluster into a protein. The research provides an excellent training environment for students at the undergraduate and graduate students as well as postdoctoral fellows, equipping them with the expertise required to become leaders in research at the chemistry-biology interface. Furthermore, by developing the curriculum for an undergraduate biochemistry lecture and laboratory course and a K-12 STEM outreach program both intimately connected with the research plan, Professor Perlstein's work broadens interest in biochemistry and biotechnology research which forms a significant portion of the job opportunities in the Boston area. The specific goal of this project is to uncover why the yeast cytosolic iron sulfur cluster scaffolding proteins require ATP hydrolysis for their activity. Since the yeast enzyme and its homologs in the Mrp/Nbp35 family of cluster scaffolding ATPases form a distinct subfamily within the deviant Walker A family of P-loop NTPases, Dr. Perlstein and her group hypothesize that ATP hydrolysis is utilized to induce a conformational change within the scaffold that is used to coordinate assembly of a nascent cluster on the scaffold with the transfer of this cluster to a recipient. This nucleotide driven switch could impact cluster assembly and/or transfer functions of the scaffold via direct allosteric communication between the cluster scaffolding site and the ATPase site, via regulation of dynamic interactions with other cluster biogenesis proteins, or via a combination of these two mechanisms. By determining how ATPase mutants affect cluster scaffolding activities in vitro, identifying other factors that interact with the scaffold, and elucidating how kinetic mechanism of ATP hydrolysis is affected by scaffolded cluster or other cluster assembly factors, the researchers expect to uncover how nucleotide hydrolysis is exploited by this large family of ATP hydrolyzing iron sulfur cluster scaffolds to coordinate assembly of a nascent cluster within the scaffolding site with the subsequent trafficking steps required to mobilize the cluster from scaffold to target. This research is integrated with an educational plan aimed at exposing K-12 students to research and career opportunities at the chemistry-biology interface via collaboration with Boston University's Learning Resource Network. Furthermore, Professor Perlstein leads the development of an Intensive Biochemistry course for undergraduate chemistry and biology majors tailored to the needs of students planning to pursue research careers.

波士顿大学的 Deborah Perlstein 教授获得了生命过程化学项目颁发的职业奖，正在研究铁和硫簇如何组装并转移到真核细胞细胞质中适当的蛋白质中。簇生物合成对于细胞生长和分裂至关重要，因为需要铁硫簇的蛋白质在 DNA 复制和修复以及许多其他重要的细胞功能中发挥着核心作用。尽管其重要性不容置疑，但科学家们对这些铁硫簇如何合成并最终转化为适当的蛋白质/酶的分子细节知之甚少。 Perlstein 教授的实验室正在结合遗传、生物化学和生物物理方法来阐明蛋白质支架上簇的组装过程以及最终将簇插入蛋白质所需的后续步骤。该研究为本科生、研究生以及博士后提供了良好的培训环境，使他们具备成为化学-生物学领域研究领导者所需的专业知识。 此外，通过开发与研究计划密切相关的本科生生物化学讲座和实验室课程以及 K-12 STEM 外展计划，Perlstein 教授的工作扩大了人们对生物化学和生物技术研究的兴趣，这构成了波士顿地区就业机会的重要组成部分。该项目的具体目标是揭示为什么酵母胞质铁硫簇支架蛋白需要 ATP 水解才能发挥其活性。 由于簇支架 ATP 酶的 Mrp/Nbp35 家族中的酵母酶及其同源物在 P 环 NTP 酶的异常 Walker A 家族中形成了一个独特的亚家族，Perlstein 博士和她的团队假设 ATP 水解用于诱导支架内的构象变化，该变化用于协调支架上新生簇的组装以及该簇的转移。 集群到接收者。这种核苷酸驱动的开关可以通过簇支架位点和 ATP 酶位点之间的直接变构通讯、通过调节与其他簇生物发生蛋白的动态相互作用或通过这两种机制的组合来影响支架的簇组装和/或转移功能。通过确定 ATPase 突变体如何影响体外簇支架活性，识别与支架相互作用的其他因素，并阐明支架簇或其他簇组装因素如何影响 ATP 水解的动力学机制，研究人员期望揭示这一大家族的 ATP 水解铁硫簇支架如何利用核苷酸水解来协调新生的组装 脚手架站点内的集群，以及将集群从脚手架移动到目标所需的后续贩运步骤。 这项研究与一项教育计划相结合，旨在通过与波士顿大学学习资源网络的合作，让 K-12 学生获得化学-生物学界面的研究和职业机会。此外，Perlstein 教授还负责为化学和生物学专业的本科生开发强化生物化学课程，以满足计划从事研究职业的学生的需求。