Elucidating the Roles of E3 Ligases and Deubiquitinating Enzymes in Mitophagy by Orthogonal Ubiquitin Transfer and Linkage-Specific Ubiquitin Probes

通过正交泛素转移和连接特异性泛素探针阐明 E3 连接酶和去泛素化酶在线粒体自噬中的作用

• 批准号: 2109051
• 负责人: Jun Yin
• 金额: $ 67.8万
• 依托单位: Georgia State University Research Foundation, Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2109051&HistoricalAwards=false
• 关键词: Elucidating; Roles; E3; Ligases; Deubiquitinating

The Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Jun Yin from Georgia State University and Dr. Ashton Cropp of Virginia Commonwealth University to investigate the roles of protein modification by ubiquitin (UB) as a fundamental mechanism for the cell to cope with oxidative damage. UB is a small protein mediating protein interaction for the regulation of various cellular processes, including those carried out by mitochondria. Mitochondria are “powerhouses” in the cell for energy production, but at the same time, they are “heavy polluters” that release reactive oxygen species (ROS) into their local environments, which inflicts damage on cells. During the process of aging, cells are less capable of managing ROS produced by mitochondria, and the accumulated damage could result in brain damage and heart failure at the end of the human life span. For self-protection, cells initiate a process called mitophagy with enzymes such as E3 UB ligases, where flagging the damaged mitochondria with UB takes place so damaged mitochondria can be isolated and removed before they release a substantial amount of damaging ROS in the cell. The joint teams of Yin and Cropp will assemble an integrated technology platform with a combined use of protein engineering, reactivity-based chemical probe design, and protein analysis methods to elucidate the mechanisms of mitophagy regulation that result from protein modification by UB. Their work will define the cellular steps underpinning ROS response and reveal how the breakdown of such processes may contribute to human aging. The integrated outreach program of the project will provide substantial training opportunities to K-12 students from local communities in Atlanta, GA and Richmond, VA. The highly diverse student body in the urban areas of the two cities will benefit from early exposure to the development of science and technology and the rewarding career opportunities in chemically driven biological research. Protein modification by UB plays an essential role in sensing damaged mitochondria and signaling their removal by mitophagy. Various E3 UB ligases have been found to be recruited to damaged mitochondria and decorate them with UB chains to induce mitophagy. Currently, the ubiquitination targets of the E3s are not well characterized, so the E3-regulaued mitophagy pathways are undefined. Furthermore, E3s can assemble a network of UB chains of diverse linkages around damaged mitochondria, but little is known about how the UB chains are trimmed and edited by deubiquitinating enzymes (DUBs) to counteract the UB chain extension reaction catalyzed by the E3s. The joint team from the Yin and Cropp laboratories plans to fill these knowledge gaps by profiling the substrates of various E3s at the initiation phase of mitophagy with an innovative proteomic platform known as orthogonal UB transfer (OUT). OUT will enable the identification of the direct substrates of the E3s to map the associated cell signaling pathways regulating mitophagy. By comparing the substrate profiles of various E3s, the team will differentiate the roles of E3s in mitophagy regulation. Furthermore, the team has developed an innovative method to synthesize linkage-specific UB chains and UB-substrate conjugates based on unnatural amino acid (UAA) incorporation and plans to use the UB conjugates as probes to capture DUBs that regulate mitophagy. By accomplishing the research plan, the team expects to map the protein ubiquitination pathways regulating mitophagy and identify DUBs interacting with the E3s in editing diverse linkages of UB chains for proper encoding of the mitophagy signals. The significance of the work is to develop powerful technologies to map cell signaling pathways mediated by UB transfer, elucidate the actions of E3s and DUBs in mitophagy signaling, and decipher the cellular mechanisms underlying mitochondria quality control to neutralize the damage by ROS.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.

化学系生命过程化学项目资助格鲁吉亚州立大学的Jun Yin博士和弗吉尼亚联邦大学的Ashton Cropp博士研究泛素（UB）修饰蛋白质的作用，作为细胞科普氧化损伤的基本机制。UB是一种介导蛋白质相互作用的小蛋白，用于调节各种细胞过程，包括线粒体进行的过程。线粒体是细胞中产生能量的“发电站”，但同时，它们也是“重污染者”，将活性氧（ROS）释放到其局部环境中，对细胞造成损害。在衰老过程中，细胞管理线粒体产生的ROS的能力降低，累积的损伤可能导致人类寿命结束时的脑损伤和心力衰竭。为了自我保护，细胞用酶如E3 UB连接酶启动一个称为线粒体自噬的过程，其中用UB标记受损的线粒体，因此受损的线粒体可以在细胞中释放大量破坏性ROS之前被分离和去除。Yin和Cropp的联合团队将组装一个综合技术平台，结合使用蛋白质工程，基于反应性的化学探针设计和蛋白质分析方法，以阐明UB修饰蛋白质导致的线粒体自噬调控机制。他们的工作将定义支持ROS反应的细胞步骤，并揭示这些过程的分解如何导致人类衰老。该项目的综合推广计划将为来自佐治亚州亚特兰大和弗吉尼亚州里士满当地社区的K-12学生提供大量培训机会。这两个城市的城市地区高度多样化的学生群体将受益于早期接触科学和技术的发展，以及化学驱动生物研究的有益职业机会。UB对蛋白质的修饰在感知受损线粒体和通过线粒体自噬将其清除的信号中起着至关重要的作用。已经发现各种E3 UB连接酶被募集到受损的线粒体并用UB链装饰它们以诱导线粒体自噬。目前，E3的泛素化靶点还没有得到很好的表征，因此E3调节的线粒体自噬途径还不确定。此外，E3可以在受损的线粒体周围组装具有不同连接的UB链网络，但关于UB链如何被去泛素化酶（DUBs）修剪和编辑以抵消E3催化的UB链延伸反应知之甚少。来自Yin和Cropp实验室的联合团队计划通过使用称为正交UB转移（OUT）的创新蛋白质组学平台在线粒体自噬的起始阶段分析各种E3的底物来填补这些知识空白。OUT将能够鉴定E3的直接底物，以绘制调节线粒体自噬的相关细胞信号通路。通过比较各种E3的底物谱，研究小组将区分E3在线粒体自噬调控中的作用。此外，该团队还开发了一种基于非天然氨基酸（UAA）掺入合成连接特异性UB链和UB底物缀合物的创新方法，并计划使用UB缀合物作为探针来捕获调节线粒体自噬的DUB。通过完成研究计划，该团队希望绘制调节线粒体自噬的蛋白质泛素化途径，并确定DUB与E3相互作用，编辑UB链的不同连接，以正确编码线粒体自噬信号。这项工作的意义在于开发强大的技术来定位UB转移介导的细胞信号通路，阐明E3和DUBs在线粒体自噬信号传导中的作用，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进行评估，被认为值得支持的搜索.

项目成果

Effects of UBE3A on Cell and Liver Metabolism through the Ubiquitination of PDHA1 and ACAT1.

• DOI: 10.1021/acs.biochem.2c00624
• 发表时间: 2023-04-04
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Peng, Kangli;Wang, Shirong;Liu, Ruochuan;Zhou, Li;Jeong, Geon H.;Jeong, In Ho;Liu, Xianpeng;Kiyokawa, Hiroaki;Xue, Bingzhong;Zhao, Bo;Shi, Hang;Yin, Jun
• 通讯作者: Yin, Jun

Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

• DOI: 10.1002/advs.202206068
• 发表时间: 2023-07
• 期刊: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Inhibition of the Ubiquitin Transfer Cascade by a Peptidomimetic Foldamer Mimicking the E2 N-Terminal Helix

• DOI: 10.1021/acs.jmedchem.2c01459
• 发表时间: 2022-12-26
• 期刊: JOURNAL OF MEDICINAL CHEMISTRY
• 影响因子: 7.3
• 作者: Zhou，Li;Jeong，In Ho;Huang，Bo
• 通讯作者: Huang，Bo