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

化学部生命过程化学计划资助佐治亚州立大学的尹俊博士和弗吉尼亚联邦大学的阿什顿·克罗普博士研究泛素(UB)修饰蛋白质的作用，作为细胞应对氧化损伤的基本机制。UB是一种小分子蛋白质，参与调节包括线粒体在内的各种细胞过程的相互作用。线粒体是细胞中产生能量的“动力源”，但与此同时，它们也是“重污染者”，会向当地环境中释放活性氧种(ROS)，从而对细胞造成损害。在衰老过程中，细胞管理线粒体产生的ROS的能力较弱，累积的损伤可能会导致脑损伤和人类寿命结束时的心力衰竭。为了自我保护，细胞用E3 UB连接酶等酶启动一个称为有丝分裂的过程，在这个过程中用UB标记受损的线粒体，这样受损的线粒体就可以被分离并移除，然后它们就会在细胞中释放大量的破坏性ROS。Yen和Cropp的联合团队将组合使用蛋白质工程、基于反应性的化学探针设计和蛋白质分析方法来组装一个集成的技术平台，以阐明UB修饰蛋白质导致的有丝分裂调控机制。他们的工作将定义支撑ROS反应的细胞步骤，并揭示这些过程的崩溃可能如何促进人类衰老。该项目的综合外展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。这两个城市城区高度多样化的学生群体将受益于早期接触到科学技术的发展和在化学驱动的生物研究方面的有益职业机会。UB对蛋白质的修饰在感知受损线粒体和通过有丝分裂去除线粒体的信号中起着至关重要的作用。已发现多种E3UB连接酶被招募到受损的线粒体上，并用UB链修饰它们以诱导有丝分裂。目前，E3的泛素化靶点还没有得到很好的描述，因此E3调节的有丝分裂吞噬途径还不确定。此外，E3可以在受损的线粒体周围组装一个由不同连接组成的UB链网络，但关于UB链是如何被去泛素酶(DUBS)修剪和编辑以抵消E3催化的UB链延伸反应的知之甚少。来自Yen和Cropp实验室的联合团队计划通过在有丝分裂的初始阶段使用一种名为正交UB转移(OUT)的创新蛋白质组平台来分析各种E3的底物，以填补这些知识空白。OUT将能够识别E3的直接底物，以绘制调节有丝分裂的相关细胞信号通路。通过比较不同E3的底物谱，该团队将区分E3在有丝分裂调控中的作用。此外，该团队还开发了一种创新的方法来合成基于非天然氨基酸(UAA)掺入的连接特异性UB链和UB底物连接物，并计划使用UB连接物作为探针来捕获调节有丝分裂的DUB。通过完成这项研究计划，该团队希望绘制出调节有丝分裂的蛋白质泛素化途径，并在编辑UB链的不同连接时识别与E3相互作用的DUB，以正确编码有丝分裂信号。这项工作的意义是开发强大的技术来绘制由UB转移介导的细胞信号通路，阐明E3和DUB在有丝分裂信号转导中的作用，并破译线粒体质量控制的细胞机制以中和ROS的损害。这一奖项反映了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。

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.