Expanding Mechanistic Insights into Protein Ubiquitylation

扩展对蛋白质泛素化的机制见解

• 批准号: 10330645
• 负责人: Rachel E Klevit
• 金额: $ 68.76万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330645
• 关键词: Address; Affect; BARD1 gene; BRCA1 gene; Biochemical; Biochemical Reaction; Biophysics; Cell physiology; Cells; DNA Damage; Data; Development; Enzymes; Family; Functional disorder; Genetic Transcription; Human; Human Genome; Knowledge; Lysine; Modification; Process; Protein Engineering; Proteins; Reaction; Research; Site; Structure; System; Translations; Tumor Suppressor Proteins; Ubiquitin; Work; cell growth regulation; human disease; insight; malignant breast neoplasm; member; protein function; response; stem; therapeutic target; ubiquitin-protein ligase

PROJECT SUMMARY Covalent attachment of ubiquitin (Ub) to other proteins is among the most widespread and diverse modes of eukaryotic cellular regulation. The modification occurs on practically every protein in a cell at some point in its lifetime and is itself highly diverse. The type of ubiquitylation determines a product’s fate and a protein may undergo different modes of ubiquitylation depending on cellular circumstances. The origins of this diversity stem from the protein machinery responsible for Ub attachment. A trio of enzymes, E1, E2, and E3 coordinate the process, with several E1s, dozens of E2s, and many hundreds of E3s encoded in the human genome. Over the past 20 years, we have asked fundamental questions about how E2s and E3s work and have contributed to the structural, biochemical, and mechanistic understandings of the field. Our work began with the breast cancer tumor suppressor, BRCA1/BARD1 that was among the earliest RING-type E3 ligases to be identified. Over the years, we have expanded to study numerous E3s and E2s, making many unexpected discoveries along the way. The wide reach of protein ubiquitylation in cellular function means that dysfunction of components is associated with myriad human diseases and developmental issues. Such associations make the Ub system attractive for therapeutic targeting. Direct targeting of the ubiquitylation machinery as well as efforts to re-engineer protein ubiquitylation machinery to selectively target a specific cellular protein are both proving to be powerful strategies. Such translational efforts rely implicitly on mechanistic understanding and reveal the power of well- grounded structure/function research. Despite the apparent maturity of the field, there is still much we do not understand at a fundamental level. We do not know the full range of biochemical reactions carried out by the ~30 human E2s as fully one-quarter of these are uncharacterized. Existing data reveal that not all E2s carry out the presumed reaction that attaches Ub to lysine sidechains, implying the existence of ubiquitylated species that have yet to be investigated in cells. Second, understanding of how E2/E3s carry out mono-ubiquitylation is lacking. Unlike poly-ubiquitylation, attachment of a single Ub (mono-Ub) tends to occur in a site- selective manner implying that substrates to be mono-ubiquitylated are handled differently from those destined to have chains built upon them. Third, lack of knowledge regarding how mono- Ub attachment affects the structure and function of proteins limits understanding of how the modification regulates critical cellular processes including transcription, translation, and DNA damage response, among others.

项目总结 泛素(Ub)与其他蛋白质的共价结合是最广泛和最广泛的 真核细胞调控的多种模式。这种修改几乎发生在每一次 蛋白质存在于细胞生命周期的某一时刻，本身就是高度多样化的。泛素化的类型 决定一种产品的命运，蛋白质可能经历不同的泛素化模式 这取决于手机的环境。这种多样性的起源源于蛋白质 负责Ub附着的机器。三种酶，E1，E2和E3协调 进程，在人类中编码了几个E1、数十个E2和数百个E3 基因组。在过去的20年里，我们问了一些基本的问题，比如E2和 E3S工作，并对结构、生化和机械方面的理解做出了贡献 在赛场上。我们的工作始于乳腺癌肿瘤抑制基因BRCA1/BARD1，即 是最早发现的环型E3连接酶之一。多年来，我们已经扩展到 研究大量的E3和E2，在此过程中有许多意想不到的发现。 蛋白质泛素化在细胞功能中的广泛应用意味着 这些成分与无数的人类疾病和发育问题有关。是这样的 协会使Ub系统对治疗靶向具有吸引力。直接瞄准 泛素化机制以及重新设计蛋白质泛素化机制以 事实证明，选择性地针对特定的细胞蛋白质都是有效的策略。是这样的 翻译努力隐含地依赖于机械性的理解，并揭示了井的力量 接地结构/功能研究。尽管该油田表面上已经成熟，但仍有 许多我们在根本层面上不理解的东西。我们不知道全系列的 由大约30个人类雌二醇组进行的生化反应，其中整整四分之一是 没有特征的。现有数据显示，并不是所有的E2都会做出假定的反应 将Ub连接到赖氨酸侧链上，意味着存在尚未发生泛素化的物种 在细胞中进行研究。第二，对E2/E3如何进行单一泛素化的理解是 缺乏。与多泛素化不同，单个Ub(Mono-Ub)的附着往往发生在一个位置- 选择性方式意味着要进行单一泛素化的底物的处理方式不同于 那些注定要戴上锁链的人。第三，缺乏关于单核细胞如何- UB附着影响蛋白质的结构和功能限制了人们对 修饰调控关键的细胞过程，包括转录、翻译和DNA 损害反应，以及其他。