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.

项目概要 泛素 (Ub) 与其他蛋白质的共价连接是最广泛和最常见的连接方式之一。 真核细胞调节的多种模式几乎发生在每一种细胞上。 蛋白质在其生命周期的某个时刻存在于细胞中，并且其本身具有高度多样性。 决定产品的命运，蛋白质可能会经历不同的泛素化模式 取决于细胞环境，这种多样性的起源源于蛋白质。 负责 Ub 附着的机制 三种酶 E1、E2 和 E3 协调 过程中，人类编码了几个 E1、几十个 E2 和数百个 E3 在过去的 20 年里，我们提出了有关 E2s 和基因组的基本问题。 E3 发挥作用并促进了对结构、生化和机制的理解 我们的工作始于乳腺癌肿瘤抑制因子 BRCA1/BARD1。 多年来，我们已将其扩展到最早的 RING 型 E3 连接酶之一。 研究了大量的 E3 和 E2，一路上取得了许多意想不到的发现。 蛋白质泛素化在细胞功能中的广泛影响意味着 成分与多种人类疾病和发育问题有关。 这种关联使得 Ub 系统对于直接靶向治疗具有吸引力。 泛素化机制以及重新设计蛋白质泛素化机制的努力 选择性地靶向特定的细胞蛋白都被证明是有效的策略。 转化努力隐含地依赖于机械理解并揭示了良好的力量 尽管该领域明显成熟，但仍然存在基础结构/功能研究。 我们从根本上不了解很多东西，我们不了解全部内容。 由约 30 个人类 E2 进行的生化反应，其中四分之一是 现有数据表明，并非所有 E2 都会发生假定的反应。 将 Ub 连接到赖氨酸侧链上，这意味着存在尚未被泛素化的物种 其次，了解 E2/E3 如何进行单泛素化。 与多泛素化不同，单个 Ub（单 Ub）的附着往往发生在一个位点上。 选择性方式意味着要单泛素化的底物的处理方式与 第三，缺乏关于如何单一化的知识。 Ub 附着影响蛋白质的结构和功能限制了对蛋白质如何 修饰调节关键的细胞过程，包括转录、翻译和 DNA 损害响应等。