Investigations into ubiquitin binding proteins using structure guided reactivity

使用结构引导反应性研究泛素结合蛋白

• 批准号: 10538227
• 负责人: Rishi Patel
• 金额: $ 4.68万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538227
• 关键词: 26S proteasome; Address; Affect; Affinity; Amides; Amines; Attenuated; Azides; Binding; Binding Proteins; Binding Sites; Biochemical; Biology; Biophysics; C-terminal; Cell physiology; Chemicals; Chemistry; Code; Collaborations; Complex; Coupled; Cysteine; DNA Repair; Data; Deubiquitination; Development; Disease; Elements; Enzymes; Eukaryotic Cell; Genetic Code; Genus Mycobacterium; Goals; Health; Human; Inflammatory Response; Investigation; Knowledge; Laboratories; Lead; Legionella; Life; Ligation; Link; Liquid Chromatography; Lysine; Malignant Neoplasms; Mediating; Methods; Modeling; Modification; Molecular; Molecular Conformation; NF-kappa B; Nerve Degeneration; Organism; Pacific Northwest; Pathogenesis; Peptides; Phenylalanine; Polymers; Polyubiquitin; Post-Translational Protein Processing; Protein Chemistry; Proteins; Proteome; Proteomics; Reader; Regulation; Reporting; Research; Role; Salmonella; Scheme; Signal Transduction; Site; Specificity; Structure; Surface; System; Tyrosine; Ubiquitin; Ubiquitin C; Ubiquitin family; Ubiquitination; Virulence; X-Ray Crystallography; analog; aqueous; base; crosslink; experimental study; insight; intein; interest; mutation screening; protein complex; protein function; protein protein interaction; receptor; reconstitution; tandem mass spectrometry; ubiquitin-protein ligase

PROJECT SUMMARY Ubiquitin (Ub) is a principal post-translational modifier crucial to eukaryotic biology. The Ub system relays an intricate control over cellular processes through its attachment and detachment on substrate protein, giving rise to a “ubiquitin code”. Deregulation in this code has severe consequences, primarily in disease pathogenesis. In contrast to other post-translational modifications being singular, ubiquitination comes in many different flavors. Subsequent attachment of the C-terminus of one Ub moiety onto a substrate lysine residue or one of eight amines (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63) of another Ub moiety gives rise to distinct Ub linkages that are recognized by the decoders or readers of the “ubiquitin code”. These Ub receptors facilitate important functions such as inflammatory response and DNA damage repair. Research over the past few decades has provided critical insight into the writers that establish and erasers that remove the “ubiquitin code”, yet our understanding of these Ub-receptors and Ub-interacting proteins is limited in comparison. This proposal describes the use of Ub-based probes for profiling UBDs and Ub-dependent protein-protein interactions using expanded protein chemistry. In two aims, we leverage genetic code expansion and liquid chromatography-tandem mass spectrometry to address the goal of this project: to develop a means to identify and structurally characterize Ub-dependent protein-protein interactions. Aim 1. Identify transiently interacting UBDs of eukaryotic origin using photoaffinity biotinylated Ub probes and structurally characterize promising candidates using chemo-selective ligation and X-ray crystallography. Aim 2. Develop a means to perform chemical (poly)ubiquitination using the traceless Staudinger ligation for access to homogenous (poly)Ub probes and (poly)ubiquitinated substrate protein. This will be applied to the atypical Lys29 ubiquitination of the 26S proteasome receptor Rpn13, a regulatory effect of the E3 ligase UBE3C. These approaches in Ub chemical biology will allow us to study Ub-dependent protein-protein interactions and their biophysical counterpart by reconstituting the elements needed to chemically trap noncovalent interactions, a feature currently limiting the study of the Ub system. Taken together, we seek to describe how these Ub-based interactions biochemically attenuate function and corroborate these findings using structural evidence.

项目概要 泛素 (Ub) 是对真核生物学至关重要的主要翻译后修饰因子。 通过其在底物蛋白上的附着和分离来复杂地控制细胞过程，从而产生 该代码的放松管制会产生严重后果，主要是在疾病发病机制中。 与其他单一的翻译后修饰相比，泛素化有多种不同的形式。 随后将一个 Ub 部分的 C 末端附着到底物赖氨酸残基或八个之一上 另一个 Ub 部分的胺（Met1、Lys6、Lys11、Lys27、Lys29、Lys33、Lys48、Lys63）产生不同的 Ub 这些 Ub 受体促进了“泛素代码”的解码器或读取器识别的连接。 过去几年对炎症反应和DNA损伤修复等重要功能的研究。 几十年来，我们对建立和删除“泛素代码”的作者提供了批判性的见解， 然而，相比之下，我们对这些 Ub 受体和 Ub 相互作用蛋白的了解是有限的。 描述了使用基于 Ub 的探针来分析 UBD 和 Ub 依赖性蛋白质-蛋白质相互作用 使用扩展蛋白质化学有两个目标，我们利用遗传密码扩展和液体。 色谱-串联质谱法来解决该项目的目标：开发一种方法来识别 目标 1. 识别瞬时相互作用。 使用光亲和生物素化 Ub 探针对真核起源的 UBD 进行结构表征，有前景 目标 2. 开发一种执行方法。 使用无痕施陶丁格连接进行化学（多）泛素化以获得同质（多）Ub 探针 和（多）泛素化底物蛋白 这将应用于 26S 的非典型 Lys29 泛素化。 蛋白酶体受体 Rpn13，对 Ub 化学中的 E3 连接酶 UBE3C 具有调节作用。 生物学将使我们能够研究 Ub 依赖性蛋白质-蛋白质相互作用及其生物物理对应物 重建化学捕获非共价相互作用所需的元素，这一特征目前限制了 综上所述，我们试图描述这些基于 Ub 的生物化学相互作用。 削弱功能并使用结构证据证实这些发现。