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

基本信息

批准号：
10677607
负责人：
Rishi Patel
金额：
$ 4.77万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10677607
关键词：
26S proteasome Address Affect Affinity Amides Amines Attenuated Azides Binding Binding Proteins Binding Sites Biochemical Biology Biophysics Biotinylation 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 Legionella Life Ligation Link Liquid Chromatography Lysine Malignant Neoplasms Maps Mediating Methods Modeling Modification Molecular Molecular Conformation NF-kappa B Nerve Degeneration Organism Pacific Northwest Pathogenesis Peptides Phenylalanine Polymers Post-Translational Protein Processing Protein Chemistry Proteins Proteome Proteomics Reader Regulation Reporting Research Role Salmonella Scheme Shapes Signal Induction Signal Transduction Site Specificity Structure Surface System Tyrosine Ubiquitin Ubiquitin family Ubiquitination Virulence X-Ray Crystallography analog aqueous crosslink experimental study insight intein interest mutation screening protein complex protein function protein protein interaction receptor reconstitution tandem mass spectrometry ubiquitin-protein ligase

26S proteasome Address Affect Affinity Amides Amines Attenuated Azides Binding Binding Proteins Binding Sites Biochemical Biology Biophysics Biotinylation 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 Legionella Life Ligation Link Liquid Chromatography Lysine Malignant Neoplasms Maps Mediating Methods Modeling Modification Molecular Molecular Conformation NF-kappa B Nerve Degeneration Organism Pacific Northwest Pathogenesis Peptides Phenylalanine Polymers Post-Translational Protein Processing Protein Chemistry Proteins Proteome Proteomics Reader Regulation Reporting Research Role Salmonella Scheme Shapes Signal Induction Signal Transduction Site Specificity Structure Surface System Tyrosine Ubiquitin Ubiquitin family Ubiquitination Virulence X-Ray Crystallography analog aqueous crosslink experimental study insight intein interest mutation screening protein complex protein function protein protein interaction receptor reconstitution tandem mass spectrometry ubiquitin-protein ligase

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项目摘要

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.

项目摘要泛素（UB）是对真核生物学至关重要的主要后翻译修饰剂。 UB系统中继通过其附着和脱离底物蛋白质对细胞过程的复杂控制，产生到“泛素代码”。在此代码中，放松管制具有严重的后果，主要是疾病发病机理。在与其他翻译后的修饰形成鲜明对比，泛素化具有许多不同的口味。随后将一个UB部分的C端附着在底物赖氨酸居住或八个 Amines（Met1，Lys6，Lys11，Lys27，Lys29，Lys33，Lys48，Lys63）的另一个UB部分产生了独特的UB “泛素代码”的解码器或读者识别的链接。这些UB受体有助于重要功能，例如炎症反应和DNA损伤修复。过去几个研究几十年来，已经为删除“泛素代码”的作家提供了重要的见解但是，相比之下，我们对这些UB受体和UB相互作用蛋白的理解受到限制。这个建议描述使用基于UB的问题用于分析UBD和UB依赖性蛋白质 - 蛋白质相互作用使用扩展的蛋白质化学。在两个目标中，我们利用遗传代码扩展和液体色谱串联质谱法以解决该项目的目标：开发一种识别的方法并在结构上表征了UB依赖性蛋白质 - 蛋白质相互作用。目标1。识别瞬时交互使用光接性生物素化的UB问题和结构表征前景的真核形象的UBD 使用化学选择性连接和X射线晶体学的候选者。瞄准2。发展一种手段使用无可迹纹刺uding的化学（聚）泛素化，以获取均匀（poly）UB问题和（聚）泛素化的底物蛋白。这将应用于26S的非典型Lys29泛素化蛋白酶体受体RPN13，E3连接酶UBE3C的调节作用。这些方法中的UB化学方法生物学将使我们能够研究UB依赖性的蛋白质 - 蛋白质相互作用及其生物物理对应物重新建立化学诱捕非共价相互作用所需的元素，该功能当前限制了 UB系统的研究。综上所述，我们试图描述这些基于UB的互动如何生化使用结构证据来衰减功能并证实这些发现。