Detection and quantitation of branched ubiquitin in polyubiquitinated proteins

多泛素化蛋白中分支泛素的检测和定量

• 批准号: 10058026
• 负责人: Robert Cohen
• 金额: $ 22.5万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-11 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058026
• 关键词: Acute; Adopted; Amines; Biochemical; Cell Cycle Regulation; Cells; Chemicals; Cleaved cell; Code; Color; Complex; DNA Damage; DNA Repair; Detection; Deubiquitinating Enzyme; Digestion; Dipeptides; Escherichia coli; Eukaryotic Cell; Genetic; Growth; Histone Code; In Vitro; Infection; Isomerism; Isotope Labeling; Isotopes; Light; Link; Lysine; Maintenance; Mass Spectrum Analysis; Mediating; Membrane; Methods; Names; Nucleosomes; Peptide Hydrolases; Peptides; Polymers; Polyubiquitin; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Proteolysis; Proteomics; Research; Research Personnel; Resolution; Signal Transduction; Site; Stress; Structure; Testing; Transcriptional Activation; Translations; Trypsin; Ubiquitin; Ubiquitination; Vesicle; Viral; amino group; experimental study; improved; ion mobility; monomer; polypeptide; protein degradation; response; tool; trafficking

ABSTRACT Ubiquitin (Ub) is a highly conserved eukaryotic protein that is attached to other cellular proteins as a post- translational modification (PTM). Ubiquitination provides signals used to regulate essential processes that in- clude, to name just a few, intracellular protein degradation, transcription activation, DNA damage repair, cell- cycle control, and membrane trafficking. This enormous diversity of functions is made possible by the structural diversity of Ub signals, which can take the form of one or more monoUb units or, because Ub itself can be ubiquitinated, linear or branched multi-Ub polymers (“polyUb”). Adding to this complexity is the fact that Ub has 8 different sites used for Ub–Ub attachments, and mixtures of Ub–Ub linkage types and even branched Ub units are found within polyUb chains. Thus, an enormous variety of distinct polyUb structures are possible. In- deed, a wealth of genetic, biochemical, and proteomic studies support the idea that structurally distinct (poly)Ub signals mediate different interactions and functions in cells. In analogy with the concept that a “histone code” underlies nucleosome PTM functions, the existence of a “ubiquitin code” has been proposed. This idea now underlies much of the research in the Ub field, but experi- ments to test and explore it have been severely limited by the enormous variety of possible polyUb structures. Thus, the precise structures are rarely known for mixed-linkage forms of polyUb, which can be assembled into many topologically distinct isomers. This problem is particularly acute for branched polyUb chains where, with few exceptions, the linkages that comprise branched Ub units are invisible to current methods of analysis. De- termination of whether or not a ubiquitin code exists — and if it does, decoding it — will require new tools to identify the complex polyUb signals that can be found at different sites on different proteins. This proposal is to develop strategies and materials that will enable a comprehensive proteomic analy- sis of branching in polyUb, which by current methods can be studied only qualitatively and only for a small subset of polyUb isoforms. Complementary methods of chemical derivatization and proteolysis will be developed to facilitate analysis of Ub–Ub linkages by mass spectrometry. By successful completion of the pro- posed Aims, we will establish the means to identify and quantify all polyUb branch points where two ubiquitins are conjugated directly to another Ub. These methods are a prerequisite to distinguish and characterize differ- ent polyUb structures and to decode their functions, and to identify the specific machinery used for branched polyUb assembly and disassembly.

抽象的 泛素 (Ub) 是一种高度保守的真核蛋白，作为后缀蛋白附着在其他细胞蛋白上。 翻译修饰（PTM）。泛素化提供了用于调节基本过程的信号， 仅举几例，包括细胞内蛋白质降解、转录激活、DNA 损伤修复、细胞- 循环控制和膜运输。这种巨大的功能多样性是通过结构实现的 Ub 信号的多样性，可以采用一个或多个 monoUb 单元的形式，或者，因为 Ub 本身可以是 泛素化、线性或支化多泛素聚合物（“polyUb”）。增加这种复杂性的是，Ub 有 用于 Ub-Ub 连接的 8 个不同位点，以及 Ub-Ub 连接类型的混合物，甚至支链 Ub 单元存在于多聚泛素链中。因此，多种不同的聚泛素结构是可能的。在- 行为，大量的遗传、生化和蛋白质组学研究支持这样的观点：结构上不同 (多)泛素信号介导细胞中不同的相互作用和功能。 与“组蛋白密码”是核小体 PTM 功能基础的概念类似， “泛素代码”被提出。这个想法现在成为 Ub 领域许多研究的基础，但经验 测试和探索它的方法受到了多种可能的聚泛素结构的严重限制。 因此，混合连接形式的多聚泛素的精确结构很少为人所知，而多聚泛素可以组装成 许多拓扑不同的异构体。这个问题对于支化多聚泛素链来说尤其严重，其中， 除极少数例外，构成支化 Ub 单元的连接对于当前的分析方法来说是不可见的。德- 终止泛素代码是否存在——如果存在，对其进行解码——将需要新的 用于识别复杂多聚泛素信号的工具，这些信号可以在不同蛋白质的不同位点找到。 该提案旨在开发能够进行全面蛋白质组分析的策略和材料 PolyUb 中的支化现象，通过目前的方法只能定性研究，并且只能针对 PolyUb 同工型的一小部分。化学衍生化和蛋白水解的补充方法将是 开发用于促进通过质谱分析 Ub-Ub 连接。通过成功完成亲 提出的目标，我们将建立识别和量化两个泛素所在的所有多聚泛素分支点的方法 直接与另一个 Ub 缀合。这些方法是区分和表征差异的先决条件 进入polyUb结构并解码其功能，并识别用于分支的特定机制 PolyUb 组装和拆卸。