Detection and quantitation of branched ubiquitin in polyubiquitinated proteins

摘要 泛素(ubiquitin，Ub)是一种高度保守的真核蛋白质，作为一种细胞内信号转导蛋白与其他细胞蛋白结合。 翻译修饰(PTM)。泛素化提供了信号，用于调节在- 包括细胞内蛋白质降解、转录激活、DNA损伤修复、细胞- 周期控制和膜贩运。这种巨大的功能多样性是由结构上的 Ub信号的分集，其可以采取一个或多个单Ub单元的形式，或者因为Ub本身可以是 泛素化、线状或支化的多Ub聚合物(“PolyUb”)。增加这种复杂性的是Ub具有 用于Ub-Ub连接的8个不同站点，以及Ub-Ub链接类型的混合，甚至分支Ub 在PolyUb链中可以找到单元。因此，各种不同的PolyUb结构是可能的。在- 大量的遗传、生化和蛋白质组学研究支持这种结构上截然不同的观点 (Poly)Ub信号调节细胞内不同的相互作用和功能。 类似于核小体PTM功能的基础是组蛋白密码的概念，存在一个 “泛素码”已经被提出。这一想法现在是Ub领域的大部分研究的基础，但实验- 测试和探索它的努力受到了可能的PolyUb结构的巨大变化的严重限制。 因此，对于可以组装成的混合连接形式的PolyUb，精确的结构很少为人所知 许多拓扑上截然不同的异构体。这个问题对于分支PolyUb链尤其严重，在这种情况下， 少数例外情况是，目前的分析方法看不到组成分支Ub单元的联系。去- 终止泛素代码是否存在--如果存在，解码它--将需要新的 识别复杂的PolyUb信号的工具，这些信号可以在不同蛋白质的不同位置找到。 这项建议是开发战略和材料，使全面的蛋白质组分析- PolyUb中分支的SIS，按照目前的方法只能定性地研究并且仅针对 PolyUb亚型的一小部分。化学衍生化和蛋白质分解的补充方法将是 开发的目的是便于通过质谱学分析Ub-Ub联系。通过顺利完成PRO- 设定目标，我们将建立识别和量化所有PolyUb分支点的方法， 直接与另一个Ub结合。这些方法是区分和表征差异的先决条件- ENT PolyUb结构并对其功能进行解码，并识别用于分支的特定机器 PolyUb组装和拆卸。

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.