Detection and quantitation of branched ubiquitin in polyubiquitinated proteins

摘要 泛素（Ub）是一种高度保守的真核生物蛋白质，其作为一种后连接蛋白与其他细胞蛋白质连接。 翻译修饰（PTM）。泛素化提供了用于调节重要过程的信号， 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由结构 Ub信号的多样性，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 泛素化的、线性或支化的多Ub聚合物（“polyUb”）。增加这种复杂性的是， 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，甚至分支Ub 在polyUb链中发现了单元。因此，各种不同的polyUb结构是可能的。在- 事实上，大量的遗传学、生物化学和蛋白质组学研究都支持这一观点，即结构上不同的 （poly）Ub信号在细胞中介导不同的相互作用和功能。 与“组蛋白密码”是核小体PTM功能的基础的概念类似， “泛素编码”已经被提出。这个想法现在是UB领域大部分研究的基础，但经验- 由于可能的polyUb结构种类繁多，测试和探索它的努力受到了严重限制。 因此，混合连接形式的polyUb的精确结构很少为人所知，其可以组装成 许多拓扑上不同的异构体。这个问题对于支链polyUb链特别严重，其中， 除了少数例外，包括分支的Ub单元的连接是不可见的，目前的分析方法。去- 终止是否存在泛素代码-如果存在，解码它-将需要新的 用于识别复杂polyUb信号的工具，这些信号可以在不同蛋白质的不同位点找到。 这项建议是发展战略和材料，使全面的蛋白质组学分析， polyUb中分支的姐妹，通过目前的方法只能定性地研究，并且只能用于 polyUb亚型的小子集。化学衍生和蛋白水解的补充方法将在 开发用于通过质谱法促进Ub-Ub连接的分析。通过圆满完成亲- 提出的目标，我们将建立方法来识别和量化所有polyUb分支点，其中两个泛素 直接与另一个Ub结合。这些方法是区分和表征不同- 进入polyUb结构，并解码其功能，并确定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.