Research Project
10261524
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.
