Development of linkage-specific ubiquitin binding elements

DESCRIPTION provided by applicant Identification quantification and isolation of low abundance proteins from complex mixtures is at best a difficult task This is especially the case for proteins carrying a post translational modification PTM that affects their half life or regulatory properties Examples of such PTMs include phosphorylation glycosylation especially O GlcNAcylation and ubiquitylation In many instances one PTM can abrogate or enhance other PTMs on the same protein providing exquisite mechanisms for control of the proteinandapos s activity andquot Fishingandquot a protein with one particular PTM out of the pool of possible modifie proteins becomes nearly impossible without selective tools A further complication in the case of ubiquitylation is the presence of multiple types of Ub Ub linkages in polyubiquitin chains Ubiquitin Ub is attached via isopeptide bonds to lysine residues in the target protein These Ub moieties can then serve as substrates for the conjugation of additional Ubs again through the formation of isopeptide bonds between the C terminus of one Ub and any of seven lysines in the target Ub The general consensus in the field is that chains with different linkages convey different meanings to the cell and hence determine the ultimate fate of the protein be it degradation translocation phosphorylation etc The precise information encoded in different chain linkages is largely unknown due to the lack of specific reagents that recognize different linkages The goal of this proposal is to develop tools that allow the selective identification quantification and isolation of proteins modified by polyubiquitin chains containing different linkages This will be accomplished using information encoded in the human genome that allows the cell to discriminate between different linkages i e Ub binding domains UbDs In Phase I we probed protein microarrays covering of the human proteome in order to identify novel UbDs exhibiting at least partial selectivity In Phase II we will use these UbDs to construct higher avidity reagents capable of linkage specific discrimination Both ubiquitylation and de ubiquitylation have been linked to cancer inflammation and neurological diseases hence the tools developed in this grant will have a major impact on our ability to dissect these disease processes PUBLIC HEALTH RELEVANCE Ubiquitinylation and de ubiquitinylation are post translational modifications that affect the function of many proteins and whose dysregulation has been implicated in many disease processes from cancer to inflammation to neurological degeneration Study of these modifications is difficult due to their low abundance and the complexity of polyubiquitin chains This grant proposes to generate specific reagents that will distinguish between different forms of polyubiquitin enabling their detection and isolation from complex mixtures Use of these tools will dramatically enhance our understanding of many diseases