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    Dara P. Dowlatshahi.
    The covalent attachment of ubiquitin (Ub), a 76-amino-acid protein, to another protein is a highly occurring and conserved post-translational modification. Ub functions in a plethora of diverse signaling pathways including proteasomal degradation, endocytosis and trafficking of membrane receptors and DNA repair when conjugated to substrate proteins. Ub is unique in that it can also be conjugated to itself to create an assortment of polyubiquitin chains, through either its amino terminus or any of its seven internal lysines, where a large part of its diverse signaling is ascribed to these polyubiquitin chains. Previous work has identified proteins, which specifically recognize these different types of Ub linkages to contain domains or motifs termed Ub binding domains (UBDs). Studies on the various and diverse roles of Ub signaling along with its own diversity of conjugates present on substrates and their recognition by UBDs have uncovered the existence of a Ub syntax or code. The main goal of the work entailed in this thesis was to contribute to decryption of this code by identifying new linkage specific polyubiquitin binding proteins and characterizing their function and mechanism of polyubiquitin recognition. We developed and used a K63 linkage specific polyubiquitin affinity reagent in conjunction with shotgun LC-MS/MS to identify novel polyubiquitin binding proteins. We found that the majority of polyubiquitin dependent identified proteins were previously known Ub binders as well as a few previously unidentified Ub interacting proteins, demonstrating this as a valid approach for the identification of polyubiquitin binding proteins. One such previously unidentified protein we identified was ALIX, a a component of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery known to be involved in Human Immunodeficiency Virus (HIV) budding. We characterized ALIX and found that it binds directly and selectively to K63-linked polyubiquitin via two potential Ub binding sites on a single [alpha]-helical surface within a coiled-coil region, where mutation of these sites impaired retroviral release. Our study demonstrates ALIX as the first example of a K63 chain specific polyubiquitin receptor in the endosomal sorting pathway that supplies evidence for a functional link between K63 polyubiquitin binding and ESCRT function, specifically in retrovirus budding. In summary the findings in our study contribute to the understanding of the role of K63-linked polyubiquitin in the ESCRT pathway. Our work also demonstrates the power of using unbiased affinity capture and tandem mass spectrometry to identify polyubiquitin receptor proteins to further decryption of the Ub code.
    Digital Access   2012