Summary: | The ubiquitin-proteasome pathway (UPP) is the principal mechanism for the selective degradation of short-lived proteins. The proximal signal for UPP-mediated proteolysis is the covalent modification of target proteins with multiple ubiquitin polypeptides. Protein-ubiquitin conjugation is catalyzed by ubiquitinating enzymes, which assemble the polyubiquitin degradation signal on a target protein. It is postulated that removal of ubiquitin by deubiquitinating enzymes may also regulate protein targeting to the UPP. However, in comparison to ubiquitinating enzymes, relatively little is known about the functions or regulation of deubiquitinating enzymes. Dub-1 and Dub-2 are closely related deubiquitinating enzymes that were initially identified in hematopoietic cell lines as cytokine-inducible proteins. To gain insights into the substrate(s) and function(s) of the Dub enzymes, I examined the expression of Dub-1 and Dub-2 mRNA and protein, and investigated a potential role for these enzymes in regulation of signal transduction through the nuclear factor-kappa B (NF-kB) family of transcription factors. Expression analyses indicated that Dub-1 is expressed in the developing murine limb bud and in interleukin-3-stimulated FL5.12 pro-B cells. To investigate whether the principal inhibitory protein of NF-kB, IkBa, is a substrate for Dub-1 and Dub-2, a novel in vitro deubiquitination assay was established using polyubiquitinated IkBa as the substrate. In addition, I provide evidence for an IkBa-directed deubiquitinating activity in cytoplasmic lysates from a panel of cell lines. Using this and other complementary assays, I show that Dub-1 and Dub-2 do not deubiquitinate IkBa, do not stabilize IkBa, and do not modulate NF-kB activity. In addition, I show that Dub-1, but not the closely homologous Dub-2, is degraded via the UPP in HEK-293T cells. The UPP-mediated degradation of Dub-1 does not require an intact Dub-1 catalytic domain, thus indicating that this process does not proceed via Dub-1-catalyzed transfer of ubiquitin from a substrate to itself. Overall, these studies provide valuable insights as to the regulation of Dub-1 and Dub-2 that may help elucidate the substrate(s) and biological role(s) of these enzymes. Furthermore, the reagents generated for this dissertation will be useful for the study of Dub biochemistry and IkBa deubiquitination.
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