Abstract
The attachment of ubiquitin to substrate proteins is a key process in regulating cellular events such as cell cycle progression, signal transduction, differentiation, apoptosis, and the clearance of misfolded or aberrant proteins. Like other post-translational modifications, ubiquitination is also reversible. Deconjugation is performed by a family of cysteine- or metallo-proteases collectively known as deubiquitinating enzymes (DUBs). Approximately 100 putative DUBs have been identified in the human genome but only a minority of them has been functionally characterized. The aim of this thesis has been to study the function of selected DUBs in disease-relevant cellular pathways.
Screen of the canonical Wnt-signaling pathway with an RNA interference (RNAi) library targeting the human DUBs identified the ubiquitin-specific protease (USP)-4 as a negative regulator. USP4 interacts with two known components in the pathway: the Nemo like kinase (Nlk) and the T-cell factor 4 (TCF4). NLK promotes nuclear accumulation of USP4 where a subpopulation of TCF4 is a substrate of USP4-dependent deubiquitination. Using a yeast-2 hybrid strategy to search for relevant interactions, we identified the proteasome as a binding partner of USP4. USP4 interacts with the S9 subunit of the 19S regulatory particle (RP) through an N-terminal ubiquitin-like (UBL) domain that resembles, but is functionally distinct from, the UBLs of hHR23a/b and Ubiquilin-1. S9 is as an essential proteasome subunit that may regulate the structural integrity of the 26S complex. Thus, USP4 may play a role in the dynamics of ubiquitination at the proteasome.
A bioinformatics strategy was used to search for membrane-associated DUBs. We found that a putative transmembrane domain targets USP19 to the endoplasmic reticulum (ER). USP19 is a target of the unfolded protein response and rescues ERAD substrate from proteasomal degradation. Moreover, USP19 interacts with the E3 ligases seven in absentia homolog (SIAH) 1 and SIAH2 that mediate USP19 ubiquitination and degradation by the proteasome. Bioinformatics and biochemical analysis revealed the presence in USP19 of a SIAH-interacting motif that is found in a subset of SIAH targets and may function as a degradation signal. A non-enzymatic role of USP19 in the regulation of the reposed to hypoxia was suggested by the finding that wild-type and catalytic mutant USP19 interact with the hypoxia-inducible factor-1α (HIF-1α). In the absence of USP19, cells fail to mount a proper response to hypoxia.
