Functional studies of deubiquitinating enzymes

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.

List of scientific papers

I. Zhao Bin, Schlesiger C, Masucci MG, Lindsten K. The ubiquitin specific protease 4 (USP4) is a new player in the Wnt signalling pathway. J Cell Mol Med. 2009 Aug;13(8B):1886-9.
https://doi.org/10.1111/j.1582-4934.2009.00682.x

II. Zhao Bin, Velasco K, Sompallae R, Pfirrmann T, Lindsten K. The ubiquitin specific protease 4 (USP4) interacts with the proteasome via an internal ubiquitin-like (Ubl) domain. [Manuscript]

III. Hassink GC, Zhao Bin, Sompallae R, Altun M, Gastaldello S, Zinin NV, Masucci MG, Lindsten K. The ER-resident ubiquitin-specific protease 19 participates in the UPR and rescues ERAD substrates. EMBO Rep. 2009 Jul;10(7):755-61.
https://doi.org/10.1038/embor.2009.69

IV. Velasco K, Zhao Bin, Callegari S, Altun M, Liu H, Hassink G, Lindsten K. A SIAH-interacting motif in the ubiquitin specific protease 19 (USP19) regulates its stability. [Manuscript]

V. Altun M, Zhao Bin, Velasco K, Liu H, Hassink G, Paschke J, Pereira T, Lindsten K. Ubiquitin-specific protease 19 (USP19) regulates hypoxia-inducible factor 1α (HIF-1α) during hypoxia. J Biol Chem. 2012 Jan;287(3):1962-9.
https://doi.org/10.1074/jbc.M111.305615