title: Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel-Lindau tumor suppressor protein.
abstract: von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of the VHL gene. Recent studies suggest that VHL protein (pVHL) is a component of an E3 ubiquitin ligase, but the detailed biological function of pVHL remains to be determined. To further elucidate the biological functions of pVHL, we searched pVHL-interacting proteins using yeast two-hybrid screening. A novel protein named VHL-interacting deubiquitinating enzyme 1 (VDU1) was identified as being able to directly interact with pVHL in vitro and in vivo. We have determined the full-length cDNA of this enzyme, which includes two putative subtypes. Type I consists of 942 amino acids, and type II consists of 911 amino acids with predicted molecular masses of 107 and 103 kDa, respectively. We have also cloned a mouse homologue of this enzyme. Sequence analysis reveals that this protein is conserved between human and mouse and contains the signature motifs of the ubiquitin-specific processing protease family. Enzymatic function studies demonstrate its deubiquitinating activity. We have determined that the VDU1-interacting region in pVHL is located in its beta-domain, and several naturally occurring mutations located in this domain disrupt the interaction between pVHL and VDU1 protein. Co-immunoprecipitation demonstrates that VDU1 can be recruited into the pVHL-elongin C-elongin B complex. Finally, we demonstrate that VDU1 is able to be ubiquitinated via a pVHL-dependent pathway for proteasomal degradation, and VHL mutations that disrupt the interaction between VDU1 and pVHL abrogate the ubiquitination of VDU1. Our findings indicate that VDU1, a novel ubiquitin-specific processing protease, is a downstream target for ubiquitination and degradation by pVHL E3 ligase. Targeted degradation of VDU1 by pVHL could be crucial for regulating the ubiquitin-proteasome degradation pathway.
authors.affiliation: Department of Urology, University of Rochester School of Medicine, Rochester, New York 14642, USA.
authors.forename: Zaibo
authors.initials: Z
authors.lastname: Li
authors.forename: Xi
authors.initials: X
authors.lastname: Na
authors.forename: Dakun
authors.initials: D
authors.lastname: Wang
authors.forename: Susan R
authors.initials: SR
authors.lastname: Schoen
authors.forename: Edward M
authors.initials: EM
authors.lastname: Messing
authors.forename: Guan
authors.initials: G
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
chemical_list: D000596:Amino Acids; D018076:DNA, Complementary; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D005982:Glutathione Transferase; D010450:Endopeptidases; C449799:USP33 protein, human; D043222:Ubiquitin Thiolesterase; D008025:Ligases; C497446:VHL protein, human