The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1.
The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.
Hetfeld
Bettina K J
BK
Division of Molecular Biology, Department of Surgery, Charité, Universitätsmedizin Berlin, Monbijoustrasse 2, 10117 Berlin, Germany. bettina.hetfeld@charite.de
Helfrich
Annett
A
Kapelari
Barbara
B
Scheel
Hartmut
H
Hofmann
Kay
K
Guterman
Adi
A
Glickman
Michael
M
Schade
Rüdiger
R
Kloetzel
Peter-Michael
PM
Dubiel
Wolfgang
W
D000595:Amino Acid Sequence; D015153:Blotting, Western; D000075686:COP9 Signalosome Complex; D002352:Carrier Proteins / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000235:genetics; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism* / Q000648:ultrastructure; D010618:Phenanthrolines / Q000494:pharmacology; D025821:Polyubiquitin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025801:Ubiquitin / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases; D016335:Zinc Fingers / Q000235:genetics*
D002352:Carrier Proteins; D018076:DNA, Complementary; C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D010618:Phenanthrolines; C479529:RBX1 protein, human; D025801:Ubiquitin; D025821:Polyubiquitin; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex; C579226:USP15 protein, human; D064570:Ubiquitin-Specific Proteases; C025205:1,10-phenanthroline