An evolutionarily conserved cysteine protease, human bleomycin hydrolase, binds to the human homologue of ubiquitin-conjugating enzyme 9.
Bleomycin hydrolase (BH) is a highly conserved cysteine proteinase that deamidates and inactivates the anticancer drug bleomycin. Yeast BH self-assembles to form a homohexameric structure, which resembles a 20 S proteasome and may interact with other proteins. Therefore, we searched for potential human BH (hBH) partners using the yeast two-hybrid system with a HeLa cDNA library and identified the full-length human homologue of yeast ubiquitin-conjugating enzyme 9 (UBC9). Cotransformation assays using hBH deletion mutants revealed that the carboxyl terminus of hBH (amino acids 356-455), which contains two of the three essential catalytic amino acids, was not critical for protein binding in the yeast two-hybrid environment. In vitro translated human UBC9 was precipitated by glutathione S-transferase-hBH fusion protein but not by glutathione S-transferase. Efficient in vitro binding occurred in the absence of the first 24 amino acids of UBC9 and the catalytic Cys93 of UBC9. We confirmed that hBH and UBC9 interacted in vivo by affinity copurification of proteins overexpressed in mammalian cells. Using immunocytochemical analysis, hBH was colocalized with UBC9. Coexpression of hBH and UBC9 in mammalian cells did not markedly alter the bleomycin-hydrolyzing activity of hBH or apparent small ubiquitin-related modifier 1 addition. This is the first reported heteromeric interaction with hBH, and it suggests a role for hBH in intracellular protein processing and degradation.
Koldamova
R P
RP
Department of Pharmacology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Lefterov
I M
IM
DiSabella
M T
MT
Lazo
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D001483:Base Sequence; D001761:Bleomycin / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D019143:Evolution, Molecular; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D008025:Ligases / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D000903:Antibiotics, Antineoplastic; D001761:Bleomycin; D044763:Ubiquitin-Conjugating Enzymes; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9