Koldamova RP, Lefterov IM, DiSabella MT, Lazo JS

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 ...

Read More

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.

Date: Dec. 01, 1998

View in: Pubmed Google Scholar

11133