M T DiSabella | PubMed Health

PubMed1999

Human bleomycin hydrolase binds ribosomal proteins.

Bleomycin hydrolase (BH) is a cysteine proteinase that inactivates the anticancer drug bleomycin. Yeast BH forms a homohexameric structure that resembles a 20S proteasome and binds to single-stranded RNA and DNA. We now demonstrate that human BH (hBH) interacts and colocalizes with ribosomal proteins. Using a yeast two-hybrid system, we found hBH bound to human homologues of rat ribosomal proteins L11 and L29. The N-terminus of hBH (amino acids 14-175), which contains a catalytic Cys93, was critical for the binding to L11 in the two-hybrid environment. hBH precipitated 35S-labeled L11 and L29 in vitro, and hBH colocalized with L11 and L29 as determined by immunofluorescence. In addition to cytosolic bleomycin hydrolase, we found abundant bleomycin hydrolase activity associated with the ribosomal subcellular fraction by differential centrifugation. hBH was also detected by Western immunoblotting in a high-speed particulate fraction, where the majority of L11 and L29 were found. In vitro experiments showed recombinant hBH binds to Chinese hamster ovary cell microsomes. Thus, our data strongly suggest that hBH exists as both a free cytosolic and ribosome-associated protein.

Animals↗

PubMed1998

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.

Amino Acid Sequence↗