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.
Koldamova
R P
RP
Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Lefterov
I M
IM
DiSabella
M T
MT
Almonte
C
C
Watkins
S C
SC
Lazo
J S
JS
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
D012269:Ribosomal Proteins; C027624:ribosomal protein L11; C035078:ribosomal protein L29; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase