Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase.
Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.
Zimny
Jaroslaw
J
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, International Center for Public Health, Newark, New Jersey 07101, USA.
Sikora
Marta
M
Guranowski
Andrzej
A
Jakubowski
Hieronim
H
D001665:Binding Sites; D001761:Bleomycin / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D006710:Homocysteine / Q000494:pharmacology / Q000633:toxicity*; D006801:Humans; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry; D009154:Mutation; D010920:Placenta / Q000201:enzymology / Q000473:pathology; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D007783:Lactones; D011994:Recombinant Proteins; D006710:Homocysteine; D001761:Bleomycin; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase