Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Bleomycin is an anticancer drug used to treat various tumors.
• The metabolic inactivation of bleomycin is thought to involve the enzyme bleomycin hydrolase.
• Understanding the human form of this enzyme (hBH) is crucial for cancer therapy.

Purpose of the Study:

• To clone and characterize the human bleomycin hydrolase (hBH) enzyme.
• To investigate the potential role of hBH in bleomycin resistance in tumors.

Main Methods:

• Cloning of the human bleomycin hydrolase (hBH) cDNA.
• Functional expression of recombinant hBH in Spodoptera frugiperda Sf9 cells using baculovirus.
• Biochemical characterization of the recombinant hBH protein, including substrate specificity and localization studies.
• Analysis of hBH expression levels in human organs and tumor cell lines.

Main Results:

• The cDNA for hBH was cloned, predicting a 52 kDa protein with conserved active site residues of cysteine proteases.
• Recombinant hBH was functionally expressed, forming a hexamer and acting as an aminopeptidase.
• hBH shows a pH optimum of 7.2, suggesting cytosolic/nuclear localization.
• hBH was detected in most human organs at low to moderate levels, with significantly higher RNA levels in tumor cell lines.
• In vitro studies demonstrated that hBH effectively degrades both bleomycin A2 and B2.

Conclusions:

• Human bleomycin hydrolase (hBH) is a cysteine protease with aminopeptidase activity.
• The enzyme's ability to degrade bleomycin in vitro and its higher expression in tumors suggest it plays a role in bleomycin resistance.
• hBH represents a potential target for overcoming resistance to bleomycin in cancer treatment.