ABSTRACT
Cellular bio-energetic metabolism and mitochondria are recognized as potential targets for anticancer agents, due to the numerous relevant peculiarities cancer cells exhibit. Jasmonates are anticancer agents that interact directly with mitochondria. The aim of this study was to identify mitochondrial molecular targets of jasmonates. We report that jasmonates bind to hexokinase and detach it from the mitochondria and its mitochondrial anchor-the voltage-dependent anion channel (VDAC), as judged by hexokinase immunochemical and activity determinations, surface plasmon resonance analysis and planar lipid bilayer VDAC-activity analysis. Furthermore, the susceptibility of cancer cells and mitochondria to jasmonates is dependent on the expression of hexokinase, evaluated using hexokinase-overexpressing transfectants and its mitochondrial association. Many types of cancer cells exhibit overexpression of the key glycolytic enzyme, hexokinase, and its excessive binding to mitochondria. These characteristics are considered to play a pivotal role in cancer cell growth rate and survival. Thus, our findings provide an explanation for the selective effects of jasmonates on cancer cells. Most importantly, this is the first demonstration of a cytotoxic mechanism based on direct interaction between an anticancer agent and hexokinase. The proposed mechanism can serve to guide development of a new selective approach for cancer therapy.
Subject(s)
Acetates/metabolism , Cyclopentanes/metabolism , Hexokinase/metabolism , Mitochondria/metabolism , Oxylipins/metabolism , Acetates/pharmacology , Adenosine Triphosphate/metabolism , Animals , Cell Death/drug effects , Cell Death/genetics , Cyclopentanes/pharmacology , DNA Damage , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Hexokinase/genetics , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondrial Swelling/drug effects , Neoplasms/metabolism , Oxylipins/pharmacology , Protein Binding , Rats , Transfection , Tumor Cells, Cultured , Voltage-Dependent Anion Channels/metabolismABSTRACT
A beta-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65 degrees C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.
Subject(s)
Geobacillus stearothermophilus/enzymology , Multigene Family/genetics , Catalysis , Cloning, Molecular , Consensus Sequence , Escherichia coli/genetics , Glycosides/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Xylosidases/chemistry , Xylosidases/genetics , Xylosidases/metabolismABSTRACT
A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.
