title: Phosphorylation status of 72 kDa MMP-2 determines its structure and activity in response to peroxynitrite.
abstract: Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO(-)) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO(-) treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1-1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO(-). Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO(-) activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.
authors.affiliation: Department of Pediatrics, Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada ; Department of Pharmacology, Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Anna Laura
authors.initials: AL
authors.lastname: Jacob-Ferreira
authors.forename: Marcia Yuri
authors.initials: MY
authors.lastname: Kondo
authors.forename: Pravas Kumar
authors.initials: PK
authors.lastname: Baral
authors.forename: Michael N G
authors.initials: MN
authors.lastname: James
authors.forename: Andrew
authors.initials: A
authors.lastname: Holt
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Fan
authors.forename: Richard
authors.initials: R
authors.lastname: Schulz
mesh_terms: D002942:Circular Dichroism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D059748:Proteolysis; D019210:Troponin I / Q000737:chemistry
chemical_list: D016877:Oxidants; D019210:Troponin I; D030421:Peroxynitrous Acid; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D005978:Glutathione