Activation and modulation of 72 kDa matrix metalloproteinase-2 by peroxynitrite and glutathione

Matrix metalloproteinase-2 (MMP-2) has emerged as a key protease in various pathologies associated with oxidative stress, including myocardial ischemia-reperfusion, heart failure or inflammation. Peroxynitrite (ONOO⁻), an important effector of oxidative stress, was reported to activate some full length MMP zymogens, particularly in the presence of glutathione (GSH), but whether this occurs for MMP-2 is unknown. Treating MMP-2 zymogen with ONOO⁻ resulted in a concentration-dependent regulation of MMP-2, with 0.3-1 μM ONOO⁻ increasing and 30-100 μM ONOO⁻ attenuating enzyme activity. The enzyme's V_max was also significantly increased by 1 μM ONOO⁻. Comparable responses to ONOO⁻ treatment were observed using the intracellular target of MMP-2, troponin I (TnI). GSH at 100 μM attenuated the effects of ONOO⁻ on MMP-2. Mass spectrometry revealed that ONOO⁻ can oxidize and, in the presence of GSH, S-glutathiolate the MMP-2 zymogen or a synthetic peptide containing the cysteine-switch motif in the enzyme's autoinhibitory domain. These results suggest that ONOO⁻ and GSH can modulate the activity of 72 kDa MMP-2 by modifying the cysteine residue in the autoinhibitory domain of the zymogen, a process that may be relevant to pathophysiological conditions associated with increased oxidative stress.