Nitric oxide regulation of MMP-9 activation and its relationship to modifications of the cysteine switch.
Matrix metalloproteases (MMPs) are Zn-containing endopeptidases involved in the degradation of extracellular matrix components and are typically secreted in a latent (pro-MMP) form and activated either by proteolytic or oxidative disruption of a conserved cysteine switch. Several recent studies have suggested that nitric oxide (NO) can contribute to the activation of MMPs, but the mechanisms involved are incompletely understood. We investigated the ability of NO to regulate the activation of (pro)MMP-9 using a variety of NO-donor compounds and characterized modifications of the cysteine switch using a synthetic peptide (PRCGVPDLGR) representing the cysteine switch domain of MMP-9. Among the NO-donors used, only S-nitrosocysteine (SNOC) was found to be capable of modest activation of proMMP-9, but S-nitrosoglutathione (GSNO) or the NONOates, DEA-NO, SPER-NO, or DETA-NO, were ineffective. In fact, high concentrations of DETA-NO were found to inhibit MMP-9 activity, presumably by direct interaction with the active-site Zn (2+). Analysis of chemical modifications within the Cys-containing peptide, PRCGVPDLGR, revealed rapid and transient S-nitrosylation by SNOC and GSNO, and formation of mixed disulfides and dimerized peptide as major final products. Similarly, NONOates induced transient S-nitrosylation and primarily peptide dimerization. Coordination of the peptide Cys with a synthetic Zn (2+) complex, to more closely mimic the structure of the active site in proMMP-9, reduced peptide nitrosylation and oxidation by NONOates, but enhanced peptide nitrosylation by SNOC and GSNO. Collectively, our results demonstrate that NO is incapable of directly activating proMMP-9 and that S-nitrosylation of MMP-9 propeptide by NO-donors is unrelated to their ability to regulate MMP-9 activity.
McCarthy
Sean M
SM
Department of Pathology, University of Vermont, Burlington, Vermont 05405, USA.
Bove
Peter F
PF
Matthews
Dwight E
DE
Akaike
Takaaki
T
van der Vliet
Albert
A
D001671:Biochemistry / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000502:physiology; D008954:Models, Biological; D009569:Nitric Oxide / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D026403:S-Nitrosothiols / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D010455:Peptides; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D020780:Matrix Metalloproteinase 9; D005978:Glutathione; D015032:Zinc; D003545:Cysteine