Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases

The mechanism by which enzymes recognize the “uniform” collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P₃ subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.     

Cardiac Restricted Overexpression of Membrane Type-1 Matrix Metalloproteinase Causes Adverse Myocardial Remodeling following Myocardial Infarction

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.     

Evolution of disintegrin cysteine-rich and mammalian matrix-degrading metalloproteinases: Gene duplication and divergence of a common ancestor rather than convergent evolution

The evolution of the Metalloproteinase Disintegrin Cysteine-rich (MDC) gene family and that of the mammalian Matrix-degrading Metalloproteinases (MMPs) are compared. The alignment of snake venom and mammalian MDC and MMP precursor sequences generated a phylogenetic tree that grouped these proteins mainly according to their function. Based on this observation, a common ancestry is suggested for mammalian and snake venom MDCs; it is also possible that gene duplication of the already-assembled domain structure, followed by divergence of the copies, may have significantly contributed to the evolution of the functionally diverse MDC proteins. The data also suggest that the structural resemblance of the zinc-binding motif of venom MDCs and MMPs may best be explained by common ancestry and conservation of the proteolytic motifs during the divergence of the proteins rather than through convergent evolution. Correspondence to: J.M. Crampton     

Insight towards the conserved water mediated recognition of catalytic and structural Zn+2 ions in human Matrix Metalloproteinase-8 enzyme: A study by MD-simulation methods

Human matrix metalloproteinase-8 (hMMP-8) plays a important role in the progression of colorectal cancer, metastasis, multiple sclerosis and rheumetoid arthritis. Extensive MD-simulation of the PDB and solvated structures of hMMP-8 has revealed the presence of few conserved water molecules around the catalytic and structural zinc (ZnC and ZnS) ions. The coordination of two conserved water molecules (W and WS) to ZnS and the H-bonding interaction of WS to S151 have indicated the plausible involvement of that metal ion in the catalytic process. Beside this the coupling of ZnC and ZnS metal ions (ZnC – W_H (W₁)…..W₂ ….H₁₆₂ - ZnS) through two conserved hydrophilic centers (occupied by water molecules) may also provide some rational on the recognition of two zinc ions which were separated by ~13 Å in their X-ray structures. This unique recognition of both the Zn⁺² ions in the enzyme through conserved water molecules may be implemented/ exploited for the design of antiproteolytic agent using water mimic drug design protocol.     

Interstitial Collagen Catabolism

Interstitial collagen mechanical and biological properties are altered by proteases that catalyze the hydrolysis of the collagen triple-helical structure. Collagenolysis is critical in development and homeostasis but also contributes to numerous pathologies. Mammalian collagenolytic enzymes include matrix metalloproteinases, cathepsin K, and neutrophil elastase, and a variety of invertebrates and pathogens possess collagenolytic enzymes. Components of the mechanism of action for the collagenolytic enzyme MMP-1 have been defined experimentally, and insights into other collagenolytic mechanisms have been provided. Ancillary biomolecules may modulate the action of collagenolytic enzymes.     

Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.