Catalytic Domain Of MMP-12 Research Articles

Data-driven dentistry: Computational revelations redefining pulp capping.

Pulpal and periradicular diseases stem from immune reactions to microbiota, causing inflammation. Limited blood supply hampers dental pulp self-healing. Managing inflammation involves eliminating bacteria and reducing pro-inflammatory mediators especially MMP-9, which has a significant correlation with pulpitis. s. Flavonoids like Hesperidin, Baicalein, Epigallocatechin gallate, Genistein, Icariin, and Quercetin show potential for pulp capping. This in-silico study compares various Flavonoids for their anti-inflammatory effects on MMP-9, with Chlorhexidine as a control, a known MMP-9 inhibitor. Protein and Ligand Preparation: The human MMP-9 catalytic domain (PDB ID: 4XCT) structure was retrieved, and necessary modifications were made. Flavonoids from PubChem database were prepared for docking using AutoDock Vina. A grid for docking was created, and molecular dynamics simulations were conducted using Gromacs-2019.4 with GROMOS96 force field. Trajectory analysis was performed, and MM-PBSA calculation determined binding free energies. Analysis of MMP-9 and ligand interactions revealed Hesperidin's high binding affinity, forming numerous hydrogen bonds with specific amino acids. Molecular dynamics simulations confirmed stability, with RMSD, RMSF, Rg, and SASA indicating consistent complex behaviour over 100 ns. MM-PBSA calculation affirmed favourable energy contributions in MMP-9-Hesperidin interactions. MMP-9 plays a crucial role in prognosis of pulpitis. Incorporating MMP-9 inhibitors into pulp capping agents may enhance therapeutic efficacy. Hesperidin emerges as a potent MMP-9 inhibitor, warranting further in vivo validation against other agents.

Exploring natural anthraquinones as potential MMP2 inhibitors: A computational study

ObjectiveMatrix metalloproteinase-2 (MMP2) plays a significant role in cleaving extracellular matrix components, leading to many cancer cells' progression and invasion behavior. Therefore, MMP2 inhibition may hold promise for cancer treatment. Anthraquinones have shown antineoplastic effects, some of which have been used in clinical practice as anticancer drugs. This study used a computational drug discovery approach to assess the possible inhibitory effects of selected anthraquinones on MMP2. The results were then compared with that of Captopril, which was considered a standard drug. MethodsThis study used the AutoDock 4.0 tool to evaluate the binding affinity of 21 anthraquinones to the MMP2 catalytic domain. The most favorable scores based on the Gibbs free binding energy scores were given to the highest-ranked ligands. The Discovery Studio Visualizer tool illustrated interactions between MMP2 residues and top-ranked anthraquinones. ResultsA total of 12 anthraquinones were identified with ΔGbinding scores less than − 10 kcal/mol. Pulmatin (Chrysophanol-8-glucoside) was the most potent MMP2 inhibitor, with a ΔGbinding score of − 12.91 kcal/mol. This anthraquinone was able to restrict MMP2 activity within a picomolar range. ConclusionMMP2 inhibition by anthraquinones, notably Pulmatin, may be a useful therapeutic approach for cancer treatment.

Sodium hyaluronate-g-2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide with enhanced affinity towards MMP12 catalytic domain to be used as visco-supplement with increased degradation resistance

The present paper describes the functionalization of sodium hyaluronate (NaHA) with a small molecule (2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide) (MMPI) having proven inhibitory activity against membrane metalloproteins involved in inflammatory processes (i.e. MMP12). The obtained derivative (HA-MMPI) demonstrated an increased resistance to the in-vitro degradation by hyaluronidase, viscoelastic properties close to those of healthy human synovial fluid, cytocompatibility towards human chondrocytes and nanomolar affinity towards MMP 12. Thus, HA-MMPI can be considered a good candidate as viscosupplement in the treatment of knee osteoarticular disease.

Selectivity Conversion of Protease Inhibitory Antibodies.

ABSTRACT Background: Proteases are one of the largest pharmaceutical targets for drug developments. Their dysregulations result in a wide variety of diseases. Because proteolytic networks usually consist of protease family members that share high structural and catalytic homology, distinguishing them using small molecule inhibitors is often challenging. To achieve specific inhibition, this study described a novel approach for the generation of protease inhibitory antibodies. As a proof of concept, we aimed to convert a matrix metalloproteinase (MMP)-14 specific inhibitor to MMP-9 specific inhibitory antibodies with high selectivity. Methods: An error-prone single-chain Fv (scFv) library of an MMP-14 inhibitor 3A2 was generated for yeast surface display. A dual-color competitive FACS was developed for selection on MMP-9 catalytic domain (cdMMP-9) and counter-selection on cdMMP-14 simultaneously, which were fused/conjugated with different fluorophores. Isolated MMP-9 inhibitory scFvs were biochemically characterized by inhibition assays on MMP-2/-9/-12/-14, proteolytic stability tests, inhibition mode determination, competitive ELISA with TIMP-2 (a native inhibitor of MMPs), and paratope mutagenesis assays. Results: We converted an MMP-14 specific inhibitor 3A2 into a panel of MMP-9 specific inhibitory antibodies with dramatic selectivity shifts of 690–4,500 folds. Isolated scFvs inhibited cdMMP-9 at nM potency with high selectivity over MMP-2/-12/-14 and exhibited decent proteolytic stability. Biochemical characterizations revealed that these scFvs were competitive inhibitors binding to cdMMP-9 near its reaction cleft via their CDR-H3s. Conclusions: This study developed a novel approach able to convert the selectivity of inhibitory antibodies among closely related protease family members. This methodology can be directly applied for mAbs inhibiting many proteases of biomedical importance. Statement of SignificanceTo achieve high selectivity required for therapies, we developed a novel approach for the generation of protease inhibitory antibodies with nM potency and decent proteolytic stability. The methodology demonstrated here can be readily applied to many proteases of biomedical importance.

Development of Matrix Metalloproteinase-2 Inhibitors for Cardioprotection.

The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.

Identification of matrix metalloproteinase 9-interacting sequences in staphylococcal superantigen-like protein 5

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, β1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4β9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved β1-3 but not α4β9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted β1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored β1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the β1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.

Abstract P108: Circulating Matrix Metalloproteinase-2 Invades the Heart and Causes Heart Failure

Background: An increase in MMP-2 levels is reported in heart failure (HF). However the role of MMP-2 in the pathogenesis of HF remains unclear. The aim of the present study was to determine the effect of increased circulating levels of MMP-2 on heart morphology and function. Methods and Results: Purified MMP-2 catalytic domain fused to GFP (catMMP-2/GFP) or saline (control) was injected into 11-wk-old male C57BL/6 mice for four weeks. The fluorescent active protein was tracked in vivo and homed in the heart. Cardiomyocyte diameter not changed between groups (catMMP-2/GFP: 11.37 ± 0.25 μm, n=7; Control: 11.38 ± 0.13 μm, n=7; P =0.97). On the other hand, fibrosis increased in the hearts of catMMP-2/GFP mice (0.82 ± 0.05% area/field, n=7 vs Control: 0.58 ± 0.02% area/field, n=7; P< 0.05). Apoptotic stained nuclei in the left ventricle (LV) of catMMP-2/GFP injected-mice amounted to 7.24%, n=4, P<0,05, whilst the LV of control animals only exhibited 0.27%, n=4. catMMP-2/GFP localized in the heart interstitium, where increased proteolytic activity (15.13 ± 1.33 U, n=5 vs Control: 9.70 ± 0.42 U, n=5; P <0.05). Hearts of catMMP-2/GFP mice showed 25% decrease in cardiac output (13 ± 1 mL/min, n=9 vs Control: 17 ± 1 mL/min, n=9), 30% decrease in ejection fraction (40 ± 2 %, n=9 vs Control: 56 ± 2 %, n=9) and stroke volume (28 ± 2 μL, n=9 vs Control: 40 ± 2 μL, n=9), and 34% decrease in fractional shortening (18 ± 1 %, n=9 vs Control: 28 ± 1 %, n=9) ( P <0.05 for all data).Western blotting showed 40% decrease in N-cadherin in animals that received catMMP-2/GFP (0.53 ± 0.08 U, n=6 vs control: 0.89 ± 0.11 U, n=6; P <0.05). Expression of signaling proteins also changed in LV of catMMP-2/GFP mice: TGF-β1 expression increased by 30% (0.60 ± 0.07 U, n=7 vs control: 0.40 ± 0.05 U, n=7, P<0,05), pAkt/Akt decreased by 40% (0.46 ± 0.05 U, n=7 vs control: 0.76 ± 0.11 U, n=7; P <0.05), pSMAD2/total SMAD2 ratio increased (1.88 ± 0.22 U, n=6 vs control: 0.93±0.12 U, n=6, P<0,05), and pSMAD3/total SMAD3 ratio increased (1.24 ± 0.22 U, n=7 vs control: 0.33 ± 0.07 U, n=7; P <0.05). Conclusions: Circulating active MMP-2 homing to the heart interstitium degrades N-cadherin and induces TGF-β1 overexpression, leading to apoptosis, and fibrosis. This mechanism may account for heart function loss when plasma levels of MMP-2 increase.

Matrix metalloproteinase 2 fused to GFP, expressed in E. coli, successfully tracked MMP-2 distribution in vivo

Matrix Metalloproteinases (MMPs) participate in many physiological and pathological processes. One major limitation to a better understanding of the role MMPs play in these processes is the lack of well-characterized chimeric proteins and characterization of their fluorescence. The specialized literature has reported on few constructs bearing MMPs fused to the sequence of the green fluorescent protein (GFP), but none of the described constructs have been intended for expression in bacteria or for purification and use in vivo. This work has tested a recombinant reporter protein containing the MMP-2 catalytic domain fused to GFP in terms of purification efficiency, degradation of substrates in solution and in zymograms, kinetic activity, GFP fluorescence, and GFP fluorescence in whole animals after injection of the purified and lyophilized fluorescent protein. This work has also characterized rhMMP-2 (recombinant human MMP-2) and inactive clones and used them as negative controls in experiments employing catMMP-2/GFP and rhMMP-2. To our knowledge, this is the first study that has fully characterized a chimeric protein with the MMP-2 catalytic domain fused to GFP, that has efficiently purified such protein from bacteria in a single-step, and that has obtained an adequate chimeric protein for injection in animals and tracking of MMP-2 fate and activity in vivo.

New insights into the substrate specificity of macrophage elastase MMP-12.

Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.

Development of in-cell imaging assay systems for MMP-2 and MMP-9 based on trans-localizing molecular beacon proteins.

A sensitive in-cell imaging MMP-2 and MMP-9 detection systems that enables direct fluorescence detection of a target protease and its inhibition inside living cells has been developed. This in-cell imaging system utilizes the concept of fluorescent molecular beacon reporter (MBR) protein comprising a masking protein, a mitochondrial targeting sequence, a protease specific cleavage sequence and a fluorescent marker sequence, green fluorescent protein (GFP). The MBR protein is designed to change its intracellular location upon cleavage by either MMP-2 or MMP-9 from cytosol to mitochondria. Full and partial MMP-2 and MMP-9 were tested for optimal expression and activity in the cell. The activity of MMP-2 and MMP-9 was approximately 65-71%. Among MMP clones, MMP-2 catalytic domain and MMP-9 clone containing pro, catalytic and hemopexin domain were most active. Both MMP-2 and MMP-9 required divalent ions Ca and Zn for its activity and MMP-9 was more active at higher Ca/Zn ratio. With the in-cell imaging assay the protease activity can be measured in cellular environment and cellular toxicity of candidate molecules can be monitored at the same time. These are great advantage when compared to other currently used in vitro biochemical assays. The in-cell imaging assay developed in this study can be modified for other MMPs and can be used in various life science and drug discovery researches including the high throughput screening and high contents screening applications.

Structural and dynamics analysis of matrix metalloproteinases MMP-2 complexed with chemically modified tetracyclines (CMTs)

Matrix metalloproteinases (MMPs) play a critical role in physiological processes and pathological conditions such tumor invasion and metastasis. In recent years, a number of MMP inhibitors have been proposed, including the chemically modified tetracyclines (CMTs), which have been evaluated in preclinical cancer models showing promising results. This work provides insights into the structure and dynamics of the MMP-2 catalytic domain complexed with seven CMT (CMT-n), based on the analysis of molecular dynamics trajectories in solution. The comparative analysis of various relevant molecular aspects of the different complexes of MMP-2 and CMT-n derivatives was performed aiming to elucidate the effect of ligands on the enzyme structure. These include the radial distribution function of the water molecules around the catalytic zinc, the solvent accessible surface area for the inhibitors and the root-mean-square fluctuation for all amino acid residues. The results help to understand the differences in the binding modes of related compounds and, therefore, add to further design of novel tetracycline-based inhibitors for MMP enzymes.

Alendronate promotes plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin degradation sites within the MMP-9 catalytic domain

Irreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin–domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation.

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that α-elastin species cover the primed subsites, a strip across the β-sheet from β-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1' specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.

MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III

Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly 775-Leu 776- in α-2 type I collagen and -Gly 775-Ile 776- in α-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III.

Role of leukemia cell invadosome in extramedullary infiltration

AbstractAcute myelogenous leukemias (AMLs) are characterized by medullary and extramedullary invasion. We hypothesized that a supramolecular complex, the leukemia-cell invadosome, which contains certain integrins, matrix metalloproteinases (MMPs), and other as-yet unidentified proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. Here we show that the specific binding of MMP-9 to leukocyte surface β2 integrin is required for pericellular proteolysis and migration of AML-derived cells. An efficient antileukemia effect was obtained by the hexapeptide HFDDDE, a motif of the MMP-9 catalytic domain that mediates integrin binding: HFDDDE prevented proMMP-9 binding, transmigration through a human endothelial cell layer, and extracellular matrix degradation. Notably, the functional protein anchorage between β2 integrin and proMMP-9 described in this study does not involve the enzymatic active sites targeted by known MMP inhibitors. Taken together, our results provide a biochemical working definition for the human leukemia invadosome. Disruption of specific protein complexes within this supramolecular target complex may yield a new class of anti-AML drugs with anti-invasion (rather than or in addition to cytotoxic) attributes.

Design, Synthesis, Biological Evaluation, and NMR Studies of a New Series of Arylsulfones As Selective and Potent Matrix Metalloproteinase-12 Inhibitors

Overexpression of macrophage elastase (MMP-12), a member of the matrix metalloproteinases family, can be linked to tissue remodeling and degradation in some inflammatory processes, such as chronic obstructive pulmonary disease (COPD), emphysema, rheumatoid arthritis (RA), and atherosclerosis. On this basis, MMP-12 can be considered an attractive target for studying selective inhibitors that are useful in the development of new therapies for COPD and other inflammatory diseases. We report herein the design, synthesis, and in vitro evaluation of a new series of compounds, possessing an arylsulfonyl scaffold, for their potential as selective inhibitors of MMP-12. The best compound in the series showed an IC50 value of 0.2 nM, with good selectivity over MMP-1 and MMP-14. A docking study was carried out on this compound in order to investigate its binding interactions with MMP-12, and NMR studies on the complex with the MMP-12 catalytic domain were able to validate the proposed binding mode.

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Matrix metalloproteinase (MMP)-12 (or metalloelastase) efficiently hydrolyzed the gelatinase-selective alpha1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was submicromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased k(cat)/K(m) toward this substrate by decreasing K(m), just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent alpha1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the beta-sheet and adjoining loops. The alpha1(V) THP protected MMP-12 from the NMR line broadening effects of Gd .EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward alpha1(V) fluorescent THP without affecting general MMP activity. Surprisingly the alpha1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and beta-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.

Peptide Hydrolysis Catalyzed by Matrix Metalloproteinase 2: A Computational Study

The MMP-2 reaction mechanism is investigated by using different computational methodologies. First, quantum mechanical (QM) calculations are carried out on a cluster model of the active site bound to an Ace-Gly approximately Ile-Nme peptide. Along the QM reaction path, a Zn-bound water molecule attacks the Gly carbonyl group to give a tetrahedral intermediate. The breaking of the C-N bond is completed thanks to the Glu 404 residue that shuttles a proton from the water molecule to Ile-N atom. The gas-phase QM energy barrier is quite low ( approximately 14 kcal/mol), thus suggesting that the essential catalytic machinery is included in the cluster model. A similar reaction path occurs in the MMP-2 catalytic domain bound to an octapeptide substrate according to hybrid QM and molecular mechanical (QM/MM) geometry optimizations. However, the rupture of the Gly( P 1) approximately Ile( P 1') amide bond is destabilized in the static QM/MM calculations, owing to the positioning of the Ile( P 1') side chain inside the MMP-2 S 1' pocket and to the inability of simple energy miminization methodologies to properly relax complex systems. Molecular dynamics simulations show that these steric limitations are overcome easily through structural fluctuations. The energetic effect of structural fluctuations is taken into account by combining QM energies with average MM Poisson-Boltzmann free energies, resulting in a total free energy barrier of 14.8 kcal/mol in good agreement with experimental data. The rate-determining event in the MMP-2 mechanism corresponds to a H-bond rearrangement involving the Glu 404 residue and/or the Glu 404-COOH --> N-Ile( P 1') proton transfer. Overall, the present computational results and previous experimental data complement each other well in order to provide a detailed view of the MMPs catalytic mechanism.

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study ( Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020-14028 ) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala(88) and Gly(94) in the non-prime side and Tyr(145) and Thr(146) in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.

Novel Function for a Matrix Metalloprotease

As the family name clearly implies, matrix metalloproteases (MMPs) are localized to the extracellular matrix, where they function as proteases, modulating cell signaling by cleaving proteins such as growth factors or growth factor receptors. In the past few years, MMPs in some cell types have also been observed to function in the nucleus. Eguchi et al. implicate MMPs in a nuclear function that does not require its proteolytic activity, however. This alternative function was first suggested when the authors identified MMP3 in a screen for transcriptional regulators of the gene encoding connective tissue growth factor (CTGF), an insulin-like growth factor implicated in diverse physiological and morphogenetic processes, including arthritis, angiogenesis, and wound healing. MMP3 was detected in the nuclei of mouse chondrocytes both in vitro and in vivo, and in vitro knockdown of MMP3 by RNA interference suggested that MMP3 activated CTGF transcription. MMP3’s interaction with the CTGF promoter was direct and sequence-specific, as assayed by a luciferase reporter system and band-shift experiments. Furthermore, the catalytic domain of MMP3 was not required for its DNA-binding or transcriptional activator functions. Finally, binding to the CTGF enhancer was limited to MMP3; overexpression or inhibition of other MMPs did not affect CTGF expression. The results presented in this study suggest an unconventional role for MMPs in the regulation of gene expression. T. Eguchi, S. Kubota, K. Kawata, Y. Mukudai, J. Uehara, T. Ohgawara, S. Ibaragi, A. Sasaki, T. Kuboki, M. Takigawa, Novel transcription factor-like function of human matrix metalloproteinase 3 regulating the CTGF/CCN2 gene. Mol. Cell. Biol. 28 , 2391-2413 (2008). [Abstract] [Full Text]