scholarly journals Catalytic- and ecto-domains of membrane type 1-matrix metalloproteinase have similar inhibition profiles but distinct endopeptidase activities

2004 ◽  
Vol 377 (3) ◽  
pp. 775-779 ◽  
Author(s):  
Douglas R. HURST ◽  
Martin A. SCHWARTZ ◽  
Mohammad A. GHAFFARI ◽  
Yonghao JIN ◽  
Harald TSCHESCHE ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Active Site ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Vital Role ◽  
Peptide Substrates ◽  
Enzyme Active Site ◽  
Membrane Type ◽  
Function Relationship

Membrane type 1-matrix metalloproteinase (MT1-MMP/MMP-14) is a major collagenolytic enzyme that plays a vital role in development and morphogenesis. To elucidate further the structure–function relationship between the human MT1-MMP active site and the influence of the haemopexin domain on catalysis, substrate specificity and inhibition kinetics of the cdMT1-MMP (catalytic domain of MT1-MMP) and the ecto domain ΔTM-MT1-MMP (transmembrane-domain-deleted MT1-MMP) were compared. For substrate 1 [Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, where Mca stands for (7-methoxycoumarin-4-yl)acetyl- and Dpa for N-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl], the activation energy Ea was determined to be 11.2 and 12.2 kcal/mol (1 cal=4.184 J) for cdMT1-MMP and ΔTM-MT1-MMP respectively, which is consistent with kcat/KM values of 7.37 and 1.46×104 M−1·s−1. The kcat/KM values for a series of similar single-stranded peptide substrates were determined and found to correlate with a slope of 0.17 for the two enzyme forms. A triple-helical peptide substrate was predicted to have a kcat/KM of 0.87×104 M−1·s−1 for ΔTM-MT1-MMP based on the value for cdMT1-MMP of 5.12×104 M−1·s−1; however, the actual value was determined to be 2.5-fold higher, i.e. 2.18×104 M−1·s−1. These results suggest that cdMT1-MMP is catalytically more efficient towards small peptide substrates than ΔTM-MT1-MMP and the haemopexin domain of MT1-MMP facilitates the hydrolysis of triple-helical substrates. Diastereomeric inhibitor pairs were utilized to probe further binding similarities at the active site. Ratios of Ki values for the inhibitor pairs were found to correlate between the enzyme forms with a slope of 1.03, suggesting that the haemopexin domain does not significantly modify the enzyme active-site structure.

scholarly journals Molecular Dissection of the Structural Machinery Underlying the Tissue-invasive Activity of Membrane Type-1 Matrix Metalloproteinase

2008 ◽  
Vol 19 (8) ◽  
pp. 3221-3233 ◽  
Author(s):  
Xiao-Yan Li ◽  
Ichiro Ota ◽  
Ikuo Yana ◽  
Farideh Sabeh ◽  
Stephen J. Weiss
Keyword(s):  
Matrix Metalloproteinase ◽  
Type I Collagen ◽  
Cell Function ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Type I ◽  
Cell Trafficking ◽  
Membrane Type

Membrane type-1 matrix metalloproteinase (MT1-MMP) drives cell invasion through three-dimensional (3-D) extracellular matrix (ECM) barriers dominated by type I collagen or fibrin. Based largely on analyses of its impact on cell function under two-dimensional culture conditions, MT1-MMP is categorized as a multifunctional molecule with 1) a structurally distinct, N-terminal catalytic domain; 2) a C-terminal hemopexin domain that regulates substrate recognition as well as conformation; and 3) a type I transmembrane domain whose cytosolic tail controls protease trafficking and signaling cascades. The MT1-MMP domains that subserve cell trafficking through 3-D ECM barriers in vitro or in vivo, however, remain largely undefined. Herein, we demonstrate that collagen-invasive activity is not confined strictly to the catalytic, hemopexin, transmembrane, or cytosolic domain sequences of MT1-MMP. Indeed, even a secreted collagenase supports invasion when tethered to the cell surface in the absence of the MT1-MMP hemopexin, transmembrane, and cytosolic tail domains. By contrast, the ability of MT1-MMP to support fibrin-invasive activity diverges from collagenolytic potential, and alternatively, it requires the specific participation of MT-MMP catalytic and hemopexin domains. Hence, the tissue-invasive properties of MT1-MMP are unexpectedly embedded within distinct, but parsimonious, sequences that serve to tether the requisite matrix-degradative activity to the surface of migrating cells.

scholarly journals The Soluble Catalytic Domain of Membrane Type 1 Matrix Metalloproteinase Cleaves the Propeptide of Progelatinase A and Initiates Autoproteolytic Activation

1996 ◽  
Vol 271 (29) ◽  
pp. 17119-17123 ◽  
Author(s):  
Horst Will ◽  
Susan J. Atkinson ◽  
Georgina S. Butler ◽  
Bryan Smith ◽  
Gillian Murphy
Keyword(s):  
Matrix Metalloproteinase ◽  
Catalytic Domain ◽  
Membrane Type

scholarly journals Membrane interactions with membrane type 1 matrix metalloproteinase

2018 ◽  
Author(s):  
◽  
Tara Marcink
Keyword(s):  
Cell Surface ◽  
Matrix Metalloproteinase ◽  
Catalytic Domain ◽  
Membrane Surface ◽  
Membrane Binding ◽  
Tumor Cell Migration ◽  
Peripheral Protein ◽  
Membrane Type ◽  
Dynamics Simulations

Membrane type 1 matrix metalloproteinase (MT1-MMP) is essential to a myriad of extracellular activities including tumor cell migration and angiogenesis. At the cell surface, MT1-MMP is a major factor in the proteolysis of receptors, growth factors, and collagen. MT1-MMP extracellular domains bind the cell surface which can be influential in bringing these complexes together. This study uses new techniques to uncover the interactions between MT1-MMP and the cell surface. Described here is the development of techniques in protein and lipid preparations, NMR data acquisition, and structure determination by molecular dynamics simulations. Through these methods, the HPX domain was shown to bind nanodiscs by opposing tips of blade II and blade IV. The protruding part of these tips contain an EPGYPK sequence that are seen dipping into the membrane surface making contact with the lipid head groups. Blade IV membrane binding allows collagen to bind unhindered. Both blade II and blade IV membrane binding structures are shown to be favorable for homodimerization without disruption of the collagen binding site. The catalytic domain is shown to at least transiently bind membranes. This study then hypothesizes and discusses how these interactions impact both future peripheral protein membrane interaction studies and uncover similarities between the MMP family.

scholarly journals Inhibition of enzyme activity of and cell-mediated substrate cleavage by membrane type 1 matrix metalloproteinase by newly developed mercaptosulphide inhibitors

2005 ◽  
Vol 392 (3) ◽  
pp. 527-536 ◽  
Author(s):  
Douglas R. Hurst ◽  
Martin A. Schwartz ◽  
Yonghao Jin ◽  
Mohammad A. Ghaffari ◽  
Pallavi Kozarekar ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Active Site ◽  
Competitive Inhibition ◽  
Human Cancer ◽  
Calf Serum ◽  
Inhibition Mechanism ◽  
Air Oxidation ◽  
Substrate Cleavage ◽  
Membrane Type

MT1-MMP (membrane type 1 matrix metalloproteinase, or MMP-14) is a key enzyme in molecular carcinogenesis, tumour-cell growth, invasion and angiogenesis. Novel and potent MMP inhibitors with a mercaptosulphide zinc-binding functionality have been designed and synthesized, and tested against human MT1-MMP and other MMPs. Binding to the MT1-MMP active site was verified by the competitive-inhibition mechanism and stereochemical requirements. MT1-MMP preferred deep P1′ substituents, such as homophenylalanine instead of phenylalanine. Novel inhibitors with a non-prime phthalimido substituent had Ki values in the low-nanomolar range; the most potent of these inhibitors was tested and found to be stable against air-oxidation in calf serum for at least 2 days. To illustrate the molecular interactions of the inhibitor–enzyme complex, theoretical docking of the inhibitors into the active site of MT1-MMP and molecular minimization of the complex were performed. In addition to maintaining the substrate-specificity pocket (S1′ site) van der Waals interactions, the P1′ position side chain may be critical for the peptide-backbone hydrogen-bonding network. To test the inhibition of cell-mediated substrate cleavage, two human cancer-cell culture models were used. Two of the most potent inhibitors tested reached the target enzyme and effectively inhibited activation of proMMP-2 by endogenous MT1-MMP produced by HT1080 human fibrosarcoma cells, and blocked fibronectin degradation by prostate cancer LNCaP cells stably transfected with MT1-MMP. These results provide a model for mercaptosulphide inhibitor binding to MT1-MMP that may aid in the design of more potent and selective inhibitors for MT1-MMP.

scholarly journals Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the ‘aggrecanase’ and the MMP sites

1998 ◽  
Vol 333 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Frank H. BÜTTNER ◽  
Clare E. HUGHES ◽  
Daniel MARGERIE ◽  
Andrea LICHTE ◽  
Harald TSCHESCHE ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Catalytic Domain ◽  
Human Articular Cartilage ◽  
Recombinant Enzymes ◽  
Recombinant Product ◽  
Weak Activity ◽  
Membrane Type

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704–709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor ‘aggrecanase ’ catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269–20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the ‘aggrecanase ’ site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS …) and BC-14 (anti-FFGV …) to distinguish between ‘aggrecanase ’ and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong ‘aggrecanase ’ activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for ‘aggrecanase ’ activity in vivo.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of membrane type 1 matrix metalloproteinase

2014 ◽  
Vol 70 (2) ◽  
pp. 232-235 ◽  
Author(s):  
Hideaki Ogata ◽  
Elena Decaneto ◽  
Moran Grossman ◽  
Martina Havenith ◽  
Irit Sagi ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Catalytic Domain ◽  
Large Family ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Data Sets ◽  
X Ray ◽  
Cell Parameters ◽  
Membrane Type

Membrane type 1 matrix metalloproteinase (MT1-MMP) belongs to the large family of zinc-dependent endopeptidases termed MMPs that are located in the extracellular matrix. MT1-MMP was crystallized at 277 K using the vapour-diffusion method with PEG as a precipitating agent. Data sets for MT1-MMP were collected to 2.24 Å resolution at 100 K. The crystals belonged to space groupP43212, with unit-cell parametersa= 62.99,c= 122.60 Å. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (VM) of 2.90 Å3 Da−1; the solvent content is estimated to be 57.6%.

scholarly journals Degradomic and yeast 2-hybrid inactive catalytic domain substrate trapping identifies new membrane-type 1 matrix metalloproteinase (MMP14) substrates: CCN3 (Nov) and CCN5 (WISP2)

2017 ◽  
Vol 59 ◽  
pp. 23-38 ◽  
Author(s):  
Georgina S. Butler ◽  
Andrea R. Connor ◽  
Nor Eddine Sounni ◽  
Ulrich Eckhard ◽  
Charlotte J. Morrison ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Catalytic Domain ◽  
Membrane Type

scholarly journals Biochemical evidence of the interactions of membrane type-1 matrix metalloproteinase (MT1-MMP) with adenine nucleotide translocator (ANT): potential implications linking proteolysis with energy metabolism in cancer cells

2009 ◽  
Vol 420 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Ilian A. Radichev ◽  
Albert G. Remacle ◽  
Nor Eddine Sounni ◽  
Sergey A. Shiryaev ◽  
Dmitri V. Rozanov ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Matrix Metalloproteinase ◽  
Cancer Cells ◽  
Catalytic Domain ◽  
Adenine Nucleotide ◽  
Test System ◽  
Coupling Mechanism ◽  
Adenine Nucleotide Translocator ◽  
Membrane Type

Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; ΔCT) MT1-MMP–FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and ΔCT constructs. The WT and E240A constructs also interacted with α-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the ΔCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2–FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.

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