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Christopher M Overall - One of the best experts on this subject based on the ideXlab platform.

  • the collAgen binding domAin of GelAtinAse A modulAtes degrAdAtion of collAgen iv by GelAtinAse b
    Journal of Molecular Biology, 2009
    Co-Authors: Magda Gioia, Ghislain Opdenakker, Christopher M Overall, Susanna Monaco, Philippe E Van Den Steen, Diego Sbardella, Giuseppe Grasso, Stefano Marini, Massimo Coletta
    Abstract:

    SummAry Type IV collAgen remodeling plAys A criticAl role in inflAmmAtory responses, Angiogenesis And metAstAsis. Its remodeling is executed by A fAmily of mAtrix metAlloproteinAses (MMPs), of which the constitutive GelAtinAse A (MMP2) And the inducible GelAtinAse B (MMP9) Are key exAmples. Thus, in mAny pAthologicAl conditions, both GelAtinAses Act together. Kinetic dAtA Are reported for the enzymAtic processing At 37 °C of type IV collAgen from humAn plAcentA by MMP9 And its modulAtion by the fibronectin-like collAgen binding domAin (CBD) of MMP2. The α1 And α2 chAin components of type IV collAgen were cleAved by GelAtinAses And identified by mAss spectrometry As well As EdmAn sequencing. SurfAce plAsmon resonAnce interAction AssAys showed thAt CBD bound type IV collAgen At two topologicAlly distinct sites. On the bAsis of linked-function AnAlysis, we demonstrAted thAt CBD of MMP2 tuned the cleAvAge of collAgen IV by MMP9, presumAbly by inducing A ligAnd-linked structurAl chAnge on the type IV collAgen. At low concentrAtions, the CBD bound the first site And thereby AllostericAlly modulAted the binding of MMP9 to collAgen IV, thus enhAncing the collAgenolytic Activity of MMP9. At high concentrAtions, CBD binding to the second site interfered with MMP9 binding to collAgen IV, Acting As A competitive inhibitor. Interestingly, modulAtion of collAgen IV degrAdAtion by inActive forms of MMP2 Also occurred in A cell-bAsed system, reveAling thAt this interrelAtionship Affected neutrophil migrAtion Across A collAgen IV membrAne. The regulAtion of the proteolytic processing by A cAtAlyticAlly inActive domAin (i.e., CBD) suggests thAt the two GelAtinAses might cooperAte in degrAding substrAtes even when either one is inActive. This observAtion reinforces the ideA of exosite tArgets for MMP inhibitors, which should include All mAcromoleculAr substrAte recognition sites.

  • chArActerizAtion of the distinct collAgen binding helicAse And cleAvAge mechAnisms of mAtrix metAlloproteinAse 2 And 14 GelAtinAse A And mt1 mmp the differentiAl roles of the mmp hemopexin c domAins And the mmp 2 fibronectin type ii modules in collAg
    Journal of Biological Chemistry, 2004
    Co-Authors: Todd R Moore, Georgina S Butler, Christopher M Overall
    Abstract:

    AbstrAct MAtrix metAlloproteinAse-2 (MMP-2, GelAtinAse A) And membrAne type (MT)1-MMP (MMP-14) Are cooperAtive dynAmic components of A cell surfAce proteolytic Axis involved in regulAting the cellulAr signAling environment And pericellulAr collAgen homeostAsis. Although MT1-MMP exhibits type I collAgenolytic but poor gelAtinolytic Activities, MMP-2 is A potent GelAtinAse with weAk type I collAgenolytic behAvior. RecombinAnt linker/hemopexin C domAin (LCD) of MT1-MMP binds nAtive type I collAgen, blocks MT1-MMP collAgenolytic Activity in trAns, And by circulAr dichroism spectroscopy, induces locAlized structurAl perturbAtion in the collAgen. These chAnges were reflected by enhAnced cleAvAge of the MT1-LCD-bound collAgen by the collAgenAses MMP-1 And MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD Alone cAn initiAte triple helicAse Activity. In contrAst, the nAtive And denAtured collAgen binding properties of MMP-2 reside in the fibronectin type II modules, Accordingly termed the collAgen binding domAin (CBD). RecombinAnt CBD (but not the MMP-2 LCD) Also chAnged the circulAr dichroism spectrA leAding to increAsed MMP-1 And -8 cleAvAge of nAtive collAgen. However, recombinAnt CBD reduced gelAtin And collAgen cleAvAge by MMP-2 in trAns As did CBD23, which comprises the second And third fibronectin type II modules, but not the CBD23 mutAnt W316A/W374A, which neither binds gelAtin nor collAgen. This indicAtes thAt MMP-2 And MT1-MMP bind collAgen At A different site thAn MMP-1 And MMP-8. Thus, MMP-2 utilizes the CBD in cis for collAgen binding And triple helicAse Activity, which compensAtes for the lAck of collAgen binding by the MMP-2 LCD. Hence, the MMP fAmily hAs evolved two distinct mechAnisms for collAgen triple helicAse Activity using two structurAlly distinct domAins, with triple helicAse Activity occurring independent of α-chAin hydrolysis.

  • tissue inhibitor of metAlloproteinAses 4 inhibits but does not support the ActivAtion of GelAtinAse A viA efficient inhibition of membrAne type 1 mAtrix metAlloproteinAse
    Cancer Research, 2001
    Co-Authors: Heather F Bigg, Charlotte J Morrison, Georgina S Butler, Marie A Bogoyevitch, Zhiping Wang, Paul D Soloway, Christopher M Overall
    Abstract:

    The tissue inhibitors of metAlloproteinAses 1–4 (TIMPs) hAve discrete regulAtory roles in the ActivAtion of mAtrix metAlloproteinAse (MMP)-2 (GelAtinAse A), An importAnt bAsement membrAne-degrAding MMP pivotAl to tumor metAstAsis And Angiogenesis. TIMP-2 binds to both the hemopexin C domAin of proGelAtinAse A And the Active site of membrAne type-1 (MT1) MMP. This trimeric complex presents the cell surfAce-bound GelAtinAse A zymogen to A free MT1-MMP molecule for ActivAtion. To investigAte the role of TIMP-4 in the ActivAtion process, we developed A new procedure for the expression And purificAtion of recombinAnt humAn TIMP-4 from bAby hAmster kidney cells. The recombinAnt TIMP-4 wAs A potent inhibitor of GelAtinAse A {AppArent K i [ K i(App.) ] ≤ 9 pm; k on (AssociAtion rAte constAnt), 4.57 ± 0.13 × 10 6 m −1 s −1 } And wAs less dependent upon hemopexin C domAin interActions thAn TIMP-2 in its mode of binding And inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP ( K i(App.) ≤ 100 pm; k on , 3.49 ± 0.34 × 10 6 m −1 s −1 ) And blocked the concAnAvAlin A-induced cellulAr ActivAtion of proGelAtinAse A. In concAnAvAlin A-stimulAted homozygous Timp2 −/− fibroblAsts or unstimulAted MT1-MMP-trAnsfected Timp2 −/− cells, which cAnnot ActivAte proGelAtinAse A, ActivAtion wAs restored by the Addition of 0.3–5 nm TIMP-2 but not by TIMP-4, unequivocAlly showing the TIMP-2 dependency of MT1-MMP-induced ActivAtion of GelAtinAse A And the fAct thAt TIMP-4 cAnnot support ActivAtion. The dominAnce of TIMP-2 in the ActivAtion process wAs further supported by the preferentiAl binding of TIMP-2 compAred with TIMP-4 to the hemopexin C domAin of proGelAtinAse A in inhibitor mixtures And by the Ability of TIMP-2 to displAce TIMP-4 from the hemopexin C domAin. Hence, TIMP-4 regulAtes GelAtinAse A Activity by efficient inhibition of MT1-MMP-mediAted ActivAtion And by inhibiting the ActivAted enzyme And, thus, is A tumor resistAnce fActor in the peritumor stromA.

  • the mAtrix metAlloproteinAse GelAtinAse A in humAn dentine
    Archives of Oral Biology, 2000
    Co-Authors: Martinde Las S Heras, Aurora Valenzuela, Christopher M Overall
    Abstract:

    A dentine protein extrAction protocol wAs modified in order to identify mAtrix metAlloproteinAse gelAtinolytic Activities in the non-minerAlized And minerAlized phAses of humAn dentine. Dentine proteins from 24 individuAl permAnent molArs from pAtients Aged 15‐73 yeArs were sequentiAlly extrActed, first with guAnidinium chloride (G1 extrAct), then EDTA (E extrAct), And After this deminerAlizAtion step, AgAin by guAnidinium chloride (G2 extrAct) to dissociAte collAgen-AssociAted proteins. ExtrActs were AnAlysed by sodium dodecyl sulphAte‐polyAcrylAmide gel electrophoresis And the gels were processed by Western blotting And zymogrAphy to detect gelAtinolytic Activities. Active And lAtent forms of GelAtinAse A were identified in the non-minerAlized dentine frAction (G1 extrAct) of 58% of the teeth. Other gelAtinolytic species were Also detected by zymogrAphy with AppArent Mr of 92, 54 And 30 kDA. Although GelAtinAse A wAs detected in the G1 extrActs of teeth from All Ages, indicAting more recent synthesis And remodelling of the predentine, GelAtinAse A wAs never detected in Any E extrAct or in the G2 extrActs of pAtients older thAn 41 yeArs. The presence of the Active form of GelAtinAse A in minerAlized humAn dentine implicAtes this enzyme in dentine minerAlizAtion. 7 2000 Elsevier Science Ltd. All rights reserved.

  • inflAmmAtion dAmpened by GelAtinAse A cleAvAge of monocyte chemoAttrActAnt protein 3
    Science, 2000
    Co-Authors: Angus G Mcquibban, Jianghong Gong, Christopher A Mcculloch, Ian Clarklewis, Christopher M Overall
    Abstract:

    Tissue degrAdAtion by the mAtrix metAlloproteinAse GelAtinAse A is pivotAl to inflAmmAtion And metAstAses. Recognizing the cAtAlytic importAnce of substrAte-binding exosites outside the cAtAlytic domAin, we screened for extrAcellulAr substrAtes using the GelAtinAse A hemopexin domAin As bAit in the yeAst two-hybrid system. Monocyte chemoAttrActAnt protein-3 (MCP-3) wAs identified As A physiologicAl substrAte of GelAtinAse A. CleAved MCP-3 binds to CC-chemokine receptors-1, -2, And -3, but no longer induces cAlcium fluxes or promotes chemotAxis, And insteAd Acts As A generAl chemokine AntAgonist thAt dAmpens inflAmmAtion. This suggests thAt mAtrix metAlloproteinAses Are both effectors And regulAtors of the inflAmmAtory response.

Motoharu Seiki - One of the best experts on this subject based on the ideXlab platform.

  • front cell specific expression of membrAne type 1 mAtrix metAlloproteinAse And GelAtinAse A during cohort migrAtion of colon cArcinomA cells induced by hepAtocyte growth fActor scAtter fActor
    Cancer Research, 2000
    Co-Authors: Kazuki Nabeshima, Teruhiko Inoue, Yoshiya Shimao, Motoharu Seiki, Yoshifumi Itoh, Yasunori Okada, Masashi Koono
    Abstract:

    MigrAtion of tumor cells is usuAlly Assessed As single cell locomotion in vitro using Boyden chAmber type AssAys. In vivo , however, cArcinomA cells frequently invAde the surrounding tissue As coherent clusters or nests of cells. We hAve cAlled this type of movement “cohort migrAtion” And developed A two-dimensionAl in vitro cohort migrAtion model, in which humAn rectAl well-differentiAted AdenocArcinomA cells (L-10) migrAte from piled-up cell islAnds As coherent sheets of cells when stimulAted with hepAtocyte growth fActor/scAtter fActor. In this study, we exAmined whether there is A cohort migrAtion-specific wAy of expression of mAtrix metAlloproteinAses (MMP) And whether degrAdAtion of extrAcellulAr mAtrix is necessAry for this type of migrAtion. Production of membrAne-type 1-MMP (MT1-MMP) And GelAtinAse A (MMP-2) by L-10 cells wAs demonstrAted by gelAtin zymogrAphy, immunoblotting, And reverse trAnscription-PCR. When cohort migrAtion wAs induced with hepAtocyte growth fActor/scAtter fActor, MT1-MMP And MMP-2 were immunolocAlized predominAntly in the leAding edges of the front cells of migrAting cell sheets, with the following cells being negAtive. In Addition, during the cohort migrAtion on gelAtin-coAted substrAtum, the gelAtin mAtrix wAs degrAded by the cells, in A very orgAnized mAnner, cAusing rAdiAlly ArrAyed lysis of gelAtin mAtrix At the sites of leAding edges. BB94, A synthetic inhibitor specific to MMPs, tissue inhibitor of metAlloproteinAses-1 And -2, And the COOH-terminAl hemopexin-like domAin of MMP-2 inhibited the migrAtion on gelAtin mAtrix. Thus, these dAtA demonstrAte thAt gelAtin mAtrix is reorgAnized to suit cell migrAtion viA leAding-edge-of-front-cell-specific locAlizAtion of MT1-MMP And MMP-2 during cohort migrAtion And suggest thAt the reorgAnizAtion is essentiAl for this type of migrAtion.

  • processing of A precursor of 72 kilodAlton type iv collAgenAse GelAtinAse A by A recombinAnt membrAne type 1 mAtrix metAlloproteinAse
    Cancer Research, 1996
    Co-Authors: Takeshi Kinoshita, Hiroshi Sato, Takahisa Takino, Michiyasu Itoh, Toshifumi Akizawa, Motoharu Seiki
    Abstract:

    MembrAne-type 1 mAtrix metAlloproteinAse thAt is AssociAted with the proteolytic ActivAtion of proGelAtinAse A wAs expressed As A recombinAnt fusion protein in EscherichiA coli . The recombinAnt enzyme cleAved the propeptide sequence of GelAtinAse A in A sequence-specific mAnner. A mutAnt proGelAtinAse A thAt hAs A substitution of Asn66-Leu to Ile-VAl wAs not processed At All. The processing wAs blocked by tissue inhibitor of metAlloproteinAses-2 or BB-94 but not by tissue inhibitor of metAlloproteinAses-1. Thus, membrAne-type 1 mAtrix metAlloproteinAse is A direct ActivAtor of proGelAtinAse A without requiring AdditionAl proteAses.

  • cell surfAce binding And ActivAtion of GelAtinAse A induced by expression of membrAne type 1 mAtrix metAlloproteinAse mt1 mmp
    FEBS Letters, 1996
    Co-Authors: Hiroshi Sato, Yasunori Okada, Takahisa Takino, Takeshi Kinoshita, Kazushi Imai, William Stetler G Stevenson, Motoharu Seiki
    Abstract:

    GelAtinAse A is secreted As A proenzyme (proGelAtinAse A) which is ActivAted And bound on the surfAce of tumor And normAl cells. We hAve reported thAt the expression of A membrAne-type-1-mAtrix metAlloproteinAse (MT1-MMP) induces ActivAtion of proGelAtinAse A. Here we demonstrAte thAt the expression of MT1-MMP in COS-1 cells induces cell-surfAce binding of proGelAtinAse A which is consequently processed to An intermediAte form. Processing from the intermediAte to the fully Active form is dependent on the GelAtinAse A concentrAtion. These results suggest thAt the cell-surfAce binding concentrAtes the GelAtinAse A intermediAte form locAlly to Allow Autoproteolytic processing to the fully Active form.

  • mt mmp the cell surfAce ActivAtor of prommp 2 pro GelAtinAse A is expressed with its substrAte in mouse tissue during embryogenesis
    Journal of Cell Science, 1996
    Co-Authors: Hiroaki Kinoh, Yasunori Okada, Hiroshi Sato, Takahisa Takino, Yoshio Tsunezuka, Atsuhiro Kawashima, Motoharu Seiki
    Abstract:

    MAtrix metAlloproteinAses (MMPs), which degrAde the components of the extrAcellulAr mAtrix, Are key enzymes involved in the tissue remodeling of multicellulAr orgAnisms. Since MMPs Are secreted As inActive zymogens (pro-MMPs), they hAve to be ActivAted to function. We identified A membrAne-type MMP (MT-MMP) thAt ActivAted proMMP-2 (pro-GelAtinAse A = 72 kDA type IV pro-collAgenAse) And described its expression on the invAsive tumor cell surfAce. In this study we further exAmined the expression And role of MT-MMP in the ActivAtion of proMMP-2 during mouse embryogenesis. Northern blotting demonstrAted thAt MT-MMP expression wAs increAsed together with thAt of MMP-2 And its inhibitor gene, TIMP-2, in embryos depending upon the number of dAys After gestAtion, And decreAsed with mAturAtion After birth. In situ hybridizAtion And immunohistochemistry locAlized MT-MMP mRNA And protein in the cells of ossifying tissues where both MMP-2 And TIMP-2 were expressed. ActivAted MMP-2 wAs detected by gelAtin zymogrAphy in the lysAtes prepAred from the micro dissected tissues thAt expressed the three genes. The ActivAtion rAte of proMMP-2 wAs proportionAl to the expression of MMP-2 And MT-MMP. These results indicAted thAt proMMP-2 ActivAtion through its ActivAtor, MT-MMP, is A physiologicAl system used by orgAnisms to initiAte tissue remodeling on the cell surfAce.

  • enhAnced production of mAtrix metAlloproteinAses And ActivAtion of mAtrix metAlloproteinAse 2 GelAtinAse A in humAn gAstric cArcinomAs
    International Journal of Cancer, 1996
    Co-Authors: Hidehiro Nomura, Motoharu Seiki, Noboru Fujimoto, Yasunori Okada
    Abstract:

    We exAmined the production And tissue locAlizAtion of mAtrix metAlloproteinAses (MMPs) And tissue inhibitors of metAlloproteinAses (TIMPs) in gAstric cArcinomA tissues. MMP-1 (tissue collAgenAse), MMP-9 (GelAtinAse B) And TIMP-2 were immunolocAlized in cArcinomA cells And MMP-2 (GelAtinAse A) on tumor cell membrAnes, whereAs no or little immunostAining for MMP-3 (stromelysin-1) And TIMP-1 wAs seen in cArcinomA cells. StromAl cells in cArcinomA tissue were Also positively stAined for these MMPs And TIMPs. MMP-2 immunostAining wAs observed exclusively on AdvAnced gAstric cArcinomA cells And correlAted with vAsculAr invAsion by tumor cells. SAndwich enzyme immunoAssAys reveAled enhAnced production of MMP-1, MMP-2, MMP-3, MMP-9 And TIMP-1 by cArcinomA tissues. GelAtinolytic Activities were significAntly higher in cArcinomA sAmples thAn in normAl controls. Using gelAtin zymogrAphy, Active forms of MMP-2 And MMP-9 were more frequently detected in cArcinomA tissue, And the ActivAtion rAte of the zymogen of MMP-2 (proMMP-2), but not thAt of proMMP-9, correlAted well with degree of locAl invAsion And lymphAtic permeAtion. Our dAtA indicAte An enhAnced production of 4 MMPs in gAstric cArcinomA tissue And suggest thAt ActivAtion of pro-MMP-2 mAy be A key step for spreAding of gAstric cArcinomA cells.

Gillian Murphy - One of the best experts on this subject based on the ideXlab platform.

  • 129 – GelAtinAse A
    Handbook of Proteolytic Enzymes, 2020
    Co-Authors: Gillian Murphy
    Abstract:

    Publisher SummAry This chApter describes the structurAl chemistry And the biochemistry of GelAtinAse A. GelAtinAse A reAdily degrAdes the denAtured form of All collAgens thAt hAve been studied. In some instAnces, the cleAvAge of solubilized nAtive collAgens hAs been described, including type I collAgen type V collAgen And type VII collAgen. The Ability to degrAde type IV collAgen vAries with the extent of exposure of the lAtter to reducing Agents And the temperAture with the extent of loss of nAtive stAte by the prepArAtion under study. It is not At All cleAr thAt this enzyme cAn effectively cleAve nAtive type IV collAgen in vivo. Numerous other extrAcellulAr mAtrix components Are degrAded, including elAstin, lAminin-1 And lAminin-5, fibronectin And AggrecAn. PlAsminogen cleAvAge by GelAtinAse A generAtes AngiostAtin, which Acts As An Anti-Angiogenic Agent. The chemokine CCL7 MCP-3 is inActivAted by GelAtinAse A cleAvAge And the frAgment generAted Acts As A receptor AntAgonist The GelAtinAse A precursor is mAde up of the typicAl mAtrixin ArrAngement of A signAl peptide, A propeptide, A cAtAlytic unit And A C-terminAl domAin.

  • the timp2 membrAne type 1 metAlloproteinAse receptor regulAtes the concentrAtion And efficient ActivAtion of proGelAtinAse A A kinetic study
    Journal of Biological Chemistry, 1998
    Co-Authors: Georgina S Butler, Susan J Atkinson, Horst Will, Thomas Crabbe, Michael J Butler, Tatsuya Tamura, Steven Schade Van Westrum, John Clements, Mariepia Dortho, Gillian Murphy
    Abstract:

    AbstrAct We hAve used C-terminAl domAin mutAnts to further define the role of interActions of proGelAtinAse A And membrAne type 1 mAtrix metAlloproteinAse (MT1 MMP) in the binding of TIMP2 And in the cell-AssociAted ActivAtion of proGelAtinAse A. Soluble constructs of MT1 MMP were used to demonstrAte thAt binding with TIMP2 occurs primArily through N-terminAl domAin interActions, leAving the C-terminAl domAin free for interActions with proGelAtinAse A. The rAte of Autolytic ActivAtion of proGelAtinAse A initiAted by MT1 MMP cleAvAge could be potentiAted by concentrAtion of the proenzyme by binding to hepArin. Residues 568–631 of the proGelAtinAse A C-terminAl domAin Are importAnt in formAtion of the hepArin binding site, since replAcement of this region with the corresponding stromelysin-1 sequence Abolished binding to hepArin And the potentiAtion of ActivAtion. The sAme region of GelAtinAse A wAs required for binding of lAtent And Active enzyme to TIMP2, but residues 418–474 were not importAnt. A similAr pAttern wAs seen using cell membrAne-AssociAted MT1 MMP; residues 568–631 were required for binding And ActivAtion of proGelAtinAse A, whereAs residues 418–474 were not. Neither region wAs required for ActivAtion in solution. The Addition of TIMP2 to HT1080 membrAne prepArAtions expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiAtion of proGelAtinAse A ActivAtion. This effect wAs dependent upon TIMP2 binding to MT1 MMP rAther thAn At An independent membrAne site. Together, the dAtA suggest thAt TIMP2 forms A receptor with MT1 MMP thAt regulAtes the concentrAtion And efficient generAtion of functionAlly Active GelAtinAse A.

  • cellulAr mechAnisms for humAn procollAgenAse 3 mmp 13 ActivAtion evidence thAt mt1 mmp mmp 14 And GelAtinAse A mmp 2 Are Able to generAte Active enzyme
    Journal of Biological Chemistry, 1996
    Co-Authors: Vera Knäuper, Bryan J Smith, Carlos Lopezotin, Susan J Atkinson, R M Hembry, Heather Stanton, Horst Will, Gillian Murphy
    Abstract:

    AbstrAct GelAtinAse A And membrAne-type metAlloproteinAse (MT1-MMP) were Able to process humAn procollAgenAse-3 (Mr 60,000) to the fully Active enzyme (Tyr85 N terminus; Mr 48,000). MT1-MMP ActivAted procollAgenAse-3 viA A Mr 56,000 intermediAte (Ile36 N terminus) to 48,000 which wAs the result of the cleAvAge of the Glu84-Tyr85 peptide bond. We hAve estAblished thAt the ActivAtion rAte of procollAgenAse-3 by MT1-MMP wAs enhAnced in the presence of proGelAtinAse A, thereby demonstrAting A unique new ActivAtion cAscAde consisting of three members of the mAtrix metAlloproteinAse fAmily. In Addition, procollAgenAse-3 cAn be ActivAted by plAsmin, which cleAved the Lys38-Glu39 And Arg76-Cys77 peptide bonds in the propeptide domAin. Autoproteolysis then resulted in the releAse of the rest of the propeptide domAin generAting Tyr85 N-terminAl Active collAgenAse-3. However, plAsmin cleAved the C-terminAl domAin of collAgenAse-3 which results in the loss of its collAgenolytic Activity. ConcAnAvAlin A-stimulAted fibroblAsts expressing MT1-MMP And fibroblAst-derived plAsmA membrAnes were Able to process humAn procollAgenAse-3 viA A Mr 56,000 intermediAte form to the finAl Mr 48,000 Active enzyme which, by AnAlogy with proGelAtinAse A ActivAtion, mAy represent A model system for in vivo ActivAtion. Inhibition experiments using tissue inhibitor of metAlloproteinAses, plAsminogen ActivAtor inhibitor-2, or Aprotinin demonstrAted thAt ActivAtion in the cellulAr model system wAs due to MT1-MMP/GelAtinAse A And excluded the pArticipAtion of serine proteinAses such As plAsmin during procollAgenAse-3 ActivAtion. We hAve estAblished thAt proGelAtinAse A cAn considerAbly potentiAte the ActivAtion rAte of procollAgenAse-3 by crude plAsmA membrAne prepArAtions from concAnAvAlin A-stimulAted fibroblAsts, thus confirming our results using purified proGelAtinAse A And MT1-MMP. This new ActivAtion cAscAde mAy be significAnt in humAn breAst cAncer pAthology, where All three enzymes hAve been implicAted As plAying importAnt roles.

  • Assessment of the role of the fibronectin like domAin of GelAtinAse A by AnAlysis of A deletion mutAnt
    Journal of Biological Chemistry, 1994
    Co-Authors: Gillian Murphy, Susan J Atkinson, Quang Nguyen, Mark I Cockett, J A Allan, C G Knight, Frances Willenbrock, Andrew J P Docherty
    Abstract:

    AbstrAct The properties of A deletion mutAnt deltA V191-Q364 of GelAtinAse A, which represents the removAl of the fibronectin-like type II repeAts defined by exons 5-7, were compAred with those of full-length GelAtinAse A. Both enzymes underwent self-ActivAtion over A similAr time course in the presence of 4-Aminophenylmercuric AcetAte. The fully Active enzymes hAd similAr kcAt/Km vAlues for the cleAvAge of An octApeptide substrAte, but the deletion mutAnt hAd 50% of the Activity of wild type GelAtinAse A AgAinst betA-cAsein And 10% of the Activity AgAinst gelAtin. The cleAvAge pAttern for gelAtin wAs similAr for both enzymes but differed for type IV collAgen. CompArison of the rAtes of AssociAtion of the tissue inhibitors of metAlloproteinAse (TIMP)-1 And TIMP-2 And their N-terminAl domAins to both forms of GelAtinAse indicAted thAt the fibronectin-like domAin plAys little role in TIMP binding. The deletion mutAnt fAiled to bind to collAgen, while the wild type GelAtinAse bound tightly, indicAting thAt the fibronectin-like domAin is the sole site of collAgen binding. Both GelAtinAses could be ActivAted by concAnAvAlin A-ActivAted fibroblAsts, suggesting thAt the fibronectin-like domAin is not required for the membrAne-mediAted ActivAtion process.

  • the Activity of the tissue inhibitors of metAlloproteinAses is regulAted by c terminAl domAin interActions A kinetic AnAlysis of the inhibition of GelAtinAse A
    Biochemistry, 1993
    Co-Authors: Frances Willenbrock, Mark I Cockett, Andrew J P Docherty, Thomas Crabbe, Patrick M Slocombe, Christopher W Sutton, Mark Oshea, Keith Brocklehurst, Ian Phillips, Gillian Murphy
    Abstract:

    : The cloning And expression of the full-length tissue inhibitor of metAlloproteinAse 2 (TIMP-2), deltA 187-194TIMP-2, And deltA 128-194TIMP-2 And the purificAtion of these inhibitors And A cleAved version of TIMP-2 lAcking nine C-terminAl Amino Acids (deltA 186-194TIMP-2) Are described. The mechAnism of inhibition of GelAtinAse A by the TIMPs wAs investigAted by compAring the kinetics of AssociAtion of TIMP-1, TIMP-2, the C-terminAl deletions, And the mutAnts of both TIMPs which consisted of the N-terminAl domAin only. The full-length TIMPs inhibited GelAtinAse A rApidly with AssociAtion constAnts of 3.2 x 10(6) M-1 s-1 for TIMP-1 And 2.1 x 10(7) M-1 s-1 for TIMP-2 At I = 0.2. The C-terminAl peptide of TIMP-2 is proposed to exist As An exposed "tAil" responsible for binding to proGelAtinAse A And for increAsing the rAte of inhibition of Active GelAtinAse A through electrostAtic interActions with the C-terminAl domAin of the enzyme. The C-terminAl domAins of both TIMP-1 And TIMP-2 pArticipAte in low-Affinity interActions with the C-terminAl domAin of GelAtinAse A which increAse the rAte of AssociAtion by A fActor of About 100 in both cAses.

Yasunori Okada - One of the best experts on this subject based on the ideXlab platform.

  • zymogrAphic AnAlysis of circulAting And tissue forms of colon cArcinomA GelAtinAse A mmp 2 And b mmp 9 sepArAted by mono And two dimensionAl electrophoresis
    Matrix Biology, 2001
    Co-Authors: Ida Pucciminafra, S Minafra, Giampiero La Rocca, Marilisa Barranca, Simona Fontana, Giuseppina Alaimo, Yasunori Okada
    Abstract:

    AbstrAct GelAtinAse A (MMP-2) And GelAtinAse B (MMP-9) plAy A key role in the proteolytic cAscAde leAding to ECM degrAdAtion during invAsion And metAstAsis. The enzyme Activity is regulAted both At the intrA- And extrA-cellulAr level. ExtrAcellulAr regulAtion is Achieved mAinly through the bAlAnce between proenzyme ActivAtion And inhibition, which AppeArs to be Altered in cAncer pAtients. One of the mechAnisms of MMP inhibition is the binding of the enzymes to AppropriAte tissue inhibitors (TIMP). In the recent literAture, it hAs been suggested thAt MMP-2 And/or MMP-9 Are indeed over-produced in mAny cArcinomAs, while the identity of the vArious enzymAtic forms (lAtent, ActivAted And enzyme/inhibitor complexes) remAins to be elucidAted. In this study we hAve AnAlyzed the circulAting forms of MMP-9 And MMP-2 in serum sAmples of pAtients with colon cArcinomA, As well As the enzymAtic Activities present in tissue extrActs from surgicAl frAgments (primAry tumor And its pAired heAlthy tissue). Proteins were sepArAted by meAns of mono-dimensionAl or bidimensionAl electrophoresis, And the enzymes detected by gelAtin zymogrAphy And immunologicAl AssAys. The results of densitometric AnAlyses demonstrAte thAt proMMP-9, but not proMMP-2, is significAntly higher in the oncologic serA vs. the normAl serA. In Addition, severAl oligomeric circulAting And tissue forms of MMP-9 Are preferentiAlly found in the oncologic sAmples, both in mono- And second-dimension zymogrAms. The ActivAted forms of MMP-2 And MMP-9 Are uniquely present in the primAry tumor extrActs, thus confirming the involvement of the tissue microenvironment in GelAtinAse ActivAtion And function.

  • gliomA cell extrAcellulAr mAtrix metAlloproteinAse inducer emmprin cd147 stimulAtes production of membrAne type mAtrix metAlloproteinAses And ActivAted GelAtinAse A in co cultures with brAin derived fibroblAsts
    Cancer Letters, 2000
    Co-Authors: Tetsuro Sameshima, Masashi Koono, Kazuki Nabeshima, Yasunori Okada, Bryan P Toole, Kiyotaka Yokogami, Tomokazu Goya, Shinichiro Wakisaka
    Abstract:

    AbstrAct ExtrAcellulAr mAtrix metAlloproteinAse inducer (EMMPRIN) Also cAlled CD147, bAsigin or M6 in the humAn is A member of the immunoglobulin superfAmily thAt is enriched on the surfAce of tumor cells And stimulAtes AdjAcent stromAl cells to produce severAl mAtrix metAlloproteinAses (MMPs). In this study, we hAve demonstrAted thAt coculturing of EMMPRIN-expressing humAn glioblAstomA multiforme cells (U251) with brAin-derived humAn fibroblAsts not only stimulAtes production, but Also ActivAtion of pro-GelAtinAse A (proMMP-2), An enzyme thAt is enriched in mAlignAnt gliomAs And most likely cruciAl to tumor progression. Production of membrAne types 1 And 2-MMPs (MT1-MMP And MT2-MMP), which Are ActivAtors of proMMP-2, wAs Also stimulAted in these cocultures. StimulAtion of MMP-2, MT1-MMP And MT2-MMP production wAs inhibited by Anti-EMMPRIN monoclonAl Antibody in A dose-dependent mAnner. Thus, we hAve shown, for the first time, thAt EMMPRIN cAuses increAsed expression of MT1-MMP And MT2-MMP, As well As increAsed production And ActivAtion of MMP-2.

  • front cell specific expression of membrAne type 1 mAtrix metAlloproteinAse And GelAtinAse A during cohort migrAtion of colon cArcinomA cells induced by hepAtocyte growth fActor scAtter fActor
    Cancer Research, 2000
    Co-Authors: Kazuki Nabeshima, Teruhiko Inoue, Yoshiya Shimao, Motoharu Seiki, Yoshifumi Itoh, Yasunori Okada, Masashi Koono
    Abstract:

    MigrAtion of tumor cells is usuAlly Assessed As single cell locomotion in vitro using Boyden chAmber type AssAys. In vivo , however, cArcinomA cells frequently invAde the surrounding tissue As coherent clusters or nests of cells. We hAve cAlled this type of movement “cohort migrAtion” And developed A two-dimensionAl in vitro cohort migrAtion model, in which humAn rectAl well-differentiAted AdenocArcinomA cells (L-10) migrAte from piled-up cell islAnds As coherent sheets of cells when stimulAted with hepAtocyte growth fActor/scAtter fActor. In this study, we exAmined whether there is A cohort migrAtion-specific wAy of expression of mAtrix metAlloproteinAses (MMP) And whether degrAdAtion of extrAcellulAr mAtrix is necessAry for this type of migrAtion. Production of membrAne-type 1-MMP (MT1-MMP) And GelAtinAse A (MMP-2) by L-10 cells wAs demonstrAted by gelAtin zymogrAphy, immunoblotting, And reverse trAnscription-PCR. When cohort migrAtion wAs induced with hepAtocyte growth fActor/scAtter fActor, MT1-MMP And MMP-2 were immunolocAlized predominAntly in the leAding edges of the front cells of migrAting cell sheets, with the following cells being negAtive. In Addition, during the cohort migrAtion on gelAtin-coAted substrAtum, the gelAtin mAtrix wAs degrAded by the cells, in A very orgAnized mAnner, cAusing rAdiAlly ArrAyed lysis of gelAtin mAtrix At the sites of leAding edges. BB94, A synthetic inhibitor specific to MMPs, tissue inhibitor of metAlloproteinAses-1 And -2, And the COOH-terminAl hemopexin-like domAin of MMP-2 inhibited the migrAtion on gelAtin mAtrix. Thus, these dAtA demonstrAte thAt gelAtin mAtrix is reorgAnized to suit cell migrAtion viA leAding-edge-of-front-cell-specific locAlizAtion of MT1-MMP And MMP-2 during cohort migrAtion And suggest thAt the reorgAnizAtion is essentiAl for this type of migrAtion.

  • cell surfAce binding And ActivAtion of GelAtinAse A induced by expression of membrAne type 1 mAtrix metAlloproteinAse mt1 mmp
    FEBS Letters, 1996
    Co-Authors: Hiroshi Sato, Yasunori Okada, Takahisa Takino, Takeshi Kinoshita, Kazushi Imai, William Stetler G Stevenson, Motoharu Seiki
    Abstract:

    GelAtinAse A is secreted As A proenzyme (proGelAtinAse A) which is ActivAted And bound on the surfAce of tumor And normAl cells. We hAve reported thAt the expression of A membrAne-type-1-mAtrix metAlloproteinAse (MT1-MMP) induces ActivAtion of proGelAtinAse A. Here we demonstrAte thAt the expression of MT1-MMP in COS-1 cells induces cell-surfAce binding of proGelAtinAse A which is consequently processed to An intermediAte form. Processing from the intermediAte to the fully Active form is dependent on the GelAtinAse A concentrAtion. These results suggest thAt the cell-surfAce binding concentrAtes the GelAtinAse A intermediAte form locAlly to Allow Autoproteolytic processing to the fully Active form.

  • mt mmp the cell surfAce ActivAtor of prommp 2 pro GelAtinAse A is expressed with its substrAte in mouse tissue during embryogenesis
    Journal of Cell Science, 1996
    Co-Authors: Hiroaki Kinoh, Yasunori Okada, Hiroshi Sato, Takahisa Takino, Yoshio Tsunezuka, Atsuhiro Kawashima, Motoharu Seiki
    Abstract:

    MAtrix metAlloproteinAses (MMPs), which degrAde the components of the extrAcellulAr mAtrix, Are key enzymes involved in the tissue remodeling of multicellulAr orgAnisms. Since MMPs Are secreted As inActive zymogens (pro-MMPs), they hAve to be ActivAted to function. We identified A membrAne-type MMP (MT-MMP) thAt ActivAted proMMP-2 (pro-GelAtinAse A = 72 kDA type IV pro-collAgenAse) And described its expression on the invAsive tumor cell surfAce. In this study we further exAmined the expression And role of MT-MMP in the ActivAtion of proMMP-2 during mouse embryogenesis. Northern blotting demonstrAted thAt MT-MMP expression wAs increAsed together with thAt of MMP-2 And its inhibitor gene, TIMP-2, in embryos depending upon the number of dAys After gestAtion, And decreAsed with mAturAtion After birth. In situ hybridizAtion And immunohistochemistry locAlized MT-MMP mRNA And protein in the cells of ossifying tissues where both MMP-2 And TIMP-2 were expressed. ActivAted MMP-2 wAs detected by gelAtin zymogrAphy in the lysAtes prepAred from the micro dissected tissues thAt expressed the three genes. The ActivAtion rAte of proMMP-2 wAs proportionAl to the expression of MMP-2 And MT-MMP. These results indicAted thAt proMMP-2 ActivAtion through its ActivAtor, MT-MMP, is A physiologicAl system used by orgAnisms to initiAte tissue remodeling on the cell surfAce.

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  • tissue inhibitor of metAlloproteinAses 4 inhibits but does not support the ActivAtion of GelAtinAse A viA efficient inhibition of membrAne type 1 mAtrix metAlloproteinAse
    Cancer Research, 2001
    Co-Authors: Heather F Bigg, Charlotte J Morrison, Georgina S Butler, Marie A Bogoyevitch, Zhiping Wang, Paul D Soloway, Christopher M Overall
    Abstract:

    The tissue inhibitors of metAlloproteinAses 1–4 (TIMPs) hAve discrete regulAtory roles in the ActivAtion of mAtrix metAlloproteinAse (MMP)-2 (GelAtinAse A), An importAnt bAsement membrAne-degrAding MMP pivotAl to tumor metAstAsis And Angiogenesis. TIMP-2 binds to both the hemopexin C domAin of proGelAtinAse A And the Active site of membrAne type-1 (MT1) MMP. This trimeric complex presents the cell surfAce-bound GelAtinAse A zymogen to A free MT1-MMP molecule for ActivAtion. To investigAte the role of TIMP-4 in the ActivAtion process, we developed A new procedure for the expression And purificAtion of recombinAnt humAn TIMP-4 from bAby hAmster kidney cells. The recombinAnt TIMP-4 wAs A potent inhibitor of GelAtinAse A {AppArent K i [ K i(App.) ] ≤ 9 pm; k on (AssociAtion rAte constAnt), 4.57 ± 0.13 × 10 6 m −1 s −1 } And wAs less dependent upon hemopexin C domAin interActions thAn TIMP-2 in its mode of binding And inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP ( K i(App.) ≤ 100 pm; k on , 3.49 ± 0.34 × 10 6 m −1 s −1 ) And blocked the concAnAvAlin A-induced cellulAr ActivAtion of proGelAtinAse A. In concAnAvAlin A-stimulAted homozygous Timp2 −/− fibroblAsts or unstimulAted MT1-MMP-trAnsfected Timp2 −/− cells, which cAnnot ActivAte proGelAtinAse A, ActivAtion wAs restored by the Addition of 0.3–5 nm TIMP-2 but not by TIMP-4, unequivocAlly showing the TIMP-2 dependency of MT1-MMP-induced ActivAtion of GelAtinAse A And the fAct thAt TIMP-4 cAnnot support ActivAtion. The dominAnce of TIMP-2 in the ActivAtion process wAs further supported by the preferentiAl binding of TIMP-2 compAred with TIMP-4 to the hemopexin C domAin of proGelAtinAse A in inhibitor mixtures And by the Ability of TIMP-2 to displAce TIMP-4 from the hemopexin C domAin. Hence, TIMP-4 regulAtes GelAtinAse A Activity by efficient inhibition of MT1-MMP-mediAted ActivAtion And by inhibiting the ActivAted enzyme And, thus, is A tumor resistAnce fActor in the peritumor stromA.

  • specific high Affinity binding of tissue inhibitor of metAlloproteinAses 4 timp 4 to the cooh terminAl hemopexin like domAin of humAn GelAtinAse A timp 4 binds proGelAtinAse A And the cooh terminAl domAin in A similAr mAnner to timp 2
    Journal of Biological Chemistry, 1997
    Co-Authors: Heather F Bigg, Bjorn Steffensen, Christopher M Overall
    Abstract:

    AbstrAct The binding properties of the newly described tissue inhibitor of metAlloproteinAses-4 (TIMP-4) to proGelAtinAse A And to the COOH-terminAl hemopexin-like domAin (C domAin) of the enzyme were exAmined. We present evidence for the first time of A specific, high Affinity interAction between TIMP-4 And the C domAin of humAn GelAtinAse A And show thAt TIMP-4 binds both proGelAtinAse A And the C domAin in A similAr mAnner to thAt of TIMP-2. SAturAble binding of recombinAnt C domAin to TIMP-4 And to TIMP-2 but not to TIMP-1 wAs demonstrAted using A microwell protein binding AssAy. The recombinAnt collAgen binding domAin of GelAtinAse A, comprised of the three fibronectin type II-like repeAts, did not bind to TIMP-4, indicAting thAt binding is mediAted selectively by the C domAin. Binding to TIMP-4 wAs of high Affinity with An AppArent K d of 1.7 × 10−7 m but slightly weAker thAn thAt to TIMP-2 (AppArent K d of 0.66 × 10−7 m). Affinity chromAtogrAphy confirmed the TIMP-4-C domAin interAction And Also showed thAt the complex could not be disrupted by 1 m NACl or 10% dimethyl sulfoxide, thereby further demonstrAting the tight binding. To verify the biologicAl significAnce of this interAction, binding of full-length proGelAtinAse A to TIMP-4 wAs investigAted. TIMP-4 And TIMP-2 but not TIMP-1 bound specificAlly to purified TIMP-2-free humAn recombinAnt full-length proGelAtinAse A And to full-length rAt proenzyme from the conditioned culture medium of ROS 17/2.8 cells. PreincubAtion of the C domAin with TIMP-2 wAs found to reduce subsequent binding to TIMP-4 in A concentrAtion-dependent mAnner. Competition between TIMP-2 And TIMP-4 for A common or overlApping binding sites on the GelAtinAse A C domAin mAy occur; AlternAtively TIMP-2 mAy prevent the binding of TIMP-4 by steric hindrAnce or induction of A conformAtionAl chAnge in the C domAin. We propose thAt the binding of proGelAtinAse A to TIMP-4 represents A third TIMP-proGelAtinAse interAction in Addition to thAt of proGelAtinAse A with TIMP-2 And proGelAtinAse B with TIMP-1 described previously. This new phenomenon mAy be of importAnt physiologicAl significAnce in modulAting the cell surfAce ActivAtion of proGelAtinAse A.