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1.
Biomolecules ; 12(8)2022 08 19.
Article in English | MEDLINE | ID: mdl-36009039

ABSTRACT

Homomultimerization of MT1-MMP (membrane type 1 matrix metalloproteinase) through the hemopexin, transmembrane, and cytoplasmic domains plays a very important role in the activation of proMMP-2 and the degradation of pericellular collagen. MT1-MMP is overexpressed in many types of cancers, and it is considered to be a key enzyme in facilitating cancer cell migration. Since the oligomerization of MT1-MMP is important for its proteolytic activity in promoting cancer invasion, we have further investigated the multimerization by using heterologously expressed MT1-MMP ectodomains in insect cells to gain additional mechanistic insight into this process. We show that the whole ectodomain of MT1-MMP can form dimers and higher-order oligomeric complexes. The enzyme is secreted in its active form and the multimeric complex assembly is mediated by the catalytic domain. Blocking the prodomain removal determines the enzyme to adopt the monomeric structure, suggesting that the prodomain prevents the MT1-MMP oligomerization process. The binding affinity of MT1-MMP to type I collagen is dependent on the oligomeric state. Thus, the monomers have the weakest affinity, while the binding strength increases proportionally with the complexity of the multimers. Collectively, our experimental results indicate that the catalytic domain of MT1-MMP is necessary and sufficient to mediate the formation of multimeric structures.


Subject(s)
Matrix Metalloproteinase 14 , Metalloendopeptidases , Catalytic Domain , Enzyme Activation , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinases, Membrane-Associated/metabolism , Metalloendopeptidases/metabolism , Protein Structure, Tertiary
2.
Proc Natl Acad Sci U S A ; 108(14): 5578-83, 2011 Apr 05.
Article in English | MEDLINE | ID: mdl-21415364

ABSTRACT

Proteolytic release from the cell surface is an essential activation event for many growth factors and cytokines. TNF-α converting enzyme (TACE) is a membrane-bound metalloprotease responsible for solubilizing many pathologically significant membrane substrates and is an attractive therapeutic target for the treatment of cancer and arthritis. Prior attempts to antagonize cell-surface TACE activity have focused on small-molecule inhibition of the metalloprotease active site. Given the highly conserved nature of metalloprotease active sites, this paradigm has failed to produce a truly specific TACE inhibitor and continues to obstruct the clinical investigation of TACE activity. We report the bespoke development of a specific TACE inhibitory human antibody using "two-step" phage display. This approach combines calculated selection conditions with antibody variable-domain exchange to direct individual antibody variable domains to desired epitopes. The resulting "cross-domain" human antibody is a previously undescribed selective TACE antagonist and provides a unique alternative to small-molecule metalloprotease inhibition.


Subject(s)
ADAM Proteins/antagonists & inhibitors , Antibodies/pharmacology , Drug Design , Immunoglobulin Variable Region/metabolism , Models, Molecular , ADAM Proteins/metabolism , ADAM17 Protein , Animals , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin Variable Region/genetics , Mice , Mutagenesis , Peptide Library , Protein Structure, Tertiary/genetics , Surface Plasmon Resonance
3.
J Cell Sci ; 123(Pt 23): 4182-93, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-21062896

ABSTRACT

Membrane-type-1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion, with elevated levels correlating with a poor prognosis in cancer. MT1-MMP-mediated transcriptional regulation of genes in cancer cells can contribute to tumour growth, although this is poorly understood at a mechanistic level. In this study, we investigated the mechanism by which MT1-MMP regulates the expression of VEGF-A in breast cancer cells. We discovered that MT1-MMP regulates VEGFR-2 cell surface localisation and forms a complex with VEGFR-2 and Src that is dependent on the MT1-MMP hemopexin domain and independent of its catalytic activity. Although the localisation of VEGFR-2 was independent of the catalytic and intracellular domain of MT1-MMP, intracellular signalling dependent on VEGFR-2 activity leading to VEGF-A transcription still required the MT1-MMP catalytic and intracellular domain, including residues Y573, C574 and DKV582. However, there was redundancy in the function of the catalytic activity of MT1-MMP, as this could be substituted with MMP-2 or MMP-7 in cells expressing inactive MT1-MMP. The signalling cascade dependent on the MT1-MMP-VEGFR-2-Src complex activated Akt and mTOR, ultimately leading to increased VEGF-A transcription.


Subject(s)
Gene Expression Regulation , Matrix Metalloproteinase 14/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Amino Acid Sequence , Cell Line, Tumor , Humans , Matrix Metalloproteinase 14/chemistry , Matrix Metalloproteinase 14/genetics , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins pp60(c-src)/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics
4.
Protein Eng Des Sel ; 22(3): 175-88, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19022801

ABSTRACT

An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.


Subject(s)
Immunoglobulin Fab Fragments/immunology , Immunoglobulin kappa-Chains/immunology , Immunoglobulin lambda-Chains/immunology , Receptors, IgG/immunology , Recombinant Fusion Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Bispecific/genetics , Antibodies, Bispecific/immunology , Antibodies, Bispecific/metabolism , Antibody Affinity , Base Sequence , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Flow Cytometry , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/metabolism , Immunoglobulin lambda-Chains/genetics , Immunoglobulin lambda-Chains/metabolism , Ki-1 Antigen/metabolism , Killer Cells, Natural , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Myeloma Proteins/metabolism , Protein Folding , Receptors, IgG/genetics , Receptors, IgG/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Solubility
5.
Invest Ophthalmol Vis Sci ; 49(7): 2812-22, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18408187

ABSTRACT

PURPOSE: Tissue inhibitor of metalloprotease (TIMP)-3 is an inhibitor of matrix metalloprotease (MMP) and regulates angiogenesis. In the eye, TIMP3 is tightly associated with Bruch's membrane. In this study, the authors analyzed mice lacking TIMP3 for retinal abnormalities. METHODS: Mice with targeted disruption of the Timp3 gene were generated (Timp3(-/-)) and bred into C57/Bl6 and CD1 backgrounds. Eyes were analyzed by light and electron microscopy. Vasculature was examined by scanning laser ophthalmoscopy, corrosion casts, and whole mount preparations. MMP activity was assessed by in situ zymography, angiogenic potential was evaluated by tube formation, and aortic ring assays and signaling pathways were studied by immunoblotting. RESULTS: TIMP3-deficient mice develop abnormal vessels with dilated capillaries throughout the choroid. Enhanced MMP activity in the choroid region of Timp3(-/-) eyes was detected when compared with controls. Timp3(-/-)-derived tissue showed an increased angiogenic activity over wild-type, an effect that could specifically be inhibited by recombinant TIMP3. Moreover, the antiangiogenic property of TIMP3 was demonstrated to reside within the C-terminal domain. When VEGFR2 inhibitor was added to Timp3(-/-) aortic explants, endothelial sprout formation was markedly reduced, which provided evidence for an unbalanced VEGF-mediated angiogenesis in Timp3(-/-) animals. Finally, angiogenic signaling pathways are activated in Timp3(-/-)-derived cells. CONCLUSIONS: These findings suggest that the distinct choroidal phenotype in mice lacking TIMP3 may be the result of a local disruption of extracellular matrix and angiogenic homeostasis, and they support an important role of TIMP3 in the regulation of choroidal vascularization.


Subject(s)
Choroid/blood supply , Neovascularization, Physiologic/physiology , Tissue Inhibitor of Metalloproteinase-3/physiology , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiopathology , Blood Vessels/abnormalities , Blood Vessels/pathology , Bruch Membrane/pathology , Capillaries/growth & development , Choroid/enzymology , Eye/metabolism , In Vitro Techniques , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Knockout , Neovascularization, Pathologic/etiology , Neovascularization, Physiologic/drug effects , Phenotype , Pigment Epithelium of Eye/pathology , Protein Structure, Tertiary/physiology , Recombinant Proteins/pharmacology , Retina/pathology , Signal Transduction , Tissue Inhibitor of Metalloproteinase-3/deficiency , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors
6.
Matrix Biol ; 27(5): 381-92, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18295466

ABSTRACT

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular degeneration of late onset. A key feature of the disease is the thickening of Bruch's membrane, an ECM structure located between the RPE and the choroid. SFD is caused by mutations in the gene encoding the ECM-associated tissue inhibitor of metalloproteases-3 (TIMP3). We have recently generated two Timp3 gene-targeted mouse lines, one deficient for the murine gene (Timp3-/-) and one carrying an SFD-related S156C mutation. Based on extracts and cell cultures derived from tissues of these animals we now evaluated TIMP3 functionality and its contribution to SFD. We show that the activity levels of TIMP3 target proteases including TACE, ADAMTS4/5 and aggrecan-cleaving MMPs are similar in Timp3S156/+ and Timp3S156C/S156C mice when compared to controls. In Timp3-/- mice, a significant enhancement of enzyme activity was observed for TACE but not for ADAMTS4/5 and MMPs indicating a compensatory effect of other inhibitors regulating the latter two groups of proteases. Fibrin bead assays show that angiogenesis in Timp3S156/+ and Timp3S156C/S156C mice is not altered whereas increased formation of capillary tubes was observed in Timp3-/- animals over controls. Rescue experiments using recombinant proteins demonstrate that the inhibitory activities of TIMP3 towards TACE and aggrecan-cleaving MMPs as well as the anti-angiogenic properties of TIMP3 are not impaired by SFD mutation S156C. We finally demonstrate that wild-type and S156C-TIMP3 proteins block the binding of VEGF to its receptor VEGFR2 to a similar extent. Taken together, this study shows that S156C-TIMP3 retains its known functional properties suggesting that causes other than an imbalance in protease or angiogenic activities represent the primary molecular defect underlying SFD.


Subject(s)
Macular Degeneration/genetics , Mutation , Tissue Inhibitor of Metalloproteinase-3/genetics , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM17 Protein , ADAMTS4 Protein , ADAMTS5 Protein , Aggrecans/genetics , Aggrecans/metabolism , Amino Acid Substitution , Animals , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Liver/metabolism , Macular Degeneration/metabolism , Macular Degeneration/physiopathology , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Mice , Mice, Knockout , Neovascularization, Physiologic/genetics , Neovascularization, Physiologic/physiology , Procollagen N-Endopeptidase/genetics , Procollagen N-Endopeptidase/metabolism , Recombinant Proteins/metabolism , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/genetics , Tissue Inhibitor of Metalloproteinase-2/metabolism , Tissue Inhibitor of Metalloproteinase-3/metabolism , Tissue Inhibitor of Metalloproteinase-3/physiology , Tissue Inhibitor of Metalloproteinases/genetics , Tissue Inhibitor of Metalloproteinases/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Tissue Inhibitor of Metalloproteinase-4
7.
Biol Chem ; 385(2): 137-43, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15101556

ABSTRACT

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.


Subject(s)
DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Molecular Chaperones/chemistry , Periplasmic Proteins/chemistry , Amino Acid Sequence , Circular Dichroism , Crystallization , Crystallography, X-Ray , DNA-Binding Proteins/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Hot Temperature , Molecular Chaperones/genetics , Molecular Sequence Data , Periplasm/metabolism , Periplasmic Proteins/genetics , Protein Denaturation , Protein Renaturation , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
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