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Methods Mol Biol ; 2043: 75-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463904


A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc-dependent metalloproteinases that are involved in the maintenance of cartilage extracellular matrix (ECM) and are currently considered the major aggrecanases in the development of osteoarthritis. In this chapter we describe the establishment and cultivation of cell lines expressing ADAMTS-4,-5 and their domain deletion mutants; the collection of medium containing expressed ADAMTS-4,-5; the subsequent purification of this medium through anti-FLAG affinity chromatography; and the characterization of ADAMTS-4,-5 activity using synthetic Förster resonance energy transfer (FRET) peptide substrates.

Hepatology ; 61(1): 238-48, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25131778


UNLABELLED: Due to its ability to inhibit prometastatic matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP)-1 has been thought to suppress tumor metastasis. However, elevated systemic levels of TIMP-1 correlate with poor prognosis in cancer patients, suggesting a metastasis-stimulating role of TIMP-1. In colorectal cancer patients, tumor as well as plasma TIMP-1 levels were correlated with synchronous liver metastasis or distant metastasis-associated disease relapse. In mice, high systemic TIMP-1 levels increased the liver susceptibility towards metastasis by triggering the formation of a premetastatic niche. This promoted hepatic metastasis independent of origin or intrinsic metastatic potential of tumor cells. High systemic TIMP-1 led to increased hepatic SDF-1 levels, which in turn promoted recruitment of neutrophils to the liver. Both inhibition of SDF-1-mediated neutrophil recruitment and systemic depletion of neutrophils reduced TIMP-1-induced increased liver susceptibility towards metastasis. This indicates a crucial functional role of neutrophils in the TIMP-1-induced premetastatic niche. CONCLUSION: Our results identify TIMP-1 as an essential promoter of hepatic premetastatic niche formation.

Carcinoma/secundário , Quimiocina CXCL12/metabolismo , Neoplasias Hepáticas/secundário , Infiltração de Neutrófilos , Receptores CXCR4/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Carcinoma/sangue , Linhagem Celular Tumoral , Humanos , Fígado/imunologia , Fígado/metabolismo , Neoplasias Hepáticas/sangue , Camundongos , Camundongos Endogâmicos , Células NIH 3T3 , Inibidor Tecidual de Metaloproteinase-1/sangue
ChemistryOpen ; 2(3): 99-105, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24551546


The synthesis and matrix metalloproteinase (MMP) inhibitory activity of a cyclam-marimastat conjugate and its metal complexes are described. The conjugate, synthesized with a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition ("click" reaction), contains two zinc-binding groups (ZBGs). The metal complexation behavior with copper(II) and zinc(II) was investigated using UV/Vis spectrophotometry and (1)H NMR spectroscopy, respectively, demonstrating that the first equivalent of the metal ion was chelated by the cyclam-triazole moiety rather than the hydroxamic acid site. Thus, the corresponding mononuclear metal-cyclam complexes were successfully prepared with one equivalent of the metal salt. Both the cyclam-marimastat conjugate and its metal complexes exhibited slightly reduced potency against MMP-1, but essentially identical inhibitory activity against MMP-3. The conjugate and its metal complexes displayed little or no cytotoxicity, further supporting their potential suitability for imaging MMP localization and activity. To the best of our knowledge, this is the first report that describes the incorporation of metal complexes into an MMP inhibitor without influencing the preexisting ZBG, and the first report of the evaluation of structures containing more than one ZBG as MMP inhibitors.

Biochem J ; 431(1): 113-22, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20645923


We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) of aggrecan, gst-IGD-flag, these reactive-site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra alanine residues can be attached to the N-terminus before the Ki (app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [-1] position produced inhibitors as potent as the alanine mutant. The mutants N-TIMP-3(T2G), [-1A]N-TIMP-3 and [-2A]N-TIMP-3 were effective inhibitors of aggrecan degradation, but not of collagen degradation in both IL-1α (interleukin-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [-1A]N-TIMP-3 mutant has additional stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.

Proteínas ADAM/antagonistas & inibidores , Pró-Colágeno N-Endopeptidase/antagonistas & inibidores , Pró-Colágeno N-Endopeptidase/química , Inibidor Tecidual de Metaloproteinase-3/química , Inibidor Tecidual de Metaloproteinase-3/genética , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAMTS4 , Agrecanas/metabolismo , Animais , Cartilagem Articular/metabolismo , Domínio Catalítico , Células Cultivadas , Humanos , Mutação , Pró-Colágeno N-Endopeptidase/genética , Suínos , Inibidor Tecidual de Metaloproteinase-3/metabolismo