RESUMEN
Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin α3ß1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to α3ß1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the α3ß1-binding domain and the growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43-Ala66, which shows α3ß1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to α3ß1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that possesses both angio-inhibitory and, using a murine xenograft model of Kaposi's sarcoma, anti-tumorigenic activity in vivo. Thus, both the α3ß1-binding and the angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B-C loop).
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Integrina alfa3beta1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Secuencia de Aminoácidos , Animales , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Ratones , Ratones Desnudos , Análisis por Micromatrices , Datos de Secuencia Molecular , Neovascularización Patológica/metabolismo , Péptidos/administración & dosificación , Péptidos/síntesis química , Péptidos/farmacología , Unión Proteica , Sarcoma de Kaposi/patología , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/química , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
In view of the importance of information transfer mediated throughout the cell by recognition, phosphorylation or dephosphorylation of kinases, their adapters, or substrates, this method was developed. The method provides a potent research tool for rapidly generating and testing these substrates as modeled by synthetic peptide arrays. The peptides or phosphorylated peptides are automatically generated on the inner surfaces of microplate wells, covalently linked to a polylysine polymer so that they are in a sterically favorable conformation, immediately available for in situ testing. Products up to 18 amino acids long have shown excellent mass spectral homogeneity. Thus, determinate peptide libraries can be ready for testing in as little as 2 days after the conception of an experiment. The process can be easily automated using robotic liquid handlers and is extremely rapid, sensitive, and economical. Optionally, the method can be upgraded to a higher throughput level using more powerful workstations with greater capacity, such as the Biomek FX, or any similar robotics capable of transfer-from-file logic to guide synthesis cycles.