RESUMO
Protein-protein interactions are governed by a variety of structural features. The sequence specificities of such interactions are usually easier to establish than the "topological specificities," whereby interactions may be classified based on recognition of distinct three-dimensional structural motifs. Approaches to explore topological specificities have been based primarily on assembly of mini-proteins with well defined secondary, tertiary, and/or quarternary structures. The present chapter focuses on three approaches for constructing topologically well defined mini-proteins: template-assembled synthetic proteins (TASPs), disulfide-stabilized structures, and peptide-amphiphiles (PAs). Specific examples are given for applying each approach to explore topologically-dependent protein-protein interactions. TASPs are utilized to identify a metastatic melanoma receptor that binds to the alpha1(IV)1263-1277 region of basement membrane (type IV) collagen. A disulfide-stabilized structure incorporating a sarafotoxin (SRT) 6b model was examined as a matrix metalloproteinase (MMP)-3 inhibitor. PAs were developed as (a) fluorogenic triple-helical or polyPro II substrates for MMPs and aggrecanase members of the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family and (b) glycosylated and nonglycosylated ligands for metastatic melanoma cells. Topologically constrained mini-proteins have proved to be quite versatile, helping to define critical primary, secondary, and tertiary structural elements that modulate enzyme and receptor functions.
Assuntos
Proteínas/síntese química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Dissulfetos/química , Estabilidade de Medicamentos , Humanos , Ligantes , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Biologia Molecular/métodos , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Inibidores Teciduais de Metaloproteinases/síntese química , Inibidores Teciduais de Metaloproteinases/química , Inibidores Teciduais de Metaloproteinases/farmacologia , Venenos de Víboras/síntese química , Venenos de Víboras/química , Venenos de Víboras/farmacologiaRESUMO
Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related secreted proteins that limit matrix metalloproteinase (MMP) activity and also have direct effects on cell growth. We used the highly efficient adenoviral delivery system to overexpress individual TIMPs from the cytomegalovirus immediate early promoter in rat aortic smooth muscle cells. Overexpression of TIMP-1, -2, or -3, or a synthetic MMP inhibitor similarly inhibited SMC chemotaxis and invasion through reconstituted basement membrane. TIMP-1 overexpression did not effect cell proliferation. By contrast, TIMP-2 caused a dose-dependent reduction in proliferation, an effect not mimicked by a synthetic MMP inhibitor. TIMP-3 overexpression induced DNA synthesis, and promoted SMC death by apoptosis, a phenotype reproduced by adding TIMP-3 to uninfected cells, but not by a synthetic MMP inhibitor. Our study is the first to compare systematically the effect of overexpression of three TIMPs in any cell. We found similar effects on invasion mediated by inhibition of MMP activity, but widely divergent effects on proliferation and death through actions of TIMP-2 and -3 independent of MMP inhibition. These findings have important implications for the physiological roles of TIMPs and their use in gene therapy.