RESUMO
Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like beta-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF beta-chain (HGF beta) and competitively inhibited binding to Met with an IC(50) of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC(50) of approximately 20 microM. The 2D (1)H-NMR structure of HB10 revealed a beta-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF beta mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF beta domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Biblioteca de Peptídeos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Soluções , Relação Estrutura-AtividadeRESUMO
High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.
Assuntos
Serina Endopeptidases/química , Sequência de Aminoácidos , Chaperoninas/química , Escherichia coli/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Ligantes , Dados de Sequência Molecular , Domínios PDZ , Peptídeos/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The Tat protein controls transcription in lentiviruses such as HIV. A cyclic peptide analog of the RNA binding domain of the bovine immunodeficiency virus (BIV) Tat protein is shown to bind specifically to its target RNA stem loop. NMR data indicate a similar mode of binding of linear and cyclic peptides.