RESUMO
Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle alpha-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with gamma-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC.
Assuntos
Artérias/lesões , Artérias/patologia , Células da Medula Óssea/patologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Animais , Artérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Células da Medula Óssea/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Notch3 , Receptor Notch4 , Proteínas Repressoras/biossíntese , Proteínas Serrate-Jagged , Transdução de Sinais , Túnica Íntima/lesões , Túnica Íntima/metabolismo , Túnica Íntima/patologiaRESUMO
The AcrAB-TolC system exports a wide variety of drugs and toxic compounds, and confers intrinsic drug tolerance on Escherichia coli. The crystal structures suggested that AcrB and TolC directly dock with each other. However, biochemical and biophysical evidence of their interaction has been contradictory until recently. In this study, we examine the interaction sites by means of in vivo disulfide cross-linking between cysteine residues introduced by site-directed mutagenesis at the tops of the vertical hairpins of AcrB and the bottoms of the coiled coils of polyhistidine-tagged TolC molecules, which are structurally predicted docking sites. The AcrB-TolC complex formed through disulfide cross-linking was detected when a specific pair of mutants was coexpressed in E. coli. Our observations suggested that the AcrB-TolC complex may be formed through a two-step mechanism via transient tip-to-tip interaction of AcrB and TolC. The cross-linking was not affected by AcrA, the substrate, or a putative proton coupling site mutation.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Transporte/química , Dissulfetos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Complexos Multiproteicos/química , Substituição de Aminoácidos/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Tolerância a Medicamentos/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Histidina/química , Histidina/genética , Histidina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação Puntual , Ligação Proteica/genética , Estrutura Quaternária de Proteína/genética , Estrutura Secundária de Proteína/genéticaRESUMO
MiniANP is a synthetic pentadecapeptide analogue of atrial natriuretic polypeptide (ANP). We have used the proline-scanning mutagenesis and the analogue peptides with shorter backbones to characterize the turn-like conformation at residue 6-9 and an extended structure of Gly5-Gly6 as the receptor-bound structure of miniANP. A docking study of miniANP at the binding site of the type A natriuretic peptide receptor (NPR-A) supported the deduced conformation in the receptor-bound structure.
Assuntos
Fator Natriurético Atrial/química , Fator Natriurético Atrial/farmacologia , Prolina/química , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/genética , Células CHO , Cricetinae , Dados de Sequência Molecular , MutagêneseRESUMO
Analogues of mini atrial natriuretic peptide (miniANP) that provide conformational properties related to biological activity were designed on the basis of the structure revealed by NMR and restrained molecular dynamics (rMD) simulation, and an analogue with a high level of biological activity was successfully obtained. MiniANP is a cyclic pentadecapeptide analogue of atrial natriuretic polypeptide (ANP). The conformation of miniANP analyzed by NMR and rMD simulation indicated that positive phi angles are preferred for Gly(5) and Gly(6), which is typical for D-amino acids. On the basis of the structural information, [D-Ala(5)]miniANP, [D-Ala(6)]miniANP, and [D-Ala(5) D-Ala(6)]miniANP were synthesized. The biological activity of [D-Ala(5)]miniANP was stronger than that of miniANP, confirming that Gly(5) of miniANP takes a positive phi angle on binding to the receptor. Conformational analysis of these analogue peptides by NMR suggested that a turnlike conformation at residues 6-9 and a proximate pair formed by side chains of Phe(4) and Ile(11) are important for the biological activity.