RESUMO
The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Catálise , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Proteína Smad7 , Transativadores/genética , Transativadores/fisiologia , Transfecção , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.
Assuntos
Endotélio Vascular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator de Crescimento Transformador beta/metabolismo , Transporte Ativo do Núcleo Celular , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II , Animais , Sítios de Ligação , Biotinilação , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Endoglina , Ativação Enzimática , Citometria de Fluxo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica , Fosforilação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Fatores de Tempo , Transativadores/metabolismoRESUMO
OBJECTIVE: To determine if patients with hereditary hemorrhagic telangiectasia (HHT) show alterations in transforming growth factor (TGF)-beta and its pathways. METHODS: Blood samples were obtained from HHT patients and controls, while endothelial cells were derived from umbilical veins of newborns (HUVEC) from HHT families. TGF-beta1 in plasma, or secreted by HUVEC, and plasma endoglin levels were measured by ELISA. Cellular levels of endoglin and receptor Smad proteins were tested by metabolic labeling and immunoprecipitation, mRNA levels for endoglin and TGF-beta1 by real-time PCR, and receptor Smad phosphorylation by Western blotting. RESULTS: TGF-beta1 and endoglin plasma levels analyzed in 197 individuals showed an inverse correlation with age. Circulating levels of TGF-beta1 were reduced in HHT1 patients (with Endoglin mutations) compared to control, but not in HHT2 patients (with ALK1 mutations). Endoglin levels were unchanged in plasma but decreased in activated monocytes and HUVEC with an HHT1 genotype. These HUVEC also expressed reduced levels of endoglin and TGF-beta1 mRNA, secreted less TGF-beta1, and showed normal receptor Smad expression and phosphorylation. CONCLUSIONS: Decreased plasma TGF-beta1 levels in HHT1 patients correlate with reduced production by endothelial cells. The lower endoglin expression in these cells may alter the regulation of TGF-beta1 via Smad-independent pathways.
