RESUMO
Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.
Assuntos
Proteínas Culina/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Fator de Transcrição STAT2/metabolismo , Fatores de Transcrição/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Proteínas Culina/genética , Elonguina , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Vírus Sinciciais Respiratórios/genética , Fator de Transcrição STAT1/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
The aqueous humour of the eye is a rich source of growth factors, many of which have been shown to be lens cell mitogens; however, the identity of the endogenous mitogen(s) for lens cells is still unknown. As a first approach to identify the mechanisms by which these aqueous humour-derived growth factors induce lens cell proliferation, the present study set out to examine MAPK/ERK1/2 and PI3-K/Akt signalling associated with lens cell proliferation. Using a lens explant system, we examined the effects of different lens mitogens (aqueous humour, FGF, PDGF, IGF and EGF) using 5'-2'-bromo-deoxyuridine incorporation. In addition, we adopted immunolabelling techniques to compare the roles that the ERK1/2 and PI3-K signalling pathways play in regulating lens cell proliferation. We showed that the aqueous humour, and all the other growth factors examined, could activate ERK1/2 and PI3-K/Akt signalling. By targeting these pathways using specific pharmacological inhibitors, we were able to show that both ERK1/2 and PI3-K signalling are required for growth factor-induced lens cell proliferation, and that there was a strong correlation between the spatial distribution of proliferating cells in lens explants with ERK1/2 labelling. Furthermore, our blocking studies confirmed that PI3-K/Akt signalling can act upstream of ERK1/2, potentiating ERK1/2 phosphorylation in growth factor-induced lens cell proliferation. A better understanding of the signalling pathways required for aqueous humour-induced lens cell proliferation may ultimately allow us to identify the mitogen(s) that are important for regulating lens cell proliferation in situ.
Assuntos
Humor Aquoso/metabolismo , Substâncias de Crescimento/farmacologia , Cristalino/citologia , Sistema de Sinalização das MAP Quinases , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Cristalino/enzimologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Estimulação QuímicaRESUMO
Signal transduction through the T cell receptor (TCR) and a costimulatory molecule, CD28, results in the stimulation of multiple signaling pathways, leading to the activation of several transcription factors including activator protein-1 (AP-1), nuclear factor of activated T cells (NF-AT), and nuclear factor kappa B (NF-kappaB). The molecular mechanisms by which NF-kappaB is activated by TCR-CD28 have only recently become known. New findings indicate that the adaptor molecules CARMA1 and Bcl10 are essential to the process. Additionally, a critical role for MALT1/paracaspase has been identified. MALT1, CARMA1, and Bcl10 form a tripartite protein complex, in which Bcl10 is thought to facilitate the oligomerization of MALT1 monomers. Overexpression of MALT1, as observed in a subset of lymphoma patients, leads to the potent activation of NF-kappaB, suggesting that MALT1 might stimulate (directly or indirectly) the kinase complex [IKK, inhibitor of NF-kappaB (IkappaB) kinase] responsible for activating cytoplasmic NF-kappaB for translocation into the nucleus. Moreover, the MALT1-CARMA1-Bcl10 complex is responsible for ubiquitination of NEMO, a step that appears to be critical for TCR-induced NF-kappaB activation but not for induction mediated by other stimuli such as TNF or IL-1.