RESUMO
SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.
Assuntos
HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Regulação Alostérica , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Hidrólise , Modelos Biológicos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/genética , Células Mieloides/virologia , Nucleosídeo-Trifosfatase/genética , Estrutura Terciária de Proteína , Transcrição Reversa , Proteína 1 com Domínio SAM e Domínio HD , Nucleotídeos de Timina/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação ViralRESUMO
Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), a Mendelian inflammatory disease which displays remarkable clinical and biochemical overlap with congenital viral infection. SAMHD1 (SAM domain and HD domain-containing protein 1) has also been defined as an HIV-1 restriction-factor that, through a novel triphosphohydrolase activity, inhibits early stage HIV-1 replication in myeloid-derived dendritic cells (MDDCs), macrophages and resting CD4+ T-cells. The potent activity of SAMHD1 is likely to be the subject of a variety of regulatory mechanisms. Knowledge of proteins that interact with SAMHD1 may not only enhance our understanding of the pathogenesis of AGS, but may also provide further details on the link between the regulation of cellular dNTPs and HIV-1 restriction. In the present study, we used a yeast two-hybrid screen and pull-down analysis followed by MS to identify the eukaryotic elongation factor 1A1 (eEF1A1) as a potential interaction partner of SAMHD1. This interaction was confirmed by unbiased co-immunoprecipitation and demonstrated in situ by a proximity ligation assay (PLA). We show that this interaction is enhanced in mutant SAMHD1 cell lines and suggest that eEF1A1 may mediate SAMHD1 turnover by targeting it to the proteosome for degradation through association with Cullin4A and Rbx1.
Assuntos
Doenças Autoimunes do Sistema Nervoso/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Malformações do Sistema Nervoso/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Fibroblastos/patologia , Humanos , Imunoprecipitação , Monócitos/metabolismo , Monócitos/patologia , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Fator 1 de Elongação de Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteína 1 com Domínio SAM e Domínio HD , Transdução de Sinais , Elongação da Transcrição Genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The C-terminal domain (CTD) of the large subunit of RNA polymerase II is a platform for mRNA processing factors and links gene transcription to mRNA capping, splicing and polyadenylation. Pcf11, an essential component of the mRNA cleavage factor IA, contains a CTD-interaction domain that binds in a phospho-dependent manner to the heptad repeats within the RNA polymerase II CTD. We show here that the phosphorylated CTD exists as a dynamic disordered ensemble in solution and, by induced fit, it assumes a structured conformation when bound to Pcf11. In addition, we detected cis-trans populations for the CTD prolines, and found that only the all-trans form is selected for binding. These data suggest that the recognition of the CTD is regulated by independent site-specific modifications (phosphorylation and proline cis-trans isomerization) and, probably, by the local concentration of suitable binding sites.