RESUMO
Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.
Assuntos
Caspases/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Ribonucleases/metabolismo , Células Th17/citologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Genes rel/genética , Células HEK293 , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Fatores Reguladores de Interferon/genética , Interleucina-6/genética , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas Nucleares/genética , Proteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Células Th17/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3' UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem-loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1-3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.
Assuntos
Inativação Gênica , Proteínas I-kappa B/genética , Proteínas Nucleares/genética , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Ribonucleico , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Células HeLa , Humanos , Elongação Traducional da Cadeia Peptídica , Domínios Proteicos , RNA Mensageiro/metabolismo , Receptor EphB3 , Ribonucleases/química , Ribossomos/metabolismo , Fatores de Transcrição/químicaRESUMO
Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.
Assuntos
Capsídeo/metabolismo , Enterovirus Humano C/fisiologia , Infecções por Enterovirus/metabolismo , Glutationa/metabolismo , Montagem de Vírus/fisiologia , Glutationa/antagonistas & inibidores , Células HeLa , HumanosRESUMO
DCs develop from multipotent progenitors (MPPs), which commit into DC-restricted common dendritic cell progenitors (CDPs). CDPs further differentiate into classical DCs (cDCs) and plasmacytoid DCs (pDCs). Here, we studied the impact of histone acetylation on DC development in C57BL/6 mice by interfering with histone acetylation and deacetylation, employing histone deacetylase (HDAC) inhibitors. We observed that commitment of MPPs into CDPs was attenuated by HDAC inhibition and that pDC development was specifically blocked. Gene expression profiling revealed that HDAC inhibition prevents establishment of a DC-specific gene expression repertoire. Importantly, protein levels of the core DC transcription factor PU.1 were reduced in HDAC inhibitor-treated cells and consequently PU.1 recruitment at PU.1 target genes Fms-like tyrosine kinase 3 (Flt3), interferon regulatory factor 8 (IRF8), and PU.1 itself was impaired. Thus, our results demonstrate that attenuation of PU.1 expression by HDAC inhibition causes reduced expression of key DC regulators, which results in attenuation of DC development. We propose that chromatin modifiers, such as HDACs, are required for establishing a DC gene network, where Flt3/STAT3 signaling drives PU.1 and IRF8 expression and DC development. Taken together, our study identifies HDACs as critical regulators of DC lineage commitment and development.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/enzimologia , Histona Desacetilases/metabolismo , Acetilação , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Células Dendríticas/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Histona Desacetilases/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/enzimologia , Células-Tronco/metabolismo , Transativadores/genética , Transativadores/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3'-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3'-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3'-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Receptores OX40/genética , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/citologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/antagonistas & inibidores , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Sequências Repetidas Invertidas , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/imunologia , Conformação de Ácido Nucleico , Cultura Primária de Células , Ligação Proteica , Estabilidade de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/imunologia , Receptores OX40/antagonistas & inibidores , Receptores OX40/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Repressoras/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/imunologiaRESUMO
The synthesis of the subunits of the C1 complex (C1q, C1s, C1r), and its regulator C1 inhibitor (C1-Inh) by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture enzyme-linked immunosorbent assay, we show here for the first time that, in addition to C1q, peripheral blood monocytes, and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, dendritic cell, and T cell activities, and its implications in host defense and tolerance.
RESUMO
Dendritic cells (DC) develop from hematopoietic stem cells, which is guided by instructive signals through cytokines. DC development progresses from multipotent progenitors (MPP) via common DC progenitors (CDP) into DC. Flt3 ligand (Flt3L) signaling via the Flt3/Stat3 pathway is of pivotal importance for DC development under steady state conditions. Additional factors produced during steady state or inflammation, such as TGF-ß1 or GM-CSF, also influence the differentiation potential of MPP and CDP. Here, we studied how gp130, GM-CSF and TGF-ß1 signaling influence DC lineage commitment from MPP to CDP and further into DC. We observed that activation of gp130 signaling promotes expansion of MPP. Additionally, gp130 signaling inhibited Flt3L-driven DC differentiation, but had little effect on GM-CSF-driven DC development. The inflammatory cytokine GM-CSF induces differentiation of MPP into inflammatory DC and blocks steady state DC development. Global transcriptome analysis revealed a GM-CSF-driven gene expression repertoire that primes MPP for differentiation into inflammatory DC. Finally, TGF-ß1 induces expression of DC-lineage affiliated genes in MPP, including Flt3, Irf-4 and Irf-8. Under inflammatory conditions, however, the effect of TGF-ß1 is altered: Flt3 is not upregulated, indicating that an inflammatory environment inhibits steady state DC development. Altogether, our data indicate that distinct cytokine signals produced during steady state or inflammation have a different outcome on DC lineage commitment and differentiation.