RESUMO
Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.
Assuntos
Fator 1 de Ribosilação do ADP/imunologia , Autofagia/imunologia , Proteínas de Transporte/imunologia , Interferon gama/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Família da Proteína 8 Relacionada à Autofagia , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , Simulação por Computador , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , GTP Fosfo-Hidrolases/imunologia , GTP Fosfo-Hidrolases/metabolismo , Edição de Genes , Immunoblotting , Imunoprecipitação , Interferon gama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus-induced activation of the NLRP3 inflammasome. Infection with an RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3-dependent necrosis. Together our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus-induced activation of the NLRP3 inflammasome and establish a direct link between inflammation and cell-death signaling pathways.
Assuntos
Proteínas de Transporte/imunologia , Inflamassomos/imunologia , Infecções por Vírus de RNA/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Dinaminas/imunologia , Ensaio de Imunoadsorção Enzimática , GTP Fosfo-Hidrolases/imunologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Mitocondriais/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Vírus de RNA , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
The upregulation of interferon (IFN)-inducible GTPases in response to pathogenic insults is vital to host defense against many bacterial, fungal, and viral pathogens. Several IFN-inducible GTPases play key roles in mediating inflammasome activation and providing host protection after bacterial or fungal infections, though their role in inflammasome activation after viral infection is less clear. Among the IFN-inducible GTPases, the expression of immunity-related GTPases (IRGs) varies widely across species for unknown reasons. Here, we report that IRGB10, but not IRGM1, IRGM2, or IRGM3, is required for NLRP3 inflammasome activation in response to influenza A virus (IAV) infection in mice. While IRGB10 functions to release inflammasome ligands in the context of bacterial and fungal infections, we found that IRGB10 facilitates endosomal maturation and nuclear translocation of IAV, thereby regulating viral replication. Corresponding with our in vitro results, we found that Irgb10-/- mice were more resistant to IAV-induced mortality than WT mice. The results of our study demonstrate a detrimental role of IRGB10 in host immunity in response to IAV and a novel function of IRGB10, but not IRGMs, in promoting viral translocation into the nucleus.
Assuntos
GTP Fosfo-Hidrolases/imunologia , Inflamassomos/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Infecções por Orthomyxoviridae/imunologia , Replicação Viral/imunologia , Animais , GTP Fosfo-Hidrolases/genética , Inflamassomos/genética , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Replicação Viral/genéticaRESUMO
Toxoplasma gondii infects virtually any nucleated cell and resides inside a non-phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of immunity regulated GTPases (IRGs) and guanylate binding proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine-threonine kinases ROP17 and ROP18, and a pseudo-kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM. Here, we characterise GRA60, a novel dense granule effector, which localises to the vacuolar space and PVM and contributes to virulence of RH in mice, suggesting a role in the subversion of host cell defence mechanisms. Members of the host cell IRG defence system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and found associated to ROP18, a member of the virulence complex.
Assuntos
Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Toxoplasma/imunologia , Toxoplasma/metabolismo , Toxoplasmose/imunologia , Toxoplasmose/metabolismo , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , DNA de Protozoário , Fibroblastos/parasitologia , Prepúcio do Pênis/parasitologia , GTP Fosfo-Hidrolases/imunologia , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes , Interações Hospedeiro-Parasita , Humanos , Imunidade , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/genética , Vacúolos/metabolismo , VirulênciaRESUMO
Interferon (IFN)-induced guanosine triphosphate hydrolysing enzymes (GTPases) have been identified as cornerstones of IFN-mediated cell-autonomous defence. Upon IFN stimulation, these GTPases are highly expressed in various host cells, where they orchestrate anti-microbial activities against a diverse range of pathogens such as bacteria, protozoan and viruses. IFN-induced GTPases have been shown to interact with various host pathways and proteins mediating pathogen control via inflammasome activation, destabilising pathogen compartments and membranes, orchestrating destruction via autophagy and the production of reactive oxygen species as well as inhibiting pathogen mobility. In this mini-review, we provide an update on how the IFN-induced GTPases target pathogens and mediate host defence, emphasising findings on protection against bacterial pathogens.
Assuntos
Bactérias/imunologia , Infecções Bacterianas/imunologia , GTP Fosfo-Hidrolases/imunologia , Imunidade Inata/imunologia , Interferons/imunologia , Animais , Bactérias/patogenicidade , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , GTP Fosfo-Hidrolases/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferons/metabolismo , Transdução de Sinais/imunologia , Virulência/imunologiaRESUMO
Human IFNs are secreted cytokines shown to stimulate the expression of over one thousand genes. These IFN-inducible genes primarily encode four major protein families, known as IFN-stimulated GTPases (ISGs), namely myxovirus-resistance proteins, guanylate-binding proteins (GBPs), p47 immunity-related GTPases and very large inducible guanosine triphosphate hydrolases (GTPases). These families respond specifically to type I or II IFNs and are well reported in coordinating immunity against some well known as well as newly discovered viral, bacterial and parasitic infections. A growing body of evidence highlights the potential contributory and regulatory roles of ISGs in dysregulated inflammation and autoimmune diseases. Our focus was to draw attention to studies that demonstrate increased expression of ISGs in the serum and affected tissues of patients with RA, SS, lupus, IBD and psoriasis. In this review, we analysed emerging literature describing the potential roles of ISGs, particularly the GBP family, in the context of autoimmunity. We also highlighted the promise and implications for therapeutically targeting IFNs and GBPs in the treatment of rheumatic diseases.
Assuntos
Autoimunidade , GTP Fosfo-Hidrolases/imunologia , Inflamação/imunologia , Interferons/imunologia , Doenças Reumáticas/imunologia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Inflamação/metabolismo , Doenças Reumáticas/metabolismoRESUMO
Immunity-related GTPases (IRGs) are a family of large interferon-inducible GTPases that function in effective host defense against invading pathogens. IRGs have been extensively studied in mammals for their roles in the elimination of intracellular pathogens; however, their homologs in lower vertebrates are not well known. In this study, an IRG from obscure puffer (Takifugu obscurus), ToIRG, was identified and further characterized for its functional activity. The ToIRG gene encodes a protein of 396 amino acids containing a typical N-terminal GTPase domain with three conserved motifs. Phylogenetic analysis revealed that it has a closer evolutionary relationship with mammalian GKS IRGs. Gene expression profile analysis revealed that ToIRG was ubiquitously expressed in all tested healthy tissues of obscure puffer and upregulated in response to Aeromonas hydrophila or Edwardsiella tarda challenge. The subcellular localization of ToIRG is characterized as condensed forms around the nucleus. Importantly, an antimicrobial assay in vitro suggested that ToIRG enhanced the ability of host cells to resist both intracellular (E. tarda) and extracellular pathogens (A. hydrophila). Taken together, these results provide the functional characterization of obscure puffer IRGs in immune defense, which is the first study to reveal the function of IRGs in bony fish and will provide important insights into the evolutionary divergence of IRGs.
Assuntos
Doenças dos Peixes/imunologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Takifugu/genética , Takifugu/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , GTP Fosfo-Hidrolases/química , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Filogenia , Alinhamento de Sequência/veterináriaRESUMO
BACKGROUND: GTPase of immunity-associated protein 5 (GIMAP5) is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 single nucleotide polymorphisms have been linked to an increased risk for asthma, whereas loss of Gimap5 in mice has been associated with severe CD4+ T cell-driven immune pathology. OBJECTIVE: We sought to identify the molecular and cellular mechanisms by which Gimap5 deficiency predisposes to allergic airway disease. METHODS: CD4+ T-cell polarization and development of pathogenic CD4+ T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite-induced airway inflammation was assessed by using a complete Gimap5 LOF (Gimap5sph/sph) and conditional Gimap5fl/flCd4Cre/ert2 mice. RESULTS: GIMAP5 LOF mutations in both mice and human subjects are associated with spontaneous polarization toward pathogenic TH17 and TH2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient TH cell differentiation is associated with increased DNA damage, particularly during TH1-polarizing conditions. DNA damage in Gimap5-deficient CD4+ T cells could be controlled by TGF-ß, thereby promoting TH17 polarization. When challenged with house dust mite in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness), with increased development of TH2, TH17, and pathogenic TH17/TH2 cells. CONCLUSION: Activation of Gimap5-deficient CD4+ T cells is associated with increased DNA damage and reduced survival that can be overcome by TGF-ß. This leads to selective survival of pathogenic TH17 cells but also TH2 cells in human subjects and mice, ultimately promoting allergic airway disease.
Assuntos
Asma/imunologia , GTP Fosfo-Hidrolases/deficiência , Mutação com Perda de Função , Células Th17/imunologia , Células Th2/imunologia , Animais , Asma/genética , Asma/patologia , GTP Fosfo-Hidrolases/imunologia , Proteínas de Ligação ao GTP , Humanos , Camundongos , Camundongos Transgênicos , Células Th17/patologia , Células Th2/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
Background: Soluble ST2 (interleukin 1 receptor-like 1) (sST2) is involved in inflammatory diseases and increased in heart failure (HF). We herein investigated sST2 effects on oxidative stress and inflammation in human cardiac fibroblasts and its pathological role in human aortic stenosis (AS).Methods and results: Using proteomics and immunodetection approaches, we have identified that sST2 down-regulated mitofusin-1 (MFN-1), a protein involved in mitochondrial fusion, in human cardiac fibroblasts. In parallel, sST2 increased nitrotyrosine, protein oxidation and peroxide production. Moreover, sST2 enhanced the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1ß and monocyte chemoattractant protein-1 (CCL-2). Pharmacological inhibition of transcriptional factor nuclear factor κB (NFκB) restored MFN-1 levels and improved oxidative status and inflammation in cardiac fibroblasts. Mito-Tempo, a mitochondria-specific superoxide scavenger, as well as Resveratrol, a general antioxidant, attenuated oxidative stress and inflammation induced by sST2. In myocardial biopsies from 26 AS patients, sST2 up-regulation paralleled a decrease in MFN-1. Cardiac sST2 inversely correlated with MFN-1 levels and positively associated with IL-6 and CCL-2 in myocardial biopsies from AS patients.Conclusions: sST2 affected mitochondrial fusion in human cardiac fibroblasts, increasing oxidative stress production and inflammatory markers secretion. The blockade of NFκB or mitochondrial reactive oxygen species restored MFN-1 expression, improving oxidative stress status and reducing inflammatory markers secretion. In human AS, cardiac sST2 levels associated with oxidative stress and inflammation. The present study reveals a new pathogenic pathway by which sST2 promotes oxidative stress and inflammation contributing to cardiac damage.
Assuntos
Estenose da Valva Aórtica/imunologia , Fibroblastos/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Estresse Oxidativo , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Biomarcadores , Células Cultivadas , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Dinâmica Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/imunologia , Miocárdio/imunologia , Miocárdio/patologiaRESUMO
GTPases of immunity-associated proteins (GIMAPs) are expressed in lymphocytes and regulate survival/death signaling and cell development within the immune system. We found that human GIMAP6 is expressed primarily in T cell lines. By sorting human peripheral blood mononuclear cells and performing quantitative RT-PCR, GIMAP6 was found to be expressed in CD3+ cells. In Jurkat cells that had been knocked down for GIMAP6, treatment with hydrogen peroxide, FasL, or okadaic acid significantly increased cell death/apoptosis. Exogenous expression of GMAP6 protected Huh-7 cells from apoptosis, suggesting that GIMAP6 is an anti-apoptotic protein. Furthermore, knockdown of GIMAP6 not only rendered Jurkat cells sensitive to apoptosis but also accelerated T cell activation under phorbol 12-myristate 13-acetate/ionomycin treatment conditions. Using this experimental system, we also observed a down-regulation of p65 phosphorylation (Ser-536) in GIMAP6 knockdown cells, indicating that GIMAP6 might display anti-apoptotic function through NF-κB activation. The conclusion from the study on cultured T cells was corroborated by the analysis of primary CD3+ T cells, showing that specific knockdown of GIMAP6 led to enhancement of phorbol 12-myristate 13-acetate/ionomycin-mediated activation signals. To characterize the biochemical properties of GIMAP6, we purified the recombinant GIMAP6 to homogeneity and revealed that GIMAP6 had ATPase as well as GTPase activity. We further demonstrated that the hydrolysis activity of GIMAP6 was not essential for its anti-apoptotic function in Huh-7 cells. Combining the expression data, biochemical properties, and cellular features, we conclude that GIMAP6 plays a role in modulating immune function and that it does this by controlling cell death and the activation of T cells.
Assuntos
Adenosina Trifosfatases/imunologia , Proteínas Reguladoras de Apoptose/imunologia , GTP Fosfo-Hidrolases/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Adenosina Trifosfatases/genética , Proteínas Reguladoras de Apoptose/genética , GTP Fosfo-Hidrolases/genética , Humanos , Ionomicina/farmacologia , Células Jurkat , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologiaRESUMO
Guanylate-binding proteins (GBP) are a family of dynamin-related large GTPases which are expressed in response to interferons and other pro-inflammatory cytokines. GBPs mediate a broad spectrum of innate immune functions against intracellular pathogens ranging from viruses to bacteria and protozoa. Several binding partners for individual GBPs have been identified and several different mechanisms of action have been proposed depending on the organisms, the cell type and the pathogen used. Many of these anti-pathogenic functions of GBPs involve the recruitment to and the subsequent destruction of pathogen containing vacuolar compartments, the assembly of large oligomeric innate immune complexes such as the inflammasome, or the induction of autophagy. Furthermore, GBPs often cooperate with immunity-related GTPases (IRGs), another family of dynamin-related GTPases, to exert their anti-pathogenic function, but since most IRGs have been lost in the evolution of higher primates, the anti-pathogenic function of human GBPs seems to be IRG-independent. GBPs and IRGs share biochemical and structural properties with the other members of the dynamin superfamily such as low nucleotide affinity and a high intrinsic GTPase activity which can be further enhanced by oligomerisation. Furthermore, GBPs and IRGs can interact with lipid membranes. In the case of three human and murine GBP isoforms this interaction is mediated by C-terminal isoprenylation. Based on cell biological studies, and in analogy to the function of other dynamins in membrane scission events, it has been postulated that both GBPs and IRGs might actively disrupt the outer membrane of pathogen-containing vacuole leading to the detection and destruction of the pathogen by the cytosolic innate immune system of the host. Recent evidence, however, indicates that GBPs might rather function by mediating membrane tethering events similar to the dynamin-related atlastin and mitofusin proteins, which mediate fusion of the ER and mitochondria, respectively. The aim of this review is to highlight the current knowledge on the function of GBPs in innate immunity and to combine it with the recent progress in the biochemical characterisation of this protein family.
Assuntos
Citoplasma/imunologia , Proteínas de Ligação ao GTP/fisiologia , Imunidade Inata , Animais , Autofagia/imunologia , Citoplasma/microbiologia , Citoplasma/parasitologia , Citoplasma/virologia , GTP Fosfo-Hidrolases/imunologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interferons/imunologia , Interferons/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Vacúolos/parasitologia , Vacúolos/virologiaRESUMO
Dendritic cells (DCs) have the striking ability to cross-present exogenous antigens in association with major histocompatibility complex (MHC) class I to CD8(+) T cells. However, the intracellular pathways underlying cross-presentation remain ill defined. Current models involve cytosolic proteolysis of antigens by the proteasome and peptide import into endoplasmic reticulum (ER) or phagosomal lumen by the transporters associated with antigen processing (TAP1 and TAP2). Here, we show that DCs expressed an ER-resident 47 kDa immune-related GTPase, Igtp (Irgm3). Igtp resides on ER and lipid body (LB) membranes where it binds the LB coat component ADFP. Inactivation of genes encoding for either Igtp or ADFP led to defects in LB formation in DCs and severely impaired cross-presentation of phagocytosed antigens to CD8(+) T cells but not antigen presentation to CD4(+) T cells. We thus define a new role for LB organelles in regulating cross-presentation of exogenous antigens to CD8(+) T lymphocytes in DCs.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Lipídeos/imunologia , Fagocitose , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Retículo Endoplasmático/imunologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Perilipina-2RESUMO
Major advances have been made in melanoma treatment with the use of molecularly targeted therapies and immunotherapies, and numerous regimens are now approved by the US Food and Drug Administration for patients with stage IV disease. However, therapeutic resistance remains an issue to both classes of agents, and reliable biomarkers of therapeutic response and resistance are lacking. Mechanistic insights are being gained through preclinical studies and translational research, offering potential strategies to enhance responses and survival in treated patients. A comprehensive understanding of the immune effects of common mutations at play in melanoma is critical, as is an appreciation of the molecular mechanisms contributing to therapeutic resistance to immunotherapy. These mechanisms and the interplay between them are discussed herein. Cancer 2017;123:2130-42. © 2017 American Cancer Society.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Melanoma/imunologia , PTEN Fosfo-Hidrolase/imunologia , Proteínas Proto-Oncogênicas B-raf/imunologia , Neoplasias Cutâneas/imunologia , Evasão Tumoral/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Antígeno CTLA-4/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , Humanos , Imidazóis/administração & dosagem , Imunoterapia , Indóis/administração & dosagem , Ipilimumab , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Terapia de Alvo Molecular , Mutação , Nivolumabe , Oximas/administração & dosagem , PTEN Fosfo-Hidrolase/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Sulfonamidas/administração & dosagem , Evasão Tumoral/genética , Vemurafenib , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/imunologiaRESUMO
The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.
Assuntos
Morte Celular/imunologia , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica de Plantas/imunologia , Oryza/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal/imunologia , Apoptose/imunologia , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/imunologia , Imunidade Inata/imunologia , Imunoprecipitação , Proteínas de Plantas , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/imunologia , UbiquitinaçãoRESUMO
The intracellular parasite Toxoplasma gondii enjoys a wide host range and is adept at surviving in both naive and activated macrophages. Previous studies have emphasized the importance of the active serine-threonine protein kinase rhoptry protein 18 (ROP18), which targets immunity-related GTPases (IRGs), in mediating macrophage survival and acute virulence of T. gondii in mice. Here, we demonstrate that ROP18 exists in a complex with the pseudokinases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein 7 (GRA7). Individual deletion mutant gra7 or rop18 was partially attenuated for virulence in mice, whereas the combined gra7rop18 mutant was avirulent, suggesting these proteins act together in the same pathway. The virulence defect of the double mutant was mirrored by increased recruitment of IRGs and clearance of the parasite in IFN-γ-activated macrophages in vitro. GRA7 was shown to recognize a conserved feature of IRGs, binding directly to the active dimer of immunity-related GTPase a6 in a GTP-dependent manner. Binding of GRA7 to immunity-related GTPase a6 led to enhanced polymerization, rapid turnover, and eventual disassembly. Collectively, these studies suggest that ROP18 and GRA7 act in a complex to target IRGs by distinct mechanisms that are synergistic.
Assuntos
Antígenos de Protozoários/imunologia , GTP Fosfo-Hidrolases/imunologia , Regulação da Expressão Gênica , Proteínas de Protozoários/imunologia , Toxoplasma/patogenicidade , Animais , Dimerização , Feminino , Deleção de Genes , Guanosina Trifosfato/metabolismo , Interações Hospedeiro-Parasita , Hidrólise , Imunidade Inata , Interferon gama/imunologia , Macrófagos/citologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/imunologia , Fatores de Tempo , VirulênciaRESUMO
BACKGROUND: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. RESULTS: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. CONCLUSIONS: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.
Assuntos
GTP Fosfo-Hidrolases/imunologia , Proteínas de Ligação ao GTP/imunologia , Lisossomos/imunologia , Animais , Autofagia , Linhagem Celular , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/genética , Deleção de Genes , Imunidade Inata , Infecções/genética , Infecções/imunologia , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.
Assuntos
Encephalitozoon cuniculi/imunologia , Encefalitozoonose/imunologia , GTP Fosfo-Hidrolases/imunologia , Interferon gama/imunologia , Animais , Sobrevivência Celular , Encephalitozoon cuniculi/crescimento & desenvolvimento , Encefalitozoonose/microbiologia , Fibroblastos , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/genética , Imunidade Inata , Membranas Intracelulares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Fagossomos/imunologia , Vacúolos/imunologiaRESUMO
Nod-like receptor family, pyrin domain-containing 3 (NLRP3), is involved in the early stages of the inflammatory response by sensing cellular damage or distress due to viral or bacterial infection. Activation of NLRP3 triggers its assembly into a multimolecular protein complex, termed "NLRP3 inflammasome." This event leads to the activation of the downstream molecule caspase-1 that cleaves the precursor forms of proinflammatory cytokines, such as interleukin 1 beta (IL-1ß) and IL-18, and initiates the immune response. Recent studies indicate that the reactive oxygen species produced by mitochondrial respiration is critical for the activation of the NLRP3 inflammasome by monosodium urate, alum, and ATP. However, the precise mechanism by which RNA viruses activate the NLRP3 inflammasome is not well understood. Here, we show that loss of mitochondrial membrane potential [ΔΨ(m)] dramatically reduced IL-1ß secretion after infection with influenza, measles, or encephalomyocarditis virus (EMCV). Reduced IL-1ß secretion was also observed following overexpression of the mitochondrial inner membrane protein, uncoupling protein-2, which induces mitochondrial proton leakage and dissipates ΔΨ(m). ΔΨ(m) was required for association between the NLRP3 and mitofusin 2, a mediator of mitochondrial fusion, after infection with influenza virus or EMCV. Importantly, the knockdown of mitofusin 2 significantly reduced the secretion of IL-1ß after infection with influenza virus or EMCV. Our results provide insight into the roles of mitochondria in NLRP3 inflammasome activation.
Assuntos
Proteínas de Transporte/imunologia , GTP Fosfo-Hidrolases/imunologia , Inflamassomos/imunologia , Infecções por Vírus de RNA/imunologia , Análise de Variância , Animais , Interleucina-1beta/imunologia , Canais Iônicos/metabolismo , Potencial da Membrana Mitocondrial/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2RESUMO
Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , GTP Fosfo-Hidrolases/imunologia , Malária Cerebral/imunologia , Plasmodium berghei/imunologia , Transferência Adotiva , Animais , Antígenos de Protozoários/imunologia , Encéfalo/imunologia , Encéfalo/parasitologia , Encéfalo/patologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Proliferação de Células/genética , Quimiocina CCL2/biossíntese , Quimiocina CCL3/biossíntese , Quimiocina CCL4/biossíntese , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Inflamação/genética , Inflamação/imunologia , Interferon Tipo I/biossíntese , Interferon gama/imunologia , Interleucina-6/biossíntese , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genéticaRESUMO
GTPase immune-associated proteins (Gimap) genes encode evolutionarily conserved GTP-binding proteins that are preferentially expressed in immune cells. Specific members have been shown to be involved in lymphocyte development, or are associated with inflammatory and autoimmune diseases. However, the function of these proteins remains poorly understood, both at the cellular and molecular levels. A new study in this issue of the European Journal of Immunology [Eur. J. Immunol. 2014. 44: 561-572] points to the distinct but partly overlapping functions of two members of this family, Gimap3 and Gimap5, and offers new insight into their potential functions in T cells.