RESUMO
Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.
Assuntos
Transplante de Medula Óssea , Fator Estimulador de Colônias de Granulócitos , Animais , Camundongos , Citocinas , Interleucina-1 , Fator de Necrose Tumoral alfa/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismoRESUMO
RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.
RESUMO
RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Disponibilidade Biológica , Linhagem Celular , Doença Crônica , Desenho de Fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Esclerose Múltipla/tratamento farmacológico , Pirazóis/farmacocinética , Ratos , Retinose Pigmentar/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.
Assuntos
Carcinoma Ductal Pancreático/imunologia , Tolerância Imunológica/imunologia , Macrófagos/imunologia , Neoplasias Pancreáticas/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Tolerância Imunológica/genética , Células L , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Transcrição STAT1/metabolismo , Células Th1/citologia , Células Th17/citologiaRESUMO
Therapies that suppress RIPK1 kinase activity are emerging as promising therapeutic agents for the treatment of multiple inflammatory disorders. The ability to directly measure drug binding of a RIPK1 inhibitor to its target is critical for providing insight into pharmacokinetics, pharmacodynamics, safety and clinical efficacy, especially for a first-in-class small-molecule inhibitor where the mechanism has yet to be explored. Here, we report a novel method for measuring drug binding to RIPK1 protein in cells and tissues. This TEAR1 (Target Engagement Assessment for RIPK1) assay is a pair of immunoassays developed on the principle of competition, whereby a first molecule (ie, drug) prevents the binding of a second molecule (ie, antibody) to the target protein. Using the TEAR1 assay, we have validated the direct binding of specific RIPK1 inhibitors in cells, blood and tissues following treatment with benzoxazepinone (BOAz) RIPK1 inhibitors. The TEAR1 assay is a valuable tool for facilitating the clinical development of the lead RIPK1 clinical candidate compound, GSK2982772, as a first-in-class RIPK1 inhibitor for the treatment of inflammatory disease.
Assuntos
Anticorpos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Células HT29 , Humanos , Imunoensaio , Macaca fascicularis , Masculino , Ligação Proteica/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
GSK2982772 is a highly selective inhibitor of receptor-interacting protein kinase 1 (RIPK1) being developed to treat chronic inflammatory diseases. This first-in-human study evaluated safety, tolerability, pharmacokinetics (PK), and exploratory pharmacodynamics (PD) of GSK2982772 administered orally to healthy male volunteers. This was a Phase I, randomized, placebo-controlled, double-blind study. In Part A, subjects received single ascending doses of GSK2982772 (0.1-120 mg) or placebo in a crossover design during each of 4 treatment periods. In Part B, subjects received repeat doses of GSK2982772 (20 mg once daily [QD] to up to 120 mg twice daily [BID]) or placebo for 14 days. Part C was an open-label relative bioavailability study comparing 20-mg tablets vs capsules. Safety, tolerability, pharmacokinetics (PK), RIPK1 target engagement (TE), and pharmacodynamics (PD) were assessed. The most common adverse events (AEs) were contact dermatitis and headache. Most AEs were mild in intensity, and there were no deaths or serious AEs. The PK of GSK2982772 was approximately linear over the dose range studied (up to 120 mg BID). There was no evidence of drug accumulation upon repeat dosing. Greater than 90% RIPK1 TE was achieved over a 24-hour period for the 60-mg and 120-mg BID dosing regimens. Single and repeat doses of GSK2982772 were safe and well tolerated. PK profiles showed dose linearity. The high levels of RIPK1 TE support progression into Phase II clinical trials for further clinical development.
Assuntos
Inibidores de Proteínas Quinases/administração & dosagem , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adulto , Área Sob a Curva , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacocinética , Adulto JovemRESUMO
RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Colite Ulcerativa/tratamento farmacológico , Inflamação/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Animais , Benzazepinas/química , Benzazepinas/farmacologia , Colite Ulcerativa/imunologia , Citocinas/imunologia , Cães , Haplorrinos , Humanos , Inflamação/imunologia , Camundongos , Simulação de Acoplamento Molecular , Coelhos , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Suínos , Porco Miniatura , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.
Assuntos
Apoptose , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Técnicas de Introdução de Genes , Células HT29 , Humanos , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Necrose/enzimologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidoresRESUMO
The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1ß production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12(-/-) mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12(-/-) mice showed a slight decrease in IL-1ß and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12(-/-) mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12(-/-) mice. Likewise, Nlrp12(-/-) mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.
Assuntos
Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Klebsiella pneumoniae/fisiologia , Mycobacterium tuberculosis/fisiologia , Animais , Células da Medula Óssea/citologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.
RESUMO
Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Imunidade Inata , Inflamassomos/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Proteólise , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células HEK293 , Humanos , Inflamassomos/metabolismo , Proteínas NLR , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de ProteínaRESUMO
In vitro data suggest that a subgroup of NLR proteins, including NLRP12, inhibits the transcription factor NF-κB, although physiologic and disease-relevant evidence is largely missing. Dysregulated NF-κB activity is associated with colonic inflammation and cancer, and we found Nlrp12(-/-) mice were highly susceptible to colitis and colitis-associated colon cancer. Polyps isolated from Nlrp12(-/-) mice showed elevated noncanonical NF-κB activation and increased expression of target genes that were associated with cancer, including Cxcl13 and Cxcl12. NLRP12 negatively regulated ERK and AKT signaling pathways in affected tumor tissues. Both hematopoietic- and nonhematopoietic-derived NLRP12 contributed to inflammation, but the latter dominantly contributed to tumorigenesis. The noncanonical NF-κB pathway was regulated upon degradation of TRAF3 and activation of NIK. NLRP12 interacted with both NIK and TRAF3, and Nlrp12(-/-) cells have constitutively elevated NIK, p100 processing to p52 and reduced TRAF3. Thus, NLRP12 is a checkpoint of noncanonical NF-κB, inflammation, and tumorigenesis.
Assuntos
Transformação Celular Neoplásica/patologia , Colite/metabolismo , Colite/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Colite/genética , Neoplasias do Colo/genética , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Among the 22 members of the nucleotide binding-domain, leucine rich repeat-containing (NLR) family, less than half have been functionally characterized. Of those that have been well studied, most form caspase-1 activating inflammasomes. NLRP12 is a unique NLR that has been shown to attenuate inflammatory pathways in biochemical assays and mediate the lymph node homing of activated skin dendritic cells in contact hypersensitivity responses. Since the mechanism between these two important observations remains elusive, we further evaluated the contribution of NLRP12 to organ specific adaptive immune responses by focusing on the lung, which, like skin, is exposed to both exogenous and endogenous inflammatory agents. In models of allergic airway inflammation induced by either acute ovalbumin (OVA) exposure or chronic house dust mite (HDM) antigen exposure, Nlrp12(-/-) mice displayed subtle differences in eosinophil and monocyte infiltration into the airways. However, the overall development of allergic airway disease and airway function was not significantly altered by NLRP12 deficiency. Together, the combined data suggest that NLRP12 does not play a vital role in regulating Th2 driven airway inflammation using common model systems that are physiologically relevant to human disease. Thus, the allergic airway inflammation models described here should be appropriate for subsequent studies that seek to decipher the contribution of NLRP12 in mediating the host response to agents associated with asthma exacerbation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Hipersensibilidade Respiratória/genética , Doenças Respiratórias/genética , Animais , Antígenos de Dermatophagoides/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Feminino , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/metabolismo , Doenças Respiratórias/metabolismo , Doenças Respiratórias/patologiaRESUMO
Nucleotide-binding domain leucine-rich repeat (NLR) proteins are regulators of inflammation and immunity. Although first described 8 y ago, a physiologic role for NLRP12 has remained elusive until now. We find that murine Nlrp12, an NLR linked to atopic dermatitis and hereditary periodic fever in humans, is prominently expressed in dendritic cells (DCs) and neutrophils. Nlrp12-deficient mice exhibit attenuated inflammatory responses in two models of contact hypersensitivity that exhibit features of allergic dermatitis. This cannot be attributed to defective Ag processing/presentation, inflammasome activation, or measurable changes in other inflammatory cytokines. Rather, Nlrp12(-/-) DCs display a significantly reduced capacity to migrate to draining lymph nodes. Both DCs and neutrophils fail to respond to chemokines in vitro. These findings indicate that NLRP12 is important in maintaining neutrophils and peripheral DCs in a migration-competent state.
Assuntos
Quimiotaxia de Leucócito/imunologia , Células Dendríticas/imunologia , Dermatite de Contato/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Mieloides/imunologia , Animais , Células Dendríticas/metabolismo , Dermatite de Contato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell-cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor-ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunidade/imunologia , Semaforinas/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Proliferação de Células , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Semaforinas/antagonistas & inibidores , Transdução de SinaisRESUMO
The RIG-like helicase (RLH) family of intracellular receptors detect viral nucleic acid and signal through the mitochondrial antiviral signalling adaptor MAVS (also known as Cardif, VISA and IPS-1) during a viral infection. MAVS activation leads to the rapid production of antiviral cytokines, including type 1 interferons. Although MAVS is vital to antiviral immunity, its regulation from within the mitochondria remains unknown. Here we describe human NLRX1, a highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family member (known as NLR) that localizes to the mitochondrial outer membrane and interacts with MAVS. Expression of NLRX1 results in the potent inhibition of RLH- and MAVS-mediated interferon-beta promoter activity and in the disruption of virus-induced RLH-MAVS interactions. Depletion of NLRX1 with small interference RNA promotes virus-induced type I interferon production and decreases viral replication. This work identifies NLRX1 as a check against mitochondrial antiviral responses and represents an intersection of three ancient cellular processes: NLR signalling, intracellular virus detection and the use of mitochondria as a platform for anti-pathogen signalling. This represents a conceptual advance, in that NLRX1 is a modulator of pathogen-associated molecular pattern receptors rather than a receptor, and identifies a key therapeutic target for enhancing antiviral responses.
Assuntos
Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Vírus/imunologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Interferon beta/metabolismo , Camundongos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , NF-kappa B/metabolismo , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-kappaB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-kappaB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1.
Assuntos
Trifosfato de Adenosina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Trifosfato de Adenosina/análise , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos CD40/farmacologia , Linhagem Celular , Quimiocinas/análise , Citocinas/análise , DNA Complementar/genética , Ativação Enzimática , Escherichia coli/genética , Hemaglutininas/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Rim/citologia , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Mutação , Testes de Precipitina , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Fatores de Tempo , TransfecçãoRESUMO
Monarch-1/NLRP12 is expressed in myeloid cells and functions as a negative regulator of inflammation by inducing proteasome-mediated degradation of NF-kappaB-inducing kinase. Monarch-1 is a member of the CATERPILLER gene family, also known as the nucleotide-binding domain leucine-rich repeat gene family. This family shares strong structural homology to major immune regulators expressed in lower organisms, including plants. In plants, these disease-resistance proteins (R proteins) sense pathogenic insult and initiate a protective response to limit pathogen growth. To perform this role, many R proteins require the highly conserved chaperone molecule, heat shock protein (Hsp) 90. Using a two-dimensional gel/mass spectrometry system, we detected the association of the nucleotide-binding domain leucine-rich repeat protein Monarch-1 with heat shock proteins. Further analysis indicates that analogous to plant R proteins, Hsp90 is required for Monarch-1 activity. In human monocytes, Monarch-1 associates with Hsp90, and these complexes are sensitive to treatment with specific Hsp90 inhibitors. Disruption of these complexes results in rapid degradation of Monarch-1 via the proteasome and prevents Monarch-1-induced proteolysis of NF-kappaB-inducing kinase. This demonstrates that Hsp90 is a critical regulator of Monarch-1 anti-inflammatory activity.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Cultivadas , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Monócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Quinase Induzida por NF-kappaBRESUMO
One of the most important advances in human immunology in the last decade has been the characterization of evolutionarily conserved molecular mediators important in controlling innate immunity. A prime example of this is the discovery of the mammalian Toll-like receptor family. Toll molecules were first discovered in Drosophila and were found to protect the organism from fungal infection. In mammals, Toll-like receptors respond to a wide variety of microbial products and serve as a bridge between innate and adaptive immunity. In the last 4 years, another important family of molecules has been discovered, and it is evolutionarily conserved from plants to humans. This family was first christened CATERPILLER by our laboratory, and is also known as NBD-LRR or NLR. CATERPILLER family members have rapidly gained prominence as important regulators of inflammatory responses to pathogens and their products. This article discusses some of the members of this family and their role in human disease.