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1.
Nat Immunol ; 17(3): 250-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26642356

RESUMO

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1ß (IL-1ß) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.


Assuntos
Proteínas de Transporte/imunologia , Macrófagos/imunologia , Mitose/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Caspase 1 , Cromatografia em Gel , Ensaio de Unidades Formadoras de Colônias , Citocinas , Proteínas do Citoesqueleto , Células Dendríticas , Encefalomielite Autoimune Experimental/imunologia , Feminino , Citometria de Fluxo , Células HEK293 , Humanos , Imunoprecipitação , Técnicas In Vitro , Inflamassomos/genética , Inflamassomos/imunologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Monócitos , Quinases Relacionadas a NIMA , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio , Medula Espinal/imunologia
2.
EMBO Rep ; 25(8): 3601-3626, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38956225

RESUMO

Signals emanating from the T-cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T-cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T-cell function. Through forward genetic screening in mice, we discover that loss-of-function mutations in LDL receptor-related protein 10 (Lrp10) cause naive and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. T-cell activation induces Lrp10 expression, which post-translationally suppresses IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhances T-cell homeostatic expansion through IL7R signaling. Lrp10-deficient mice are also intrinsically resistant to syngeneic tumors. This phenotype depends on dense tumor infiltration of CD8 T cells, which display increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T-cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy.


Assuntos
Linfócitos T CD8-Positivos , Homeostase , Receptores de Interleucina-7 , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Receptores de Interleucina-7/metabolismo , Receptores de Interleucina-7/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Transdução de Sinais , Ativação Linfocitária/imunologia , Camundongos Knockout , Camundongos Endogâmicos C57BL , Memória Imunológica , Neoplasias/imunologia , Neoplasias/genética , Humanos
3.
Immunity ; 45(4): 761-773, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27692612

RESUMO

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.


Assuntos
Inflamassomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , RNA Nuclear Pequeno/farmacologia , Animais , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , Quinases Relacionadas a NIMA/metabolismo , Quinona Redutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor 7 Toll-Like/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260399

RESUMO

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.


Assuntos
Mutação em Linhagem Germinativa/genética , Leucócitos/metabolismo , Aprendizado de Máquina , Meiose/genética , Algoritmos , Animais , Automação , Feminino , Citometria de Fluxo , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Probabilidade , Reprodutibilidade dos Testes , Software
5.
Proc Natl Acad Sci U S A ; 116(23): 11380-11389, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31097594

RESUMO

LPS-responsive beige-like anchor (LRBA) protein deficiency in humans causes immune dysregulation resulting in autoimmunity, inflammatory bowel disease (IBD), hypogammaglobulinemia, regulatory T (Treg) cell defects, and B cell functional defects, but the cellular and molecular mechanisms responsible are incompletely understood. In an ongoing forward genetic screen for N-ethyl-N-nitrosourea (ENU)-induced mutations that increase susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice, we identified two nonsense mutations in Lrba Although Treg cells have been a main focus in LRBA research to date, we found that dendritic cells (DCs) contribute significantly to DSS-induced intestinal inflammation in LRBA-deficient mice. Lrba-/- DCs exhibited excessive IRF3/7- and PI3K/mTORC1-dependent signaling and type I IFN production in response to the stimulation of the Toll-like receptors (TLRs) 3, TLR7, and TLR9. Substantial reductions in cytokine expression and sensitivity to DSS in LRBA-deficient mice were caused by knockout of Unc93b1, a chaperone necessary for trafficking of TLR3, TLR7, and TLR9 to endosomes. Our data support a function for LRBA in limiting endosomal TLR signaling and consequent intestinal inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colite/metabolismo , Endossomos/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Colite/induzido quimicamente , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Sulfato de Dextrana/farmacologia , Feminino , Inflamação/metabolismo , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 115(37): E8698-E8706, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30150374

RESUMO

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment.


Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Melanoma Experimental/terapia , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Sinergismo Farmacológico , Humanos , Imunoterapia/métodos , Estimativa de Kaplan-Meier , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Células THP-1 , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo
7.
Plant J ; 97(6): 1168-1182, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30536697

RESUMO

Many quantitative traits are composites of other traits that contribute differentially to genetic variation. Quantitative trait locus (QTL) mapping of these composite traits can benefit by incorporating the mechanistic process of how their formation is mediated by the underlying components. We propose a dissection model by which to map these interconnected components traits under a joint likelihood setting. The model can test how a composite trait is determined by pleiotropic QTLs for its component traits or jointly by different sets of QTLs each responsible for a different component. The model can visualize the pattern of time-varying genetic effects for individual components and their impacts on composite traits. The dissection model was used to map two composite traits, stemwood volume growth decomposed into its stem height, stem diameter and stem form components for Euramerican poplar adult trees, and total lateral root length constituted by its average lateral root length and lateral root number components for Euphrates poplar seedlings. We found the pattern of how QTLs for different components contribute to phenotypic variation in composite traits. The detailed understanding of the genetic machineries of composite traits will not only help in the design of molecular breeding in plants and animals, but also shed light on the evolutionary processes of quantitative traits under natural selection.


Assuntos
Herança Multifatorial , Populus/genética , Locos de Características Quantitativas/genética , Mapeamento Cromossômico , Fenótipo , Caules de Planta/genética , Plântula/genética , Árvores , Madeira/genética
8.
Brief Bioinform ; 19(6): 1430-1439, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28575183

RESUMO

Heterochrony is known as a developmental change in the timing or rate of ontogenetic events across phylogenetic lineages. It is a key concept synthesizing development into ecology and evolution to explore the mechanisms of how developmental processes impact on phenotypic novelties. A number of molecular experiments using contrasting organisms in developmental timing have identified specific genes involved in heterochronic variation. Beyond these classic approaches that can only identify single genes or pathways, quantitative models derived from current next-generation sequencing data serve as a more powerful tool to precisely capture heterochronic variation and systematically map a complete set of genes that contribute to heterochronic processes. In this opinion note, we discuss a computational framework of genetic mapping that can characterize heterochronic quantitative trait loci that determine the pattern and process of development. We propose a unifying model that charts the genetic architecture of heterochrony that perceives and responds to environmental perturbations and evolves over geologic time. The new model may potentially enhance our understanding of the adaptive value of heterochrony and its evolutionary origins, providing a useful context for designing new organisms that can best use future resources.


Assuntos
Simulação por Computador , Locos de Características Quantitativas , Animais , Fenótipo
9.
Proc Natl Acad Sci U S A ; 113(7): E884-93, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26831104

RESUMO

Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.


Assuntos
Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/agonistas , Peptidomiméticos/farmacologia , Receptor 4 Toll-Like/agonistas , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
10.
J Am Chem Soc ; 140(43): 14440-14454, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30272974

RESUMO

A screen conducted with nearly 100000 compounds and a surrogate functional assay for stimulation of an immune response that measured the release of TNF-α from treated human THP-1 myeloid cells differentiated along the macrophage line led to the discovery of the diprovocims. Unique to these efforts and of special interest, the screening leads for this new class of activators of an immune response came from a compound library designed to promote cell-surface receptor dimerization. Subsequent comprehensive structure-activity relationship studies improved the potency 800-fold over that of the screening leads, providing diprovocim-1 and diprovocim-2. The diprovocims act by inducing cell-surface toll-like receptor (TLR)-2 dimerization and activation with TLR1 (TLR1/TLR2 agonist), bear no structural similarity to any known natural or synthetic TLR agonist, and are easy to prepare and synthetically modify, and selected members are active in both human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full agonist activity at extraordinarily low concentrations (EC50 = 110 pM) in human THP-1 cells, being more potent than the naturally derived TLR1/TLR2 agonist Pam3CSK4 or any other known small molecule TLR agonist.


Assuntos
Antineoplásicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma Experimental/patologia , Camundongos , Conformação Molecular , Células THP-1
11.
Proc Natl Acad Sci U S A ; 112(5): E440-9, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25605905

RESUMO

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.


Assuntos
Mutação Puntual , Alelos , Animais , Feminino , Genes Letais , Ligação Genética , Masculino , Camundongos , Linhagem , Fenótipo , Locos de Características Quantitativas
13.
Blood ; 119(24): 5769-71, 2012 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-22550345

RESUMO

TNFα is a powerful inflammatory stimulus, central both to the control of infection, and as an agent of inflammatory disease. The most potent inducers of TNFα secretion signal through the Toll-like receptors, and we describe here a chemically-induced mutation that impairs this response in macrophages. A missense mutation was revealed in the gene encoding the inactive rhomboid protease iRhom2, which was not complemented by a null allele of the same gene. Neither the missense nor the null allele affected TLR-induced secretion of IL-6. Moreover, unlike a mutation in TNFα, the iRhom2 missense mutation did not cause enhanced susceptibility to colitis induced by dextran sodium sulfate. These results establish a specific role for iRhom2 in the secretion of TNFα, and present a new target for the modulation of inflammation.


Assuntos
Proteínas de Transporte/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Alelos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Análise Mutacional de DNA , Genes Recessivos/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação/genética , Receptores Toll-Like/metabolismo
14.
J Innate Immun ; 16(1): 226-247, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38527452

RESUMO

INTRODUCTION: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αß) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients. METHODS: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-ß was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αß, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αßR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction. RESULTS: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αß by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αß-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αß-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-ß pretreatment enhances the subsequent induction of IFN-αß in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αß overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice. CONCLUSION: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects.


Assuntos
Adenoviridae , Citocinas , Fator Regulador 3 de Interferon , Lipopolissacarídeos , Macrófagos , Camundongos Knockout , Animais , Camundongos , Lipopolissacarídeos/imunologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Macrófagos/imunologia , Citocinas/metabolismo , Camundongos Endogâmicos C57BL , Fator Regulador 7 de Interferon/metabolismo , Fator Regulador 7 de Interferon/genética , Vetores Genéticos , Infecções por Adenoviridae/imunologia , Interferon Tipo I/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Interferon beta/metabolismo
15.
PLoS Pathog ; 7(5): e1002057, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21625535

RESUMO

The signaling of Toll-like receptors (TLRs) is the host's first line of defense against microbial invasion. The mitochondrion is emerging as a critical platform for antiviral signal transduction. The regulatory role of mitochondria for TLR signaling remains to be explored. Here, we show that the mitochondrial outer-membrane protein MARCH5 positively regulates TLR7 signaling. Ectopic expression or knockdown of MARCH5 enhances or impairs NF-κB-mediated gene expression, respectively. MARCH5 interacts specifically with TANK, and this interaction is enhanced by R837 stimulation. MARCH5 catalyzes the K63-linked poly-ubiquitination of TANK on its Lysines 229, 233, 280, 302 and 306, thus impairing the ability of TANK to inhibit TRAF6. Mislocalization of MARCH5 abolishes its action on TANK, revealing the critical role of mitochondria in modulating innate immunity. Arguably, this represents the first study linking mitochondria to TLR signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Citocinas/análise , Células HEK293 , Humanos , Imunidade Inata , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Quinolinas , Interferência de RNA , RNA Interferente Pequeno , Receptores de Reconhecimento de Padrão , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 7 Toll-Like/agonistas , Receptores Toll-Like/agonistas , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
16.
bioRxiv ; 2023 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-38106103

RESUMO

Signals emanating from the T cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T cell function. Through forward genetic screening in mice, we discovered that loss-of-function mutations in LDL receptor related protein 10 ( Lrp10 ) caused naïve and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. Lrp10 was induced with T cell activation and its expression post-translationally suppressed IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhanced T cell homeostatic expansion through IL7R signaling. Lrp10 -deficient mice were also intrinsically resistant to syngeneic tumors. This phenotype depended on dense tumor infiltration of CD8 T cells that displayed increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy.

17.
J Cell Biol ; 178(2): 231-44, 2007 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-17620405

RESUMO

As a latent transcription factor, nuclear factor kappaB (NF-kappaB) translocates from the cytoplasm into the nucleus upon stimulation and mediates the expression of genes that are important in immunity, inflammation, and development. However, little is known about how it is regulated inside the nucleus. By a two-hybrid approach, we identify a prefoldin-like protein, ubiquitously expressed transcript (UXT), that is expressed predominantly and interacts specifically with NF-kappaB inside the nucleus. RNA interference knockdown of UXT leads to impaired NF-kappaB activity and dramatically attenuates the expression of NF-kappaB-dependent genes. This interference also sensitizes cells to apoptosis by tumor necrosis factor-alpha. Furthermore, UXT forms a dynamic complex with NF-kappaB and is recruited to the NF-kappaB enhanceosome upon stimulation. Interestingly, the UXT protein level correlates with constitutive NF-kappaB activity in human prostate cancer cell lines. The presence of NF-kappaB within the nucleus of stimulated or constitutively active cells is considerably diminished with decreased endogenous UXT levels. Our results reveal that UXT is an integral component of the NF-kappaB enhanceosome and is essential for its nuclear function, which uncovers a new mechanism of NF-kappaB regulation.


Assuntos
Elementos Facilitadores Genéticos/genética , NF-kappa B/metabolismo , Transcrição Gênica , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Luciferases/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Chaperonas Moleculares , NF-kappa B/genética , Proteínas de Neoplasias , Neoplasias da Próstata/patologia , Interferência de RNA , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Técnicas do Sistema de Duplo-Híbrido
18.
J Med Chem ; 65(13): 9230-9252, 2022 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-35767437

RESUMO

The diprovocims, a new class of toll-like receptor (TLR) agonists, bear no similarity to prior TLR agonists, act through a well-defined mechanism (TLR1/TLR2 agonist), exhibit exquisite structure-activity relationships, and display in vivo adjuvant activity. They possess potent and efficacious agonist activity toward human TLR1/TLR2 but modest agonism toward the murine receptor. A manner by which diprovocims can be functionalized without impacting hTLR1/TLR2 activity is detailed, permitting future linkage to antigenic, targeting, or delivery moieties. Improvements in both potency and its low efficacy in the murine system were also achieved, permitting more effective use in animal models while maintaining the hTLR1/TLR2 activity. The prototypical member diprovocim-X exhibits the excellent potency/efficacy of diprovocim-1 in human cells, displays substantially improved potency/efficacy in mouse macrophages, and serves as an adjuvant in mice when coadministered with a nonimmunogenic antigen, indicating stimulation of the adaptive as well as innate immune response.


Assuntos
Receptor 1 Toll-Like , Receptor 2 Toll-Like , Imunidade Adaptativa , Adjuvantes Imunológicos/farmacologia , Animais , Ciclopropanos , Humanos , Camundongos , Pirrolidinas , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas
19.
J Immunol ; 182(6): 3782-92, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19265157

RESUMO

Virus infection induces host antiviral responses including induction of type I IFNs. Transcription factor IFN regulatory factor 3 (IRF3) plays an essential role and is tightly regulated in this process. Herein we report that TRIM21 (tripartite motif-containing 21) is significantly induced and interacts with IRF3 upon RNA virus infection. Ectopic expression or knockdown of TRIM21 could respectively enhance or impair IRF3-mediated gene expression. Mechanistically, TRIM21 interferes with the interaction between Pin1 (peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1) and IRF3, thus preventing IRF3 ubiquitination and degradation. A conserved motif in the B 30.2 domain of TRIM21 is critical for its modulation of IRF3 function, while the RING finger is dispensable. Host antiviral responses are significantly boosted or crippled in the presence or absence of TRIM21. Our results identify TRIM21 as an essential modulator of IRF3 stability and demonstrate that it positively regulates the strength and duration of primary antiviral response, thus further strengthening the notion that the TRIM family is evolutionarily integrated with innate immunity.


Assuntos
Antivirais/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fator Regulador 3 de Interferon/metabolismo , Proteínas Nucleares/fisiologia , Infecções por Respirovirus/virologia , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Evolução Molecular , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/fisiologia , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/metabolismo , Ribonucleoproteínas , Vírus Sendai/imunologia , Ubiquitinação/imunologia
20.
Nat Commun ; 12(1): 1379, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654074

RESUMO

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Herpesvirus Humano 1/fisiologia , Humanos , Interleucina-6/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Inibidor de NF-kappaB alfa/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo
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