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 â¼55% 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 , SoftwareRESUMO
Dendritic cells (DCs), which are known to support immune activation during infection, may also regulate immune homeostasis in resting animals. Here we show that mice lacking the ubiquitin-editing molecule A20 specifically in DCs spontaneously showed DC activation and population expansion of activated T cells. Analysis of DC-specific epistasis in compound mice lacking both A20 and the signaling adaptor MyD88 specifically in DCs showed that A20 restricted both MyD88-independent signals, which drive activation of DCs and T cells, and MyD88-dependent signals, which drive population expansion of T cells. In addition, mice lacking A20 specifically in DCs spontaneously developed lymphocyte-dependent colitis, seronegative ankylosing arthritis and enthesitis, conditions stereotypical of human inflammatory bowel disease (IBD). Our findings indicate that DCs need A20 to preserve immune quiescence and suggest that A20-dependent DC functions may underlie IBD and IBD-associated arthritides.
Assuntos
Colite/imunologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espondilite Anquilosante/imunologia , Ubiquitina-Proteína Ligases/genética , Animais , Colite/patologia , Colite/prevenção & controle , Doença de Crohn/genética , Cisteína Endopeptidases , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Predisposição Genética para Doença , Homeostase/imunologia , Humanos , Doenças Linfáticas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Esplenomegalia/genética , Espondilite Anquilosante/patologia , Espondilite Anquilosante/prevenção & controle , Linfócitos T/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A20 is an anti-inflammatory protein linked to multiple human autoimmune diseases and lymphomas. A20 possesses a deubiquitinating motif and a zinc finger, ZF4, that binds ubiquitin and supports its E3 ubiquitin ligase activity. To understand how these activities mediate A20's physiological functions, we generated two lines of gene-targeted mice, abrogating either A20's deubiquitinating activity (Tnfaip3(OTU) mice) or A20's ZF4 (Tnfaip3(ZF4) mice). Both Tnfaip3(OTU) and Tnfaip3(ZF4) mice exhibited increased responses to TNF and sensitivity to colitis. A20's C103 deubiquitinating motif restricted both K48- and K63-linked ubiquitination of receptor interacting protein 1 (RIP1). A20's ZF4 was required for recruiting A20 to ubiquitinated RIP1. A20(OTU) proteins and A20(ZF4) proteins complemented each other to regulate RIP1 ubiquitination and NFκB signaling normally in compound mutant Tnfaip3(OTU/ZF4) cells. This complementation involved homodimerization of A20 proteins, and we have defined an extensive dimerization interface in A20. These studies reveal how A20 proteins collaborate to restrict TNF signaling.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Cisteína Endopeptidases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Multimerização Proteica , Transdução de Sinais/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Dedos de Zinco/genéticaRESUMO
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 fármacosRESUMO
The SMCR8-WDR41-C9ORF72 complex is a regulator of autophagy and lysosomal function. Autoimmunity and inflammatory disease have been ascribed to loss-of-function mutations of Smcr8 or C9orf72 in mice. In humans, autoimmunity has been reported to precede amyotrophic lateral sclerosis caused by mutations of C9ORF72 However, the cellular and molecular mechanisms underlying autoimmunity and inflammation caused by C9ORF72 or SMCR8 deficiencies remain unknown. Here, we show that splenomegaly, lymphadenopathy, and activated circulating T cells observed in Smcr8-/- mice were rescued by triple knockout of the endosomal Toll-like receptors (TLRs) TLR3, TLR7, and TLR9. Myeloid cells from Smcr8-/- mice produced excessive inflammatory cytokines in response to endocytosed TLR3, TLR7, or TLR9 ligands administered in the growth medium and in response to TLR2 or TLR4 ligands internalized by phagocytosis. These defects likely stem from prolonged TLR signaling caused by accumulation of LysoTracker-positive vesicles and by delayed phagosome maturation, both of which were observed in Smcr8-/- macrophages. Smcr8-/- mice also showed elevated susceptibility to dextran sodium sulfate-induced colitis, which was not associated with increased TLR3, TLR7, or TLR9 signaling. Deficiency of WDR41 phenocopied loss of SMCR8. Our findings provide evidence that excessive endosomal TLR signaling resulting from prolonged ligand-receptor contact causes inflammatory disease in SMCR8-deficient mice.
Assuntos
Proteína C9orf72/metabolismo , Proteínas de Transporte/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Toll-Like/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteínas Relacionadas à Autofagia , Proteína C9orf72/genética , Proteínas de Transporte/genética , Colite/induzido quimicamente , Sulfato de Dextrana , Regulação da Expressão Gênica , Hematopoese/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Mutação , Transdução de Sinais/imunologia , Receptores Toll-Like/genéticaRESUMO
Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9-targeted (Gatmc/c ) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/c colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury.
Assuntos
Colite/prevenção & controle , Creatina/farmacologia , Homeostase/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Amidinotransferases/genética , Amidinotransferases/metabolismo , Animais , Sistemas CRISPR-Cas , Colite/genética , Colite/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Creatina/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologiaRESUMO
A20 is a ubiquitin modifying enzyme that restricts NF-kappaB signals and protects cells against tumor necrosis factor (TNF)-induced programmed cell death. Given recent data linking A20 (TNFAIP3) with human B cell lymphomas and systemic lupus erythematosus (SLE), we have generated mice bearing a floxed allele of Tnfaip3 to interrogate A20's roles in regulating B cell functions. A20-deficient B cells are hyperresponsive to multiple stimuli and display exaggerated NF-kappaB responses to CD40-induced signals. Mice expressing absent or hypomorphic amounts of A20 in B cells possess elevated numbers of germinal center B cells, autoantibodies, and glomerular immunoglobulin deposits. A20-deficient B cells are resistant to Fas-mediated cell death, probably due to increased expression of NF-kappaB-dependent antiapoptotic proteins such as Bcl-x. These findings show that A20 can restrict B cell survival, whereas A20 protects other cells from TNF-induced cell death. Our studies demonstrate how reduced A20 expression predisposes to autoimmunity.
Assuntos
Autoimunidade , Linfócitos B/citologia , Linfócitos B/imunologia , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Linfócitos B/enzimologia , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Linhagem da Célula , Sobrevivência Celular , Cisteína Endopeptidases/deficiência , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
We describe a metabolic disorder characterized by lipodystrophy, hepatic steatosis, insulin resistance, severe diabetes, and growth retardation observed in mice carrying N-ethyl-N-nitrosourea (ENU)-induced mutations. The disorder was ascribed to a mutation of kelch repeat and BTB (POZ) domain containing 2 (Kbtbd2) and was mimicked by a CRISPR/Cas9-targeted null allele of the same gene. Kbtbd2 encodes a BTB-Kelch family substrate recognition subunit of the Cullin-3-based E3 ubiquitin ligase. KBTBD2 targeted p85α, the regulatory subunit of the phosphoinositol-3-kinase (PI3K) heterodimer, causing p85α ubiquitination and proteasome-mediated degradation. In the absence of KBTBD2, p85α accumulated to 30-fold greater levels than in wild-type adipocytes, and excessive p110-free p85α blocked the binding of p85α-p110 heterodimers to IRS1, interrupting the insulin signal. Both transplantation of wild-type adipose tissue and homozygous germ line inactivation of the p85α-encoding gene Pik3r1 rescued diabetes and hepatic steatosis phenotypes of Kbtbd2-/- mice. Kbtbd2 was down-regulated in diet-induced obese insulin-resistant mice in a leptin-dependent manner. KBTBD2 is an essential regulator of the insulin-signaling pathway, modulating insulin sensitivity by limiting p85α abundance.
Assuntos
Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Dieta/efeitos adversos , Predisposição Genética para Doença , Resistência à Insulina , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/transplante , Animais , Glicemia , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Culina/metabolismo , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica , Estudos de Associação Genética , Genótipo , Insulina/sangue , Resistência à Insulina/genética , Lipodistrofia/etiologia , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Obesidade/etiologia , Obesidade/patologia , Fenótipo , Ligação Proteica , Transporte Proteico , Transdução de Sinais , UbiquitinaçãoRESUMO
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 QuantitativasRESUMO
Muramyl dipeptide (MDP), a product of bacterial cell-wall peptidoglycan, activates innate immune cells by stimulating nucleotide-binding oligomerization domain containing 2 (NOD2) -dependent activation of the transcription factor NFkappaB and transcription of proinflammatory genes. A20 is a ubiquitin-modifying enzyme that restricts tumor necrosis factor (TNF) receptor and Toll-like receptor (TLR) -induced signals. We now show that MDP induces ubiquitylation of receptor- interacting protein 2 (RIP2) in primary macrophages. A20-deficient cells exhibit dramatically amplified responses to MDP, including increased RIP2 ubiquitylation, prolonged NFkappaB signaling, and increased production of proinflammatory cytokines. In addition, in vivo responses to MDP are exaggerated in A20-deficient mice and in chimeric mice bearing A20-deficient hematopoietic cells. These exaggerated responses occur independently of the TLR adaptors MyD88 and TRIF as well as TNF signals. These findings indicate that A20 directly restricts NOD2 induced signals in vitro and in vivo, and provide new insights into how these signals are physiologically restricted.
Assuntos
Cisteína Endopeptidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Macrófagos/metabolismo , Camundongos , Camundongos Mutantes , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina/metabolismoRESUMO
Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to tumour necrosis factor (TNF)-induced programmed cell death, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD (Fas-associated death domain-containing protein) in TNF-induced signalling complexes, preventing caspase 8 cleavage and programmed cell death. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1's anti-apoptotic activity. These studies provide insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/fisiologia , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/química , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Alinhamento de Sequência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
During endosomal recycling, Sorting Nexin 17 (SNX17) facilitates the transport of numerous membrane cargo proteins by tethering them to the Retriever complex. Despite its importance, the mechanisms underlying this interaction have remained elusive. Here, we report the structure of the Retriever-SNX17 complex determined using cryogenic electron microscopy (cryo-EM). Our structure reveals that the C-terminal tail of SNX17 engages with a highly conserved interface between the VPS35L and VPS26C subunits of Retriever. Through comprehensive biochemical, cellular, and proteomic analyses, we demonstrate that disrupting this interface impairs the Retriever-SNX17 interaction, subsequently affecting the recycling of SNX17-dependent cargos and altering the composition of the plasma membrane proteome. Intriguingly, we find that the SNX17-binding pocket on Retriever can be utilized by other ligands that share a consensus acidic C-terminal tail motif. By showing how SNX17 is linked to Retriever, our findings uncover a fundamental mechanism underlying endosomal trafficking of critical cargo proteins and reveal a mechanism by which Retriever can engage with other regulatory factors.
RESUMO
Intestinal immunity is dependent on barrier function to maintain quiescence. The mechanisms for the maintenance of this barrier are not fully understood. Delta 4-desaturase, sphingolipid 2 (DEGS2) is a lipid desaturase and hydroxylase that catalyzes the synthesis of ceramide and phytoceramide from dihydroceramide. Using a forward genetic approach, we found and validated a mutation in Degs2 as causative of increasing susceptibility to colitis and altering the phytoceramide balance in the colon. DEGS2 is expressed in the intestinal epithelium, and the colitis phenotype is dependent on the non-hematopoietic compartment of the mouse. In the absence of DEGS2, the colon lacks phytoceramides and accumulates large amounts of the precursor lipid dihydroceramide. In response to dextran sodium sulfate (DSS)-induced colitis, colonic epithelial cells in DEGS2-deficient mice had increased cell death and decreased proliferation compared to those in wild-type mice. These findings demonstrate that DEGS2 is needed to maintain epithelial integrity, protect against DSS-induced colitis and maintain lipid balance in vivo.
Assuntos
Colite , Animais , Camundongos , Ceramidas , Oxigenases de Função Mista , Inflamação , Ácidos Graxos DessaturasesRESUMO
A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.
Assuntos
Mucosa Intestinal , Intestinos , Proteínas de Membrana , Animais , Camundongos , Microbioma Gastrointestinal , Glicosilação , Células Caliciformes/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , Proteínas de Membrana/metabolismo , Muco , Celulas de Paneth/metabolismoRESUMO
Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell-specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor ß (IL-2Rß) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2's mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Imunidade Celular , Estresse Oxidativo , RNA de Transferência/metabolismo , Linfócitos T/imunologia , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , Feminino , Deleção de Genes , Infecções por Herpesviridae/imunologia , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muromegalovirus , Ligação Proteica , Biossíntese de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transdução de SinaisRESUMO
T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Animais , Linfócitos B/imunologia , Transplante de Medula Óssea , Diferenciação Celular , Feminino , Centro Germinativo/imunologia , Células HEK293 , Humanos , Imunoglobulinas/sangue , Imunoterapia Adotiva , Masculino , Camundongos Transgênicos , Mutação , Proteína Quinase D2 , Proteínas Quinases/genéticaAssuntos
Antivirais/uso terapêutico , Crioglobulinemia/etiologia , Hepatite C/complicações , Oligopeptídeos/uso terapêutico , Vasculite do Sistema Nervoso Central/etiologia , Crioglobulinemia/tratamento farmacológico , Glomerulonefrite/etiologia , Hepatite C/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/etiologia , Vasculite do Sistema Nervoso Central/tratamento farmacológicoRESUMO
The primary goals of modern genetics are to identify disease-causing mutations and to define the functions of genes in biological processes. Two complementary approaches, reverse and forward genetics, can be used to achieve this goal. Reverse genetics is a gene-driven approach that comprises specific gene targeting followed by phenotypic assessment. Conversely, forward genetics is a phenotype-driven approach that involves the phenotypic screening of organisms with randomly induced mutations followed by subsequent identification of the causative mutations (i.e., those responsible for phenotype). In this article, we focus on how forward genetics in mice can be used to explore dermatologic disease. We outline mouse mutagenesis with the chemical N-ethyl-N-nitrosourea and the strategy used to instantaneously identify mutations that are causative of specific phenotypes. Furthermore, we summarize the types of phenotypic screens that can be performed to explore various aspects of dermatologic disease.
Assuntos
Testes Genéticos/métodos , Projetos de Pesquisa , Dermatopatias/genética , Animais , Cruzamento/métodos , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidade , Mutação/efeitos dos fármacos , Fenótipo , Transdução de Sinais/genética , Pele/patologia , Dermatopatias/diagnóstico , Dermatopatias/patologiaRESUMO
Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a member of the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) transcription factor family. ARNT2 heterodimerizes with several members of the family, including single-minded homolog-1 (SIM1) and neuronal PAS domain protein 4 (NPAS4), primarily in neurons of the central nervous system. We screened 64,424 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea (ENU)-mutagenized great-grandsires for weight abnormalities. Among 17 elevated body weight phenotypes identified and mapped, one strongly correlated with an induced missense mutation in Arnt2 using a semidominant model of inheritance. Causation was confirmed by CRISPR/Cas9 gene targeting to recapitulate the original ENU allele, specifying Arg74Cys (R74C). The CRISPR/Cas9-targeted (Arnt2R74C/R74C) mice demonstrated hyperphagia and increased adiposity as well as hepatic steatosis and abnormalities in glucose homeostasis. The mutant ARNT2 protein showed decreased transcriptional activity when coexpressed with SIM1. These findings establish a requirement for ARNT2-dependent genes in the maintenance of the homeostatic feeding response, necessary for prevention of obesity and obesity-related diseases.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diabetes Mellitus/genética , Fígado Gorduroso/genética , Predisposição Genética para Doença , Hiperfagia/genética , Mutação/genética , Obesidade/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peso Corporal , Etilnitrosoureia , Feminino , Glucose/metabolismo , Células HEK293 , Homeostase , Homozigoto , Humanos , Hipotálamo/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
Myosin ID (MYO1D) is a member of the class I myosin family. We screened 48,649 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). We found and validated mutations in Myo1d as a cause of increased susceptibility to DSS-induced colitis. MYO1D is produced in the intestinal epithelium, and the colitis phenotype is dependent on the nonhematopoietic compartment of the mouse. Moreover, MYO1D appears to couple cytoskeletal elements to lipid in an ATP-dependent manner. These findings demonstrate that MYO1D is needed to maintain epithelial integrity and protect against DSS-induced colitis.