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1.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34260399

RESUMEN

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.


Asunto(s)
Mutación de Línea Germinal/genética , Leucocitos/metabolismo , Aprendizaje Automático , Meiosis/genética , Algoritmos , Animales , Automatización , Femenino , Citometría de Flujo , Masculino , Ratones Endogámicos C57BL , Fenotipo , Probabilidad , Reproducibilidad de los Resultados , Programas Informáticos
2.
Nat Immunol ; 12(12): 1184-93, 2011 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-22019834

RESUMEN

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.


Asunto(s)
Colitis/inmunología , Proteínas de Unión al ADN/genética , Células Dendríticas/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espondilitis Anquilosante/inmunología , Ubiquitina-Proteína Ligasas/genética , Animales , Colitis/patología , Colitis/prevención & control , Enfermedad de Crohn/genética , Cisteína Endopeptidasas , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Predisposición Genética a la Enfermedad , Homeostasis/inmunología , Humanos , Enfermedades Linfáticas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Transducción de Señal , Esplenomegalia/genética , Espondilitis Anquilosante/patología , Espondilitis Anquilosante/prevención & control , Linfocitos T/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina-Proteína Ligasas/metabolismo
3.
Immunity ; 38(5): 896-905, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23602765

RESUMEN

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.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Cisteína Endopeptidasas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Multimerización de Proteína , Transducción de Señal/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Dedos de Zinc/genética
4.
Proc Natl Acad Sci U S A ; 116(23): 11380-11389, 2019 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-31097594

RESUMEN

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.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colitis/metabolismo , Endosomas/metabolismo , Transducción de Señal/fisiología , Linfocitos T Reguladores/metabolismo , Animales , Autoinmunidad/fisiología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Colitis/inducido químicamente , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Sulfato de Dextran/farmacología , Femenino , Inflamación/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos
5.
Proc Natl Acad Sci U S A ; 115(49): E11523-E11531, 2018 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-30442666

RESUMEN

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.


Asunto(s)
Proteína C9orf72/metabolismo , Proteínas Portadoras/metabolismo , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores Toll-Like/metabolismo , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Proteínas Relacionadas con la Autofagia , Proteína C9orf72/genética , Proteínas Portadoras/genética , Colitis/inducido químicamente , Sulfato de Dextran , Regulación de la Expresión Génica , Hematopoyesis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Mutación , Transducción de Señal/inmunología , Receptores Toll-Like/genética
6.
Proc Natl Acad Sci U S A ; 114(7): E1273-E1281, 2017 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-28137860

RESUMEN

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.


Asunto(s)
Colitis/prevención & control , Creatina/farmacología , Homeostasis/efectos de los fármacos , Intestinos/efectos de los fármacos , Amidinotransferasas/genética , Amidinotransferasas/metabolismo , Animales , Sistemas CRISPR-Cas , Colitis/genética , Colitis/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Creatina/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Mucosa Intestinal/metabolismo , Masculino , Ratones Endogámicos C57BL , Mutación Missense , Sustancias Protectoras/metabolismo , Sustancias Protectoras/farmacología
7.
Immunity ; 33(2): 181-91, 2010 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-20705491

RESUMEN

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.


Asunto(s)
Autoinmunidad , Linfocitos B/citología , Linfocitos B/inmunología , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Linfocitos B/enzimología , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Linaje de la Célula , Supervivencia Celular , Cisteína Endopeptidasas/deficiencia , Homeostasis , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa
8.
Proc Natl Acad Sci U S A ; 113(42): E6418-E6426, 2016 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-27708159

RESUMEN

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.


Asunto(s)
Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Dieta/efectos adversos , Predisposición Genética a la Enfermedad , Resistencia a la Insulina , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/trasplante , Animales , Glucemia , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Proteínas Cullin/metabolismo , Modelos Animales de Enfermedad , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado Graso/patología , Regulación de la Expresión Génica , Estudios de Asociación Genética , Genotipo , Insulina/sangre , Resistencia a la Insulina/genética , Lipodistrofia/etiología , Lipodistrofia/metabolismo , Lipodistrofia/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Obesidad/etiología , Obesidad/patología , Fenotipo , Unión Proteica , Transporte de Proteínas , Transducción de Señal , Ubiquitinación
9.
Proc Natl Acad Sci U S A ; 112(5): E440-9, 2015 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-25605905

RESUMEN

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.


Asunto(s)
Mutación Puntual , Alelos , Animales , Femenino , Genes Letales , Ligamiento Genético , Masculino , Ratones , Linaje , Fenotipo , Sitios de Carácter Cuantitativo
10.
Immunity ; 28(3): 381-90, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18342009

RESUMEN

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.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Transducción de Señal/fisiología , Ubiquitinación/fisiología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Animales , Macrófagos/metabolismo , Ratones , Ratones Mutantes , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina/metabolismo
11.
Nature ; 457(7231): 906-9, 2009 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19060883

RESUMEN

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.


Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/fisiología , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al ADN/química , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Alineación de Secuencia , Factor de Necrosis Tumoral alfa/metabolismo
12.
bioRxiv ; 2024 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-38559023

RESUMEN

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.

13.
Dis Model Mech ; 16(9)2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37589563

RESUMEN

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.


Asunto(s)
Colitis , Animales , Ratones , Ceramidas , Oxigenasas de Función Mixta , Inflamación , Ácido Graso Desaturasas
14.
Nat Commun ; 14(1): 3652, 2023 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-37339972

RESUMEN

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.


Asunto(s)
Mucosa Intestinal , Intestinos , Proteínas de la Membrana , Animales , Ratones , Microbioma Gastrointestinal , Glicosilación , Células Caliciformes/metabolismo , Aparato de Golgi/metabolismo , Homeostasis , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , Proteínas de la Membrana/metabolismo , Moco , Células de Paneth/metabolismo
15.
Science ; 372(6543)2021 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-33986151

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inmunidad Celular , Estrés Oxidativo , ARN de Transferencia/metabolismo , Linfocitos T/inmunología , Animales , Proteínas de Ciclo Celular/genética , Proliferación Celular , Femenino , Eliminación de Gen , Infecciones por Herpesviridae/inmunología , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muromegalovirus , Unión Proteica , Biosíntesis de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismo , Transducción de Señal
16.
Sci Immunol ; 5(43)2020 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-31980486

RESUMEN

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.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteínas Quinasas/inmunología , Proteínas Proto-Oncogénicas c-bcl-6/inmunología , Animales , Linfocitos B/inmunología , Trasplante de Médula Ósea , Diferenciación Celular , Femenino , Centro Germinal/inmunología , Células HEK293 , Humanos , Inmunoglobulinas/sangre , Inmunoterapia Adoptiva , Masculino , Ratones Transgénicos , Mutación , Proteína Quinasa D2 , Proteínas Quinasas/genética
18.
J Invest Dermatol ; 139(9): 1848-1853.e1, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31445571

RESUMEN

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.


Asunto(s)
Pruebas Genéticas/métodos , Proyectos de Investigación , Enfermedades de la Piel/genética , Animales , Cruzamiento/métodos , Modelos Animales de Enfermedad , Etilnitrosourea/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mutagénesis/efectos de los fármacos , Mutágenos/toxicidad , Mutación/efectos de los fármacos , Fenotipo , Transducción de Señal/genética , Piel/patología , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/patología
19.
Dis Model Mech ; 11(12)2018 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-30563851

RESUMEN

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.


Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diabetes Mellitus/genética , Hígado Graso/genética , Predisposición Genética a la Enfermedad , Hiperfagia/genética , Mutación/genética , Obesidad/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Peso Corporal , Etilnitrosourea , Femenino , Glucosa/metabolismo , Células HEK293 , Homeostasis , Homocigoto , Humanos , Hipotálamo/patología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Transcripción Genética
20.
Cell Rep ; 24(9): 2342-2355, 2018 08 28.
Artículo en Inglés | MEDLINE | ID: mdl-30157428

RESUMEN

Physiologic microbe-host interactions in the intestine require the maintenance of the microbiota in a luminal compartment through a complex interplay between epithelial and immune cells. However, the roles of mucosal myeloid cells in this process remain incompletely understood. In this study, we identified that decreased myeloid cell phagocytic activity promotes colon tumorigenesis. We show that this is due to bacterial accumulation in the lamina propria and present evidence that the underlying mechanism is bacterial induction of prostaglandin production by myeloid cells. Moreover, we show that similar events in the normal colonic mucosa lead to reductions in Tuft cells, goblet cells, and the mucus barrier of the colonic epithelium. These alterations are again linked to the induction of prostaglandin production in response to bacterial penetration of the mucosa. Altogether, our work highlights immune cell-epithelial cell interactions triggered by the microbiota that control intestinal immunity, epithelial differentiation, and carcinogenesis.


Asunto(s)
Carcinogénesis/metabolismo , Células Epiteliales/inmunología , Intestinos/fisiopatología , Microbiota/fisiología , Células Mieloides/metabolismo , Animales , Humanos , Ratones
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