RESUMEN
Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities.
Asunto(s)
Alelos , Proteínas de Ciclo Celular/genética , Distrofias de Conos y Bastones/genética , Efecto Fundador , Pérdida Auditiva/genética , Infertilidad Masculina/genética , Mutación Missense , Adolescente , Proteínas de Ciclo Celular/química , Cilios/metabolismo , Cilios/ultraestructura , Distrofias de Conos y Bastones/diagnóstico , Análisis Mutacional de ADN , Femenino , Fibroblastos/metabolismo , Genotipo , Pérdida Auditiva/diagnóstico , Humanos , Infertilidad Masculina/diagnóstico , Masculino , Persona de Mediana Edad , Modelos Moleculares , Linaje , Fenotipo , Conformación Proteica , Relación Estructura-Actividad , Síndrome , Secuenciación del ExomaRESUMEN
Sorsby fundus dystrophy (SFD) is a macular degeneration caused by mutations in TIMP3, the majority of which introduce a novel cysteine. However, the exact molecular mechanisms underlying SFD remain unknown. We aimed to provide novel insights into the functional consequences of a distinct N-terminal mutation. Haplotype reconstruction in three SFD families revealed that the identified c.113C>G, p.(Ser38Cys) mutation is a founder in Belgian and northern French families with a late-onset SFD phenotype. Functional consequences of the p.(Ser38Cys) mutation were investigated by high-resolution Western blot analysis of wild type and mutant TIMP3 using patient fibroblasts and in vitro generated proteins, and by molecular modeling of TIMP3 and its interaction partners. We could not confirm a previous hypothesis on dimerization of mutant TIMP3 proteins. However, we identified aberrant intramolecular disulfide bonding. Our data provide evidence for disruption of the established Cys36-Cys143 disulfide bond and formation of a novel Cys36-Cys38 bond, possibly associated with increased glycosylation of the protein. In conclusion, we propose a novel pathogenetic mechanism underlying the p.(Ser38Cys) TIMP3 founder mutation involving intramolecular disulfide bonding. These results provide new insights into the pathogenesis of SFD and other retinopathies linked to mutations in TIMP3, such as age-related macular degeneration.
Asunto(s)
Efecto Fundador , Degeneración Macular/diagnóstico , Degeneración Macular/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Inhibidor Tisular de Metaloproteinasa-3/química , Inhibidor Tisular de Metaloproteinasa-3/genética , Anciano , Disulfuros , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Haplotipos , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Peso Molecular , Linaje , Conformación Proteica , Relación Estructura-Actividad , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.His325Tyr), in RCBTB1. In affected individuals, it was found to segregate with retinitis pigmentosa (RP), goiter, primary ovarian insufficiency, and mild intellectual disability. Subsequent analysis of WES data in different cohorts uncovered four additional homozygous missense mutations in five unrelated families in whom iRD segregates with or without syndromic features. Ocular phenotypes ranged from typical RP starting in the second decade to chorioretinal dystrophy with a later age of onset. The five missense mutations affect highly conserved residues either in the sixth repeat of the RCC1 domain or in the BTB1 domain. A founder haplotype was identified for mutation c.919G>A (p.Val307Met), occurring in two families of Mediterranean origin. We showed ubiquitous mRNA expression of RCBTB1 and demonstrated predominant RCBTB1 localization in human inner retina. RCBTB1 was very recently shown to be involved in ubiquitination, more specifically as a CUL3 substrate adaptor. Therefore, the effect on different components of the CUL3 and NFE2L2 (NRF2) pathway was assessed in affected individuals' lymphocytes, revealing decreased mRNA expression of NFE2L2 and several NFE2L2 target genes. In conclusion, our study puts forward mutations in RCBTB1 as a cause of autosomal-recessive non-syndromic and syndromic iRD. Finally, our data support a role for impaired ubiquitination in the pathogenetic mechanism of RCBTB1 mutations.
Asunto(s)
Alelos , Factores de Intercambio de Guanina Nucleótido/genética , Mutación Missense/genética , Distrofias Retinianas/genética , Ubiquitinación/genética , Adolescente , Adulto , Edad de Inicio , Niño , Consanguinidad , Proteínas Cullin/metabolismo , Exoma/genética , Femenino , Efecto Fundador , Genes Recesivos , Haplotipos/genética , Homocigoto , Humanos , Linfocitos/metabolismo , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Linaje , Fenotipo , ARN Mensajero/genética , Retina/metabolismo , Síndrome , TurquíaRESUMEN
PURPOSE: RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease. METHODS: Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members. RESULTS: Biallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry. CONCLUSION: This study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.
Asunto(s)
Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Retinitis Pigmentosa/genética , Factores de Transcripción/genética , Adulto , Análisis Mutacional de ADN/métodos , Proteínas del Ojo/metabolismo , Proteínas del Ojo/fisiología , Femenino , Genes Recesivos/genética , Estudios de Asociación Genética/métodos , Genotipo , Haplotipos/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Mutación Missense/genética , Linaje , Fenotipo , Retina/metabolismo , Retina/patología , Enfermedades de la Retina/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Población Blanca/genéticaRESUMEN
The original version of this Article contained an incorrect version of Fig. 3, which included two variants initially shown in black text in Fig. 3a that the authors removed from the final manuscript. The correct version of Fig. 3 without the two variants now appears in the PDF and HTML versions of the Article.
RESUMEN
The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host-pathogen interactions.
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Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Factor 6 de Ribosilación del ADP , Animales , Biomarcadores , Activación Enzimática , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Humanos , Fagocitosis/genética , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , VirulenciaRESUMEN
The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.
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Mutagénesis/genética , Mapeo de Interacción de Proteínas/métodos , Animales , Humanos , Unión ProteicaRESUMEN
PURPOSE: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases. METHODS: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46,XY DSD. Additional cases with 46,XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-ß (ER-ß) was performed in an 8-week-old human male embryo. RESULTS: We identified a homozygous ESR2 variant, c.541_543del p.(Asn181del), located in the highly conserved DNA-binding domain of ER-ß, in an individual with syndromic 46,XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-ß, were found in unrelated, nonsyndromic 46,XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-ß immunostaining was positive in the developing intestine and eyes. CONCLUSION: Our study supports a role for ESR2 as a novel candidate gene for 46,XY DSD.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY/genética , Receptor beta de Estrógeno/genética , Adolescente , Alelos , Sustitución de Aminoácidos/genética , Niño , Mapeo Cromosómico/métodos , Receptor beta de Estrógeno/metabolismo , Femenino , Frecuencia de los Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Mutación/genética , Conformación Proteica , Relación Estructura-Actividad , Secuenciación del Exoma/métodos , Adulto JovenRESUMEN
PURPOSE: We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD). METHODS: Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of mutation were investigated using luciferase assays, localization studies, and RNA-seq. RESULTS: We identified a novel heterozygous NR5A1 mutation, c.274C>T p.(Arg92Trp), in three unrelated patients. The Arg92 residue is highly conserved and located in the Ftz-F1 region, probably involved in DNA-binding specificity and stability. There were no consistent changes in transcriptional activation or subcellular localization. Transcriptomics in patient-derived lymphocytes showed upregulation of MAMLD1, a direct NR5A1 target previously associated with 46,XY DSD. In gonads of affected individuals, ovarian FOXL2 and testicular SRY-independent SOX9 expression observed. CONCLUSIONS: We propose NR5A1, previously associated with 46,XY DSD and 46,XX primary ovarian insufficiency, as a novel gene for 46,XX (ovo)testicular DSD. We hypothesize that p.(Arg92Trp) results in decreased inhibition of the male developmental pathway through downregulation of female antitestis genes, thereby tipping the balance toward testicular differentiation in 46,XX individuals. In conclusion, our study supports a role for NR5A1 in testis differentiation in the XX gonad.Genet Med 19 4, 367-376.
Asunto(s)
Proteínas de Unión al ADN/genética , Secuenciación del Exoma/métodos , Perfilación de la Expresión Génica/métodos , Proteínas Nucleares/genética , Trastornos Ovotesticulares del Desarrollo Sexual/genética , Análisis de Secuencia de ARN/métodos , Factor Esteroidogénico 1/genética , Factores de Transcripción/genética , Femenino , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Masculino , Modelos Moleculares , Mutación Missense , Ovario/metabolismo , Trastornos Ovotesticulares del Desarrollo Sexual/metabolismo , Linaje , Polimorfismo de Nucleótido Simple , Factor Esteroidogénico 1/química , Factor Esteroidogénico 1/metabolismo , Testículo/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. We here provide genetic and biochemical evidence that the metabolic and immune functions of leptin can be uncoupled at the receptor level. First, homozygous mutant fatt/fatt mice carry a spontaneous splice mutation causing deletion of the leptin receptor (LR) immunoglobulin-like domain (IGD) in all LR isoforms. These mice are hyperphagic and morbidly obese, but display only minimal changes in size and cellularity of the thymus, and cellular immune responses are unaffected. These animals also displayed liver damage in response to concavalin A comparable to wild-type and heterozygous littermates. Second, treatment of healthy mice with a neutralizing nanobody targeting IGD induced weight gain and hyperinsulinaemia, but completely failed to block development of experimentally induced autoimmune diseases. These data indicate that leptin receptor deficiency or antagonism profoundly affects metabolism, with little concomitant effects on immune functions.
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Leptina/inmunología , Leptina/metabolismo , Receptores de Leptina/metabolismo , Análisis de Varianza , Animales , Artritis Experimental/patología , Secuencia de Bases , Western Blotting , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cartilla de ADN/genética , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/patología , Citometría de Flujo , Células HEK293 , Humanos , Células MCF-7 , Masculino , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Glicoproteína Mielina-Oligodendrócito/toxicidad , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Receptores de Leptina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Eliminación de Secuencia/genéticaRESUMEN
Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal(-/-) mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.
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Factores de Ribosilacion-ADP/metabolismo , Macrófagos/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Animales , Línea Celular , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Ratones , Ratones Noqueados , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Interleucina/genética , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiologíaRESUMEN
The mechanisms controlling the steady-state cell surface levels of cytokine receptors, and consequently the cellular response to cytokines, remain poorly understood. The number of surface-exposed receptors is a dynamic balance of de novo synthesis, transport to the plasma membrane, internalization, recycling, degradation and ectodomain shedding. We previously reported that the E3 ubiquitin ligase RING finger protein 41 (RNF41) inhibits basal lysosomal degradation and enhances ectodomain shedding of JAK2-associated cytokine receptors. Ubiquitin-specific protease 8 (USP8), an RNF41-interacting deubiquitylating enzyme (DUB) stabilizes RNF41 and is involved in trafficking of various transmembrane proteins. The present study identifies USP8 as a substrate of RNF41 and reveals that loss of USP8 explains the aforementioned RNF41 effects. RNF41 redistributes and ubiquitylates USP8, and reduces USP8 levels. In addition, USP8 knockdown functionally matches the effects of RNF41 ectopic expression on the model leptin and leukemia inhibitory factor (LIF) receptors. Moreover, RNF41 indirectly destabilizes the ESCRT-0 complex through suppression of USP8. Collectively, our findings demonstrate that RNF41 controls JAK2-associated cytokine receptor trafficking by acting as a key regulator of USP8 and ESCRT-0 stability. Balanced reciprocal cross-regulation of RNF41 and USP8 thus determines whether receptors are sorted for lysosomal degradation or recycling, this way regulating basal cytokine receptor levels.
Asunto(s)
Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Receptores de Citocinas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Movimiento Celular/fisiología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Transporte de Proteínas , Receptores de Citocinas/genética , Transducción de Señal , Transfección , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Upon activation, Toll-like receptor 4 (TLR4) binds adapter proteins, including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like) for its signal transduction. TLR4 and the adapter proteins each contain a Toll/Il-1 receptor domain (TIR domain). In this study we used random mutagenesis and the mammalian two-hybrid method MAPPIT (mammalian protein-protein interaction trap) to identify mutations in Mal that disrupt its interaction with TLR4 and/or MyD88. Our study shows that four potential binding sites and the AB-loop in the Mal TIR domain all contribute to formation of the TLR4-Mal-MyD88 complex. Mutations in the symmetrical back-to-back Mal homodimer interface affect Mal homodimerization and interaction with MyD88 and TLR4. Our data suggest that Mal dimerization may lead to formation of potential binding platforms on the top and the side of the Mal dimer that bind MyD88 or TLR4. Mutations that affect the interaction of Mal with MyD88 also affect NF-κB activation induced by Mal overexpression. In MAPPIT, co-expression of the MyD88 TIR domain enhances Mal dimerization and Mal binding to TLR4. Similarly, co-expression of Mal and the MyD88 TIR domain strongly promotes dimerization of the TLR4 intracellular domain in MAPPIT. The different types of TIR-TIR interactions in the TLR4-Mal-MyD88 complex thus show cooperative binding in MAPPIT. We present plausible models for the TIR-TIR interactions in the TLR4-Mal-MyD88 complex.
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Sitios de Unión , Modelos Biológicos , Factor 88 de Diferenciación Mieloide , Multimerización de Proteína/fisiología , Receptor Toll-Like 4 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación , Factor 88 de Diferenciación Mieloide/química , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Mapeo Peptídico/métodos , Unión Proteica , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Receptor Toll-Like 4/química , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The adipocyte-derived hormone/cytokine leptin acts as a metabolic switch, connecting the body's nutritional status to high energy consuming processes such as reproduction and immune responses. Inappropriate leptin responses can promote autoimmune diseases and tumorigenesis. In this review we discuss the current strategies to modulate leptin signaling and the possibilities for their use in research and therapy.
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Leptina/antagonistas & inhibidores , Obesidad/metabolismo , Adipocitos/metabolismo , Humanos , Leptina/inmunología , Transducción de SeñalAsunto(s)
Artritis/genética , Diferenciación Celular/genética , Mutación con Ganancia de Función/genética , Células Germinativas/patología , Mutación/genética , Factor 88 de Diferenciación Mieloide/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Huesos/patología , Proteína C-Reactiva/genética , Línea Celular , Exoma/genética , Femenino , Humanos , Inflamación/genética , Monocitos/patologíaRESUMEN
OBJECTIVE: The peroxisome proliferator-activated receptor α (PPARα) is a transcription factor driving target genes involved in fatty acid ß-oxidation. To what extent various PPARα interacting proteins may assist its function as a transcription factor is incompletely understood. An ORFeome-wide unbiased mammalian protein-protein interaction trap (MAPPIT) using PPARα as bait revealed a PPARα-ligand-dependent interaction with the orphan nuclear receptor estrogen-related receptor α (ERRα). The goal of this study was to characterize the nature of the interaction in depth and to explore whether it was of physiological relevance. METHODS: We used orthogonal protein-protein interaction assays and pharmacological inhibitors of ERRα in various systems to confirm a functional interaction and study the impact of crosstalk mechanisms. To characterize the interaction surfaces and contact points we applied a random mutagenesis screen and structural overlays. We pinpointed the extent of reciprocal ligand effects of both nuclear receptors via coregulator peptide recruitment assays. On PPARα targets revealed from a genome-wide transcriptome analysis, we performed an ERRα chromatin immunoprecipitation analysis on both fast and fed mouse livers. RESULTS: Random mutagenesis scanning of PPARα's ligand-binding domain and coregulator profiling experiments supported the involvement of (a) bridging coregulator(s), while recapitulation of the interaction in vitro indicated the possibility of a trimeric interaction with RXRα. The PPARα·ERRα interaction depends on 3 C-terminal residues within helix 12 of ERRα and is strengthened by both PGC1α and serum deprivation. Pharmacological inhibition of ERRα decreased the interaction of ERRα to ligand-activated PPARα and revealed a transcriptome in line with enhanced mRNA expression of prototypical PPARα target genes, suggesting a role for ERRα as a transcriptional repressor. Strikingly, on other PPARα targets, including the isolated PDK4 enhancer, ERRα behaved oppositely. Chromatin immunoprecipitation analyses demonstrate a PPARα ligand-dependent ERRα recruitment onto chromatin at PPARα-binding regions, which is lost following ERRα inhibition in fed mouse livers. CONCLUSIONS: Our data support the coexistence of multiple layers of transcriptional crosstalk mechanisms between PPARα and ERRα, which may serve to finetune the activity of PPARα as a nutrient-sensing transcription factor.
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Receptor Relacionado con Estrógeno ERRalfa , PPAR alfa , Receptores de Estrógenos , PPAR alfa/metabolismo , PPAR alfa/genética , Animales , Ratones , Receptores de Estrógenos/metabolismo , Receptores de Estrógenos/genética , Humanos , Regulación de la Expresión Génica , Células HEK293 , Masculino , Ratones Endogámicos C57BL , Unión Proteica , Hígado/metabolismoRESUMEN
Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.
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Modelos Moleculares , Multimerización de Proteína/fisiología , Receptor Toll-Like 4/química , Animales , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Factor 88 de Diferenciación Mieloide/química , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Mapeo Peptídico , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Receptor Toll-Like 10/química , Receptor Toll-Like 10/genética , Receptor Toll-Like 10/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.
Asunto(s)
Anticuerpos/farmacología , Leptina/antagonistas & inhibidores , Nanoestructuras/química , Receptores de Leptina/antagonistas & inhibidores , Tejido Adiposo , Animales , Anticuerpos/química , Camélidos del Nuevo Mundo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Hiperinsulinismo , Hígado/anatomía & histología , Ratones , Ratones Endogámicos C57BL , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Tamaño de los Órganos , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Aumento de PesoRESUMEN
Protein mutagenesis is essential for unveiling the molecular mechanisms underlying protein function in health, disease, and evolution. In the past decade, deep mutational scanning methods have evolved to support the functional analysis of nearly all possible single-amino acid changes in a protein of interest. While historically these methods were developed in lower organisms such as E. coli and yeast, recent technological advancements have resulted in the increased use of mammalian cells, particularly for studying proteins involved in human disease. These advancements will aid significantly in the classification and interpretation of variants of unknown significance, which are being discovered at large scale due to the current surge in the use of whole-genome sequencing in clinical contexts. Here, we explore the experimental aspects of deep mutational scanning studies in mammalian cells and report the different methods used in each step of the workflow, ultimately providing a useful guide toward the design of such studies.
Asunto(s)
Escherichia coli , Proteínas , Animales , Humanos , Mutación , Proteínas/genética , Mutagénesis , Aminoácidos , Mamíferos/genéticaRESUMEN
The glucocorticoid receptor α (GRα) is a member of the nuclear receptor superfamily and functions as a glucocorticoid (GC)-responsive transcription factor. GR can halt inflammation and kill off cancer cells, thus explaining the widespread use of glucocorticoids in the clinic. However, side effects and therapy resistance limit GR's therapeutic potential, emphasizing the importance of resolving all of GR's context-specific action mechanisms. Fortunately, the understanding of GR structure, conformation, and stoichiometry in the different GR-controlled biological pathways is now gradually increasing. This information will be crucial to close knowledge gaps on GR function. In this review, we focus on the various domains and mechanisms of action of GR, all from a structural perspective.