RESUMO
The protein MSTO1 has been localized to mitochondria and linked to mitochondrial morphology, but its specific role has remained unclear. We identified a c.22G > A (p.Val8Met) mutation of MSTO1 in patients with minor physical abnormalities, myopathy, ataxia, and neurodevelopmental impairments. Lactate stress test and myopathological results suggest mitochondrial dysfunction. In patient fibroblasts, MSTO1 mRNA and protein abundance are decreased, mitochondria display fragmentation, aggregation, and decreased network continuity and fusion activity. These characteristics can be reversed by genetic rescue. Short-term silencing of MSTO1 in HeLa cells reproduced the impairment of mitochondrial morphology and dynamics observed in the fibroblasts without damaging bioenergetics. At variance with a previous report, we find MSTO1 to be localized in the cytoplasmic area with limited colocalization with mitochondria. MSTO1 interacts with the fusion machinery as a soluble factor at the cytoplasm-mitochondrial outer membrane interface. After plasma membrane permeabilization, MSTO1 is released from the cells. Thus, an MSTO1 loss-of-function mutation is associated with a human disorder showing mitochondrial involvement. MSTO1 likely has a physiologically relevant role in mitochondrial morphogenesis by supporting mitochondrial fusion.
Assuntos
Ataxia/genética , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Dinâmica Mitocondrial , Doenças Musculares/genética , Mutação , Adulto , Ataxia/patologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Doenças Musculares/patologia , Adulto JovemRESUMO
BACKGROUND: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype. METHODS: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues. RESULTS: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and ß-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy. CONCLUSION: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.
Assuntos
AMP Desaminase/genética , Hipercolesterolemia/genética , Síndrome Nefrótica/genética , Animais , HDL-Colesterol/sangue , Expressão Gênica , Estudos de Associação Genética , Hipercolesterolemia/sangue , Glomérulos Renais/patologia , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Síndrome Nefrótica/sangue , Proteinúria/sangue , Proteinúria/genéticaRESUMO
RXR signaling is predicted to have a major impact in macrophages, but neither the biological consequence nor the genomic basis of its ligand activation is known. Comprehensive genome-wide studies were carried out to map liganded RXR-mediated transcriptional changes, active binding sites, and cistromic interactions in the context of the macrophage genome architecture. The macrophage RXR cistrome has 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features, 387 liganded RXR-bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin-limited functional domains. These findings were molecularly validated using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and we show that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other, forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program.
Assuntos
Elementos Facilitadores Genéticos , Macrófagos/metabolismo , Neovascularização Fisiológica/fisiologia , Receptores X de Retinoides/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histonas/metabolismo , Ligantes , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Compostos Orgânicos/farmacologia , RNA/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Activating mutations in the KRAS gene occur frequently in human tumors, including colorectal carcinomas; most mutations occur in codons 12 and 13. Mutations in KRAS have been associated with poor response to anti-epidermal growth factor receptor antibodies. Therefore, an accurate and readily available analysis of KRAS mutational status is needed. The aim of this study was to evaluate concordance between KRAS assays performed by 6 different laboratories. METHODS: Forty formalin-fixed paraffin-embedded colorectal cancer tumor samples were obtained. Sample sections were submitted for KRAS mutation analysis to 5 independent commercial laboratories (Agencourt, Gentris, Genzyme, HistoGeneX, and Invitek) and to the Amgen DNA Sequencing Laboratory for direct polymerase chain reaction sequencing. The assay used by Invitek is no longer commercially available and has been replaced by an alternative technique. Results from the commercial services were compared with those from Amgen direct sequencing by kappa statistics. RESULTS: KRAS mutations were observed in codon 12 and/or 13 in 20 of 40 (50%) samples in Amgen direct sequencing assays. Results from HistoGeneX (kappa = 0.95), Genzyme (kappa = 0.94), and Agencourt (kappa = 0.94) were in almost perfect agreement with these results, and the results from Gentris were in substantial agreement with the results from Amgen (kappa = 0.75). The Invitek allele-specific assay demonstrated slight agreement (kappa = 0.13). CONCLUSIONS: This study provides data on the comparability of KRAS mutational analyses. The results suggest that most (but not all) commercial services provide analysis that is accurate and comparable with direct sequencing.
Assuntos
Adenocarcinoma/genética , Carcinoma/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Mutação , Proteínas Proto-Oncogênicas/genética , Kit de Reagentes para Diagnóstico , Proteínas ras/genética , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/patologia , Carcinoma/terapia , Códon , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Feminino , Fixadores , Formaldeído , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Inclusão em Parafina , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas p21(ras) , Reprodutibilidade dos Testes , Fixação de TecidosRESUMO
We conducted a genome-wide screen using the mutagen N-ethyl-N-nitrosourea to identify recessive mutations in genes that lead to altered lipid traits in mice. We screened 7,546 G3 mice that were of mixed C57BL/6J (B6) x C3.SW-H2(b)/SnJ (C3) genomes and identified three pedigrees with differences in plasma HDL-cholesterol. Genome scan analyses mapped three distinct loci to chromosomes 3, 4, and 7. An S1748L missense mutation was identified in ABCA1 in one pedigree with undetectable levels of HDL-cholesterol and resulted in reduced protein levels. This phenotype was completely penetrant, semi-dominant, and cosegregated with high plasma triglycerides. Mice in a second pedigree had very high levels of plasma total cholesterol and HDL-cholesterol (up to 800 mg/dl total cholesterol). Despite a high degree of phenotype lability and reduced penetrance, an I68N missense mutation was identified in the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). Finally, a second high HDL-cholesterol pedigree of mice, again with a highly labile phenotype and reduced penetrance, was mapped to a 7 Mb locus on chromosome 3. These results illustrate the use of a hybrid background for simultaneous screening and mapping of mutagenized pedigrees of mice and identification of three novel alleles of HDL-cholesterol phenotypes.
Assuntos
HDL-Colesterol/sangue , HDL-Colesterol/genética , Genes Recessivos , Mutação , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Colesterol/deficiência , Mapeamento Cromossômico , DNA/genética , Etilnitrosoureia/toxicidade , Feminino , Testes Genéticos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese , Mutagênicos/toxicidade , Mutação de Sentido Incorreto , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D(4) (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid.