RESUMEN
Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.
Asunto(s)
Amiloidosis , Apolipoproteínas A , Nefritis Intersticial , Insuficiencia Renal Crónica , Humanos , Persona de Mediana Edad , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/genética , Nefritis Intersticial/complicaciones , Mutación , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/complicacionesRESUMEN
The development of resistance to previously effective treatments has been a challenge for health care providers and a fear for patients undergoing cancer therapy. This is an unfortunately frequent occurrence for patients undergoing targeted therapy for tumours harboring the activating V600E mutation of the BRAF gene. Since the initial identification of the BRAF mutation in 2002, a series of small molecular inhibitors that target the BRAFV600E have been developed, but intrinsic and acquired resistance to these drugs has presented an ongoing challenge. More recently, improvements in therapy have been achieved by combining the use of BRAF inhibitors with other drugs, such as inhibitors of the downstream effector mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) kinase (MEK). Despite improved success in response rates and in delaying resistance using combination therapy, ultimately, the acquisition of resistance remains a concern. Recent research articles have shed light on some of the underlying mechanisms of this resistance and have proposed numerous strategies that might be employed to overcome or avoid resistance to targeted therapies. This review will explore some of the resistance mechanisms, compare what is known in melanoma cancer to colorectal cancer, and discuss strategies under development to manage the development of resistance.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Sustitución de Aminoácidos , Codón , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
BACKGROUND: Many plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants. HYPOTHESIS/PURPOSE: Here we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine. STUDY DESIGN: This work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA). RESULTS: Of the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine. CONCLUSION: The results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds.
Asunto(s)
Alcaloides/farmacología , Antioxidantes/farmacología , Apigenina/farmacología , Benzodioxoles/farmacología , NADPH Oxidasas/antagonistas & inhibidores , Piperidinas/farmacología , Extractos Vegetales/farmacología , Alcamidas Poliinsaturadas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estilbenos/farmacología , Línea Celular , Humanos , Peróxido de Hidrógeno , NADP/antagonistas & inhibidores , Oxidación-Reducción , Triterpenos Pentacíclicos , Resveratrol , Triterpenos/metabolismo , Triterpenos/farmacologíaRESUMEN
NADPH oxidase (NOX) enzymes show great potential as therapeutic pharmacological targets. This Forum revolves around the roles of specific NOX isoforms in oxidative stress-mediated pathologies, available NOX antagonists/agonists as well as the potential side effects of NOX inhibition and the requisite identification of novel oxidative biomarkers as a measure of NOX activity in patients. In addition, an original article reports the discovery of a novel small molecule NOX2 inhibitor. Finally an attractive and innovative therapeutic approach for modulating NOX activity through the inhibition of the proton channel Hv1 is discussed.
Asunto(s)
Descubrimiento de Drogas , NADPH Oxidasas/uso terapéutico , Animales , Humanos , Canales Iónicos/metabolismo , Estrés OxidativoRESUMEN
Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient's cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.
Asunto(s)
Antineoplásicos/farmacología , Embrión no Mamífero/efectos de los fármacos , Genómica/métodos , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Pez Cebra/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Células Cultivadas , Niño , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas c-akt/genética , Receptor Notch1/genética , Transducción de Señal , Trasplante Heterólogo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.
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Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Quinasas Quinasa Quinasa PAM/genética , Neoplasias Gástricas/genética , Antígenos CD , Cadherinas/genética , Análisis Mutacional de ADN , Femenino , Ligamiento Genético , Genotipo , Humanos , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Neoplasias Gástricas/patologíaRESUMEN
IMPORTANCE: Retinal detachment with avascularity of the peripheral retina, typically associated with familial exudative vitreoretinopathy (FEVR), can result from mutations in KIF11, a gene recently identified to cause microcephaly, lymphedema, and chorioretinal dysplasia (MLCRD) as well as chorioretinal dysplasia, microcephaly, and mental retardation (CDMMR). Ophthalmologists should be aware of the range of presentations for mutations in KIF11 because the phenotypic distinction between FEVR and MLCRD/CDMMR portends management implications in patients with these conditions. OBJECTIVE: To identify gene mutations in patients who present with a FEVR phenotype and explore the spectrum of ocular and systemic abnormalities caused by KIF11 mutations in a cohort of patients with FEVR or microcephaly in conjunction with chorioretinopathy or FEVR. DESIGN, SETTING, AND PARTICIPANTS: Clinical data and DNA were collected from each participant between 1998 and 2013 from the clinical practices of ophthalmologists and clinical geneticists internationally. Twenty-eight FEVR probands with diagnoses made by the referring physician and without a known FEVR gene mutation, and 3 with microcephaly and chorioretinopathy, were included. At least 1 patient in each pedigree manifested 1 or more of the following: macular dragging, partial retinal detachment, falciform folds, or total retinal detachment. EXPOSURES: Whole-exome sequencing was conducted on affected members in multiplex pedigrees, and Sanger sequencing of the 22 exons of the KIF11 gene was performed on singletons. Clinical data and history were collected and reviewed. MAIN OUTCOMES AND MEASURES: Identification of mutations in KIF11. RESULTS: Four novel heterozygous KIF11 mutations and 1 previously published mutation were identified in probands with FEVR: p.A218Gfs*15, p.E470X, p.R221G, c.790-1G>T, and the previously described heterozygous p.R47X. Documentation of peripheral avascular areas on intravenous fluorescein angiography was possible in 2 probands with fibrovascular proliferation demonstrating phenotypic overlap with FEVR. CONCLUSIONS AND RELEVANCE: Mutations in KIF11 cause a broader spectrum of ocular disease than previously reported, including retinal detachment. The KIF11 gene likely plays a role in retinal vascular development and mutations in this gene can lead to clinical overlap with FEVR. Cases of FEVR should be carefully inspected for the presence of microcephaly as a marker for KIF11-related disease to enhance the accuracy of the prognosis and genetic counseling.
Asunto(s)
Cinesinas/genética , Linfedema/genética , Microcefalia/genética , Mutación , Displasia Retiniana/genética , Secuencia de Bases , Niño , Preescolar , Análisis Mutacional de ADN , Electrofisiología , Exones/genética , Enfermedades Hereditarias del Ojo , Facies , Vitreorretinopatías Exudativas Familiares , Femenino , Angiografía con Fluoresceína , Humanos , Linfedema/diagnóstico , Masculino , Microcefalia/diagnóstico , Datos de Secuencia Molecular , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Enfermedades de la Retina/diagnóstico , Enfermedades de la Retina/genética , Displasia Retiniana/diagnósticoRESUMEN
Pediatric intracranial calcification may be caused by inherited or acquired factors. We describe the identification of a novel rearrangement in which a downstream pseudogene translocates into exon 9 of OCLN, resulting in band-like brain calcification and advanced chronic kidney disease in early childhood. SNP genotyping and read-depth variation from whole exome sequencing initially pointed to a mutation in the OCLN gene. The high degree of identity between OCLN and two pseudogenes required a combination of multiplex ligation-dependent probe amplification, PCR, and Sanger sequencing to identify the genomic rearrangement that was the underlying genetic cause of the disease. Mutations in exon 3, or at the 5-6 intron splice site, of OCLN have been reported to cause brain calcification and polymicrogyria with no evidence of extra-cranial phenotypes. Of the OCLN splice variants described, all make use of exon 9, while OCLN variants that use exons 3, 5, and 6 are tissue specific. The genetic rearrangement we identified in exon 9 provides a plausible explanation for the expanded clinical phenotype observed in our individuals. Furthermore, the lack of polymicrogyria associated with the rearrangement of OCLN in our patients extends the range of cranial defects that can be observed due to OCLN mutations.
Asunto(s)
Encéfalo/fisiopatología , Calcinosis/fisiopatología , Reordenamiento Génico , Riñón/fisiopatología , Ocludina/genética , Canadá , Preescolar , Mapeo Cromosómico , Variaciones en el Número de Copia de ADN , Exoma , Exones , Femenino , Eliminación de Gen , Genotipo , Homocigoto , Humanos , Intrones , Malformaciones del Desarrollo Cortical/genética , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Ocludina/metabolismo , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Empalme del ARN , Análisis de Secuencia de ADNRESUMEN
Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.
Asunto(s)
Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/enzimología , Degeneración Nerviosa/patología , Sistema Nervioso Periférico/patología , Ubiquitina-Proteína Ligasas/metabolismo , Acrilamida/farmacología , Animales , Axones/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Línea Celular , Enfermedad de Charcot-Marie-Tooth/complicaciones , Enfermedad de Charcot-Marie-Tooth/patología , Modelos Animales de Enfermedad , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Mutantes , Mutagénesis/genética , Mutación/genética , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/enzimología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/enzimología , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , TransfecciónRESUMEN
BACKGROUND AND AIM: Our objective was to identify the molecular genetic basis of an Alagille-like condition not linked to JAG1 or NOTCH2 in two related sibships. METHODS: Because of common ancestry, and an autosomal recessive mode of inheritance, it was hypothesized that all affected and no unaffected individuals would be homozygous for the same haplotype in the region of the causative gene. Single nucleotide polymorphism arrays were therefore used to genotype 3 affected individuals from two sibships, their mothers and four unaffected siblings, to identify regions of homozygosity. Genes within the largest regions were prioritized and sequenced for mutations. Mutant RNA transcripts were also sequenced. RESULTS: A novel splice acceptor site mutation in the ATP8B1 gene was identified (a G-C preceding exon 16 resulting in a 4 bp deletion and frameshift from the 5' end of exon 16). This result was unexpected because ATP8B1 mutations are associated with progressive familial intrahepatic cholestasis type 1 (PFIC1). Intrahepatic bile duct paucity, cardiac anomalies, renal tubular acidosis and hypothyroidism led to an initial diagnosis of Alagille syndrome. However, in retrospect, abnormal sweat chloride, normal gamma-glutamyl transferase, normal to low cholesterol, and an autosomal recessive mode of inheritance were consistent with PFIC1. Renal tubular acidosis, hypothyroidism and cardiac anomalies have not previously been associated with PFIC1. CONCLUSION: This work expands the phenotypic spectrum of PFIC1, and highlights the overlap in clinical phenotype between Alagille syndrome and PFIC1. Knowledge of the causative mutation allows for carrier testing and prenatal diagnosis in this community.
Asunto(s)
Adenosina Trifosfatasas/genética , Colestasis Intrahepática/genética , Mutación del Sistema de Lectura , Sitios de Empalme de ARN/genética , Niño , Colestasis Intrahepática/diagnóstico , Femenino , Marcadores Genéticos , Genotipo , Técnicas de Genotipaje , Humanos , Masculino , Fenotipo , Polimorfismo de Nucleótido Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , HermanosRESUMEN
Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H(2)O(2) release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H(2)O(2) release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (nâ=â279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10-8) and 54 suggestive associations (p<1.00×10-5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and â¼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H(2)O(2) release was observed in Down Syndrome (DS) individuals (p<2.88×10-12). Taken together, our results show strong evidence of genetic control of H(2)O(2) in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.
Asunto(s)
Peróxido de Hidrógeno/química , Adulto , Anciano , Línea Celular Tumoral , Estudios de Cohortes , Ligamiento Genético , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Peróxido de Hidrógeno/metabolismo , Recién Nacido , Persona de Mediana Edad , Modelos Genéticos , NADPH Oxidasas/metabolismo , Fenotipo , Polimorfismo de Nucleótido Simple , Especies Reactivas de Oxígeno , Análisis de Secuencia de ADNRESUMEN
NOX enzymes are major ROS generators in pathological states and represent a promising pharmacological target. In this issue of Chemistry & Biology, Smith and colleagues developed an original and elegant cell-free assay to identify specific Nox2 inhibitors in a high throughput manner.
RESUMEN
In mammals, the NADPH oxidase family of enzymes comprises seven members: NOXs 1-5, DUOX1, and DUOX2. All of these enzymes function to move an electron across cellular membranes, transferring it to oxygen to generate the superoxide anion. This generation of reactive oxygen species has important physiological and pathophysiological roles. NOX5 is perhaps the least well understood of these NOX isoforms, in part because the gene is not present in mice or rats. In recent years, however, there has been a rapid increase in our understanding of the NOX5 gene, the structural and biochemical aspects of the NOX5 enzyme, the role NOX5 plays in health and disease, and the development of novel NOX inhibitors. This review takes a look back at some historical aspects of the discovery of NOX5 and summarizes our current understanding of the enzyme.
Asunto(s)
Proteínas de la Membrana/fisiología , NADPH Oxidasas/fisiología , Secuencia de Aminoácidos , Animales , Evolución Molecular , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , NADPH Oxidasa 5 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/química , NADPH Oxidasas/genética , Especificidad de Órganos , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , Superóxidos/metabolismoRESUMEN
BACKGROUND: Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH: Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS: Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.
Asunto(s)
NADPH Oxidasas/antagonistas & inhibidores , Triterpenos/farmacología , Animales , Células CHO , Línea Celular , Línea Celular Transformada , Cricetinae , Citosol/efectos de los fármacos , Citosol/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Compuestos Onio/farmacología , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Triterpenos Pentacíclicos , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Dominios Homologos src/efectos de los fármacosRESUMEN
Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/enzimología , Mutagénesis Insercional , Mutación , Ubiquitina-Proteína Ligasas/genética , Secuencia de Bases , Canadá , Enfermedad de Charcot-Marie-Tooth/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo de Nucleótido Simple , Sitios de Empalme de ARN , Empalme del ARN , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx(-/-)) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx(-/-) mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.
Asunto(s)
Calnexina/genética , Calnexina/fisiología , Enfermedades Desmielinizantes/metabolismo , Vaina de Mielina/metabolismo , Animales , Animales Recién Nacidos , Calnexina/metabolismo , Membrana Celular/metabolismo , Electrofisiología/métodos , Retículo Endoplásmico/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pliegue de Proteína , Nervio Ciático/metabolismo , Nervio Ciático/ultraestructura , Médula Espinal/metabolismo , Médula Espinal/ultraestructuraRESUMEN
Autosomal-recessive cutis laxa type 2 (ARCL2) is a multisystem disorder characterized by the appearance of premature aging, wrinkled and lax skin, joint laxity, and a general developmental delay. Cutis laxa includes a family of clinically overlapping conditions with confusing nomenclature, generally requiring molecular analyses for definitive diagnosis. Six genes are currently known to mutate to yield one of these related conditions. We ascertained a cohort of typical ARCL2 patients from a subpopulation isolate within eastern Canada. Homozygosity mapping with high-density SNP genotyping excluded all six known genes, and instead identified a single homozygous region near the telomere of chromosome 17, shared identically by state by all genotyped affected individuals from the families. A putative pathogenic variant was identified by direct DNA sequencing of genes within the region. The single nucleotide change leads to a missense mutation adjacent to a splice junction in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). Bioinformatic analysis predicted a pathogenic effect of the variant on splice donor site function. Skipping of the associated exon was confirmed in RNA from blood lymphocytes of affected homozygotes and heterozygous mutation carriers. Exon skipping leads to deletion of the reductase functional domain-coding region and an obligatory downstream frameshift. PYCR1 plays a critical role in proline biosynthesis. Pathogenicity of the genetic variant in PYCR1 is likely, given that a similar clinical phenotype has been documented for mutation carriers of another proline biosynthetic enzyme, pyrroline-5-carboxylate synthase. Our results support a significant role for proline in normal development.
Asunto(s)
Cutis Laxo/genética , Pirrolina Carboxilato Reductasas/genética , Secuencia de Aminoácidos , Canadá , Niño , Cutis Laxo/patología , Análisis Mutacional de ADN , Femenino , Genes Recesivos , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Prolina/biosíntesis , Pirrolina Carboxilato Reductasas/química , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation.
Asunto(s)
Haplotipos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos B , Secuencia de Bases , Línea Celular Transformada , Humanos , NADPH Oxidasa 2 , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , ARN MensajeroRESUMEN
The superoxide-generating NADPH oxidase NOX1 is thought to be involved in signaling by the angiotensin II-receptor AT1R. However, underlying signaling steps are poorly understood. In this study, we investigated the effect of AngII on aortic smooth muscle from wild-type and NOX1-deficient mice. NOX1-deficient cells showed decreased basal ROS generation and did not produce ROS in response to AngII. Unexpectedly, AngII-dependent Ca(2+) signaling was markedly decreased in NOX1-deficient cells. Immunostaining demonstrated that AT1R was localized on the plasma membrane in wild-type, but intracellularly in NOX1-deficient cells. Immunohistochemistry and immunoblotting showed a decreased expression of AT1R in the aorta of NOX1-deficient mice. To investigate the basis of the abnormal AT1R targeting, we studied caveolin expression and phosphorylation. The amounts of total caveolin and of caveolae were not different in NOX1-deficient mice, but a marked decrease occurred in the phosphorylated form of caveolin. Exogenous H(2)O(2) or transfection of a NOX1 plasmid restored AngII responses in NOX1-deficient cells. Based on these findings, we propose that NOX1-derived reactive oxygen species regulate cell-surface expression of AT1R through mechanisms including caveolin phosphorylation. The lack cell-surface AT1R expression in smooth muscle could be involved in the decreased blood pressure in NOX1-deficient mice.