RESUMO
BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder resulting from pathogenic variants in three distinct genes, with most of the variants occurring in the electron transfer flavoprotein-ubiquinone oxidoreductase gene (ETFDH). Recent evidence of potential founder variants for MADD in the South African (SA) population, initiated this extensive investigation. As part of the International Centre for Genomic Medicine in Neuromuscular Diseases study, we recruited a cohort of patients diagnosed with MADD from academic medical centres across SA over a three-year period. The aim was to extensively profile the clinical, biochemical, and genomic characteristics of MADD in this understudied population. METHODS: Clinical evaluations and whole exome sequencing were conducted on each patient. Metabolic profiling was performed before and after treatment, where possible. The recessive inheritance and phase of the variants were established via segregation analyses using Sanger sequencing. Lastly, the haplotype and allele frequencies were determined for the two main variants in the four largest SA populations. RESULTS: Twelve unrelated families (ten of White SA and two of mixed ethnicity) with clinically heterogeneous presentations in 14 affected individuals were observed, and five pathogenic ETFDH variants were identified. Based on disease severity and treatment response, three distinct groups emerged. The most severe and fatal presentations were associated with the homozygous c.[1067G > A];c.[1067G > A] and compound heterozygous c.[976G > C];c.[1067G > A] genotypes, causing MADD types I and I/II, respectively. These, along with three less severe compound heterozygous genotypes (c.[1067G > A];c.[1448C > T], c.[740G > T];c.[1448C > T], and c.[287dupA*];c.[1448C > T]), resulting in MADD types II/III, presented before the age of five years, depending on the time and maintenance of intervention. By contrast, the homozygous c.[1448C > T];c.[1448C > T] genotype, which causes MADD type III, presented later in life. Except for the type I, I/II and II cases, urinary metabolic markers for MADD improved/normalised following treatment with riboflavin and L-carnitine. Furthermore, genetic analyses of the most frequent variants (c.[1067G > A] and c.[1448C > T]) revealed a shared haplotype in the region of ETFDH, with SA population-specific allele frequencies of < 0.00067-0.00084%. CONCLUSIONS: This study reveals the first extensive genotype-phenotype profile of a MADD patient cohort from the diverse and understudied SA population. The pathogenic variants and associated variable phenotypes were characterised, which will enable early screening, genetic counselling, and patient-specific treatment of MADD in this population.
Assuntos
Deficiência Múltipla de Acil Coenzima A Desidrogenase , Humanos , Pré-Escolar , Deficiência Múltipla de Acil Coenzima A Desidrogenase/diagnóstico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/tratamento farmacológico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Mutação/genética , África do Sul , Genótipo , Riboflavina/uso terapêutico , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/uso terapêutico , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismoRESUMO
MAPK-activating death domain protein (MADD) deficiency is associated with a broad clinical spectrum ranging from mild developmental impairment to fatal multisystem disorder. We report an additional case of severe form with some overlapping and unreported systemic features in a growth-restricted full-term male newborn. The novel findings include corpus callosum agenesis, bilateral adrenal agenesis, scrotal aplasia, and abnormal skin pigmentation. Microscopic changes are only remarkable in thyroid gland that shows decreased, variously sized follicles with absent or non-vacuolated pale colloid. This unique constellation of birth defects is associated with a novel homozygous in-frame MADD gene deletion (NM_003682.4: c.4853_4855delGCT:p.Cys1618del). This case report expands the phenotypic and genetic spectrum of MADD deficiency.
Assuntos
Agenesia do Corpo Caloso , Fatores de Troca do Nucleotídeo Guanina , Recém-Nascido , Humanos , Masculino , Domínio de Morte , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genéticaRESUMO
The PIDDosome is a multiprotein complex that includes p53-induced protein with a death domain 1 (PIDD1), receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD), and caspase-2, the activation of which is driven by PIDDosome assembly. In addition to the key role of the PIDDosome in the regulation of cell differentiation, tissue homeostasis, and organogenesis and regeneration, caspase-2, RAIDD and PIDD1 engagement in neuronal development was shown. Here, we focus on the involvement of PIDDosome components in neurodegenerative disorders, including retinal neuropathies, different types of brain damage, and Alzheimer's disease (AD), Huntington's disease (HD), and Lewy body disease. We also discuss pathogenic variants of PIDD1, RAIDD, and caspase-2 that are associated with intellectual, behavioral, and psychological abnormalities, together with prospective PIDDosome inhibition strategies and their potential clinical application.
Assuntos
Proteína Adaptadora de Sinalização CRADD , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Humanos , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/genética , Caspase 2/metabolismo , Estudos Prospectivos , Apoptose/fisiologiaRESUMO
PIDDosome formation followed by caspase-2 activation is critical for genotoxic stress-induced apoptotic cell death. Failure of proper caspase-2 activation causes a neurodevelopmental disorder and intellectual disability. R815W, R862W, and Q863stop mutations in p53-induced protein with a death domain (PIDD), a component of the PIDDosome, also lead to this disorder. However, the molecular mechanisms underlying this pathogenesis remain elusive. In this study, we analyzed the molecular mechanisms underlying the pathogenesis of the PIDD DD pathogenic variants R815W, R862W, and Q863stop. We determined that these mutations prevented the interaction between PIDD and RIP-associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD), a molecule that mediates PIDDosome formation. The disruption of this interaction affects PIDDosome formation and caspase-2 activation.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Transtornos do Neurodesenvolvimento , Humanos , Apoptose/genética , Caspase 2/genética , Caspase 2/metabolismo , Proteína Adaptadora de Sinalização CRADD/genética , Proteína Adaptadora de Sinalização CRADD/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Transtornos do Neurodesenvolvimento/genéticaRESUMO
OBJECTIVE: Multiple genetic variants have been studied for years to try to find an association with polycystic ovary syndrome (PCOS). This meta-analysis will investigate if there are associations between increased risk of PCOS and rs6165 polymorphism in follicle stimulating hormone receptor (FSHR) gene and rs2479106 polymorphism in differentially expressed in Differentially Expressed in Normal and Neoplastic Development Isoform 1A (DENND1A) gene. METHODS: Studies were identified from PubMed library, and case-control studies with correct polymorphisms and available genotype frequencies were included. The statistical analysis is done in Review Manager 5.3, and odds ratio (OR) with corresponding 95% confidence interval (CI) was calculated to see if any association with PCOS exists. RESULTS: In the study of FSHR gene, eight articles with 1539 cases and 1877 controls were included. No relations were found between PCOS and rs6165 polymorphism in neither the allelic (OR=1.07, 95% CI=0.97-1.19, p=0.18), recessive (OR=1.21, 95% CI=0.98-1.50, p=0.07) nor the dominant (OR=1.05, 95% CI=0.91-1.20, p=0.53) model. The rs2479106 polymorphism in DENND1A gene included 10 studies with 3627 cases and 20325 controls. Only the Asian subgroup in the recessive model (OR=1.84, 95% CI=1.19-2.85, p=0.006) showed a positive relation with PCOS, while associations were not found within the overall results in the allelic (OR=1.09, 95% CI=0.98-1.21, p=0.10), recessive (OR=1.26, 95% CI=0.73-2.19, p=0.41) or the dominant (OR=1.31, 95% CI=1.00-1.71, p=0.05) model. CONCLUSIONS: This meta-analysis suggests that rs2479106 polymorphism in DENND1A gene is associated with increased risk of PCOS in the Asian population. No relations were found with increased risk of PCOS and rs6165 polymorphism in FSHR gene.
Assuntos
Síndrome do Ovário Policístico , Feminino , Humanos , Síndrome do Ovário Policístico/epidemiologia , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único/genética , Predisposição Genética para Doença/genética , Receptores do FSH/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Estudos de Casos e ControlesRESUMO
Polycystic ovarian syndrome (PCOS) is a hormonal disorder that causes enlargement of ovaries and follicular maturation arrest, which lacks efficient treatment. N2, a semi-natural triterpenoid from the neem family, was already reported to have antioxidant and antiinflammatory properties in our previous report. This study investigated the anti-androgenic property of N2 on testosterone-induced oxidative stress in Chinese Hamster Ovarian cells (CHO) and PCOS zebrafish model. The testosterone exposure disrupted the antioxidant enzymes and ROS level and enhanced the apoptosis in both CHO cells and PCOS zebrafish. However, N2 significantly protected the CHO cells from ROS and apoptosis. N2 improved the Gonado somatic index (GSI) and upregulated the expression of the SOD enzyme in zebrafish ovaries. Moreover, the testosterone-induced follicular maturation arrest was normalized by N2 treatment in histopathology studies. In addition, the gene expression studies of Tox3 and Denndla in zebrafish demonstrated that N2 could impair PCOS condition. Furthermore, to confirm the N2 activity, the in-silico studies were performed against PCOS susceptible genes Tox3 and Dennd1a using molecular docking and molecular dynamic simulations. The results suggested that N2 alleviated the oxidative stress and apoptosis in-vitro and in-vivo and altered the expression of PCOS key genes.
Assuntos
Síndrome do Ovário Policístico , Feminino , Humanos , Animais , Cricetinae , Síndrome do Ovário Policístico/patologia , Cricetulus , Peixe-Zebra/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Células CHO , Simulação de Acoplamento Molecular , Transdução de Sinais , Testosterona , Estresse Oxidativo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismoRESUMO
Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an inborn metabolic disorder that affects fatty acid oxidation and the catabolism of branched-chain amino acids, vitamins B and energy metabolism. In this study, the induced pluripotent stem cell (iPSC) line LZUSHi002-A from PBMCs of a 10-year-old male patient with ETFDH mutations using the episomal plasmids was established, which is an ideal in vitro model to understand the exact pathogenesis of MADD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Proteínas Ferro-Enxofre , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Masculino , Humanos , Criança , Células-Tronco Pluripotentes Induzidas/metabolismo , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Riboflavina/genética , Riboflavina/metabolismo , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Mutação/genética , Ácidos Graxos/metabolismo , Vitaminas , Aminoácidos de Cadeia Ramificada/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismoRESUMO
The death fold domain-containing protein PIDD1 has recently attracted renewed attention as a regulator of the orphan cell death-related protease, Caspase-2. Caspase-2 can activate p53 to promote cell cycle arrest in response to centrosome aberrations, and its activation requires formation of the PIDDosome multi-protein complex containing multimers of PIDD1 and the adapter RAIDD/CRADD at its core. However, PIDD1 appears to be able to engage with multiple client proteins to promote an even broader range of biological responses, such as NF-κB activation, translesion DNA synthesis or cell death. PIDD1 shows features of inteins, a class of self-cleaving proteins, to create different polypeptides from a common precursor protein that allow it to serve these diverse functions. This review summarizes structural information and molecular features as well as recent experimental advances that highlight the potential pathophysiological roles of this unique death fold protein to highlight its drug-target potential.
Assuntos
Proteína Adaptadora de Sinalização CRADD , Caspase 2 , Apoptose/fisiologia , Proteína Adaptadora de Sinalização CRADD/genética , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/genética , Caspase 2/metabolismo , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular , Morte Celular , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Humanos , InflamaçãoRESUMO
To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy.
Assuntos
Proteínas Ferro-Enxofre , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Acil Coenzima A/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Seguimentos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteínas Ferro-Enxofre/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/diagnóstico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/tratamento farmacológico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Debilidade Muscular/patologia , Músculo Esquelético/metabolismo , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Estudos Retrospectivos , Riboflavina/genética , Riboflavina/uso terapêuticoRESUMO
Disordered inflammation and apoptosis are closely related to diseases, and inflammation can also promote cell apoptosis, where growing evidence has shown that circular RNAs (circRNAs) play important roles. Lipopolysaccharide (LPS) is the main component of the cytoderm of gram-negative bacterium, which can cause inflammatory responses in macrophages. We constructed an inflammatory model by exposing chicken macrophage cell lines (also known as HD11) to LPS for in vitro experiments. In this study, we validated a novel circRNA-circNFIC-which was dramatically up-regulated in tissues infected by coccidia and cells exposed to LPS. Besides, circNFIC could significantly promote the expression levels of pro-inflammation factors, including (IL-1ß, TNFα, and IFNγ) and pro-apoptosis maker genes (caspase 3 and caspase 8) in HD11 exposed to LPS or not. In terms of mechanism, circNFIC exerted notable effects on DENND1B to regulate cell inflammation and apoptosis by sponging miR-30e-3p. The molecular functions played by miR-30e-3p and DENND1B have been explored, respectively. In addition, the effects of circNFIC knockdown suppressing the expression of pro-inflammatory and pro-apoptosis functions could be reversed by a miR-30e-3p inhibitor. On the whole, circNFIC promoted cell inflammation and apoptosis via the miR-30e-3p/DENND1B axis.
Assuntos
Apoptose , Proteínas Aviárias/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , MicroRNAs/metabolismo , RNA Circular/genética , Animais , Proteínas Aviárias/genética , Linhagem Celular , Galinhas , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , MicroRNAs/genética , Fatores de Transcrição NFI/genética , RNA Circular/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Several monogenic causes for isolated dystonia have been identified, but they collectively account for only a small proportion of cases. Two genome-wide association studies have reported a few potential dystonia risk loci; but conclusions have been limited by small sample sizes, partial coverage of genetic variants, or poor reproducibility. OBJECTIVE: To identify robust genetic variants and loci in a large multicenter cervical dystonia cohort using a genome-wide approach. METHODS: We performed a genome-wide association study using cervical dystonia samples from the Dystonia Coalition. Logistic and linear regressions, including age, sex, and population structure as covariates, were employed to assess variant- and gene-based genetic associations with disease status and age at onset. We also performed a replication study for an identified genome-wide significant signal. RESULTS: After quality control, 919 cervical dystonia patients compared with 1491 controls of European ancestry were included in the analyses. We identified one genome-wide significant variant (rs2219975, chromosome 3, upstream of COL8A1, P-value 3.04 × 10-8 ). The association was not replicated in a newly genotyped sample of 473 cervical dystonia cases and 481 controls. Gene-based analysis identified DENND1A to be significantly associated with cervical dystonia (P-value 1.23 × 10-6 ). One low-frequency variant was associated with lower age-at-onset (16.4 ± 2.9 years, P-value = 3.07 × 10-8 , minor allele frequency = 0.01), located within the GABBR2 gene on chromosome 9 (rs147331823). CONCLUSION: The genetic underpinnings of cervical dystonia are complex and likely consist of multiple distinct variants of small effect sizes. Larger sample sizes may be needed to provide sufficient statistical power to address the presumably multi-genic etiology of cervical dystonia. © 2021 International Parkinson and Movement Disorder Society.
Assuntos
Estudo de Associação Genômica Ampla , Torcicolo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Frequência do Gene , Predisposição Genética para Doença/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos Testes , Torcicolo/genéticaRESUMO
The PIDDosome is a multiprotein complex, composed by the p53-induced death domain protein 1 (PIDD1), the bipartite linker protein CRADD (also known as RAIDD) and the proform of caspase-2 that induces apoptosis in response to DNA damage. In the recent years, biallelic pathogenic variants in CRADD have been associated with a neurodevelopmental disorder (MRT34; MIM 614499) characterized by pachygyria with a predominant anterior gradient, megalencephaly, epilepsy and intellectual disability. More recently, biallelic pathogenic variants in PIDD1 have been described in a few families with apparently nonsydnromic intellectual disability. Here, we aim to delineate the genetic and radio-clinical features of PIDD1-related disorder. Exome sequencing was carried out in six consanguineous families. Thorough clinical and neuroradiological evaluation was performed for all the affected individuals as well as reviewing all the data from previously reported cases. We identified five distinct novel homozygous variants (c.2584C>T p.(Arg862Trp), c.1340G>A p.(Trp447*), c.2116_2120del p.(Val706Hisfs*30), c.1564_1565delCA p.(Gln522fs*44), and c.1804_1805del p.(Gly602fs*26) in eleven subjects displaying intellectual disability, behaviorial and psychiatric features, and a typical anterior-predominant pachygyria, remarkably resembling the CRADD-related neuroimaging pattern. In summary, we outlin`e the phenotypic and molecular spectrum of PIDD1 biallelic variants supporting the evidence that the PIDD1/CRADD/caspase-2 signaling is crucial for normal gyration of the developing human neocortex as well as cognition and behavior.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Lisencefalia/genética , Adolescente , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Feminino , Genes Recessivos , Humanos , Deficiência Intelectual/patologia , Lisencefalia/patologia , Masculino , Mutação , Linhagem , SíndromeRESUMO
Rab proteins coordinate inter-organellar vesicle-mediated transport, facilitating intracellular communication, protein recycling, and signaling processes. Dysfunction of Rab proteins or their direct interactors leads to a wide range of diseases with diverse manifestations. We describe seven individuals from four consanguineous Arab Muslim families with an infantile-lethal syndrome, including failure to thrive (FTT), chronic diarrhea, neonatal respiratory distress, variable pituitary dysfunction, and distal arthrogryposis. Exome sequencing analysis in the independent families, followed by an internal gene-matching process using a local exome database, identified a homozygous splice-site variant in MADD (c.2816 + 1 G > A) on a common haplotype. The variant segregated with the disease in all available family members. Determination of cDNA sequence verified single exon skipping, resulting in an out-of-frame deletion. MADD encodes a Rab guanine nucleotide exchange factor (GEF), which activates RAB3 and RAB27A/27B and is thus a crucial regulator of neuromuscular junctions and endocrine secretory granule release. Moreover, MADD protects cells from caspase-mediated TNF-α-induced apoptosis. The combined roles of MADD and its downstream effectors correlate with the phenotypic spectrum of disease, and call for additional studies to confirm the pathogenic mechanism and to investigate possible therapeutic avenues through modulation of TNF-α signaling.
Assuntos
Artrogripose/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Insuficiência de Crescimento/genética , Pleiotropia Genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Artrogripose/patologia , Consanguinidade , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Insuficiência de Crescimento/patologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Lactente , Masculino , Linhagem , Síndrome do Desconforto Respiratório do Recém-Nascido/patologia , SíndromeRESUMO
Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Proteína de Domínio de Morte Associada a Fas/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Domínio Catalítico , Clonagem Molecular , Microscopia Crioeletrônica , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Morte Celular Regulada/genética , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PIDD1 encodes p53-Induced Death Domain protein 1, which acts as a sensor surveilling centrosome numbers and p53 activity in mammalian cells. Early results also suggest a role in DNA damage response where PIDD1 may act as a cell-fate switch, through interaction with RIP1 and NEMO/IKKg, activating NF-κB signaling for survival, or as an apoptosis-inducing protein by activating caspase-2. Biallelic truncating mutations in CRADD-the protein bridging PIDD1 and caspase-2-have been reported in intellectual disability (ID), and in a form of lissencephaly. Here, we identified five families with ID from Iran, Pakistan, and India, with four different biallelic mutations in PIDD1, all disrupting the Death Domain (DD), through which PIDD1 interacts with CRADD or RIP1. Nonsense mutations Gln863* and Arg637* directly disrupt the DD, as does a missense mutation, Arg815Trp. A homozygous splice mutation in the fifth family is predicted to disrupt splicing upstream of the DD, as confirmed using an exon trap. In HEK293 cells, we show that both Gln863* and Arg815Trp mutants fail to co-localize with CRADD, leading to its aggregation and mis-localization, and fail to co-precipitate CRADD. Using genome-edited cell lines, we show that these three PIDD1 mutations all cause loss of PIDDosome function. Pidd1 null mice show decreased anxiety, but no motor abnormalities. Together this indicates that PIDD1 mutations in humans may cause ID (and possibly lissencephaly) either through gain of function or secondarily, due to altered scaffolding properties, while complete loss of PIDD1, as modeled in mice, may be well tolerated or is compensated for.
Assuntos
Proteína Adaptadora de Sinalização CRADD , Deficiência Intelectual , Animais , Proteína Adaptadora de Sinalização CRADD/genética , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/genética , Caspase 2/metabolismo , Domínio de Morte , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Células HEK293 , Humanos , Índia , Deficiência Intelectual/genética , Camundongos , MutaçãoRESUMO
Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.
Assuntos
Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/metabolismo , Centrossomo/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Proteína Adaptadora de Sinalização CRADD/genética , Caspase 2/genética , Diferenciação Celular , Cisteína Endopeptidases/genética , Dano ao DNA , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Centriole copy number is tightly maintained by the once-per-cycle duplication of these organelles. Centrioles constitute the core of centrosomes, which organize the microtubule cytoskeleton and form the poles of the mitotic spindle. Centrosome amplification is frequently observed in tumors, where it promotes aneuploidy and contributes to invasive phenotypes. In non-transformed cells, centrosome amplification triggers PIDDosome activation as a protective response to inhibit cell proliferation, but how extra centrosomes activate the PIDDosome remains unclear. Using a genome-wide screen, we identify centriole distal appendages as critical for PIDDosome activation in cells with extra centrosomes. The distal appendage protein ANKRD26 is found to interact with and recruit the PIDDosome component PIDD1 to centriole distal appendages, and this interaction is required for PIDDosome activation following centrosome amplification. Furthermore, a recurrent ANKRD26 mutation found in human tumors disrupts PIDD1 localization and PIDDosome activation in cells with extra centrosomes. Our data support a model in which ANKRD26 initiates a centriole-derived signal to limit cell proliferation in response to centrosome amplification.
Assuntos
Caspase 2/metabolismo , Centrossomo/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Caspase 2/genética , Ciclo Celular , Diferenciação Celular , Cisteína Endopeptidases/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Deficiências do Desenvolvimento/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Doenças do Sistema Nervoso/genética , Humanos , Mutação , Fenótipo , Transporte Proteico/genética , Transdução de Sinais/genéticaRESUMO
The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.
Assuntos
Processamento Alternativo , Relógios Circadianos/genética , Exocitose , Glucose/metabolismo , Secreção de Insulina/genética , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/fisiologia , Células Cultivadas , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/fisiologia , Obesidade/genética , Obesidade/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Polycystic ovary syndrome is characterized by reproductive and metabolic disturbances throughout the female lifespan. Therefore, this study aimed to determine whether genome-wide association studies (GWAS)-identified risk variants for PCOS could confer risk of metabolic syndrome (MS) or insulin resistance (IR). Fifteen independent SNPs mapping to 11 GWAS loci genotyped in a total of 2,082 Han Chinese women independent of previous GWAS and phenotype-genotype correlations were assessed. The CC group for rs12478601 in THADA was associated with decreased rate of MS after adjustment for age (23.2 vs. 27%, P = 0.042, OR = 0.81). Using a dominant model, the GG+AG group for rs2059807 in INSR was associated with increased risk of MS after adjustment for age (26.8 vs. 22.5%, P = 0.023, OR = 1.27). The GG + GT group for rs4784165 in TOX3 was found to be associated with an increased rate of IR after adjustment for age and BMI(53.3 vs. 48.5%, P = 0.027, OR = 1.27). The GG+AG group for rs2479106 in DENND1A was associated with a decreased rate of IR (48.3 vs. 53.6%, adjusted P = 0.039, OR = 0.80). After exclusion of PCOS cases with a family history of diabetes, hypertension, or dyslipidemia, the phenotype-genotype correlations between the genes INSR and TOX3 and MS or IR were still significant (P < 0.05). Three SNPs (rs13429458 in THADA, rs10818854 in DENND1A, and rs2059807 in INSR) were significantly associated with IR; however, their association was not significant after adjustment for age and BMI. This genotype-phenotype study thus provides clues that THADA, INSR, TOX3, and DENND1A play a role in PCOS possibly through a metabolic disorder-related pathway.