RESUMO
BACKGROUND: A trio exome sequencing study identified a previously unreported NLRP1 gene variant resulting in a p.Leu813Pro substitution of the LRR (leucine-rich repeats) domain of the NLRP1 protein (NACHT, LRR and PYD domains-containing protein 1). This homozygous mutation was shared by two sisters with different clinical presentation: the younger sister had generalized inflammatory nodules with keratotic plugs, clinically resembling multiple keratoacanthomas, while the older had manifestations of familial keratosis lichenoides chronica. OBJECTIVES: To analyse the consequences of this NLRP1 variant in two siblings with a different clinical spectrum of severity. METHODS: To demonstrate the pathogenicity, p.Leu813Pro was recombinantly expressed, and its effect on inflammasome assembly was assessed. Exome sequencing and RNA-Seq were performed to identify factors with potentially modifying effects on the severity of the skin manifestation between each sibling. RESULTS: The variant p.Leu813Pro triggered activation of the NLRP1 inflammasome leading to ASC (apoptosis-associated speck-like protein containing a CARD) speck formation and interleukin (IL)-1ß release. The more severely affected sister had several additional genomic variants associated with atopy and psoriasis that were not present in her sibling. IL-5 and IL-17 emerged as dominant cytokines driving prominent inflammation in the skin of the severely affected sibling. CONCLUSIONS: To the best of our knowledge, this is the first report of a NLRP1 variant that leads to a different clinical spectrum of severity within the same sibship. IL-5 and IL-17 were the main cytokines expressed in the inflammatory lesions of the severely affected patient and might be regarded as disease modifying factors, and therefore may be considered as therapeutic targets.
Assuntos
Proteínas Reguladoras de Apoptose , Inflamassomos , Feminino , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Citocinas/metabolismo , Mutação com Ganho de Função , Inflamassomos/metabolismo , Interleucina-17/metabolismo , Interleucina-5/genética , Interleucina-5/metabolismo , Proteínas NLR/genética , Proteínas NLR/metabolismo , Fenótipo , IrmãosRESUMO
Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.
Assuntos
Cirrose Hepática , Proteínas Supressoras de Tumor , Adulto , Animais , Criança , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Síndrome , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genéticaRESUMO
Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.
Assuntos
COVID-19 , Proteases 3C de Coronavírus , Células Epiteliais , Inflamassomos , Proteínas NLR , SARS-CoV-2 , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Caspase 3/metabolismo , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Células Epiteliais/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Pulmão/metabolismo , Pulmão/virologia , Proteínas NLR/genética , Proteínas NLR/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidadeRESUMO
Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Inflamassomos/química , Inflamassomos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Caspase 1/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Inflamassomos/genética , Inflamação , Simulação de Acoplamento Molecular , Mutação , Proteínas NLR , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Transdução de SinaisRESUMO
Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131 This cleavage triggers N-glycine-mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cisteína Endopeptidases/metabolismo , Imunidade Inata , Inflamassomos/metabolismo , Mucosa Respiratória/virologia , Rhinovirus/enzimologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Reguladoras de Apoptose/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Glutamina/química , Glutamina/metabolismo , Glicina/química , Glicina/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-18/metabolismo , Proteínas NLR , ProteóliseRESUMO
Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Regulação da Expressão Gênica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoproteção/efeitos dos fármacos , Doxorrubicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Genes Precoces , Peróxido de Hidrogênio/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Fosforilação/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
The neuronal RNA binding protein NOVA regulates splicing, shuttles to the cytoplasm, and co-localizes with target transcripts in dendrites, suggesting links between splicing and local translation. Here we identified >200 transcripts showing NOVA-dependent changes in abundance, but, surprisingly, HITS-CLIP revealed NOVA binds these RNAs in introns rather than 3' UTRs. This led us to discover NOVA-regulated splicing of cryptic exons within these introns. These exons triggered nonsense mediated decay (NMD), as UPF1 and protein synthesis were required for NOVA's effect on RNA levels. Their regulation was dynamic and physiologically relevant. The NMD exons were regulated by seizures, which also induced changes in Nova subcellular localization and mediated large changes in synaptic proteins, including proteins implicated in familial epilepsy. Moreover, Nova haploinsufficient mice had spontaneous epilepsy. The data reveal a hidden means of dynamic RNA regulation linking electrical activity to splicing and protein output, and of mediating homeostatic excitation/inhibition balance in neurons.DOI:http://dx.doi.org/10.7554/eLife.00178.001.
Assuntos
Antígenos de Neoplasias/fisiologia , Éxons , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/fisiologia , Convulsões/metabolismo , Sinapses/metabolismo , Regiões 3' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Encéfalo/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Antígeno Neuro-Oncológico Ventral , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Frações SubcelularesRESUMO
Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4(+) cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.
Assuntos
Fraturas Ósseas/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Osteoblastos/transplante , Osteogênese Imperfeita/cirurgia , Receptores CXCR4/metabolismo , Animais , Matriz Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxia , Colágeno Tipo I/deficiência , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/imunologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/imunologia , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Fatores de Tempo , Regulação para CimaRESUMO
Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.
Assuntos
Líquido Amniótico/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ácido Valproico/farmacologia , Líquido Amniótico/citologia , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Genoma Humano , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem , Cinética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Transgenes , Inativação do Cromossomo X/efeitos dos fármacosRESUMO
The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.