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1.
iScience ; 25(10): 105066, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36093378

RESUMO

Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.

2.
Science ; 376(6599): eabm6380, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35587511

RESUMO

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.


Assuntos
Toxinas Bacterianas , Síndrome de Cri-du-Chat , Endopeptidases , Haploinsuficiência , Proteínas Hemolisinas , Infecções Estafilocócicas , Staphylococcus aureus , Toxinas Bacterianas/imunologia , Síndrome de Cri-du-Chat/genética , Síndrome de Cri-du-Chat/imunologia , Endopeptidases/genética , Haploinsuficiência/genética , Haploinsuficiência/imunologia , Proteínas Hemolisinas/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Celular/genética , Necrose , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/patologia
3.
Cancers (Basel) ; 14(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35158811

RESUMO

Human Herpesvirus 8 (HHV-8) is associated with three main severe orphan malignancies, Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL), which present few therapeutic options. We identified the antimalarial primaquine diphosphate (PQ) as a promising therapeutic candidate for HHV-8-associated PEL and KS. Indeed, PQ strongly reduced cell viability through caspase-dependent apoptosis, specifically in HHV-8-infected PEL cells. Reactive oxygen species (ROS)- and endoplasmic reticulum (ER) stress-mediated apoptosis signaling pathways were found to be part of the in vitro cytotoxic effect of PQ. Moreover, PQ treatment had a clinically positive effect in a nonobese diabetic (NOD)/SCID xenograft PEL mouse model, showing a reduction in tumor growth and an improvement in survival. Finally, an exploratory proof-of-concept clinical trial in four patients harboring severe KS was conducted, with the main objectives to assess the efficacy, the safety, and the tolerability of PQ, and which demonstrated a positive efficacy on Kaposi's sarcoma-related lesions and lymphedema.

4.
Front Immunol ; 12: 716469, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34434197

RESUMO

The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory response. In the first part of this review, we discuss the NF-κB inducers, signaling pathways, and regulators involved in immune homeostasis as well as detail the importance of post-translational regulation by ubiquitination in NF-κB function. We also indicate the stages of central and peripheral tolerance where NF-κB plays a fundamental role. With respect to central tolerance, we detail how NF-κB regulates medullary thymic epithelial cell (mTEC) development, homeostasis, and function. Moreover, we elaborate on its role in the migration of double-positive (DP) thymocytes from the thymic cortex to the medulla. With respect to peripheral tolerance, we outline how NF-κB contributes to the inactivation and destruction of autoreactive T and B lymphocytes as well as the differentiation of CD4+-T cell subsets that are implicated in immune tolerance. In the latter half of the review, we describe the contribution of NF-κB to the pathogenesis of autoimmunity and autoinflammation. The recent discovery of mutations involving components of the pathway has both deepened our understanding of autoimmune disease and informed new therapeutic approaches to treat these illnesses.


Assuntos
Autoimunidade , Inflamação/metabolismo , NF-kappa B/metabolismo , Animais , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade/genética , Proteínas de Transporte/metabolismo , Gerenciamento Clínico , Suscetibilidade a Doenças , Predisposição Genética para Doença , Humanos , Proteínas de Checkpoint Imunológico/genética , Tolerância Imunológica , Inflamação/etiologia , Inflamação/patologia , Inflamação/terapia , Terapia de Alvo Molecular , NF-kappa B/genética , Ligação Proteica , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
5.
iScience ; 20: 292-309, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31605944

RESUMO

CEP55 regulates the final critical step of cell division termed cytokinetic abscission. We report herein that CEP55 contains two NEMO-like ubiquitin-binding domains (UBDs), NOA and ZF, which regulate its function in a different manner. In vitro studies of isolated domains showed that NOA adopts a dimeric coiled-coil structure, whereas ZF is based on a UBZ scaffold. Strikingly, CEP55 knocked-down HeLa cells reconstituted with the full-length CEP55 ubiquitin-binding defective mutants, containing structure-guided mutations either in NOACEP55 or ZFCEP55 domains, display severe abscission defects. In addition, the ZFCEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZFCEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscission and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome.

6.
Front Immunol ; 9: 1243, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971063

RESUMO

Optineurin (Optn) is a 577 aa protein encoded by the Optn gene. Mutations of Optn are associated with normal tension glaucoma and amyotrophic lateral sclerosis, and its gene has also been linked to the development of Paget's disease of bone and Crohn's disease. Optn is involved in diverse cellular functions, including NF-κB regulation, membrane trafficking, exocytosis, vesicle transport, reorganization of actin and microtubules, cell cycle control, and autophagy. Besides its role in xenophagy and autophagy of aggregates, Optn has been identified as a primary autophagy receptor, among the five adaptors that translocate to mitochondria during mitophagy. Mitophagy is a selective macroautophagy process during which irreparable mitochondria are degraded, preventing accumulation of defective mitochondria and limiting the release of reactive oxygen species and proapoptotic factors. Mitochondrial quality control via mitophagy is central to the health of cells. One of the important surveillance pathways of mitochondrial health is the recently defined signal transduction pathway involving the mitochondrial PTEN-induced putative kinase 1 (PINK1) protein and the cytosolic RING-between-RING ubiquitin ligase Parkin. Both of these proteins, when mutated, have been identified in certain forms of Parkinson's disease. By targeting ubiquitinated mitochondria to autophagosomes through its association with autophagy related proteins, Optn is responsible for a critical step in mitophagy. This review reports recent discoveries on the role of Optn in mitophagy and provides insight into its link with neurodegenerative diseases. We will also discuss the involvement of Optn in other pathologies in which mitophagy dysfunctions are involved including cancer.


Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Animais , Biomarcadores , Proteínas de Ciclo Celular , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras , Mitofagia/genética , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Transdução de Sinais
8.
J Allergy Clin Immunol ; 140(6): 1671-1682.e2, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28249776

RESUMO

BACKGROUND: Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO. OBJECTIVES: We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway. METHODS: We sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-ß, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1ß. RESULTS: We identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1ß-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1-/- cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination. CONCLUSION: We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.


Assuntos
Fibroblastos/fisiologia , Quinase I-kappa B/metabolismo , Incontinência Pigmentar/metabolismo , Pele/patologia , Ubiquitinas/metabolismo , Clonagem Molecular , Feminino , Células HEK293 , Humanos , Quinase I-kappa B/genética , Incontinência Pigmentar/genética , Masculino , Mutação/genética , NF-kappa B/metabolismo , Linhagem , Ligação Proteica , Transdução de Sinais , Ativação Transcricional , Ubiquitinação
9.
Methods ; 115: 80-90, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27713081

RESUMO

We present TrackMate, an open source Fiji plugin for the automated, semi-automated, and manual tracking of single-particles. It offers a versatile and modular solution that works out of the box for end users, through a simple and intuitive user interface. It is also easily scriptable and adaptable, operating equally well on 1D over time, 2D over time, 3D over time, or other single and multi-channel image variants. TrackMate provides several visualization and analysis tools that aid in assessing the relevance of results. The utility of TrackMate is further enhanced through its ability to be readily customized to meet specific tracking problems. TrackMate is an extensible platform where developers can easily write their own detection, particle linking, visualization or analysis algorithms within the TrackMate environment. This evolving framework provides researchers with the opportunity to quickly develop and optimize new algorithms based on existing TrackMate modules without the need of having to write de novo user interfaces, including visualization, analysis and exporting tools. The current capabilities of TrackMate are presented in the context of three different biological problems. First, we perform Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs its early development. Our TrackMate-based lineage analysis indicates the lack of a cell-specific light-sensitive mechanism. Second, we investigate the recruitment of NEMO (NF-κB essential modulator) clusters in fibroblasts after stimulation by the cytokine IL-1 and show that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, we validate the use of TrackMate for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells.


Assuntos
Rastreamento de Células/métodos , Embrião não Mamífero/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Análise de Célula Única/métodos , Software , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Algoritmos , Animais , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Caenorhabditis elegans , Rastreamento de Células/estatística & dados numéricos , Clatrina/genética , Clatrina/metabolismo , Embrião não Mamífero/metabolismo , Endocitose , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Análise de Célula Única/estatística & dados numéricos
10.
Nat Commun ; 7: 12629, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27586688

RESUMO

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.


Assuntos
Quinase I-kappa B/ultraestrutura , Incontinência Pigmentar/patologia , Microscopia/métodos , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/fisiologia , Ligação Proteica , Estrutura Secundária de Proteína , Ubiquitina/metabolismo
11.
Cytokine Growth Factor Rev ; 29: 23-33, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26976762

RESUMO

The innate immune system has evolved to detect and neutralize viral invasions. Triggering of this defense mechanism relies on the production and secretion of soluble factors that stimulate intracellular antiviral defense mechanisms. The Tank Binding Kinase 1 (TBK1) is a serine/threonine kinase in the innate immune signaling pathways including the antiviral response and the host defense against cytosolic infection by bacteries. Given the critical roles of TBK1, important regulatory mechanisms are required to regulate its activity. Among these, Optineurin (Optn) was shown to negatively regulate the interferon response, in addition to its important role in membrane trafficking, protein secretion, autophagy and cell division. As Optn does not carry any enzymatic activity, its functions depend on its precise subcellular localization and its interaction with other proteins, especially with components of the innate immune pathway. This review highlights advances in our understanding of Optn mechanisms of action with focus on the relationships between Optn and TBK1 and their implication in host defense against pathogens. Specifically, how the antiviral immune system is controlled during the cell cycle by the Optn/TBK1 axis and the physiological consequences of this regulatory mechanism are described. This review may serve to a better understanding of the relationships between the different functions of Optn, including those related to immune responses and its associated pathologies such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget's disease of bone.


Assuntos
Ciclo Celular/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Interferons/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Fator de Transcrição TFIIIA/imunologia , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/patologia , Proteínas de Ciclo Celular , Glaucoma de Ângulo Aberto/imunologia , Glaucoma de Ângulo Aberto/patologia , Humanos , Proteínas de Membrana Transportadoras , Osteíte Deformante/imunologia , Osteíte Deformante/patologia , Viroses/imunologia , Viroses/patologia
12.
Hum Mol Genet ; 25(7): 1281-93, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26769674

RESUMO

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-ß-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-ß-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-ß-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis.


Assuntos
Doenças Ósseas Metabólicas/etiologia , Fibrose Cística/complicações , Deleção de Genes , Queratina-8/genética , Osteogênese , Animais , Doenças Ósseas Metabólicas/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , NF-kappa B , Osteoblastos/metabolismo , Transdução de Sinais , Adulto Jovem , beta Catenina
13.
J Biol Chem ; 290(29): 18009-18017, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26060255

RESUMO

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased ß-catenin phosphorylation, reduced osteoblast ß-catenin expression, and altered expression of Wnt/ß-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/ß-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/ß-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , NF-kappa B/imunologia , Osteoblastos/citologia , Via de Sinalização Wnt , Animais , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos , Osteoblastos/imunologia , Osteoblastos/patologia , beta Catenina/imunologia
15.
J Exp Med ; 212(6): 939-51, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26008899

RESUMO

Inherited, complete deficiency of human HOIL-1, a component of the linear ubiquitination chain assembly complex (LUBAC), underlies autoinflammation, infections, and amylopectinosis. We report the clinical description and molecular analysis of a novel inherited disorder of the human LUBAC complex. A patient with multiorgan autoinflammation, combined immunodeficiency, subclinical amylopectinosis, and systemic lymphangiectasia, is homozygous for a mutation in HOIP, the gene encoding the catalytic component of LUBAC. The missense allele (L72P, in the PUB domain) is at least severely hypomorphic, as it impairs HOIP expression and destabilizes the whole LUBAC complex. Linear ubiquitination and NF-κB activation are impaired in the patient's fibroblasts stimulated by IL-1ß or TNF. In contrast, the patient's monocytes respond to IL-1ß more vigorously than control monocytes. However, the activation and differentiation of the patient's B cells are impaired in response to CD40 engagement. These cellular and clinical phenotypes largely overlap those of HOIL-1-deficient patients. Clinical differences between HOIL-1- and HOIP-mutated patients may result from differences between the mutations, the loci, or other factors. Our findings show that human HOIP is essential for the assembly and function of LUBAC and for various processes governing inflammation and immunity in both hematopoietic and nonhematopoietic cells.


Assuntos
Regulação da Expressão Gênica , Ubiquitina-Proteína Ligases/deficiência , Alelos , Sequência de Aminoácidos , Ligante de CD40/metabolismo , Catálise , Feminino , Fibroblastos/metabolismo , Teste de Complementação Genética , Mutação em Linhagem Germinativa , Doença de Depósito de Glicogênio Tipo IV/patologia , Homozigoto , Humanos , Síndromes de Imunodeficiência/patologia , Inflamação , Linfangiectasia/patologia , Monócitos/metabolismo , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição , Ubiquitina/química , Ubiquitina-Proteína Ligases/genética , Adulto Jovem
16.
PLoS Pathog ; 11(4): e1004877, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25923723

RESUMO

Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.


Assuntos
Imunidade Inata , Interferon beta/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Transporte Ativo do Núcleo Celular , Substituição de Aminoácidos , Proteínas de Ciclo Celular , Linhagem Celular , Enzima Desubiquitinante CYLD , Fase G2 , Genes Reporter , Humanos , Interferon beta/genética , Proteínas de Membrana Transportadoras , Mutação , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição TFIIIA/antagonistas & inibidores , Fator de Transcrição TFIIIA/genética , Proteínas Supressoras de Tumor/agonistas , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética
17.
Elife ; 42015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25599590

RESUMO

Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.


Assuntos
Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Herpesviridae/fisiologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/virologia , Doença Aguda , Animais , Células da Medula Óssea/citologia , Caspase 1/metabolismo , Compartimento Celular , Doença Crônica , Citrobacter/fisiologia , Citocinas/biossíntese , Teste de Complementação Genética , Infecções por Herpesviridae/virologia , Humanos , Imunidade Inata , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Listeriose/microbiologia , Listeriose/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis/fisiologia , Fenótipo , Rhadinovirus/fisiologia , Toxoplasma , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
J Cell Biol ; 204(2): 231-45, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24446482

RESUMO

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.


Assuntos
Quinase I-kappa B/metabolismo , Interleucina-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Quinase I-kappa B/análise , Interleucina-1/análise , Interleucina-1/fisiologia , Quinases Associadas a Receptores de Interleucina-1/análise , Quinases Associadas a Receptores de Interleucina-1/metabolismo , NF-kappa B/análise , NF-kappa B/metabolismo , Receptores de Interleucina-1/análise , Receptores de Interleucina-1/metabolismo , Receptores do Fator de Necrose Tumoral/análise , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina/fisiologia , Ubiquitinação
19.
Nat Immunol ; 13(12): 1178-86, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23104095

RESUMO

We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic autoinflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1 (RBCK1), a component of the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin 1ß (IL-1ß) was compromised in the patients' fibroblasts. By contrast, the patients' mononuclear leukocytes, particularly monocytes, were hyper-responsive to IL-1ß. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1ß responses thus differed between cell types, consistent with the unique association of autoinflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB-dependent IL-1ß responses differently in different cell types.


Assuntos
Doença de Depósito de Glicogênio Tipo IV/genética , Doenças Hereditárias Autoinflamatórias/genética , Síndromes de Imunodeficiência/genética , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/genética , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Síndromes de Imunodeficiência/metabolismo , Interleucina-1beta/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Fatores de Transcrição , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
20.
Cell Cycle ; 11(15): 2808-18, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22801549

RESUMO

This review highlights recent advances in our understanding of the mechanisms of Optineurin (Optn) action and its implication in diseases. Optn has emerged as a key player regulating various physiological processes, including membrane trafficking, protein secretion, cell division and host defense against pathogens. Furthermore, there is growing evidence for an association of Optn mutations with human diseases such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget's disease of bone. Optn functions depend on its precise subcellular localization and its interaction with other proteins. Here, we review the mechanisms that allow Optn to ensure a timely and spatially coordinated integration of different physiological processes and discuss how their deregulation may lead to different pathologies.


Assuntos
Esclerose Lateral Amiotrófica/genética , Glaucoma/genética , Osteíte Deformante/genética , Fator de Transcrição TFIIIA/fisiologia , Animais , Apoptose , Transporte Biológico , Proteínas de Ciclo Celular , Divisão Celular , Humanos , Proteínas de Membrana Transportadoras , NF-kappa B/metabolismo , Fator de Transcrição TFIIIA/genética
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