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1.
J Exp Med ; 216(9): 1986-1998, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31235509

RESUMO

IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

2.
J Clin Immunol ; 39(4): 401-413, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037583

RESUMO

MALT1 (mucosa-associated lymphoid tissue lymphoma-translocation gene 1) is an intracellular signaling protein that activates NFκB and is crucial for both the adaptive and innate immune responses. Only 6 patients with immune deficiencies secondary to inherited mutations in the MALT1 gene have been described. PURPOSE: To provide clinical and immunological insights from 2 patients diagnosed with MALT1 immunodeficiency syndrome due to a novel MALT1 mutation. METHODS: Two cousins with suspected combined immunodeficiency underwent immunological and genetic work-up, including lymphocyte phenotyping, lymphocyte activation by mitogen stimulation, and next-generation sequencing (NGS) of T cell receptor gamma chain (TRG) repertoire. Whole exome sequencing was performed to identify the underlying genetic defect. RESULTS: Clinical findings included recurrent infections, failure to thrive, lymphadenopathy, dermatitis, and autoimmunity. Immune work-up revealed lymphocytosis, low to normal levels of immunoglobulins, absence of regulatory T cells, and low Th17 cells. A normal proliferative response was induced by phytohemagglutinin and IL-2 but was diminished with anti-CD3. TRG repertoire was diverse with a clonal expansion pattern. Genetic analysis identified a novel autosomal recessive homozygous c.1799T>A; p. I600N missense mutation in MALT1. MALT1 protein expression was markedly reduced, and in vitro IL-2 production and NFκB signaling pathway were significantly impaired. CONCLUSIONS: Two patients harboring a novel MALT1 mutation presented with signs of immune deficiency and dysregulation and were found to have an abnormal T cell receptor repertoire. These findings reinforce the link between MALT1 deficiency and combined immunodeficiency. Early diagnosis is crucial, and curative treatment by hematopoietic stem cell transplantation may be warranted.

3.
J Allergy Clin Immunol ; 143(1): 173-181.e10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30248356

RESUMO

BACKGROUND: Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. OBJECTIVE: We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. METHODS: Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. RESULTS: In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. CONCLUSIONS: Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.


Assuntos
Proteínas Adaptadoras de Sinalização CARD , Dermatite Atópica , Guanilato Ciclase , Queratinócitos , Mutação com Perda de Função , Proteínas de Membrana , Mutação de Sentido Incorreto , Transdução de Sinais/genética , Adolescente , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Feminino , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Índice de Gravidade de Doença , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
4.
J Allergy Clin Immunol ; 143(4): 1482-1495, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30170123

RESUMO

BACKGROUND: Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing. OBJECTIVES: We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. METHODS: Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants. RESULTS: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. CONCLUSION: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

6.
J Exp Med ; 214(9): 2523-2533, 2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28710273

RESUMO

Patients with hypomorphic mutations in STAT3 and patients with hypermorphic mutations in STAT1 share several clinical and cellular phenotypes suggesting overlapping pathophysiologic mechanisms. We, therefore, examined cytokine signaling and CD4+ T cell differentiation in these cohorts to characterize common pathways. As expected, differentiation of Th17 cells was impaired in both cohorts. We found that STAT1 was hyperphosphorylated in response to cytokine stimulation in both cohorts and that STAT1-dependent PD-L1 up-regulation-known to inhibit Th17 differentiation in mouse models-was markedly enhanced as well. Overexpression of SOCS3 strongly inhibited phosphorylation of STAT1 and PD-L1 up-regulation, suggesting that diminished SOCS3 expression may lead to the observed effects. Defects in Th17 differentiation could be partially overcome in vitro via PD-L1 inhibition and in a mouse model of STAT3 loss-of-function by crossing them with PD-1 knockout mice. PD-L1 may be a potential therapeutic target in several genetic diseases of immune deficiency affecting cytokine signaling.


Assuntos
Antígeno B7-H1/fisiologia , Diferenciação Celular/fisiologia , Fator de Transcrição STAT1/fisiologia , Fator de Transcrição STAT3/fisiologia , Células Th17/fisiologia , Adolescente , Adulto , Animais , Criança , Citocinas/fisiologia , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/fisiopatologia , Interleucinas/fisiologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas/fisiologia , Regulação para Cima , Adulto Jovem
7.
Nat Genet ; 49(8): 1192-1201, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28628108

RESUMO

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Dermatite Atópica/genética , Mutação em Linhagem Germinativa , Guanilato Ciclase/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , Dermatite Atópica/imunologia , Feminino , Genes Dominantes , Glutamina/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Antígenos de Histocompatibilidade Menor/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Linhagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo
9.
J Biol Chem ; 290(36): 22076-84, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26224629

RESUMO

NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are intracellular proteins that regulate the NF-κB signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-κB signaling during embryogenesis.


Assuntos
Desenvolvimento Embrionário/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Dedos de Zinco/genética
10.
Blood ; 125(4): 591-9, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25359994

RESUMO

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.


Assuntos
Doenças Autoimunes/genética , Doenças Genéticas Inatas/genética , Transtornos Linfoproliferativos/genética , Fator de Transcrição STAT3/genética , Adolescente , Adulto , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Criança , Pré-Escolar , Feminino , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Humanos , Lactente , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Masculino , Mutação , Fosforilação/genética , Fosforilação/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
11.
Proc Natl Acad Sci U S A ; 111(38): 13930-5, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25201955

RESUMO

Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor with inhibitor of histone acetyltransferase activity and is a potent suppressor of p53. Although NIR deficiency in mice leads to early embryonic lethality, lymphoid-restricted deletion resulted in the absence of double-positive CD4(+)CD8(+) thymocytes, whereas bone-marrow-derived B cells were arrested at the B220(+)CD19(-) pro-B-cell stage. V(D)J recombination was preserved in NIR-deficient DN3 double-negative thymocytes, suggesting that NIR does not affect p53 function in response to physiologic DNA breaks. Nevertheless, the combined deficiency of NIR and p53 provided rescue of DN3L double-negative thymocytes and their further differentiation to double- and single-positive thymocytes, whereas B cells in the marrow further developed to the B220(+)CD19(+) pro-B-cell stage. Our results show that NIR cooperate with p53 to impose checkpoint for the generation of mature B and T lymphocytes.


Assuntos
Diferenciação Celular/imunologia , Proteínas Repressoras/imunologia , Timócitos/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Diferenciação Celular/genética , Quebras de DNA , Camundongos , Células Precursoras de Linfócitos B/citologia , Proteínas Repressoras/genética , Timócitos/citologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
12.
Proc Natl Acad Sci U S A ; 110(13): 5127-32, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23493554

RESUMO

Mutations in the TNF family of proteins have been associated with inherited forms of immune deficiency. Using an array-based sequencing assay, we identified an autosomal-dominant deficiency in TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) in a kindred with recurrent infection and impaired antibody responses to protein and polysaccharide vaccines. This mutation occurs in the sixth exon of TWEAK and results in the amino acid substitution R145C within the conserved TNF-homology domain of the full-length protein. TWEAK mutant protein formed high molecular weight aggregates under nonreducing conditions, suggesting an increased propensity for intermolecular interactions. As a result, mutant TWEAK associated with B-cell-activating factor (BAFF) protein and down-regulated the BAFF-mediated activation of the noncanonical NF-κB pathway through inhibition of p100 processing to p52, resulting in inhibition of BAFF-dependent B-cell survival and proliferation. As BAFF mediates T-cell-independent isotype switching and B-cell survival, our data implicate TWEAK as a disease-susceptibility gene for a humoral immunodeficiency.


Assuntos
Linfócitos B/imunologia , Doenças Genéticas Inatas/imunologia , Predisposição Genética para Doença , Síndromes de Imunodeficiência/imunologia , Mutação de Sentido Incorreto , Fatores de Necrose Tumoral/imunologia , Adulto , Substituição de Aminoácidos , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Criança , Pré-Escolar , Citocina TWEAK , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Masculino , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/imunologia , Fatores de Necrose Tumoral/genética
13.
Clin Immunol ; 143(2): 152-61, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22459705

RESUMO

X-linked hyper-IgM syndrome (XHM) is a combined immune deficiency disorder caused by mutations in CD40 ligand. We tested CP-870,893, a human CD40 agonist monoclonal antibody, in the treatment of two XHM patients with biliary Cryptosporidiosis. CP-870,893 activated B cells and APCs in vitro, restoring class switch recombination in XHM B cells and inducing cytokine secretion by monocytes. CP-870,893 infusions were well tolerated and showed significant activity in vivo, decreasing leukocyte concentration in peripheral blood. Although specific antibody responses were lacking, frequent dosing in one subject primed T cells to secrete IFN-g and suppressed oocyst shedding in the stool. Nevertheless, relapse occurred after discontinuation of therapy. The CD40 receptor was rapidly internalized following binding with CP-870,893, potentially explaining the limited capacity of CP-870,893 to mediate immune reconstitution. This study demonstrates that CP-870,893 suppressed oocysts shedding in XHM patients with biliary cryptosporidiosis. The continued study of CD40 agonists in XHM is warranted.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Ligante de CD40/agonistas , Criptosporidiose/tratamento farmacológico , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/tratamento farmacológico , Adolescente , Anticorpos Monoclonais Humanizados , Ligante de CD40/imunologia , Criptosporidiose/imunologia , Criptosporidiose/microbiologia , Cryptosporidium/isolamento & purificação , Cryptosporidium/fisiologia , Citocinas/imunologia , Fezes/microbiologia , Humanos , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/imunologia , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/microbiologia , Contagem de Leucócitos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
14.
J Clin Invest ; 122(1): 315-26, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22156202

RESUMO

Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB-regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.


Assuntos
Displasia Ectodérmica/imunologia , Displasia Ectodérmica/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Adolescente , Linhagem Celular , Núcleo Celular/metabolismo , Pré-Escolar , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Displasia Ectodérmica/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Rearranjo Gênico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Síndromes de Imunodeficiência/genética , Interleucina-12/genética , Interleucina-12/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-rel , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo
15.
J Biol Chem ; 286(47): 40520-30, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21931165

RESUMO

CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK signaling. Here, we show that CYLD knock-out mice have markedly increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. In vitro stimulation of CYLD-deficient naive T cells with anti-CD3/28 in the presence of TGF-ß led to a marked increase in the number of Foxp3-expressing T cells when compared with stimulated naive control CD4(+) cells. Under endogenous conditions, CYLD formed a complex with Smad7 that facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38 activity in primary T cells impaired Treg differentiation. Together, our results show that CYLD regulates TGF-ß signaling function in T cells and the development of Tregs through deubiquitination of Smad7.


Assuntos
Cisteína Endopeptidases/metabolismo , Transdução de Sinais , Proteína Smad7/metabolismo , Linfócitos T Reguladores/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Fatores de Transcrição Forkhead/genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linfonodos/imunologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Ubiquitinação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Hum Mutat ; 32(3): 318-24, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21309033

RESUMO

The covalent attachment of lysine 63-linked polyubiquitin to the zinc-finger domain of IKBKG/NEMO (also known as IKKγ) is necessary for full activation of NF-κB. Impairments of this biochemical mechanism explain the deleterious effects of hypomorphic NEMO mutations on NF-κB signaling function in humans suffering from X-linked ectodermal dysplasia and immunodeficiency. Nevertheless, the biological function of the NEMO zinc-finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity is poorly understood. Here we show that dendritic cells from patients with EDI caused by a C-terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc-finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation.


Assuntos
Células Dendríticas/metabolismo , Displasia Ectodérmica/enzimologia , Displasia Ectodérmica/genética , Quinase I-kappa B/genética , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Cultivadas , Citocinas/biossíntese , Displasia Ectodérmica/imunologia , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/imunologia , Lipopolissacarídeos/imunologia , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , NF-kappa B/metabolismo , Deleção de Sequência , Ubiquitinação , Dedos de Zinco/genética
18.
J Biol Chem ; 286(3): 2236-44, 2011 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-20959462

RESUMO

NIR (novel INHAT repressor) is a transcriptional co-repressor with inhibitor of histone acetyltransferase (INHAT) activity and has previously been shown to physically interact with and suppress p53 transcriptional activity and function. However, the mechanism by which NIR suppresses p53 is not completely understood. Using a proteomic approach, we have identified the Aurora kinase B as a novel binding partner of NIR. We show that Aurora B, NIR and p53 exist in a protein complex in which Aurora B binds to NIR, thus also indirectly associates with p53. Functionally, overexpression of Aurora B or NIR suppresses p53 transcriptional activity, and depletion of Aurora B or NIR causes p53-dependent apoptosis and cell growth arrest, due to the up-regulation of p21 and Bax. We then demonstrate that Aurora B phosphorylates multiple sites in the p53 DNA-binding domain in vitro, and this phosphorylation probably also occurs in cells. Importantly, the Aurora B-mediated phosphorylation on Ser(269) or Thr(284) significantly compromises p53 transcriptional activity. Taken together, these results provide novel insight into NIR-mediated p53 suppression and also suggest an additional way for p53 regulation.


Assuntos
Apoptose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Genética/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Aurora Quinase B , Aurora Quinases , Deleção de Genes , Células HEK293 , Humanos , Fosforilação/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/fisiologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
19.
Hum Mutat ; 29(6): 861-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18412279

RESUMO

Alterations in nuclear factor kappa B (NF-kappaB) essential modulator (NEMO; HUGO-approved symbol IKBKG) underlie most cases of ectodermal dysplasia with immune deficiency (EDI), a human disorder characterized by anhidrosis with diminished immunity. EDI has also been associated with a single heterozygous mutation at position Ser32 of the NF-kappaB inhibitor IkappaBalpha, one of two phosphorylation sites that are essential for targeting IkappaBalpha for proteasomal degradation and hence for activation of NF-kappaB. We report a novel heterozygous nonsense mutation in the IKBA (HUGO-approved symbol, NFKBIA) gene of a 1-year-old male child with EDI that introduces a premature termination codon at position Glu14. An in-frame methionine downstream of the nonsense mutation allows for reinitiation of translation. The resulting N-terminally truncated protein lacks both serine phosphorylation sites and inhibits NF-kappaB signaling by functioning as a dominant negative on NF-kappaB activity in lymphocytes and monocytes. These findings support the scanning model for translation initiation in eukaryotes and confirm the critical role of the NF-kappaB in the human immune response.


Assuntos
Códon sem Sentido , Displasia Ectodérmica/genética , Proteínas I-kappa B/genética , Síndromes de Imunodeficiência/genética , Sequência de Aminoácidos , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/genética , Lactente , Masculino , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/imunologia , Biossíntese de Proteínas , Linfócitos T/imunologia
20.
J Clin Invest ; 116(11): 3042-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17053834

RESUMO

Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here we show that the development of B cells, T cells, and myeloid cells was unaffected in CYLD-deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity resulted in enhanced NF-kappaB and JNK activity associated with increased TNF receptor-associated factor 2 (TRAF2) and NF-kappaB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were more susceptible to induced colonic inflammation and showed a dramatic increase in the incidence of tumors compared with controls in a colitis-associated cancer model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating ubiquitination in vivo.


Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colite/metabolismo , Colite/patologia , Cisteína Endopeptidases/metabolismo , Predisposição Genética para Doença/genética , NF-kappa B/metabolismo , Animais , Transformação Celular Neoplásica/genética , Colite/complicações , Colite/genética , Neoplasias do Colo/etiologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Citocinas/biossíntese , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Ligação Proteica , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina/metabolismo
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