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1.
Nat Commun ; 10(1): 3070, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296852

RESUMO

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/ultraestrutura , Guanilato Ciclase/ultraestrutura , Domínios Proteicos , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Microscopia Crioeletrônica , Guanilato Ciclase/genética , Guanilato Ciclase/imunologia , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Multimerização Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/imunologia
2.
Pediatr Rheumatol Online J ; 17(1): 38, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286971

RESUMO

BACKGROUND: Autosomal dominant gain of function mutations in caspase recruitment domain family member 14 (CARD14) is a rare condition associated with plaque-type psoriasis, generalized pustular psoriasis, palmoplantar pustular psoriasis and pityriasis rubra pilaris. Recently, a new CARD14 -associated phenotype defined as CAPE (CARD14-associated papulosquamous eruption) with clinical features of both psoriasis and pityriasis rubra pilaris was reported. We describe a family carrying a novel heterozygous mutation in CARD14 gene, with childhood-onset erythrodermic psoriasis requiring an unusual extremely high dose (up to 2 mg/kg every 8 weeks) of ustekinumab to achieve disease remission. CASE PRESENTATION: We describe a large family with three pairs of twins presenting a clinical phenotype characterized by childhood-onset erythrodermic psoriasis; in some family members is also reported psoriatic arthritis. The two probands presented poor clinical response to topic and systemic therapy with antihistamine, steroid, retinoids, cyclosporine and etanercept. After exclusion of the most common genes associated to autoinflammatory diseases (IL36RN, IL1RN, MVK, TNFRSF1A, NLRP3, NLRP12, MEFV, NOD2, PSMB8, PSTPIP1, LPIN2) we approached a new gene search by subjecting to Whole Exome Sequencing (WES) analysis five members of the family. A novel heterozygous mutation (c.446 T > G, leading to the missense amino acid substitution p.L149R) in the exon 4 of the CARD14 gene was identified in all affected members. Increasing dosages (up to 2 mg/kg every 8 weeks) of ustekinumab, a human monoclonal antibody targeting interleukin-12 (IL-12) and interleukin-23 (IL-23), allowed the complete control of the clinical manifestations, with an evident reduction of circulating Th17 and Th22 CD4+ T cell subsets. CONCLUSIONS: We describe the association of mutations of the CARD14 gene with an erythrodermic psoriasis pedigree, underlying the necessity to investigate CARD14 mutations in childhood-onset psoriasis cases and confirming the presence of CARD14 causative mutations also in erythrodermic psoriasis form, as recently reported. Also in pediatric age, ustekinumab represents a powerful therapeutic option for this rare condition, that is usually refractory to other treatments. In young children, high and frequent dosages allowed a complete control of the clinical manifestations without any severe side effects, with a long-term follow-up.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Fármacos Dermatológicos/uso terapêutico , Mutação com Ganho de Função/genética , Guanilato Ciclase/genética , Proteínas de Membrana/genética , Psoríase/tratamento farmacológico , Psoríase/genética , Ustekinumab/uso terapêutico , Criança , Dermatite Esfoliativa/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Gêmeos Dizigóticos , Sequenciamento Completo do Exoma
7.
Biomed Res Int ; 2019: 1345402, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984775

RESUMO

Remodelling of the peripheral lung tissue and fibrotic foci are the main pathologies of idiopathic pulmonary fibrosis (IPF), a disease that is difficult to treat. TGF-ß activation of peripheral lung fibroblasts is indicated as the major cause of tissue remodelling in IPF and is resulting in fibroblast hyperplasia and deposition of extracellular matrix. Soluble guanylate cyclase (sGC) stimulators combined with cyclic AMP (cAMP) activators have been reported to reduce proliferation and matrix deposition in other conditions than IPF. Therefore, this drug combination may present a novel therapeutic concept for IPF. This study investigated the effect of BAY 41-2272 and forskolin on remodelling parameters in primary human lung fibroblasts. The study determined TGF-ß induced proliferation by direct cell counts after 3 days; and deposition of collagen type-I, type III, and fibronectin. BAY 41-2272 significantly reduced TGF-ß induced fibroblast proliferation, but did not reduce viability. This inhibitory effect was further supported by forskolin. Both BAY 41-2272 and forskolin alone reduced TGF-ß induced collagen and fibronectin de novo synthesis as well as deposition. This effect was significantly stronger when the two compounds were combined. Furthermore, the TGF-ß induced expression of fibrilar α-smooth muscle actin was reduced by BAY 41-2272 and this effect was strengthened by forskolin. In addition, BAY 41-2272 and forskolin reduced TGF-ß induced ß-catenin. All effects of BAY 41-2272 were concentration dependent. The findings suggest that BAY 41-2272 in combination with cAMP stimulation may present a novel therapeutic strategy to reduce tissue remodelling in IPF.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Colforsina/farmacologia , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , AMP Cíclico/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibronectinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Cultura Primária de Células , Pirazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , beta Catenina/genética
8.
Doc Ophthalmol ; 139(1): 45-57, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30945053

RESUMO

PURPOSE: The aim of this study was to examine the ophthalmological characteristics and genotypes of patients with congenital retinal pathologies, who display a bull's-eye maculopathy in the fundus, along with a negative scotopic electroretinogram. METHODS: We analysed the results of five patients showing both a bull's-eye maculopathy, as well as a negative scotopic ERG evoked by a bright flash. Their median age was 39 years (range 11-63 years): three males and two females. All underwent a comprehensive examination with determination of distant visual acuity (ETDRS) and recording of the full-field ERG (scotopic and photopic). Fundus, OCT, and FAF images were obtained, the kinetic visual field was determined, and colour vision (D-15) was tested in most patients. Targeted gene panel sequencing was performed on peripheral blood. RESULTS: One patient carried a homozygous ABCA4 mutation and an additional heterozygous variant in CRX. Two of the five patients were shown to have a heterozygous mutation in the CRX gene, one of whom had an additional heterozygous ABCA4 mutation. Two patients had the common heterozygous mutation c.2413G>A;p.Arg838His in GUCY2D. In all of the patients, there was a reduction in the amplitude of the b-wave with a regular a-wave amplitude in the scotopic bright-flash ERG. CONCLUSIONS: The five patients with bull's-eye maculopathy along with a negative ERG had differing genotypes. Mutations were found in the CRX gene (2 patients), the ABCA4 gene (1 patient), and the GUCY2D gene (2 patients).


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Guanilato Ciclase/genética , Proteínas de Homeodomínio/genética , Degeneração Macular/genética , Degeneração Macular/fisiopatologia , Mutação , Receptores de Superfície Celular/genética , Retina/fisiopatologia , Transativadores/genética , Adulto , Criança , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Angiofluoresceinografia , Humanos , Degeneração Macular/diagnóstico , Masculino , Pessoa de Meia-Idade , Visão Noturna/fisiologia , Estimulação Luminosa , Acuidade Visual/fisiologia , Adulto Jovem
9.
eNeuro ; 6(1)2019.
Artigo em Inglês | MEDLINE | ID: mdl-30783616

RESUMO

The membrane guanylate cyclase, ROS-GC, that synthesizes cyclic GMP for use as a second messenger for visual transduction in retinal rods and cones, is stimulated by bicarbonate. Bicarbonate acts directly on ROS-GC1, because it enhanced the enzymatic activity of a purified, recombinant fragment of bovine ROS-GC1 consisting solely of the core catalytic domain. Moreover, recombinant ROS-GC1 proved to be a true sensor of bicarbonate, rather than a sensor for CO2. Access to bicarbonate differed in rods and cones of larval salamander, Ambystoma tigrinum, of unknown sex. In rods, bicarbonate entered at the synapse and diffused to the outer segment, where it was removed by Cl--dependent exchange. In contrast, cones generated bicarbonate internally from endogenous CO2 or from exogenous CO2 that was present in extracellular solutions of bicarbonate. Bicarbonate production from both sources of CO2 was blocked by the carbonic anhydrase inhibitor, acetazolamide. Carbonic anhydrase II expression was verified immunohistochemically in cones but not in rods. In addition, cones acquired bicarbonate at their outer segments as well as at their inner segments. The multiple pathways for access in cones may support greater uptake of bicarbonate than in rods and buffer changes in its intracellular concentration.


Assuntos
Bicarbonatos/metabolismo , Guanilato Ciclase/metabolismo , Receptores de Superfície Celular/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Visão Ocular/fisiologia , Acetazolamida/farmacologia , Ambystoma , Animais , Células COS , Dióxido de Carbono/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , GMP Cíclico/metabolismo , Expressão Gênica , Guanilato Ciclase/genética , Camundongos , Proteínas Recombinantes/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Visão Ocular/efeitos dos fármacos
10.
Yonsei Med J ; 60(3): 267-276, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30799589

RESUMO

PURPOSE: Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor, the prognosis of which remains poor. Recently, microRNAs have been reported to play crucial functions in multiple tumors, including HCC. However, the molecular mechanisms of miR-370 in HCC still remain largely unknown. The present study focused on the effects of miR-370 on HCC migration, invasion, and epithelial-mesenchymal transition (EMT). MATERIALS AND METHODS: We investigated the key roles and possible regulatory mechanism of miR-370 in regulating HCC metastasis with functional assays, such as transwell assay. Quantitative real-time PCR (qRT-PCR) was used to detect miR-370 and guanylylcyclase domain containing 1 (GUCD1) expression in HCC tissues and cells. Subsequently, we performed transwell assays to determine the functions of miR-370 in HCC cell invasion and migration. Western blot was used to determine protein expressions of relevant genes. Luciferase reporter assays were conducted to confirm the target gene of miR-370. RESULTS: qRT-PCR analysis demonstrated that miR-370 was dramatically downregulated in HCC. Moreover, downregulated miR-370 was found to be associated with poor survival and adverse clinicopathologic characteristics of HCC patients. Transwell assays revealed that miR-370 overexpression dramatically suppressed HCC invasion and migration. Meanwhile, miR-370 restoration prominently inhibited EMT progression in HCC cells. Luciferase reporter assays confirmed GUCD1 as a downstream target gene of miR-370. GUCD1 expression in HCC tissues was prominently increased and inversely correlated with miR-370 expression. Furthermore, GUCD1 was verified as mediating the suppressive influence of miR-370 on cell metastasis and EMT in HCC. CONCLUSION: Taken together, our study confirmed that miR-370 suppressed HCC cell metastasis and EMT via regulating GUCD1. Accordingly, the miR-370/GUCD1 axis may potentially acts as attractive therapeutic targets and novel biomarkers for HCC treatment.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Guanilato Ciclase/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Guanilato Ciclase/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Regulação para Cima/genética
12.
J Eur Acad Dermatol Venereol ; 33(5): 944-949, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30697821

RESUMO

BACKGROUND: Pityriasis rubra pilaris (PRP) is a rare chronic inflammatory dermatosis with multifactorial aetiology. It is known that particular caspase recruitment domain family member 14 (CARD14) gene mutations are associated with familial PRP and certain forms of psoriasis. Additionally, few data are available about the role of CARD14 gene variants in sporadic PRP. The clinical picture is variable for the different types of PRP, therefore choosing the adequate treatment is often difficult, furthermore there are no specific guidelines for therapy. OBJECTIVE: Our aim was to survey the efficacy of the applied therapies and to screen the CARD14 gene variants in our PRP patients. METHODS: In this retrospective study, patients diagnosed with PRP between 2006 and 2016 at our clinic were involved. Besides the follow-up study of the treatments, the genetic analysis of CARD14 gene was performed. RESULTS: We analysed 19 patients, among whom 17 were diagnosed with type I, one with type III, and one with type V PRP. The majority of the patients were successfully treated with acitretin in combination with systemic corticosteroids, and the remaining patients were treated with other systemic therapies with diverse effects. The genetic screening of CARD14 gene revealed two previously described mutations (rs114688446, rs117918077) and six polymorphisms (rs28674001, rs2066964, rs34367357, rs11653893, rs11652075, rs2289541). Ten of 19 patients carried different CARD14 genetic variants either alone or in combination. CONCLUSION: Based on our experience, we propose that acitretin and an initial combination of short-term systemic corticosteroid therapy could be a successful treatment option for PRP. Although we identified several CARD14 variants in almost half of our cases, we did not find a correlation between the therapeutic response and the genetic background. Our data support the previous observation that CARD14 genetic variants are not specific to PRP, although they may indicate chronic inflammation.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Guanilato Ciclase/genética , Proteínas de Membrana/genética , Pitiríase Rubra Pilar/genética , Pitiríase Rubra Pilar/terapia , Adulto , Idoso , Criança , Fármacos Dermatológicos/uso terapêutico , Feminino , Seguimentos , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Creme para a Pele
13.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30530863

RESUMO

Turner syndrome (TS) is a congenital disease caused by complete or partial loss of one X chromosome. Low bone mineral status is a major phenotypic characteristic of TS that can not be fully explained by X chromosome loss, suggesting other autosomal-linked mutations may also exist. Therefore, the present study aimed to detect potential genetic mutations in TS through examination of copy number variation (CNV). Seventeen patients with TS and 15 healthy volunteer girls were recruited. Array-based comparative genomic hybridization (a-CGH) was performed on whole blood genomic DNA (gDMA) from the 17 TS patients and 15 healthy volunteer girls to identify potential CNVs. The abnormal CNV of one identified gene (CARD11) was verified by quantitative PCR. All cases diagnosed had TS based on genotype examination and physical characteristics, including short stature and premature ovarian failure. Three rare CNVs, located individually at 7p22.3, 7p22.2, and Xp22.33, where six genes (TTYH3, AMZ1, GNA12, BC038729, CARD11, and SHOX (stature homeobox)) are located, were found in TS patients. Quantitative PCR confirmed the CNV of CARD11 in the genome of TS patients. Our results indicate that CARD11 gene is one of the mutated genes involved in TS disease. However, this CNV is rare and its contribution to TS phenotype requires further study.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Cromossomos Humanos X/química , Variações do Número de Cópias de DNA , Guanilato Ciclase/genética , Síndrome de Turner/genética , Adolescente , Antropometria , Proteínas Adaptadoras de Sinalização CARD/deficiência , Estudos de Casos e Controles , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Feminino , Genótipo , Guanilato Ciclase/deficiência , Humanos , Mutação , Fenótipo , Síndrome de Turner/diagnóstico , Síndrome de Turner/patologia , Adulto Jovem
14.
J Biol Chem ; 294(7): 2318-2328, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30559291

RESUMO

Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase-activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18-160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70 Å long and 30 Å wide) consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in the RetGC binding map to a localized and contiguous area on the structure, involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was validated by mutagenesis. Introducing Trp85 or Phe29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3's apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu32 to Lys) also lowered the affinity. Conversely, introducing Val in place of Cys93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3-RetGC interactions relevant for normal vision or blindness.


Assuntos
Proteínas do Olho/química , Substituição de Aminoácidos , Animais , Bovinos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Guanilato Ciclase/química , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
15.
Neurobiol Dis ; 121: 65-75, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30213732

RESUMO

The nitric oxide - guanylyl cyclase-1 - cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma - primary open angle glaucoma (POAG; GC-1-/- mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.


Assuntos
GMP Cíclico/metabolismo , Glaucoma/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glaucoma/prevenção & controle , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Camundongos Knockout , Inibidores da Fosfodiesterase 5/administração & dosagem , Ratos Sprague-Dawley , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/efeitos dos fármacos , Transdução de Sinais , Tadalafila/administração & dosagem
17.
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
18.
Front Immunol ; 9: 2695, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30515170

RESUMO

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.


Assuntos
Proteína 10 de Linfoma CCL de Células B/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Guanilato Ciclase/imunologia , Ativação Linfocitária , Multimerização Proteica/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Proteína 10 de Linfoma CCL de Células B/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Guanilato Ciclase/genética , Células HEK293 , Humanos , Células Jurkat , Multimerização Proteica/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T/citologia
19.
Biochem Soc Trans ; 46(6): 1729-1742, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30420416

RESUMO

The availability of genome sequence information and a large number of protein structures has allowed the cataloging of genes into various families, based on their function and predicted biochemical activity. Intriguingly, a number of proteins harbor changes in the amino acid sequence at residues, that from structural elucidation, are critical for catalytic activity. Such proteins have been categorized as 'pseudoenzymes'. Here, we review the role of the pseudokinase (or kinase-homology) domain in receptor guanylyl cyclases. These are multidomain single-pass, transmembrane proteins harboring an extracellular ligand-binding domain, and an intracellular domain composed of a kinase-homology domain that regulates the activity of the associated guanylyl cyclase domain. Mutations that lie in the kinase-homology domain of these receptors are associated with human disease, and either abolish or enhance cGMP production by these receptors to alter downstream signaling events. This raises the interesting possibility that one could identify molecules that bind to the pseudokinase domain and regulate the activities of these receptors, in order to alleviate symptoms in patients harboring these mutations.


Assuntos
GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Animais , Guanilato Ciclase/genética , Humanos , Mutação/genética , Mutação Puntual/genética
20.
Front Immunol ; 9: 2239, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386326

RESUMO

The CARD: BCL10: MALT1 (CBM) complex is an essential signaling node for maintaining both innate and adaptive immune responses. CBM complex components have gained considerable interest due to the dramatic effects of associated mutations in causing severe lymphomas, immunodeficiencies, carcinomas and inflammatory disease. While MALT1 and BCL10 are ubiquitous proteins, the CARD-containing proteins differ in their tissue expression. CARD14 is primarily expressed in keratinocytes. The CARD14-BCL10-MALT1 complex is activated by upstream pathogen-associated molecular pattern-recognition in vitro, highlighting a potentially crucial role in innate immune defense at the epidermal barrier. Recent findings have demonstrated how CARD14 orchestrates activation of the NF-κB and MAPK signaling pathways via recruitment of BCL10 and MALT1, leading to the upregulation of pro-inflammatory genes encoding IL-36γ, IL-8, Ccl20 and anti-microbial peptides. Following the identification of CARD14 gain-of function mutations as responsible for the psoriasis susceptibility locus PSORS2, the past years have witnessed a large volume of case reports and association studies describing CARD14 variants as causal or predisposing to a wide range of inflammatory skin disorders. Recent publications of mouse models also helped to better understand the physiological contribution of CARD14 to psoriasis pathogenesis. In this review, we summarize the clinical, genetic and functional aspects of human and murine CARD14 mutations and their contribution to psoriatic disease pathogenesis.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Heterogeneidade Genética , Guanilato Ciclase/imunologia , Proteínas de Membrana/imunologia , Mutação , Psoríase/imunologia , Animais , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/imunologia , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Psoríase/genética , Psoríase/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
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