RESUMO
Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1ß as a key mediator, orchestrating an NF-κB-mediated IL-1ß-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1ß. With the central role of IL-1ß underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1ß antagonists in PRP.
Assuntos
Anticorpos Monoclonais Humanizados , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1beta , Queratinócitos , Pitiríase Rubra Pilar , Humanos , Pitiríase Rubra Pilar/tratamento farmacológico , Pitiríase Rubra Pilar/patologia , Pitiríase Rubra Pilar/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Masculino , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Feminino , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Pele/patologia , Pele/metabolismo , Pele/efeitos dos fármacos , Interleucina-1/antagonistas & inibidores , Interleucina-1/metabolismo , Interleucina-1/genética , Pessoa de Meia-Idade , Guanilato Ciclase/metabolismo , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/genética , Adulto , Transdução de Sinais/efeitos dos fármacos , Proteínas de MembranaRESUMO
Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.
Assuntos
GMP Cíclico , Dictyostelium , Transdução de Sinais , Dictyostelium/metabolismo , Dictyostelium/genética , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Guanilato Ciclase/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genéticaRESUMO
Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with â¼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase , Guanilato Ciclase , Modelos Moleculares , Animais , Cálcio/metabolismo , Ativação Enzimática/genética , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Guanilato Ciclase/química , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Multimerização Proteica , Retina/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
Atrial natriuretic peptide (ANP) plays a central role in the regulation of blood pressure and volume. ANP activities are mediated by natriuretic peptide receptor-A (NPR-A), a single-pass transmembrane receptor harboring intrinsic guanylate cyclase activity. This study investigated the mechanism underlying NPR-A-dependent hormone recognition through the determination of the crystal structures of the NPR-A extracellular hormone-binding domain complexed with full-length ANP, truncated mutants of ANP, and dendroaspis natriuretic peptide (DNP) isolated from the venom of the green Mamba snake, Dendroaspis angusticeps. The bound peptides possessed pseudo-two-fold symmetry, despite the lack of two-fold symmetry in the primary sequences, which enabled the tight coupling of the peptide to the receptor, and evidently contributes to guanylyl cyclase activity. The binding of DNP to the NPR-A was essentially identical to that of ANP; however, the affinity of DNP for NPR-A was higher than that of ANP owing to the additional interactions between distinctive sequences in the DNP and NPR-A. Consequently, our findings provide valuable insights that can be applied to the development of novel agonists for the treatment of various human diseases.
Assuntos
Fator Natriurético Atrial , Receptores do Fator Natriurético Atrial , Receptores do Fator Natriurético Atrial/metabolismo , Receptores do Fator Natriurético Atrial/química , Receptores do Fator Natriurético Atrial/genética , Fator Natriurético Atrial/química , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/genética , Animais , Humanos , Ligação Proteica , Cristalografia por Raios X , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Venenos Elapídicos/genética , Sequência de Aminoácidos , Modelos Moleculares , Guanilato Ciclase/metabolismo , Guanilato Ciclase/química , Guanilato Ciclase/genética , Peptídeos Natriuréticos/química , Peptídeos Natriuréticos/metabolismo , Peptídeos Natriuréticos/genética , Sítios de LigaçãoAssuntos
Anticorpos Monoclonais Humanizados , Proteínas Adaptadoras de Sinalização CARD , Guanilato Ciclase , Proteínas de Membrana , Mutação , Nevo Sebáceo de Jadassohn , Humanos , Anticorpos Monoclonais Humanizados/uso terapêutico , Proteínas Adaptadoras de Sinalização CARD/genética , Guanilato Ciclase/genética , Nevo Sebáceo de Jadassohn/genética , Nevo Sebáceo de Jadassohn/patologia , Nevo Sebáceo de Jadassohn/tratamento farmacológico , Proteínas de Membrana/genética , Feminino , MasculinoRESUMO
Ion channels activity is regulated through soluble guanylate cyclase (sGC) and adenylate cyclase (AC) pathways, while phosphodiesterases (PDE) control the intracellular levels of cAMP and cGMP. Here we applied RNA transcriptome sequencing to study changes in the gene expression of the sGC, AC, and PDE isoforms in isolated rat ventricular cardiomyocytes under conditions of microgravity and hypergravity. Our results demonstrate that microgravity reduces the expression of sGC isoform genes, while hypergravity increases their expression. For a subset of AC isoforms, gene expression either increased or decreased under both microgravity and hypergravity conditions. The expression of genes encoding 10 PDE isoforms decreased under microgravity, but increased under hypergravity. However, under both microgravity and hypergravity, the gene expression increased for 7 PDE isoforms and decreased for 3 PDE isoforms. Overall, our findings indicate specific gravity-dependent changes in the expression of genes of isoforms associated with the studied enzymes.
Assuntos
Hipergravidade , Ausência de Peso , Ratos , Animais , Diester Fosfórico Hidrolases/metabolismo , Guanilil Ciclase Solúvel , Adenilil Ciclases/genética , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/genética , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , GMP Cíclico/metabolismoRESUMO
Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.
Assuntos
Evolução Molecular , Imunoterapia Adotiva , Linfoma Cutâneo de Células T , Mutação , Linfócitos T , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Citocinas/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Imunoterapia Adotiva/métodos , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/imunologia , Linfoma Cutâneo de Células T/patologia , Linfoma Cutâneo de Células T/terapia , Fosfatidilinositol 3-Quinases , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
PURPOSE: To describe the phenotype of Danish patients with genetically verified achromatopsia (ACHM) with special focus on signs of progression on structural or functional parameters, and possible genotype-phenotype correlations. METHODS: Forty-eight patients were identified, with disease-causing variants in five different genes: CNGA3, CNGB3, GNAT2, PDE6C and PDE6H. Longitudinal evaluation was possible for 11 patients and 27 patients participated in a renewed in-depth phenotyping consisting of visual acuity assessment, optical coherence tomography (OCT), fundus autofluorescence, colour vision evaluation, contrast sensitivity, mesopic microperimetry and full-field electroretinography. Foveal morphology was evaluated based on OCT images for all 48 patients using a grading system based on the integrity of the hyperreflective photoreceptor band, the inner segment ellipsoid zone (ISe). Signs of progression were evaluated based on longitudinal data and correlation with age. RESULTS: We found a statistically significant positive correlation between OCT grade and age (Spearman ρ = 0.62, p < 0.0001) and we observed changes in the foveal morphology in 2 of 11 patients with ≥5 years of follow-up. We did not find any convincing correlation between age and functional parameters (visual acuity, retinal sensitivity and contrast sensitivity) nor did we find correlation between structural and functional parameters, or any clear genotype-phenotype correlation. CONCLUSIONS: Some patients with ACHM demonstrate signs of progressive foveal changes in OCT characteristics with increasing age. This is relevant in terms of possible new treatments. However, functional characteristics, such as visual acuity, remained stable despite changing foveal structure. Thus, seen from a patient perspective, ACHM can still be considered a non-progressive condition.
Assuntos
Defeitos da Visão Cromática , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Eletrorretinografia , Fenótipo , Tomografia de Coerência Óptica , Acuidade Visual , Humanos , Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/fisiopatologia , Defeitos da Visão Cromática/diagnóstico , Masculino , Tomografia de Coerência Óptica/métodos , Feminino , Acuidade Visual/fisiologia , Adulto , Dinamarca , Adolescente , Adulto Jovem , Criança , Pessoa de Meia-Idade , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Mutação , Testes de Campo Visual , Sensibilidades de Contraste/fisiologia , Seguimentos , Angiofluoresceinografia/métodos , Proteínas do Olho/genética , Guanilato Ciclase/genética , Campos Visuais/fisiologia , Estudos de Associação Genética , Análise Mutacional de DNA , DNA/genética , Visão de Cores/fisiologiaAssuntos
Anticorpos Monoclonais Humanizados , Proteínas Adaptadoras de Sinalização CARD , Guanilato Ciclase , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Guanilato Ciclase/genética , Proteínas de Membrana/genética , Fenótipo , Dermatopatias Papuloescamosas/tratamento farmacológico , Dermatopatias Papuloescamosas/patologia , Dermatopatias Papuloescamosas/genética , Fármacos Dermatológicos/uso terapêutico , Masculino , FemininoRESUMO
Pityriasis rubra pilaris (PRP) is a rare papulosquamous reaction pattern with a significant impact on quality of life. Type I PRP is the most common PRP variant, presenting as erythematous papules emerging in a follicular distribution and later coalescing into plaques with characteristic islands of sparing; histologically, an alternating pattern of orthokeratosis and parakeratosis is considered the hallmark of PRP (checkerboard hyperkeratosis). Other PRP variants (types II-V) differ in their age of onset and clinical presentation. Type VI PRP is a rare PRP subtype associated with human immunodeficiency virus infection and is occasionally associated with diseases of the follicular occlusion tetrad. Caspase recruitment domain family, member 14 (CARD14)-associated papulosquamous eruption and facial discoid dermatitis are newly described disease states that have an important clinical overlap with PRP, creating shared conundrums with respect to diagnosis and treatment. The etiology inciting PRP often remains uncertain; PRP has been suggested to be associated with infection, malignancy, or drug/vaccine administration in some cases, although these are based on case reports and causality has not been established. Type V PRP is often due to inborn CARD14 mutations. Furthermore, recent literature has identified interleukin-23/T-helper-17 cell axis dysregulation to be a major mediator of PRP pathogenesis, paving the way for mechanism-directed therapy. At present, high-dose isotretinoin, ixekizumab, and secukinumab are systemic agents supported by single-arm prospective studies; numerous other agents have also been trialed for PRP, with variable success rates. Here, we discuss updates on clinical manifestations, present new insights into etiopathogenesis, and offer a survey of recently described therapeutic options.
Assuntos
Pitiríase Rubra Pilar , Humanos , Pitiríase Rubra Pilar/diagnóstico , Pitiríase Rubra Pilar/etiologia , Pitiríase Rubra Pilar/terapia , Estudos Prospectivos , Qualidade de Vida , Isotretinoína/uso terapêutico , Mutação , Guanilato Ciclase/genética , Proteínas de Membrana/genética , Proteínas Adaptadoras de Sinalização CARD/genéticaRESUMO
Recent advances in medical genetics elucidated the background of diseases characterized by superficial dermal and epidermal inflammation with resultant aberrant keratosis. This led to introducing the term autoinflammatory keratinization diseases encompassing entities in which monogenic mutations cause spontaneous activation of the innate immunity and subsequent disruption of the keratinization process. Originally, autoinflammatory keratinization diseases were attributed to pathogenic variants of CARD14 (generalized pustular psoriasis with concomitant psoriasis vulgaris, palmoplantar pustulosis, type V pityriasis rubra pilaris), IL36RN (generalized pustular psoriasis without concomitant psoriasis vulgaris, impetigo herpetiformis, acrodermatitis continua of Hallopeau), NLRP1 (familial forms of keratosis lichenoides chronica), and genes of the mevalonate pathway, i.e., MVK, PMVK, MVD, and FDPS (porokeratosis). Since then, endotypes underlying novel entities matching the concept of autoinflammatory keratinization diseases have been discovered (mutations of JAK1, POMP, and EGFR). This review describes the concept and pathophysiology of autoinflammatory keratinization diseases and outlines the characteristic clinical features of the associated entities. Furthermore, a novel term for NLRP1-associated autoinflammatory disease with epithelial dyskeratosis (NADED) describing the spectrum of autoinflammatory keratinization diseases secondary to NLRP1 mutations is proposed.
Assuntos
Ceratose , Psoríase , Humanos , Psoríase/genética , Psoríase/patologia , Inflamação/genética , Mutação , Imunidade Inata , Guanilato Ciclase/genética , Proteínas de Membrana , Proteínas Adaptadoras de Sinalização CARD/genética , Interleucinas/genéticaRESUMO
Abstract BACKGROUND: Recent mutation analysis identified several missense mutations in CARD14 in psoriasis. OBJECTIVES: We performed the genomic sequence analysis on CARD14 in southern Chinese Han Cantonese with Psoriasis Vulgaris (PsV) to reveal more causative missense mutations. METHODS: A total of 131 patients with PsV and 207 matched controls were included. We conducted sequence analysis of all the exon and exon-intron boundaries of CARD14 in the group of PsV patients and subsequent case control analysis of potential sequence variants of significance. RESULTS: We found five rare mutations and four of them are annotated or reported. Only the variant (c.1291C>G) has not been reported and annotated, but the variant was also found in controls. No significant difference was detected among all rare variant allele frequencies of patients and controls. CONCLUSION: None of the new definite variants were pathogenic. The other pathogenic mutations for PsV are still elusive in our cohort.
Assuntos
Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Proteínas Adaptadoras de Sinalização CARD/genética , Guanilato Ciclase/genética , Mutação de Sentido Incorreto , Proteínas de Membrana/genética , Psoríase/genética , Análise de Sequência de DNA , Povo Asiático/genética , Estudos de Casos e Controles , China , Estudos de Coortes , Frequência do Gene , Técnicas de Genotipagem , Valor Preditivo dos TestesRESUMO
PURPOSE: The expression of natriuretic peptides in the neural bundles of the anterior portion of the optic nerves and their functions in regulating vessel tone and blood flow may suggest a possible role in the pathogenesis of glaucoma. The purpose of this study was to investigate the association between normal-tension glaucoma and the genetic variations of atrial natriuretic peptide (Nppa) and natriuretic peptide receptor A (Npr1) gene. METHODS: Sixty-seven Korean normal-tension glaucoma (NTG) patients and 100 healthy subjects (as normal controls) were enrolled. DNA from peripheral blood leukocytes was extracted, and the genotypes of five polymorphisms (c.94G>A, c.454T>C, IVS1+16C>T, IVS2+701G>A, and c.-764C>G) in the Nppa gene and one polymorphism (c.1023G>C) in the Npr1 gene were determined using the restriction fragment length polymorphism and the SNaPshot methods. The genotype and allele frequencies of these polymorphisms in patients with NTG and normal controls were compared using the Fisher's exact test and the chi-square test. RESULTS: In both groups, the genotype distributions were in accordance with the Hardy-Weinberg equilibrium. There was no significant difference in the frequency of the Nppa and Npr1 alleles or genotypes in the normal-tension glaucoma group as compared to the control group. CONCLUSIONS: Nppa and Npr1 gene polymorphisms are not associated with normal-tension glaucoma, suggesting that this gene does not have an important role in the pathogenesis of optic neuropathy in this disease.