Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.595
Filtrar
1.
Nat Commun ; 10(1): 3084, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300652

RESUMO

Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.


Assuntos
Proteínas do Domínio Armadillo/ultraestrutura , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Bovinos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X
2.
PLoS Genet ; 15(6): e1008219, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31242183

RESUMO

Fes1 is a conserved armadillo repeat-containing Hsp70 nucleotide exchange factor important for growth at high temperature, proteasomal protein degradation and prion propagation. Depleting or mutating Fes1 induces a stress response and causes defects in these processes that are ascribed solely to disruption of Fes1 regulation of Hsp70. Here, we find Fes1 was essential for degradation of gluconeogenic enzymes by the vacuole import and degradation (Vid) pathway and for cell wall integrity (CWI), which is crucial for growth at high temperature. Unexpectedly, Fes1 mutants defective in physical or functional interaction with Hsp70 retained activities that support Vid and CWI. Fes1 and the Fes1 mutants bound to the Vid substrate Fbp1 in vitro and captured Slt2, a signaling kinase that regulates CWI, from cell lysates. Our data show that the armadillo domain of Fes1 binds proteins other than Hsp70, that Fes1 has important Hsp70-independent roles in the cell, and that major growth defects caused by depleting Fes1 are due to loss of these functions rather than to loss of Hsp70 regulation. We uncovered diverse functions of Fes1 beyond its defined role in regulating Hsp70, which points to possible multi-functionality among its conserved counterparts in other organisms or organelles.


Assuntos
Parede Celular/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas de Saccharomyces cerevisiae/genética , Citosol/metabolismo , Gluconeogênese/genética , Glucose/genética , Glucose/metabolismo , Proteínas de Choque Térmico HSP70/genética , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/genética , Vacúolos/genética
3.
PLoS Genet ; 15(6): e1007960, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233487

RESUMO

UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Netrinas/genética , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/genética , Animais , Orientação de Axônios/genética , Axônios/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Movimento Celular/genética , Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/metabolismo , Microtúbulos/genética , Fatores de Crescimento Neural/genética , Fenótipo , Pseudópodes/genética , Receptores de Superfície Celular/genética , Transdução de Sinais/genética
4.
Nat Commun ; 10(1): 2506, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175295

RESUMO

Although there are many known Mendelian genes linked to epileptic or developmental and epileptic encephalopathy (EE/DEE), its genetic architecture is not fully explained. Here, we address this incompleteness by analyzing exomes of 743 EE/DEE cases and 2366 controls. We observe that damaging ultra-rare variants (dURVs) unique to an individual are significantly overrepresented in EE/DEE, both in known EE/DEE genes and the other non-EE/DEE genes. Importantly, enrichment of dURVs in non-EE/DEE genes is significant, even in the subset of cases with diagnostic dURVs (P = 0.000215), suggesting oligogenic contribution of non-EE/DEE gene dURVs. Gene-based analysis identifies exome-wide significant (P = 2.04 × 10-6) enrichment of damaging de novo mutations in NF1, a gene primarily linked to neurofibromatosis, in infantile spasm. Together with accumulating evidence for roles of oligogenic or modifier variants in severe neurodevelopmental disorders, our results highlight genetic complexity in EE/DEE, and indicate that EE/DEE is not an aggregate of simple Mendelian disorders.


Assuntos
Variação Genética , Espasmos Infantis/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , DNA (Citosina-5-)-Metiltransferases/genética , Epilepsias Mioclônicas/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Lactente , Japão , Síndrome de Lennox Gastaut/genética , Modelos Logísticos , Mutação , Neurofibromina 1/genética , Polimorfismo de Nucleotídeo Único , Análise de Componente Principal , Canais de Cátion TRPM/genética , Sequenciamento Completo do Exoma
5.
Iran J Allergy Asthma Immunol ; 18(2): 225-229, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066259

RESUMO

Hyperimmunoglobulin E syndrome (HIGE) is considered as a phagocytic or a newly classified complex and heterogeneous primary immunodeficiency disease with symptoms such as increased levels of immunoglobulin E, eczema, and, recurrent lung and skin infections. In this paper, we have presented a rare case of this syndrome. A 9-year-old Iranian girl presented with a history of pruritic maculopapular rash who was eventually diagnosed as a case of HIGE. In her recent admission, she had dysphonia, stridor and huge cauliflower cutaneous lesions on her neck, finger and vocal cords, which did not respond to intravenous antibiotics, and ultimately required surgical removal.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Herpes Simples/diagnóstico , Imunoglobulinas Intravenosas/uso terapêutico , Síndrome de Job/diagnóstico , Mutação/genética , Simplexvirus/fisiologia , Pele/patologia , Prega Vocal/patologia , Antibacterianos/uso terapêutico , Criança , Resistência a Medicamentos , Disfonia , Feminino , Herpes Simples/tratamento farmacológico , Humanos , Imunoglobulina E/metabolismo , Síndrome de Job/tratamento farmacológico , Laringoscopia , Sons Respiratórios , Pele/virologia
6.
Genome Biol Evol ; 11(6): 1679-1690, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31124564

RESUMO

Polymorphic duplications in humans have been shown to contribute to phenotypic diversity. However, the evolutionary forces that maintain variable duplications across the human genome are largely unexplored. We developed a linkage-disequilibrium based method to detect insertion sites of polymorphic duplications not represented in reference genomes. This method also allows resolution of haplotypes harboring the duplications. Using this approach, we conducted genome-wide analyses and identified the insertion sites of 22 common polymorphic duplications. We found that the majority of these duplications is intrachromosomal and only one of them is an interchromosomal insertion. Further characterization of these duplications revealed significant associations to blood and skin phenotypes. On the basis of population genetics analyses, we found that the duplication of a well-characterized pigmentation-related region, including the HERC2 gene, may be selected against in European populations. We further demonstrated that the haplotype harboring this duplication significantly affects the expression of the HERC2P9 gene in multiple tissues. Our study sheds light onto the evolutionary impact of understudied polymorphic duplications in human populations and presents methodological insights for future studies.


Assuntos
Duplicação Gênica , Genoma Humano , Fatores de Troca do Nucleotídeo Guanina/genética , Polimorfismo Genético , Animais , Projeto Genoma Humano , Humanos , Pan troglodytes/genética , Polimorfismo de Nucleotídeo Único , Seleção Genética
7.
Nat Protoc ; 14(6): 1863-1883, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076662

RESUMO

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/efeitos da radiação , Engenharia de Proteínas/métodos , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/efeitos da radiação , Animais , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/efeitos da radiação , Linhagem Celular , Clonagem Molecular/métodos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Luz , Camundongos , Modelos Moleculares , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sirolimo/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
8.
Mamm Genome ; 30(3-4): 54-62, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31020388

RESUMO

Spermatogenesis-associated protein 13 (Spata13) is a guanine nucleotide exchange factor (GEF) enriched in discrete brain regions in the adult, with pronounced expression in the extended central amygdala (CeA). Loss of Spata13, also known as the adenomatous polyposis coli exchange factor Asef2, has no identifiable phenotype although it has been shown to reduce the number and size of intestinal tumours in Apc (Min/+) mice. Nevertheless, its brain-related functions have not been investigated. To pursue this, we have generated a Spata13 knockout mouse line using CRISPR-mediated deletion of an exon containing the GTPase domain that is common to multiple isoforms. Homozygous mutants were viable and appeared normal. We subjected both male and female cohorts to a comprehensive battery of behavioural tests designed to investigate particular CeA-related functions. Here, we show that Spata13 modulates social behaviour with homozygous mutants being subordinate to wildtype controls. Furthermore, female homozygotes show increased activity in home cages during the dark phase of the light-dark cycle. In summary, Spata13 modulates social hierarchy in both male and female mice in addition to affecting voluntary activity in females.


Assuntos
Ritmo Circadiano/efeitos da radiação , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Comportamento Social , Animais , Comportamento Animal/efeitos da radiação , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Hierarquia Social , Humanos , Masculino , Camundongos , Camundongos Knockout , Fotoperíodo , Predomínio Social
9.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965557

RESUMO

The regulator of chromosome condensation 1 (RCC1) is the nucleotide exchange factor for a GTPase called the Ras-related nuclear protein, and it is important for nucleo-plasmic transport, mitosis, nuclear membrane assembly, and control of chromatin agglutination during the S phase of mitosis in animals. In plants, RCC1 molecules act mainly as regulating factors for a series of downstream genes during biological processes such as the ultraviolet-B radiation (UV-B) response and cold tolerance. In this study, 56 genes were identified in upland cotton by searching the associated reference genomes. The genes were found to be unevenly distributed on 26 chromosomes, except A06, A12, D03, and D12. Phylogenetic analysis by maximum-likelihood revealed that the genes were divided into five subgroups. The RCC1 genes within the same group shared similar exon/intron patterns and conserved motifs in their encoded proteins. Most genes of the RCC1 family are expressed differently under various hormone treatments and are negatively controlled by salt stress. Gh_A05G3028 and Gh_D10G2310, which encode two proteins located in the nucleus, were strongly induced under salt treatment, while mutants of their homoeologous gene (UVR8) in Arabidopsis and VIGS (virus induced gene silencing) lines of the two genes above in G. hirsutum exhibited a salt-sensitive phenotype indicating their potential role in salt resistance in cotton. These results provide valuable reference data for further study of RCC1 genes in cotton.


Assuntos
Gossypium/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Plantas/metabolismo , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genoma de Planta/genética , Gossypium/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(4): 348-351, 2019 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-30950023

RESUMO

OBJECTIVE: To detect pathogenic mutation of DOCK6 gene in a patient with convulsive seizure and refractory epilepsy. METHODS: CytoScan HD-Array and next generation sequencing were used to detect the potential mutation in the patient. RESULTS: The proband has carried compound heterozygous mutations of c.188C>T (p.Arg63Gln) and c.5374C>T (p.Glu1792Lys) of the DOCK6 gene, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis indicated that the two amino acids are highly conserved. Based on the ACMG guidelines, the c.188C>T mutation was predicted to be likely pathogenic, while the c.5374C>T mutation was of uncertain significance. CONCLUSION: The compound heterozygous mutations of c.188C>T (p.Arg63Gln) and c.5374C>T (p.Glu1792Lys) of the DOCK6 gene probably underlie the disease in this patient.


Assuntos
Diabetes Mellitus Tipo 2 , Displasia Ectodérmica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Deformidades Congênitas dos Membros/genética , Dermatoses do Couro Cabeludo/congênito , Criança , Humanos , Mutação , Linhagem , Dermatoses do Couro Cabeludo/genética
11.
Genes Cells ; 24(5): 390-402, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30929300

RESUMO

Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Queratinas/metabolismo , Mecanotransdução Celular , Animais , Sítios de Ligação , Cães , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Mutação , Ligação Proteica
12.
Cancer Sci ; 110(6): 2004-2013, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980774

RESUMO

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.


Assuntos
Autoanticorpos/imunologia , Neoplasias Esofágicas/imunologia , Carcinoma de Células Escamosas do Esôfago/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Processamento de RNA/imunologia , Proteínas Repressoras/imunologia , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/diagnóstico , Carcinoma de Células Escamosas do Esôfago/genética , Éxons/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de RNA , Fatores de Processamento de RNA/genética , Curva ROC , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/imunologia
13.
Allergol. immunopatol ; 47(2): 152-158, mar.-abr. 2019. graf, tab
Artigo em Espanhol | IBECS | ID: ibc-180803

RESUMO

Introduction and objectives: Long-term follow up of patients with hyper IgE syndrome (HIES), as a primary immunodeficiency disorder, has been poorly investigated. This study describes common clinical and immunological features of patients with HIES in the last 10 years in Shiraz University of Medical Sciences, Shiraz, Iran. Methods and patients: In this cross-sectional study, the symptoms and medical records of 18 patients, who were diagnosed with HIES, were observed. Genetic and immunologic study was also performed. Results: Eighteen patients with the mean age of 13 years old were investigated. Ten patients were detected to have mutations in DOCK8 gene and autosomal recessive HIES (AR-HIES); and four patients were found with STAT3 mutation and autosomal dominant HIES (AD-HIES). So, 14 patients with known genetic results were considered for further data analysis. Food allergy, eczema, viral and skin infections were the major complications of AR-HIES patients. The major clinical complications of AD-HIES patients were pneumonia, skin infections and eczema. Food allergy and viral infection were significantly higher in DOCK8 deficient patients. The most common causes of hospitalization in both AR-HIES and AD-HIES patients were pneumonia, skin infections and sepsis. The most common cause of death was found to be sepsis. Conclusions; AD-HIES and AR-HIES cannot be differentiated only based on the clinical presentations. Genetic features are also necessary for better diagnosis. This study, summarizing the clinical, immunological and genetic information of the patients with AD-HIES and AR-HIES, may open a way for better diagnosis and management of HIES


No disponible


Assuntos
Humanos , Masculino , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Adulto , Hipersensibilidade Alimentar/imunologia , Síndrome de Job/imunologia , Mutação/genética , Pneumonia/imunologia , Seguimentos , Hipersensibilidade Alimentar/genética , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina/genética , Síndrome de Job/genética , Fenótipo , Pneumonia/genética , Fator de Transcrição STAT3/genética
14.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897705

RESUMO

Cardiac ß2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The ß2AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates the generation of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory cytokine, which also mediates fibrosis in several tissues, including the heart. OPN underlies several cardiovascular pathologies, including atherosclerosis and cardiac adverse remodeling. We found that the cardiotoxic hormone aldosterone transcriptionally upregulates OPN in H9c2 rat cardiac myoblasts-an effect prevented by endogenous ß2AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both ß1AR and ß2AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered ß2AR stimulation capable of completely abrogating transforming growth factor (TGF)-ß-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gαs subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac ß2AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.


Assuntos
AMP Cíclico/metabolismo , Fibrose/metabolismo , Miócitos Cardíacos/metabolismo , Osteopontina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Western Blotting , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , AMP Cíclico/genética , Fibrose/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imunoprecipitação , Osteopontina/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Adrenérgicos beta/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
15.
Pediatr Hematol Oncol ; 36(1): 28-39, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30888230

RESUMO

A major problem of hemophilia A (HA) treatment is the development of factor VIII (FVIII) inhibitor, which usually occurs shortly after initiating replacement therapy. Several studies showed the correlation between inhibitor development and polymorphisms in inflammatory and immune response genes of HA patients; however, literature data are not available to prove this association in Iranian population. The aim of this study was to investigate a possible association between FVIII inhibitor formation and the polymorphisms of 16 inflammatory and immune response genes in Iranian severe HA patients (FVIII activity < 1%). This case-control study was performed on 55 patients with severe HA inhibitors and 45 samples without inhibitors from Iranian Comprehensive Hemophilia Care center. After extraction of whole genomic DNA from blood samples and design of primers for 16 genes, the genotyping was performed by Tetra primer ARMS PCR, and the validation of single nucleotide polymorphisms was determined by DNA sequencing. The data indicated that there was a significant association between inhibitor development, and F13A1 (TT), DOCK2 (CC& CT), and MAPK9 (TT) genotypes. Moreover, a considerably increased inhibitor risk carrying T, C, and T allele for F13A1, DOCK2, and MAPK9 genes was observed in patients with inhibitors, respectively. In contrast, there was no statistically significant difference between the genotypic and allelic frequencies for other genes in patients with inhibitors compared to patients without inhibitors. These results demonstrate that only polymorphisms in F13A1, DOCK2, and MAPK9 genes are associated with the risk of developing FVIII inhibitors in Iranian HA patients.


Assuntos
Alelos , Inibidores dos Fatores de Coagulação Sanguínea/genética , Frequência do Gene , Fatores de Troca do Nucleotídeo Guanina/genética , Hemofilia A/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Polimorfismo Genético , Adulto , Fator VIII/genética , Humanos , Irã (Geográfico) , Masculino
16.
Platelets ; 30(4): 535-539, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30849270

RESUMO

This manuscript reviews pathogenic variants in RASGRP2, which are the cause of a relatively new autosomal recessive and nonsyndromic inherited platelet function disorder, referred to as platelet-type bleeding disorder-18 (BDPLT18)(OMIM:615888). To date, 18 unrelated BDPLT18 pedigrees have been reported, harboring 19 different homozygous or compound heterozygous RASGRP2 variants. Patients with this disease present with lifelong moderate to severe bleeding, with epistaxis as the most common and relevant bleeding symptom. Biologically, they exhibit normal platelet count and morphology, reduced aggregation responses to ADP, epinephrine and low-dose collagen, and impaired αIIbß3 integrin activation (fibrinogen or PAC-1 binding) in response to most agonists except PMA. Diagnosis is confirmed by genetic analysis of RASGRP2.


Assuntos
Transtornos Plaquetários/genética , Plaquetas/patologia , Variação Genética/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Hemorragia/genética , Transtornos Plaquetários/metabolismo , Hemorragia/metabolismo , Humanos
17.
Gene ; 700: 65-69, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30898718

RESUMO

INTRODUCTION: Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the combination of aplasia cutis congenita of the scalp vertex and terminal transverse limb defects. DOCK6 (Dedicator of cytokinesis 6) is one of the six identified AOS genes. METHODS: We performed targeted next-generation sequencing (NGS) of a child with an AOS phenotype. Sanger DNA sequencing further validated her lineal consanguinity. To explore the pathological features of the mutation, a minigene assay was used to investigate the effects of the mutation on splicing. RESULTS: Two compound heterozygous DOCK6 mutations (c.4106+2T>C and c.3063 C>G (p.Y1021*)) were identified in this family, and both mutations have not been reported previously. Sanger DNA sequencing indicated that the mutations were inherited maternally and paternally, respectively. The results of the minigene assay showed that the c.4106+2T>C mutation resulted in aberrant splicing and caused a four-nucleotide insertion in the transcript and a premature stop codon. CONCLUSIONS: Our findings expanded the number of reported cases of this rare disease and the mutation spectrum of DOCK6 mutations, which can serve as the basis for prenatal diagnosis and genetic counseling.


Assuntos
Displasia Ectodérmica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Deformidades Congênitas dos Membros/genética , Mutação , Dermatoses do Couro Cabeludo/congênito , Análise de Sequência de DNA/métodos , Pré-Escolar , Consanguinidade , Feminino , Genes Recessivos , Heterozigoto , Humanos , Masculino , Linhagem , Fenótipo , Dermatoses do Couro Cabeludo/genética
18.
PLoS Genet ; 15(2): e1007964, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30817801

RESUMO

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Padrões de Herança , Região do Complexo-t do Genoma , Alelos , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Herança Paterna , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Motilidade Espermática/genética , Motilidade Espermática/fisiologia , Espermatogênese/genética
19.
Int J Mol Sci ; 20(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866497

RESUMO

Prostate cancer (PCa) is a clinically heterogeneous disease and currently, accurate diagnostic and prognostic molecular biomarkers are lacking. This study aimed to identify novel DNA hypermethylation markers for PCa with future potential for blood-based testing. Accordingly, to search for genes specifically hypermethylated in PCa tissue samples and not in blood cells or other cancer tissue types, we performed a systematic analysis of genome-wide DNA methylation data (Infinium 450K array) available in the Marmal-aid database for 4072 malignant/normal tissue samples of various types. We identified eight top candidate markers (cg12799885, DOCK2, FBXO30, GRASP, HIF3A, MOB3B, PFKP, and TPM4) that were specifically hypermethylated in PCa tissue samples and hypomethylated in other benign and malignant tissue types, including in peripheral blood cells. Potential as diagnostic and prognostic biomarkers was further assessed by the quantitative methylation specific PCR (qMSP) analysis of 37 nonmalignant and 197 PCa tissue samples from an independent population. Here, all eight hypermethylated candidates showed high sensitivity (75⁻94%) and specificity (84⁻100%) for PCa. Furthermore, DOCK2, GRASP, HIF3A and PKFP hypermethylation was significantly associated with biochemical recurrence (BCR) after radical prostatectomy (RP; 197 patients), independent of the routine clinicopathological variables. DOCK2 is the most promising single candidate marker (hazard ratio (HR) (95% confidence interval (CI)): 1.96 (1.24⁻3.10), adjusted p = 0.016; multivariate cox regression). Further validation studies are warranted and should investigate the potential value of these hypermethylation candidate markers for blood-based testing also.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/genética , Metilação de DNA , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Membrana/genética , Fosfofrutoquinase-1 Tipo C/genética , Neoplasias da Próstata/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Epigênese Genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Sensibilidade e Especificidade , Análise de Sobrevida
20.
Proc Natl Acad Sci U S A ; 116(8): 2967-2976, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30728292

RESUMO

ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.


Assuntos
Transição Epitelial-Mesenquimal/genética , Proteína Forkhead Box O1/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Ativação Transcricional/genética , Proteínas rac1 de Ligação ao GTP/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA