Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.112
Filtrar
1.
BMC Gastroenterol ; 21(1): 339, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470610

RESUMO

AIM: To discover the novel ATP7B mutations in 103 southern Chinese patients with Wilson disease (WD), and to determine the spectrum and frequency of mutations in the ATP7B gene and genotype-phenotype correlation in a large-scale sample of Chinese WD patients. METHODS: One hundred three WD patients from 101 unrelated families in southern China were enrolled in this study. Genomic DNA was extracted from the peripheral blood. Direct sequencing of all 21 exons within ATP7B was performed. Subsequently, an extensive study of the overall spectrum and frequency of ATP7B mutations and genotype-phenotype correlation was performed in all Chinese patients eligible from the literature, combined with the current southern group. RESULTS: In 103 patients with WD, we identified 48 different mutations (42 missense mutations, 4 nonsense mutations and 2 frameshifts). Of these, 3 mutations had not been previously reported: c.1510_1511insA, c.2233C>A (p.Leu745Met) and c.3824T>C (p.Leu1275Ser). The c.2333G>T (p.Arg778 Leu) at exon 8, was the most common mutation with an allelic frequency of 18.8%, followed by c.2975C>T (p.Pro992Leu) at exon 13, with an allelic frequency of 13.4%. In the comprehensive study, 233 distinct mutations were identified, including 154 missense mutations, 23 nonsense mutations and 56 frameshifts. Eighty-five variants were identified as novel mutations. The c.2333G>T (p.Arg778 Leu) and c.2975C>T (p.Pro992Leu) were the most common mutations, with allelic frequencies of 28.6% and 13.0%, respectively. Exons 8, 12, 13, 16 and 18 were recognised as hotspot exons. Phenotype-genotype correlation analysis suggested that c.2333G>T (p.Arg778 Leu) was significantly associated with lower levels of serum ceruloplasmin (P = 0.034). c.2975C>T (p.Pro992Leu) was correlated with earlier age of disease onset (P = 0.002). Additionally, we found that the c.3809A>G (p.Asn1270Ser) mutation significantly indicated younger onset age (P = 0.012), and the c.3884C>T (p.Ala1295Val) mutation at exon 18 was significantly associated with hepatic presentation (P = 0.048). Moreover, the patients with mixed presentation displayed the initial WD features at an older onset age than the groups with either liver disease or neurological presentation (P = 0.039, P = 0.015, respectively). No significant difference was observed in the presence of KF rings among the three groups with different clinical manifestations. CONCLUSION: In this study, we identified three novel mutations in 103 WD patients from the southern part of China, which could enrich the previously established mutational spectrum of the ATP7B gene. Moreover, we tapped into a large-scale study of a Chinese WD cohort to characterise the overall phenotypic and genotypic spectra and assess the association between genotype and phenotype, which enhances the current knowledge about the population genetics of WD in China.


Assuntos
Proteínas de Transporte de Cátions , ATPases Transportadoras de Cobre , Degeneração Hepatolenticular , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/genética , China , ATPases Transportadoras de Cobre/genética , Análise Mutacional de DNA , Estudos de Associação Genética , Genótipo , Degeneração Hepatolenticular/genética , Humanos , Mutação
2.
Phytochemistry ; 191: 112911, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34418773

RESUMO

The pleiotropic effects of zinc deficiency on ion homeostasis have already been described in several plants. Tobacco (Nicotiana tabacum) heavy metal ATPases HMA4.1 and HMA4.2 are involved in zinc and cadmium root-to-shoot translocation. In previous research, we have shown that N. tabacum HMA4 RNAi plants and HMA4 double-nonsense mutants exhibit strongly reduced zinc and cadmium levels in leaves as well as stunted growth. In this study, the ionome and transcriptome of these lines were investigated to better characterize the effect of reduced zinc levels and to understand the impaired growth phenotype. We found that, under standard greenhouse fertilization rates, these lines accumulated up to 4- to 6-fold more phosphorus, iron, manganese, and copper than their respective controls. Under field conditions, HMA4 double-mutant plants also exhibited similar accumulation phenotypes, albeit to a lower extent. In both HMA4 RNAi plants and HMA4 mutants, transcription analysis showed a local zinc-deficiency response in leaves as well as an FIT1-mediated iron-deficiency response in roots, likely contributing to iron and manganese uptake at the root level. A phosphate-starvation response involving HHO2 was also observed in HMA4-impaired plant leaves. The high level of phosphorus observed in HMA4-impaired plants is correlated with leaf swelling and necrosis. The upregulation of aquaporin genes is in line with cellular water influx and the observed leaf swelling phenotype. These results highlight the involvement of HMA4 in zinc homeostasis and related regulatory processes that balance the micro- and macroelements in above-ground organs.


Assuntos
Cádmio , Tabaco , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Tabaco/metabolismo , Zinco/metabolismo
3.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205953

RESUMO

The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.


Assuntos
Conexinas/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores Purinérgicos P2X7/genética , Gânglio Trigeminal/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Cultura Primária de Células , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Gânglio Trigeminal/efeitos dos fármacos
4.
Nat Commun ; 12(1): 4292, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257299

RESUMO

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metilação de DNA/genética , Metilação de DNA/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Inativação Gênica
5.
Nat Commun ; 12(1): 3750, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145229

RESUMO

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. To better understand its genetic architecture, we analyze ultra-rare de novo mutations in 354 trios with bipolar disorder. For germline de novo mutations, we find significant enrichment of loss-of-function mutations in constrained genes (corrected-P = 0.0410) and deleterious mutations in presynaptic active zone genes (FDR = 0.0415). An analysis integrating single-cell RNA-sequencing data identifies a subset of excitatory neurons preferentially expressing the genes hit by deleterious mutations, which are also characterized by high expression of developmental disorder genes. In the analysis of postzygotic mutations, we observe significant enrichment of deleterious ones in developmental disorder genes (P = 0.00135), including the SRCAP gene mutated in two unrelated probands. These data collectively indicate the contributions of both germline and postzygotic mutations to the risk of bipolar disorder, supporting the hypothesis that postzygotic mutations of developmental disorder genes may contribute to bipolar disorder.


Assuntos
Adenosina Trifosfatases/genética , Transtorno Bipolar/genética , Exoma/genética , Predisposição Genética para Doença/genética , Adulto , Éxons/genética , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Exoma
6.
Nat Commun ; 12(1): 3483, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108481

RESUMO

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Adenosina Trifosfatases/química , Compostos de Boro/química , Proteínas de Saccharomyces cerevisiae/química , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Compostos de Boro/farmacologia , Resistência a Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Mutação , Nucleotídeos/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
EBioMedicine ; 69: 103436, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34157484

RESUMO

BACKGROUND: Due to the molecular mechanism complexity and heterogeneity of gastric cancer (GC), mechanistically interpretable biomarkers were required for predicting prognosis and discovering therapeutic targets for GC patients. METHODS: Based on a total of 824 GC-specific fitness genes from the Project Score database, LASSOCox regression was performed in TCGA-STAD cohort to construct a GC Prognostic (GCP) model which was then evaluated on 7 independent GC datasets. Targets prioritization was performed in GC organoids. ARGLU1 was selected to further explore the biological function and molecular mechanism. We evaluated the potential of ARGLU1 serving as a promising therapeutic target for GC using patients derived xenograft (PDX) model. FINDINGS: The 9-gene GCP model showed a statistically significant prognostic performance for GC patients in 7 validation cohorts. Perturbation of SSX4, DDX24, ARGLU1 and TTF2 inhibited GC organoids tumor growth. The results of tissue microarray indicated lower expression of ARGLU1 was correlated with advanced TNM stage and worse overall survival. Over-expression ARGLU1 significantly inhibited GC cells viability in vitro and in vivo. ARGLU1 could enhance the transcriptional level of mismatch repair genes including MLH3, MSH2, MSH3 and MSH6 by potentiating the recruitment of SP1 and YY1 on their promoters. Moreover, inducing ARGLU1 by LNP-formulated saRNA significantly inhibited tumor growth in PDX model. INTERPRETATION: Based on genome-wide functional screening data, we constructed a 9-gene GCP model with satisfactory predictive accuracy and mechanistic interpretability. Out of nine prognostic genes, ARGLU1 was verified to be a potential therapeutic target for GC. FUNDING: National Natural Science Foundation of China.


Assuntos
Biomarcadores Tumorais/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Gástricas/genética , Transcriptoma , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Neoplasias Gástricas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
8.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072847

RESUMO

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Lipoproteínas/genética , Complexos Multiproteicos/genética , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Adenosina Trifosfatases/genética , Sequência de Aminoácidos/genética , Proteínas de Bactérias/ultraestrutura , Dimerização , Transferência Ressonante de Energia de Fluorescência , Lipoproteínas/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Multimerização Proteica/genética , Subunidades Proteicas/genética
9.
J Stroke Cerebrovasc Dis ; 30(7): 105847, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33992965

RESUMO

OBJECTIVES: This retrospective study was conducted to analyze the associations between ring finger protein 213 p.R4810K variant, clinical features and long-term outcomes in patients with moyamoya disease (MMD) after encephaloduroarteriosynangiosis treatment. MATERIALS AND METHODS: A total of 2,545 patients with MMD in China were included in this study (median of follow-up duration: 32.00 months). Multiple Cox regression models were used to assess the associations between p.R4810K variant, clinical features and long-term outcomes. RESULTS: For all patients, in multivariate Cox analysis, no association was observed between p.R4810K and long-term outcomes. Pediatric onset (HR, 0.38; 95%CI, 0.25-0.59) and headache (HR, 0.26; 95%CI, 0.08-0.83) were inversely and hypertension (HR, 1.43 95%CI, 1.06-1.94), diabetes (HR, 1.55; 95%CI, 1.00-2.40), bilateral lesions (HR, 2.73; 95%CI, 1.12-6.65) and posterior cerebral artery involvement (HR, 1.44; 95%CI, 1.08-1.90) were positively associated with follow-up stroke (all P < 0.05). Pediatric onset (HR, 0.46; 95%CI, 0.26-0.82) was inversely and hyperlipidemia (HR, 1.83; 95%CI, 1.23-2.73), smoking (HR, 1.86; 95%CI, 1.13-3.07), high Suzuki angiographic stage (HR, 1.71, 95%CI, 1.09-2.70), poor admission neurologic status (HR, 8.93; 95%CI, 6.49-12.29) and follow-up stroke (HR, 8.31; 95%CI, 6.01-11.49) were positively associated with poor neurologic outcome at the last follow-up visit (all P < 0.05). The factors were not consistent in the different groups of age at onset. CONCLUSIONS: In our study, p.R4810K may play no role in long-term outcomes in Chinese MMD. Clinical features including age at onset, initial symptoms, risk factors of stroke, imaging, poor admission neurologic status were associated with poor outcomes in MMD after EDAS.


Assuntos
Adenosina Trifosfatases/genética , Revascularização Cerebral/efeitos adversos , Doença de Moyamoya/cirurgia , Polimorfismo Genético , Complicações Pós-Operatórias/genética , Ubiquitina-Proteína Ligases/genética , Adolescente , Adulto , China , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Doença de Moyamoya/diagnóstico por imagem , Doença de Moyamoya/genética , Complicações Pós-Operatórias/diagnóstico , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
10.
Front Immunol ; 12: 651656, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936072

RESUMO

Although immune dysfunction is a key feature of coronavirus disease 2019 (COVID-19), the metabolism-related mechanisms remain elusive. Here, by reanalyzing single-cell RNA sequencing data, we delineated metabolic remodeling in peripheral blood mononuclear cells (PBMCs) to elucidate the metabolic mechanisms that may lead to the progression of severe COVID-19. After scoring the metabolism-related biological processes and signaling pathways, we found that mono-CD14+ cells expressed higher levels of glycolysis-related genes (PKM, LDHA and PKM) and PPP-related genes (PGD and TKT) in severe patients than in mild patients. These genes may contribute to the hyperinflammation in mono-CD14+ cells of patients with severe COVID-19. The mono-CD16+ cell population in COVID-19 patients showed reduced transcription levels of genes related to lysine degradation (NSD1, KMT2E, and SETD2) and elevated transcription levels of genes involved in OXPHOS (ATP6V1B2, ATP5A1, ATP5E, and ATP5B), which may inhibit M2-like polarization. Plasma cells also expressed higher levels of the OXPHOS gene ATP13A3 in COVID-19 patients, which was positively associated with antibody secretion and survival of PCs. Moreover, enhanced glycolysis or OXPHOS was positively associated with the differentiation of memory B cells into plasmablasts or plasma cells. This study comprehensively investigated the metabolic features of peripheral immune cells and revealed that metabolic changes exacerbated inflammation in monocytes and promoted antibody secretion and cell survival in PCs in COVID-19 patients, especially those with severe disease.


Assuntos
COVID-19/imunologia , Glicólise/genética , Lisina/metabolismo , Monócitos/metabolismo , Análise de Célula Única/métodos , Adenosina Trifosfatases/sangue , Adenosina Trifosfatases/genética , Anticorpos/metabolismo , COVID-19/metabolismo , COVID-19/fisiopatologia , Bases de Dados Genéticas , Proteínas Ligadas por GPI/metabolismo , Ontologia Genética , Hematopoese/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Receptores de Lipopolissacarídeos/metabolismo , Lisina/genética , Proteínas de Membrana Transportadoras/sangue , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Monócitos/imunologia , Monócitos/patologia , Fosforilação Oxidativa , RNA-Seq , Receptores de IgG/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/genética
11.
Science ; 372(6545): 984-989, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34045355

RESUMO

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Evolução Biológica , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Eucariotos/genética , Genoma , Complexos Multiproteicos/genética , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/química , Algoritmos , Animais , Nucléolo Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Centrômero/ultraestrutura , Cromossomos/química , Cromossomos Humanos/química , Cromossomos Humanos/ultraestrutura , Proteínas de Ligação a DNA/química , Genoma Humano , Genômica , Heterocromatina/ultraestrutura , Humanos , Interfase , Mitose , Modelos Biológicos , Complexos Multiproteicos/química , Telômero/ultraestrutura
12.
Vet Microbiol ; 257: 109074, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33940460

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a reemerging Alphacoronavirus that causes lethal diarrhea in piglets. Coronavirus nonstructural protein 13 (nsp13) encodes helicase, which plays pivotal roles during viral replication by unwinding viral RNA. However, the biochemical characterization of PEDV nsp13 remains largely unknown. In this study, PEDV nsp13 was expressed in Escherichia coli and purified. The recombinant nsp13 possessed ATPase and helicase activities for binding and unwinding dsDNA/RNA substrates with 5'-overhangs, and Mg2+ and Mn2+ were critical for its ATPase and helicase activities. PEDV nsp13 also unwound dsDNA into ssDNA in the pH from 6.0-9.0, and used energy from all nucleoside triphosphates and deoxynucleoside triphosphates. Site-directed mutagenesis demonstrated that Lys289 (K289) of PEDV nsp13 was essential for its ATPase and helicase activities. These results provide new insights into the biochemical properties of PEDV nsp13, which is a potential target for developing antiviral drugs.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Vírus da Diarreia Epidêmica Suína/enzimologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , RNA Viral/genética , RNA Viral/metabolismo , Suínos , Doenças dos Suínos/virologia , Células Vero
13.
J Immunol ; 206(9): 1983-1990, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33879578

RESUMO

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes that hydrolyze nucleotides such as ATP, UTP, ADP, and UDP to monophosphates derivates such as AMP and UMP. The NTPDase family consists of eight enzymes, of which NTPDases 1, 2, 3, and 8 are expressed on cell membranes thereby hydrolyzing extracellular nucleotides. Cell membrane NTPDases are expressed in all tissues, in which they regulate essential physiological tissue functions such as development, blood flow, hormone secretion, and neurotransmitter release. They do so by modulating nucleotide-mediated purinergic signaling through P2 purinergic receptors. NTPDases 1, 2, 3, and 8 also play a key role during infection, inflammation, injury, and cancer. Under these conditions, NTPDases can contribute and control the pathophysiology of infectious, inflammatory diseases and cancer. In this review, we discuss the role of NTPDases, focusing on the less understood NTPDases 2-8, in regulating inflammation and immunity during infectious, inflammatory diseases, and cancer.


Assuntos
Adenosina Trifosfatases/genética , Regulação Enzimológica da Expressão Gênica , Imunidade/genética , Inflamação/genética , Família Multigênica , Neoplasias/genética , Adenosina Trifosfatases/metabolismo , Animais , Humanos , Inflamação/enzimologia , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/enzimologia , Nucleotídeos/metabolismo
14.
Comput Biol Chem ; 92: 107488, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33930741

RESUMO

Moyamoya disease (MMD), a cerebrovascular disorder caused by the RNF213 gene, is a cerebrovascular, neurological disorder leading to ischemic strokes. Our previous work suggested that RNF213 might be involved in the pro-inflammatory TNFα-mediated insulin-resistance pathway in adipocytes. Insulin resistance can lead to cerebrovascular diseases and ischemic strokes. Though p. R4810 K has been reported as the founder mutation for Asian population with this disease, there are several mutations continuously reported in clinical diagnosis. We are interested to know whether these mutations can modulate insulin resistance. Also, we are intended to understand the causalities of RNF213 and its associated mutations in MMD. For this, we have adopted a computational approach to characterize RNF213 and its naturally occurring SNPs. Clinically reported SNPs and the predicted SNPs were analyzed for their pathogenicity and effect on the biological function of the protein. To increase accuracy, this was performed through three different analysis software (PROVEAN, SIFT, and SNAP2). The mutations that were found to be deleterious in all the three platforms were further analyzed for their effect on the thermal stability of the protein through I-mutant and iStable. It was found that R4810 K and other mutations decreased the thermodynamic stability of the protein. Loss of function of RNF213 was suggested in some reports. Contrary to this, some studies reported a gain of function state due to the R4810K mutation. To understand this we have measured the ligand-binding ability of this mutated protein through COFACTOR and COACH. An increase in ligand binding is always related to the functional stability of a protein. We have observed that the R4810K mutation might increase the iron-binding efficiency of the amino acid residues. This increase in binding was further validated by analyzing the binding efficiencies by docking. Since RNF213 was previously reported as a target for Protein Tyrosine Phosphatase 1B (PTP1B), we have also analyzed whether PTP1B-binding positions are susceptible to mutations. We have re-analyzed our earlier report on the differential expression pattern of RNF213 in cancer and obese samples. We have provided a detailed analysis of the most deleterious SNPs related to RNF213. Also, we provide a prediction for the loss of function and gain of function attributes of RNF213 and its predicted causalities in MMD and insulin resistance.


Assuntos
Adenosina Trifosfatases/genética , Resistência à Insulina/genética , Doença de Moyamoya/genética , Polimorfismo de Nucleotídeo Único/genética , Ubiquitina-Proteína Ligases/genética , Humanos , Doença de Moyamoya/diagnóstico
15.
J Tradit Chin Med ; 41(2): 276-283, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33825408

RESUMO

OBJECTIVE: To compare and observe the effects of three kinds of cephalic acupuncture therapies commonly used in the clinic on promoting nerve function rehabilitation in the brain microenvironment of rats with cerebral palsy. METHODS: A negative control group, positive control group, and three cephalic acupuncture groups based on the administration of three cephalic acupuncture therapies were established. Ten experimental rats were selected from each group at 1, 2, and 3 weeks after modeling. Neuromotor function after treatment was rated according to the Basso, Beattie, and Bresnahan method. White matter fiber bundles were evaluated by head diffusion tensor imaging. The expression levels of neuron-specific enolase, microtubule-associated protein 2, and myelin basic protein in the brain tissue extract were detected by Western blot analysis and the activities of ATPases were determined using a fixed phosphorus method. RESULTS: The pathological changes in brain tissue were restored and motor function scores were increased in the mice in each cephalic acupuncture group, and the expression of neuronal growth-related proteins in the brain tissue extract was significantly increased. Additionally, the activities of ATPases in the lesion area were significant enhanced (P < 0.05). Diffusion tensor imaging revealed that the white matter fiber bundles of mice in each cephalic acupuncture group gradually increased and recovered. The nervous system structure was significantly improved. CONCLUSIONS: All three acupuncture methods promoted the rehabilitation of nerve function damaged by cerebral palsy. These effects are likely related to the improved expression of nerve growth-related proteins, enhancement of ATPase activities, and regulation of the brain microenvironment.


Assuntos
Terapia por Acupuntura , Paralisia Cerebral/terapia , Pontos de Acupuntura , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Paralisia Cerebral/diagnóstico por imagem , Paralisia Cerebral/fisiopatologia , Paralisia Cerebral/reabilitação , Imagem de Tensor de Difusão , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
16.
Nucleic Acids Res ; 49(8): 4534-4549, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33849072

RESUMO

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/metabolismo , Escherichia coli/metabolismo , Microscopia Eletrônica de Transmissão , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
17.
Am J Hum Genet ; 108(6): 1053-1068, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33909990

RESUMO

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.


Assuntos
Anormalidades Múltiplas/patologia , Adenosina Trifosfatases/genética , Anormalidades Craniofaciais/patologia , Metilação de DNA , Epigênese Genética , Transtornos do Crescimento/patologia , Comunicação Interventricular/patologia , Mutação , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Anormalidades Múltiplas/genética , Estudos de Casos e Controles , Estudos de Coortes , Anormalidades Craniofaciais/genética , Feminino , Predisposição Genética para Doença , Transtornos do Crescimento/genética , Comunicação Interventricular/genética , Humanos , Recém-Nascido , Masculino , Transtornos do Neurodesenvolvimento/genética
18.
Clin Nephrol ; 96(2): 105-111, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33769276

RESUMO

Moyamoya disease (MMD) is the most common underlying disease in Korean pediatric renovascular hypertension (RVH). The ring finger protein 213 (RNF213) p.R4810K variant is reported to be a pathologic variant in East Asian MMD. The purpose of this study was to evaluate hypertension (HTN) prevalence and clinical manifestations as well as RNF213 p.R4810K variant prevalence in Korean pediatric MMD patients. The medical records of pediatric MMD patients from January 2000 to June 2018 were retrospectively reviewed. RVH was confirmed by computer tomography angiography or renal Doppler ultrasonography. The American Academy of Pediatrics 2017 guideline for sex-, age-, and height-related blood pressure standards was used to define HTN. Of 706 patients with MMD, 40 (5.7%) had HTN. Among these patients, 22 had RVH and 12 had HTN with no evidence of renal artery stenosis (non-RVH). Patients with MMD and RVH had an MMD onset at a younger age and lower body mass index compared to those with MMD and non-RVH. Among the patients with MMD and HTN, 4 presented with HTN before developing MMD. Genetic testing for the RNF213 p.R4810K variant was performed in 32 patients with MMD and HTN. When the patient had a homozygous RNF213 p.R4810K variant, the odds ratio of RVH to non-RVH was 8.3. Our study suggests that RVH is more prevalent than non-RVH in pediatric MMD patients. Furthermore, RNF213 p.R4810K may be the cause of RVH in Korean children with MMD.


Assuntos
Adenosina Trifosfatases/genética , Hipertensão Renovascular , Doença de Moyamoya , Ubiquitina-Proteína Ligases/genética , Criança , Feminino , Predisposição Genética para Doença/genética , Humanos , Hipertensão Renovascular/epidemiologia , Hipertensão Renovascular/etiologia , Hipertensão Renovascular/genética , Masculino , Doença de Moyamoya/complicações , Doença de Moyamoya/epidemiologia , Doença de Moyamoya/genética , República da Coreia , Estudos Retrospectivos
19.
Clin Chim Acta ; 518: 43-50, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33713692

RESUMO

BACKGROUND AND AIMS: The Dubin Johnson Syndrome (DJS) occurs mostly in young adults but an early-onset of the disease has been reported in less common forms (Neonatal DJS and Infantile DJS). In this case, the clinical findings are of limit for the DJS diagnosis. Hence, the genetic testing remains the method of choice to provide an accurate diagnosis. In our study, we aimed to perform a genetic analysis for two siblings presented with an intrahepatic cholestasis before the age of 1 year to provide a molecular explanation for the developed phenotype. PATIENTS & METHODS: A Tunisian family, having two siblings, manifesting signs of a hepatopathy, was enrolled in our study. A molecular analysis was performed, using a panel-based next generation sequencing, supplying results that were the subject of computational analysis. Then, a clinical follow-up was carried out to assess the evolution of the disease. RESULTS: The genetic analysis revealed the presence of a novel missense c.4179G > T, (p.M1393I) mutation in ABCC2 gene associated with a substitution c.2789G > A (R930Q) in ATP8B1 gene. Predictive results consolidated the pathogenic effect of both variants. These results confirmed the DJS diagnosis in the studied patients. The clinical course of both patients fit well with the benign nature of DJS. CONCLUSION: We described here a novel ABCC2 mutation associated with a putative ATP8B1 modifier variant. This finding constituted the first report of a complex genotype in DJS. Hence, genetic analysis by a panel-based next generation sequencing permits an accurate diagnosis and the identification of putative variants that could influence the developed phenotype.


Assuntos
Colestase Intra-Hepática , Icterícia Idiopática Crônica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Adenosina Trifosfatases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Icterícia Idiopática Crônica/diagnóstico , Icterícia Idiopática Crônica/genética , Mutação , Fenótipo , Adulto Jovem
20.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670267

RESUMO

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Deleção de Sequência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...