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1.
Clin Transl Sci ; 16(4): 631-646, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36631939

RESUMO

The severe acute respiratory syndrome coronavirus 2, the agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has spread worldwide since it was first identified in November 2019 in Wuhan, China. Since then, progress in pathogenesis linked severity of this systemic disease to the hyperactivation of network of cytokine-driven pro-inflammatory cascades. Here, we aimed to identify molecular biomarkers of disease severity by measuring the serum levels of inflammatory mediators in a Brazilian cohort of patients with COVID-19 and healthy controls (HCs). Critically ill patients in the intensive care unit were defined as such by dependence on oxygen supplementation (93% intubated and 7% face mask), and computed tomography profiles showing ground-glass opacity pneumonia associated to and high levels of D-dimer. Our panel of mediators included HMGB1, ATP, tissue factor, PGE2 , LTB4 , and cys-LTs. Follow-up studies showed increased serum levels of every inflammatory mediator in patients with COVID-19 as compared to HCs. Originally acting as a transcription factor, HMGB1 acquires pro-inflammatory functions following secretion by activated leukocytes or necrotic tissues. Serum levels of HMGB1 were positively correlated with cys-LTs, D-dimer, aspartate aminotransferase, and alanine aminotransferase. Notably, the levels of the classical alarmin HMGB1 were higher in deceased patients, allowing their discrimination from patients that had been discharged at the early pulmonary and hyperinflammatory phase of COVID-19. In particular, we verified that HMGB1 levels above 125.4 ng/ml is the cutoff that distinguishes patients that are at higher risk of death. In conclusion, we propose the use of serum levels of HMGB1 as a biomarker of severe prognosis of COVID-19.


Assuntos
COVID-19 , Proteína HMGB1 , Humanos , Tromboplastina , COVID-19/diagnóstico , Biomarcadores , Prognóstico , Lipídeos , Trifosfato de Adenosina
2.
PLoS Negl Trop Dis ; 16(6): e0010559, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35759510

RESUMO

Epigenetic mechanisms are responsible for a wide range of biological phenomena in insects, controlling embryonic development, growth, aging and nutrition. Despite this, the role of epigenetics in shaping insect-pathogen interactions has received little attention. Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we explored the role of the Aedes aegypti histone acetyltransferase CBP (AaCBP) after infection with Zika virus (ZIKV), focusing on the two main immune tissues, the midgut and fat body. We showed that the expression and activity of AaCBP could be positively modulated by blood meal and ZIKV infection. Nevertheless, Zika-infected mosquitoes that were silenced for AaCBP revealed a significant reduction in the acetylation of H3K27 (CBP target marker), followed by downmodulation of the expression of immune genes, higher titers of ZIKV and lower survival rates. Importantly, in Zika-infected mosquitoes that were treated with sodium butyrate, a histone deacetylase inhibitor, their capacity to fight virus infection was rescued. Our data point to a direct correlation among histone hyperacetylation by AaCBP, upregulation of antimicrobial peptide genes and increased survival of Zika-infected-A. aegypti.


Assuntos
Aedes , Infecção por Zika virus , Zika virus , Aedes/genética , Animais , Epigênese Genética , Histona Acetiltransferases/genética , Histonas/genética , Mosquitos Vetores , Zika virus/fisiologia
3.
PLoS Negl Trop Dis ; 14(7): e0008332, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609727

RESUMO

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/ultraestrutura , Esquistossomose mansoni/tratamento farmacológico , Animais , Anti-Helmínticos/farmacologia , Resistência a Medicamentos , Microscopia Eletrônica de Varredura , Oviposição/efeitos dos fármacos , Praziquantel/farmacologia , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/patologia
4.
Plos Negl Trop Dis, v. 14, n. 7, e0008332, jul. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3085

RESUMO

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.

5.
Gene ; 671: 152-160, 2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-29859286

RESUMO

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.


Assuntos
Aedes/crescimento & desenvolvimento , Proteínas HMGB/química , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Aedes/genética , Aedes/metabolismo , Animais , Núcleo Celular/metabolismo , Dicroísmo Circular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Modelos Moleculares , Fosforilação , Estrutura Secundária de Proteína , Distribuição Tecidual
6.
Plos Neglect. Trop. Dis. ; 12(10): e0006873, 2018.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15675

RESUMO

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Author summary

7.
Insect Biochem Mol Biol ; 80: 32-41, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27867076

RESUMO

High Mobility Group protein 1 (HMGB1) is a non-histone, chromatin-associated nuclear protein that functions in regulating eukaryotic gene expression. We investigated the influence and mechanism of action of Aedes aegypti HMGB1 (AaHMGB1) on mosquito Rel1A-mediated transcription from target gene promoters. The DNA-binding domain (RHD) of AaRel1A was bacterially expressed and purified, and AaHMGB1 dramatically enhanced RHD binding to consensus NF-kB/Rel DNA response elements. Luciferase reporter analyses using a cecropin gene promoter showed that AaHMGB1 potentiates the transcriptional activity of AaRel1A in Aag-2 cells. Moreover, overexpression of AaHMGB1 in Aag-2 cells led to an increase in mRNA levels of antimicrobial peptide genes. In vitro GST pull-down assays revealed that the presence of DNA is a pre-requisite for assembly of a possible ternary complex containing DNA, AaHMGB1 and AaRel1A. Notably, DNA bending by AaHMGB1 enhanced the binding of AaRel1A to a DNA fragment containing a putative NF-kB/Rel response element. Importantly, AaHMGB1 was identified as a potential immune modulator in A. aegypti through AaHMGB1 overexpression or RNAi silencing in Aag-2 cells followed by bacterial challenge or through AaHMGB1 RNAi knockdown in mosquitoes followed by Dengue virus (DENV) infection. We propose a model in which AaHMGB1 bends NF-kB/Rel target DNA to recruit and allow more efficient AaRel1A binding to activate transcription of effector genes, culminating in a stronger Toll pathway-mediated response against DENV infection.


Assuntos
Aedes/genética , Proteína HMGB1/genética , Proteínas de Insetos/genética , Chaperonas Moleculares/genética , Aedes/metabolismo , Animais , DNA/genética , DNA/metabolismo , Proteína HMGB1/metabolismo , Proteínas de Insetos/metabolismo , Chaperonas Moleculares/metabolismo
8.
Sci Rep ; 6: 36444, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27805064

RESUMO

Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.


Assuntos
Aedes/genética , Metilação de DNA , Sequência de Aminoácidos , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Genoma , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Espectrometria de Massas , RNA de Transferência/química , RNA de Transferência/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do Genoma
9.
Int J Parasitol ; 46(4): 253-62, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26820302

RESUMO

The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins.


Assuntos
Proteínas HMGB/química , Schistosoma mansoni/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , DNA de Helmintos/metabolismo , Feminino , Proteínas HMGB/genética , Proteínas HMGB/fisiologia , Imuno-Histoquímica , Masculino , Especificidade de Órgãos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Schistosoma mansoni/genética
10.
PLoS Pathog ; 10(5): e1004116, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24809504

RESUMO

Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.


Assuntos
Anti-Helmínticos/uso terapêutico , Proteínas do Ovo/genética , Epigênese Genética/fisiologia , Oviparidade/genética , Schistosoma mansoni/fisiologia , Esquistossomose/prevenção & controle , Esquistossomose/transmissão , Animais , Proteínas do Ovo/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células HEK293 , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Masculino , Terapia de Alvo Molecular/métodos , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento
11.
Proc Natl Acad Sci U S A ; 110(21): 8627-31, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23641003

RESUMO

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/fisiologia , DNA de Protozoário/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Protozoários/metabolismo , Schistosoma mansoni/enzimologia , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA de Protozoário/genética , Proteínas de Drosophila/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Camundongos , Camundongos Knockout , Proteínas de Protozoários/genética , Schistosoma mansoni/genética
12.
PLoS One ; 7(7): e40192, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22802955

RESUMO

The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.


Assuntos
Proteína HMGB1/química , Proteínas de Insetos/química , Aedes , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína HMGB1/isolamento & purificação , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/metabolismo
13.
PLoS One ; 7(6): e39104, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22723938

RESUMO

BACKGROUND: 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole (DIC) is a five-membered heterocyclic compound containing a N-O bond. The anti-inflammatory effects of this compound were studied both in vitro and in vivo. PRINCIPAL FINDINGS: DIC effectively decreased TNF-α and IL-6 release from LPS-stimulated macrophages in a dose dependent manner. DIC diminished the levels of COX-2 with subsequent inhibition of PGE(2) production. DIC also compromised HMGB1 translocation from the nucleus to the cytoplasm. Moreover, DIC prevented the nuclear translocation of NF-κB and inhibited the MAPK pathway. In vivo, DIC inhibited migration of neutrophils to the peritoneal cavity of mice. CONCLUSIONS: This study presents the potential utilization of a synthetic compound, as a lead for the development of novel anti-inflammatory drugs.


Assuntos
Anti-Inflamatórios/farmacologia , Isoxazóis/farmacologia , Piridinas/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/síntese química , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Dinoprostona/biossíntese , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteína HMGB1/metabolismo , Interleucina-6/biossíntese , Isoxazóis/administração & dosagem , Isoxazóis/síntese química , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Piridinas/administração & dosagem , Piridinas/síntese química , Transdução de Sinais , Tioglicolatos/efeitos adversos , Fator de Necrose Tumoral alfa/biossíntese
14.
PLoS One ; 6(8): e23572, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21887276

RESUMO

BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.


Assuntos
Caseína Quinase II/metabolismo , DNA de Protozoário/metabolismo , Proteína HMGB1/metabolismo , Schistosoma mansoni/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citosol/metabolismo , DNA Super-Helicoidal/metabolismo , Ensaios Enzimáticos , Feminino , Granuloma/metabolismo , Proteína HMGB1/química , Proteína HMGB1/genética , Células HeLa , Humanos , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Fígado/ultraestrutura , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Mapas de Interação de Proteínas , Schistosoma mansoni/citologia , Schistosoma mansoni/ultraestrutura
15.
Biochem Biophys Res Commun ; 390(4): 1245-9, 2009 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19879244

RESUMO

Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1DeltaC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1DeltaC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.


Assuntos
Núcleo Celular/metabolismo , Proteína HMGB1/metabolismo , Histona Acetiltransferases/metabolismo , Schistosoma mansoni/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Esquistossomose mansoni/parasitologia
16.
Exp Parasitol ; 120(2): 200-4, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18655787

RESUMO

Eukaryotic LIM domain proteins contain zinc finger forming motifs rich in cysteine and histidine that enable them to interact with other proteins. A cDNA clone isolated from an adult schistosome cDNA library revealed a sequence that coded for a novel class of proteins bearing 6 LIM domains and an N-terminal PET domain, SmLIMPETin. Phylogeny reconstruction of SmLIMPETin and comparison of its sequence to invertebrate homologues and to the vertebrate four-and-a-half LIM domains protein family (FHLs), uncovered a novel LIM domain protein family, the invertebrate LIM and PET domain protein family (LIMPETin). Northern blots, RT-PCR and Western blot showed that SmLIMPETin gene was less expressed in sexually mature adult females compared to sexually immature adult females and sexually mature and immature adult males, and not expressed in schistosomula.


Assuntos
Proteínas de Transporte/química , Proteínas de Helminto/química , Schistosoma mansoni/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Regulação para Baixo , Feminino , Proteínas de Helminto/genética , Masculino , Dados de Sequência Molecular , Filogenia , Schistosoma mansoni/classificação , Schistosoma mansoni/genética
17.
Biochem Biophys Res Commun ; 371(4): 689-93, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18455507

RESUMO

The eggs produced by sexually mature female Schistosma mansoni are responsible for the pathogenesis of the disease. The eggshell precursor gene p14 is expressed only in the vitelline cells of sexually mature female worms in response to a yet unidentified male stimulus. Herein, we report the identification of a novel nuclear receptor response element in the upstream region of the p14 gene. This element contains the canonical hexameric DNA core motif, 5'-PuGGTCA, composed of an atypically spaced direct repeat (DR17). Schistosome nuclear receptors SmRXR1 and SmNR1 specifically bound to the p14-DR17 element as a heterodimer. SmRXR1, but not SmNR1, bound to the motif as a monomer. Introduction of mutations in the TCA core sequence completely abolished the binding by SmRXR1/SmNR1 heterodimer. This finding supports our hypothesis that the expression of Schistosoma mansonip14 gene is regulated through the nuclear receptor signaling pathway.


Assuntos
Regulação da Expressão Gênica , Genes de Helmintos , Proteínas de Helminto/metabolismo , Receptores de Glutamato/metabolismo , Sequências Repetitivas de Ácido Nucleico , Receptores X de Retinoides/metabolismo , Schistosoma mansoni/genética , Animais , Sequência de Bases , Dimerização , Feminino , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Schistosoma mansoni/metabolismo , Transdução de Sinais
18.
Biochem Biophys Res Commun ; 370(1): 53-6, 2008 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-18346457

RESUMO

The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation.


Assuntos
Proteínas de Helminto/metabolismo , Histona Acetiltransferases/metabolismo , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Ativação Transcricional , Acetilação , Animais , Núcleo Celular/enzimologia , Eucromatina/enzimologia , Genes de Helmintos , Proteínas de Helminto/análise , Histona Acetiltransferases/análise , Histonas/metabolismo , Camundongos , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/metabolismo , Vitelinas/metabolismo , Vitelinas/ultraestrutura
19.
Int J Parasitol ; 38(10): 1133-47, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18359485

RESUMO

The Schistosoma mansoni nuclear receptors (NR) SmRXR1 and SmNR1 have recently been shown to form a heterodimer and to bind to canonic hormone response DNA elements. Recruitment of co-regulatory proteins to NRs is required for their transcriptional and biological activities. Here, we cloned a novel S. mansoni NR co-activator, SmNCoA-62. SmNCoA-62 is highly homologous to the human Vitamin D receptor co-activator NCoA62/SKIP. SmNCoA-62 contains the SNW nuclear receptor interaction domain and a putative C-terminus transactivation domain. By using in vitro pull-down assays, we fully mapped the interaction domains of S. mansoni NR co-activators, SmNCoA-62, SmGCN5 and SmCBP1 with SmRXR1 and SmNR1, as well as the domains that mediate interactions amongst the co-activators themselves. By mutagenesis analysis, we showed that SmCBP1 LxxLL motif 2 and LxxLL motif 3, but not LxxLL motif 1, were essential to mediate the interactions of SmCBP1 with the EF domains of SmRXR1 and SmNR1. Histone acetyltransferases SmGCN5 and SmCBP1 specifically acetylated the C/D domains of SmRXR1 and SmNR1. In addition, two acetylation sites of SmNR1 were identified. SmGCN5 and SmCBP1 also acetylated SmNCoA-62 but with significant differences in their acetylation activities. Using gel shift analysis, we were able to demonstrate, in vitro, the assembly of the co-activators on the SmRXR1/SmNR1 heterodimer bound to DNA. LxxLL motifs 2 and 3 of SmCBP1 seemed to play a crucial role for the assembly of the co-activators to the DNA-bound SmRXR1/SmNR1 heterodimer.


Assuntos
Proteínas de Helminto/genética , Receptores Citoplasmáticos e Nucleares/genética , Schistosoma mansoni/genética , Acetilação , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , DNA de Helmintos/genética , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , Proteínas de Helminto/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Receptores Citoplasmáticos e Nucleares/metabolismo , Schistosoma mansoni/classificação , Schistosoma mansoni/metabolismo , Análise de Sequência de DNA/métodos , Especificidade da Espécie
20.
Acta Trop ; 108(2-3): 186-93, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18191795

RESUMO

The platyhelminth parasite Schistosoma mansoni, the causative agent of schistosomiasis, is a dioecious parasite with a complex life cycle that includes two different hosts and two free-living stages. Yet very little is known about the biochemical details connected to these different transitions. In the present work, results will be presented showing the most recent results in S. mansoni regarding the characterization of transcription factors and coactivators that act directly on the transcriptional machinery and those that are involved with chromatin remodeling. It is hoped that the information gathered here may contribute towards the understanding of crucial events in the parasite life cycle. Likewise, the development of new drugs that could interfere with oogenesis and sexual maturation may eventually profit from the information contained herein.


Assuntos
Regulação da Expressão Gênica , Schistosoma mansoni/fisiologia , Animais , Montagem e Desmontagem da Cromatina , Elementos Reguladores de Transcrição
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