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1.
Int Arch Allergy Immunol ; 181(1): 56-70, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31707382

RESUMO

INTRODUCTION: Phospholipases are enzymes that occur in many types of human cells, including mast cells, and play an important role in the molecular background of asthma pathogenesis, and the development of inflammation NF-κB activities that affect numerous biological processes has been reported in many inflammatory diseases including asthma. Vitamin D is a widely studied factor that affects many diseases, including asthma. The aim of this study is to assess the influence of 1,25-(OH)2D3 on regulation of chosen phospholipase-A2 (PLA2) expression-selected inflammation mediators. METHODS: LUVA mast cells were stimulated with 1,25(OH)2D3, and inhibitors of NF-κB p65 and ubiquitination. Expression analysis of phospholipases (PLA2G5, PLA2G10, PLA2G12, PLA2G15, PLA2G4A, PLA2G4B, PLA2G4C, PLAA, NF-κB p65, and UBC) was done utilizing real-time PCR and Western blot. Eicosanoid (LTC4, LXA4, 15[S]-HETE, and PGE2) levels and sPLA2 were also measured. RESULTS: We found that 1,25(OH)2D3 decreased the expression of PLA2G5, PLA2G15, PLA2G5,UBC, and NF-κB p65 but increased expression of PLAA and PLA2G4C (p < 0.05). Moreover, the expression of PLA2G5 and PLA2G15 decreased after inhibition of NF-κB p65 and UBC. Increased levels of released LXA4 and 15(S)-HETE, decreased levels of LTC4, and sPLA2s enzymatic activity in response to 1,25(OH)2D3 were also observed. Additionally, NF-κB p65 inhibition led to an increase in the LXA4 concentration. CONCLUSION: Future investigations will be needed to further clarify the role of 1,25(OH)2D3 in the context of asthma and the inflammatory process; however, these results confirm a variety of effects which can be caused by this vitamin. 1,25(OH)2D3-mediated action may result in the development of new therapeutic strategies for asthma treatment.


Assuntos
Asma/metabolismo , Colecalciferol/metabolismo , Inflamação/metabolismo , Mastócitos/metabolismo , NF-kappa B/metabolismo , Fosfolipases A2/metabolismo , Asma/genética , Linhagem Celular Transformada , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Lipoxinas/metabolismo , NF-kappa B/genética , Fosfolipases A2/genética , Transdução de Sinais , Ubiquitinação
2.
Nat Commun ; 10(1): 3426, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366908

RESUMO

Apolipoprotein-B (ApoB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular damage. Here we report the LipoGlo system, which uses a luciferase enzyme (NanoLuc) fused to ApoB to monitor several key determinants of lipoprotein atherogenicity including particle abundance, size, and localization. Using LipoGlo, we comprehensively characterize the lipoprotein profile of individual larval zebrafish and collect images of atherogenic lipoprotein localization in an intact organism. We report multiple extravascular lipoprotein localization patterns, as well as identify Pla2g12b as a potent regulator of lipoprotein size. ApoB-fusion proteins thus represent a sensitive and specific approach to study atherogenic lipoproteins and their genetic and small molecule modifiers.


Assuntos
Apolipoproteínas B/química , Aterosclerose/patologia , Lipoproteínas LDL/análise , Luciferases/química , Coloração e Rotulagem/métodos , Animais , Apolipoproteínas B/metabolismo , Humanos , Larva/metabolismo , Luciferases/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Mol Biol Evol ; 36(9): 1964-1974, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220860

RESUMO

Gene expression changes contribute to complex trait variations in both individuals and populations. However, the evolution of gene expression underlying complex traits over macroevolutionary timescales remains poorly understood. Snake venoms are proteinaceous cocktails where the expression of each toxin can be quantified and mapped to a distinct genomic locus and traced for millions of years. Using a phylogenetic generalized linear mixed model, we analyzed expression data of toxin genes from 52 snake species spanning the 3 venomous snake families and estimated phylogenetic covariance, which acts as a measure of evolutionary constraint. We find that evolution of toxin combinations is not constrained. However, although all combinations are in principle possible, the actual dimensionality of phylomorphic space is low, with envenomation strategies focused around only four major toxin families: metalloproteases, three-finger toxins, serine proteases, and phospholipases A2. Although most extant snakes prioritize either a single or a combination of major toxin families, they are repeatedly recruited and lost. We find that over macroevolutionary timescales, the venom phenotypes were not shaped by phylogenetic constraints, which include important microevolutionary constraints such as epistasis and pleiotropy, but more likely by ecological filtering that permits a small number of optimal solutions. As a result, phenotypic optima were repeatedly attained by distantly related species. These results indicate that venoms evolve by selection on biochemistry of prey envenomation, which permit diversity through parallelism, and impose strong limits, since only a few of the theoretically possible strategies seem to work well and are observed in extant snakes.


Assuntos
Evolução Molecular , Seleção Genética , Venenos de Serpentes/genética , Serpentes/genética , Animais , Metaloproteases/genética , Família Multigênica , Fosfolipases A2/genética , Filogenia , Serina Proteases/genética
4.
Arch Insect Biochem Physiol ; 101(3): e21559, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31062425

RESUMO

Phospholipase A2 (PLA2 ) hydrolyzes fatty acids from phospholipids at the sn-2 position. Two intracellular PLA2 s, iPLA2 A and iPLA2 B, have been found in Spodoptera exigua. Both are calcium-independent cellular PLA2 . Their orthologs have been found in other insects. These two iPLA2 s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA2 expression. Both iPLA 2 s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA2 s. Both iPLA2 s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor-κB- and Relish-responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA2 s in response to Gram-positive bacteria containing Lys-type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA2 s in response to infection with Gram-negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA2 ) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA2 expression.


Assuntos
Fenômenos Fisiológicos Bacterianos , Expressão Gênica , Imunidade Celular , Proteínas de Insetos/genética , Fosfolipases A2/genética , Spodoptera/imunologia , Animais , Fungos/fisiologia , Proteínas de Insetos/metabolismo , Fosfolipases A2/metabolismo , Transdução de Sinais , Spodoptera/enzimologia , Spodoptera/genética , Spodoptera/microbiologia
5.
Toxins (Basel) ; 11(2)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30754700

RESUMO

The equatorial spitting cobra, Naja sumatrana, is a distinct species of medically important venomous snakes, listed as WHO Category 1 in Southeast Asia. The diversity of its venom genes has not been comprehensively examined, although a few toxin sequences annotated to Naja sputatrix were reported previously through cloning studies. To investigate this species venom genes' diversity, de novo venom-gland transcriptomics of N. sumatrana from West Malaysia was conducted using next-generation sequencing technology. Genes encoding toxins represented only 60 of the 55,396 transcripts, but were highly expressed, contributing to 79.22% of total gene expression (by total FPKM) in the venom-glands. The toxin transcripts belong to 21 families, and 29 transcripts were further identified as full-length. Three-finger toxins (3FTx) composed of long, short, and non-conventional groups, constituted the majority of toxin transcripts (91.11% of total toxin FPKM), followed by phospholipase A2 (PLA2, 7.42%)-which are putatively pro-inflammatory and cytotoxic. The remaining transcripts in the 19 families were expressed at extremely low levels. Presumably, these toxins were associated with ancillary functions. Our findings unveil the diverse toxin genes unique to N. sumatrana, and provide insights into the pathophysiology of N. sumatrana envenoming.


Assuntos
Venenos Elapídicos/genética , Naja/genética , Animais , Malásia , Fosfolipases A2/genética , Filogenia , Proteínas de Répteis/genética , Transcriptoma
6.
Am J Physiol Lung Cell Mol Physiol ; 316(4): L656-L668, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30702344

RESUMO

Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme that serves important antioxidant roles by scavenging hydroperoxides and reducing peroxidized cell membranes. Prdx6 also plays a key role in cell signaling by activating the NADPH oxidase, type 2 (Nox2) through its acidic Ca2+-independent phospholipase A2 (aiPLA2) activity. Nox2 generation of O2·-, in addition to signaling, can contribute to oxidative stress and inflammation such as during sepsis-induced acute lung injury (ALI). To evaluate a possible role of Prdx6-aiPLA2 activity in the pathophysiology of ALI associated with a systemic insult, wild-type (WT) and Prdx6-D140A mice, which lack aiPLA2 but retain peroxidase activity were administered intraperitoneal LPS. LPS-treated mutant mice had increased survival compared with WT mice while cytokines in lung lavage fluid and lung VCAM-1 expression, nitrotyrosine levels, PMN infiltration, and permeability increased in WT but not in mutant mice. Exposure of mouse pulmonary microvascular endothelial cells in primary culture to LPS promoted phosphorylation of Prdx6 and its translocation to the plasma membrane and increased aiPLA2 activity as well as increased H2O2 generation, nitrotyrosine levels, lipid peroxidation, NF-κB nuclear localization, and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome assembly; these effects were not seen in Nox2 null cells, Prdx6-D140A cells, or WT cells pretreated with MJ33, an inhibitor of aiPLA2 activity. Thus aiPLA2 activity is needed for Nox2-derived oxidant stress associated with LPS exposure. Since inactivation of aiPLA2 reduced mortality and prevented lung inflammation and oxidative stress in this animal model, the aiPLA2 activity of Prdx6 could be a novel target for prevention or treatment of sepsis-induced ALI.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Peroxirredoxina VI/antagonistas & inibidores , Fosfolipases A2/metabolismo , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico/genética , Modelos Animais de Doenças , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , NADPH Oxidase 2/metabolismo , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Inibidores de Fosfolipase A2/metabolismo , Fosfolipases A2/química , Fosfolipases A2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Arch Virol ; 164(4): 1159-1171, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30809709

RESUMO

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/farmacologia , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Venenos de Serpentes/enzimologia , Animais , Antivirais/química , Cromatografia de Afinidade , Crotalus , Vírus da Dengue/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/farmacologia , Fosfolipases A2/química , Fosfolipases A2/genética , Dobramento de Proteína , Proteínas de Répteis/química , Proteínas de Répteis/genética , Venenos de Serpentes/química , Vírus da Febre Amarela/efeitos dos fármacos , Zika virus/efeitos dos fármacos
8.
Toxins (Basel) ; 11(2)2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30691065

RESUMO

Phospholipase A2 (PLA2) is a major component in snake venoms and it is found in many different isoforms. To identify transcripts encoding different PLA2 isoforms, we developed a simple, rapid procedure. Total RNA was extracted from the venoms of three cottonmouth snakes and two diamondback rattlesnakes, and further reverse-transcribed into complementary DNA (cDNA). Using one pair of cottonmouth PLA2-specific primers and a Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, we identified 27 unique full-length PLA2 transcripts, including nine sequences identical to the previously documented ones in the database and one novel GIII-like PLA2. Two common transcripts respectively encoding Asp49 and Lys49 PLA2 isoforms were identified in all three cottonmouth venoms, that contain more PLA2 transcripts than the diamondback rattlesnake venoms. The placement of cloned PLA2 transcripts in snake venom PLA2s was further discussed by phylogenetic analysis. The procedure developed in this study paves the way for accelerated acquisition of transcriptome data on any other venom toxin families. The results obtained are crucial for insight into the structure and function of PLA2 isoforms for scientific and potential therapeutic purposes.


Assuntos
Isoenzimas/genética , Fosfolipases A2/genética , Venenos de Serpentes/genética , Agkistrodon , Animais , Crotalus , Filogenia , Venenos de Serpentes/enzimologia , Transcriptoma
9.
Cancer Res Treat ; 51(1): 391-401, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29909608

RESUMO

PURPOSE: This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer (CRC) lines. Materials and Methods: We performed paired-end RNA sequencing of 28 CRC cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. RESULTS: One thousand three hundred eighty FT candidates were detected through bioinformatics filtering. We selected six candidate FTs, including four inter-chromosomal and two intrachromosomal FTs and each FT was found in at least one of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in two. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. CONCLUSION: These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.


Assuntos
Aciltransferases/genética , Neoplasias Colorretais/genética , Fatores de Transcrição NFATC/genética , Proteínas de Fusão Oncogênica/genética , Fosfolipases A2/genética , Análise de Sequência de RNA/métodos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Invasividade Neoplásica
10.
J Proteomics ; 192: 246-257, 2019 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-30243938

RESUMO

The Asiatic coral snakes are basal in the phylogeny of coral snakes. Although envenoming by the Asiatic coral snakes is rarely fatal, little is known about their venom properties and variability from the American coral snakes. Integrating reverse-phase high performance liquid chromatography and nano-liquid chromatography-tandem mass spectrometry, we showed that the venom proteome of the Malaysian banded or striped coral snake (Calliophis intestinalis) was composed of mainly phospholipases A2 (PLA2, 43.4%) and three-finger toxins (3FTx, 20.1%). Within 3FTx, the cytotoxins or cardiotoxins (CTX) dominated while the neurotoxins' content was much lower. Its subproteomic details contrasted with the 3FTx profile of most Micrurus sp., illustrating a unique dichotomy of venom phenotype between the Old and the New World coral snakes. Calliophis intestinalis venom proteome was correlated with measured enzymatic activities, and in vivo it was myotoxic but non-lethal to mice, frogs and geckos at high doses (5-10 µg/g). The venom contains species-specific toxins with distinct sequences and antigenicity, and the antibodies raised against PLA2 and CTX of other elapids showed poor binding toward its venom antigens. The unique venom proteome of C. intestinalis unveiled a repertoire of novel toxins, and the toxicity test supported the need for post-bite monitoring of myotoxic complication. SIGNIFICANCE: Malaysian banded or striped coral snake (Calliophis intestinalis) has a cytotoxin (CTX)-predominating venom proteome, a characteristic shared by its congener, the Malayan blue coral snake (Calliophis bivirgata). With little neurotoxins (NTX), it illustrates a CTX/NTX dichotomy of venom phenotype between the Old World and the New World coral snakes. The low toxicity of the venom imply that C. intestinalis bite envenoming can be managed via symptomatic relief of the mild to moderate pain with appropriate analgesia. Systemically, the serum creatine kinase level of patients should be monitored serially for potential complication of myotoxicity. The distinct antigenicity of the venom proteins implies that the empirical use of heterologous antivenom is mostly inappropriate and not recommended.


Assuntos
Cobras Corais/metabolismo , Venenos Elapídicos/metabolismo , Neurotoxinas/metabolismo , Fosfolipases A2/metabolismo , Proteoma/metabolismo , Proteínas de Répteis/metabolismo , Animais , Cobras Corais/genética , Venenos Elapídicos/genética , Lagartos , Camundongos , Neurotoxinas/genética , Fosfolipases A2/genética , Filogenia , Proteoma/genética , Coelhos , Proteínas de Répteis/genética , Especificidade da Espécie
11.
Protein Expr Purif ; 154: 33-43, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30205154

RESUMO

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.


Assuntos
Bothrops/genética , Clonagem Molecular , Venenos de Crotalídeos , DNA Complementar , Expressão Gênica , Fosfolipases A2 , Dobramento de Proteína , Animais , Venenos de Crotalídeos/biossíntese , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/imunologia , Escherichia coli/enzimologia , Escherichia coli/genética , Cavalos/imunologia , Fosfolipases A2/biossíntese , Fosfolipases A2/genética , Fosfolipases A2/imunologia , Fosfolipases A2/isolamento & purificação
12.
J Bacteriol ; 201(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30455285

RESUMO

ExoU is a potent type III secretion system effector that is injected directly into mammalian cells by the opportunistic pathogen Pseudomonas aeruginosa As a ubiquitin-activated phospholipase A2 (PLA2), ExoU exhibits cytotoxicity by cleaving membrane phospholipids, resulting in lysis of the host cells and inhibition of the innate immune response. Recently, ExoU has been established as a model protein for a group of ubiquitin-activated PLA2 enzymes encoded by a variety of bacteria. Bioinformatic analyses of homologous proteins is a powerful approach that can complement and enhance the overall understanding of protein structure and function. To conduct homology studies, it is important to have efficient and effective tools to screen and to validate the putative homologs of interest. Here we make use of an Escherichia coli-based dual expression system to screen putative ubiquitin-activated PLA2 enzymes from a variety of bacteria that are known to colonize humans and to cause human infections. The screen effectively identified multiple ubiquitin-activated phospholipases, which were validated using both biological and biochemical techniques. In this study, two new ExoU orthologs were identified and the ubiquitin activation of the rickettsial enzyme RP534 was verified. Conversely, ubiquitin was not found to regulate the activity of several other tested enzymes. Based on structural homology analyses, functional properties were predicted for AxoU, a unique member of the group expressed by Achromobacter xylosoxidans IMPORTANCE Bacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers and inflammation during infections. Identifying enzymes with a common mechanism of activation is an initial step in understanding structural and functional properties. These properties serve as critical information for the design of specific inhibitors to reduce enzymatic activity and ameliorate host cell death. In this study, we identify and verify cytotoxic PLA2 enzymes from several bacterial pathogens. Similar to the founding member of the group, ExoU, these enzymes share the property of ubiquitin-mediated activation. The identification and validation of potential toxins from multiple bacterial species provide additional proteins from which to derive structural insights that could lead to paninhibitors useful for treating a variety of infections.


Assuntos
Proteínas de Bactérias/metabolismo , Ativação Enzimática , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Ubiquitina/metabolismo , Proteínas de Bactérias/genética , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Testes Genéticos , Fosfolipases A2/genética
13.
Mol Med Rep ; 18(5): 4733-4738, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30221721

RESUMO

The aim of the present study was to investigate the acute effect and mechanism of tumor necrosis factor (TNF) on basolateral 50 pS K channels in the thick ascending limb (TAL) of the rat kidney. The TAL tubules were isolated from the rat kidney, and the activity of the 50 pS K channels was recorded using the patch­clamp technique. The results indicated that the application of TNF (10 nM) significantly activated the 50 pS K channels and the TNF effect was concentration­dependent. Inhibition of protein kinase A, phospholipase A2 and protein tyrosine kinase using pathway inhibitors (H89, AACOCF3 and Herbimycin A, respectively) did not abolish the stimulatory effect of TNF, indicating that none of these pathways mediated the TNF effect. By contrast, the phenylarsine oxide inhibitor against protein tyrosine phosphatase (PTP) decreased the activity of the 50 pS K channels and blocked the stimulatory effect of TNF on these channels. Furthermore, western blot analysis demonstrated that the application of TNF (10 nM) in the TAL increased the phosphorylation of PTP, an indication of PTP activity stimulation. Thus, it was concluded that the acute application of TNF may stimulate the basolateral 50 pS K channel in the TAL and the stimulatory effect of TNF may be mediated by the PTP­dependent pathway.


Assuntos
Túbulos Renais/metabolismo , Rim/metabolismo , Canais de Potássio/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Ácidos Araquidônicos/administração & dosagem , Arsenicais/administração & dosagem , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoquinolinas/administração & dosagem , Rim/efeitos dos fármacos , Rim/patologia , Túbulos Renais/efeitos dos fármacos , Alça do Néfron/efeitos dos fármacos , Alça do Néfron/metabolismo , Masculino , Técnicas de Patch-Clamp , Inibidores de Fosfolipase A2/administração & dosagem , Fosfolipases A2/genética , Canais de Potássio/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Rifabutina/administração & dosagem , Rifabutina/análogos & derivados , Sulfonamidas/administração & dosagem , Fator de Necrose Tumoral alfa/administração & dosagem
14.
Methods Mol Biol ; 1835: 179-189, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30109652

RESUMO

In this study, we have produced for the first time a fish phospholipase (PLA2) in heterologous system (E. coli). The Diplodus annularis PLA2 (DaPLA2) was then refolded from inclusion bodies and purified by Ni-affinity chromatography. We used the pH-stat method (with emulsified phosphatidylcholine as substrate) and the monomolecular film technique (using various glycerophospholipids substrates spread in the form of monomolecular films at the air-water interface) to access the biochemical and kinetic properties of the recombinant DaPLA2. The DaPLA2 was found to be active and stable at higher temperatures (37-50 °C) than expected. Interestingly, DaPLA2 hydrolyzes efficiently both purified phosphatidylglycerol and phosphatidylethanolamine at 20 mN/m. These analytical results corroborate with the fact that the catalytic activity of DaPLA2, measured with the pH-stat using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk, whereas the phosphatidylglycerol is a hallmark substrate for the most secreted PLA2-IB.


Assuntos
Peixes/genética , Peixes/metabolismo , Expressão Gênica , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Animais , Clonagem Molecular , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Espectrometria de Massas , Fosfolipases A2/isolamento & purificação , Redobramento de Proteína
15.
Int J Biol Macromol ; 118(Pt A): 311-319, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29920366

RESUMO

Herein we evaluated the genotoxic effects of BnSP-6, a Lys-49 phospholipase A2 (PLA2) from Bothrops pauloensis, on breast cancer cells. BnSP-6 was able to induce a higher cytotoxic and genotoxic activity in MDA-MB-231 cells, when compared to MCF10A (a non-tumorigenic breast cell line), suggesting that this protein presented a possible preference for cancer cells. BnSP-6 inhibited MDA-MB-231 proliferation at 24, 48 and 72 h. In addition, BnSP-6 induced significant increase in the percentage of TUNEL-positive cells, a marker of DNA damage. To obtain novel insight into the direct DNA damage interference in MDA-MB-231 survival and proliferation, we evaluated cell cycle progression. BnSP-6 produced a significant decrease in 2N (G1) and an increase in the G2/M phase and this capacity is likely related to the modulation of expression of progression cell cycle-associated genes (CCND1, CCNE1, CDC25A, CHEK2, E2F1, CDH-1 and NF-kB). Taken together, these results indicate that BnSP-6 induces DNA damage in breast cancer cells and is an attractive model for developing innovative therapeutic agents against breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Fosfolipases A2/farmacologia , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Bothrops/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Lisina/química , Fosfolipases A2/química , Fosfolipases A2/genética , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/química
16.
Nat Immunol ; 19(7): 755-765, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29915298

RESUMO

The cytokines IL-17A and IL-17F have 50% amino-acid identity and bind the same receptor; however, their functional differences have remained obscure. Here we found that Il17f-/- mice resisted chemically induced colitis, but Il17a-/- mice did not, and that Il17f-/- CD45RBhiCD4+ T cells induced milder colitis in lymphocyte-deficient Rag2-/- mice, accompanied by an increase in intestinal regulatory T cells (Treg cells). Clostridium cluster XIVa in colonic microbiota capable of inducing Treg cells was increased in both Il17f-/- mice and mice given transfer Il17f-/- T cells, due to decreased expression of a group of antimicrobial proteins. There was substantial production of IL-17F, but not of IL-17A, not only by naive T cells but also by various colon-resident cells under physiological conditions. Furthermore, antibody to IL-17F suppressed the development of colitis, but antibody to IL-17A did not. These observations suggest that IL-17F is an effective target for the treatment of colitis.


Assuntos
Colite/imunologia , Microbioma Gastrointestinal , Interleucina-17/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Clostridium/crescimento & desenvolvimento , Clostridium/isolamento & purificação , Colite/tratamento farmacológico , Interleucina-17/genética , Interleucina-17/fisiologia , Intestinos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipases A2/biossíntese , Fosfolipases A2/genética , Prevotella/isolamento & purificação , Ribonuclease Pancreático/biossíntese , Ribonuclease Pancreático/genética , beta-Defensinas/biossíntese
17.
Int J Biol Macromol ; 118(Pt A): 1-8, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29886171

RESUMO

The first toxin isolated from the venomous pit viper Porthidium ophryomegas is a basic pentameric phospholipase A2 (PophPLA2). Elucidation of its amino acid sequence showed that it belongs to the group IIA of secreted PLA2s, with the presence of all 14 conserved cysteine positions. The toxin displayed catalytic activity, in agreement with the presence of Asp49 in its sequence of 121 residues. SDS-PAGE analysis revealed that this toxin is pentameric in non-reducing conditions, a structural organization that has not been described for any viperid PLA2. PophPLA2 displayed moderate myotoxic (in vivo) and cytotoxic (in vitro) activities, as well as anticoagulant activity on human plasma (in vitro). PophPLA2 was not lethal, and did not induce signs of toxicity or distress in mice, when administered intravenously at a dose of up to 100 µg (5.9 µg/g). The toxin showed highest sequence identity with other PLA2s from the venoms of ancestral Asian pit viper species.


Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A2/química , Sequência de Aminoácidos/genética , Animais , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/farmacologia , Crotalinae , Humanos , Camundongos , Fosfolipases A2/genética , Fosfolipases A2/farmacologia
18.
J Lipid Res ; 59(7): 1205-1218, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29724779

RESUMO

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.


Assuntos
Lisossomos/enzimologia , Fosfolipases A2/metabolismo , Acilação , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Mutação , Fosfolipases A2/química , Fosfolipases A2/genética , Estrutura Terciária de Proteína , Especificidade por Substrato
19.
J Reprod Dev ; 64(4): 311-317, 2018 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-29710018

RESUMO

Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG) E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.


Assuntos
Dinoprosta/biossíntese , Dinoprostona/biossíntese , Endométrio/metabolismo , Resposta ao Choque Térmico/fisiologia , Temperatura Alta , Células Estromais/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Bovinos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo
20.
PLoS Biol ; 16(4): e2005504, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29668708

RESUMO

The molecular mechanisms through which dendritic cells (DCs) prime T helper 2 (Th2) responses, including those elicited by parasitic helminths, remain incompletely understood. Here, we report that soluble egg antigen (SEA) from Schistosoma mansoni, which is well known to drive potent Th2 responses, triggers DCs to produce prostaglandin E2 (PGE2), which subsequently-in an autocrine manner-induces OX40 ligand (OX40L) expression to license these DCs to drive Th2 responses. Mechanistically, SEA was found to promote PGE2 synthesis through Dectin-1 and Dectin-2, and via a downstream signaling cascade involving spleen tyrosine kinase (Syk), extracellular signal-regulated kinase (ERK), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 1 and 2 (COX-1 and COX-2). In addition, this pathway was activated independently of the actions of omega-1 (ω-1), a previously described Th2-priming glycoprotein present in SEA. These findings were supported by in vivo murine data showing that ω-1-independent Th2 priming by SEA was mediated by Dectin-2 and Syk signaling in DCs. Finally, we found that Dectin-2-/-, and to a lesser extent Dectin-1-/- mice, displayed impaired Th2 responses and reduced egg-driven granuloma formation following S. mansoni infection, highlighting the physiological importance of this pathway in Th2 polarization during a helminth infection. In summary, we identified a novel pathway in DCs involving Dectin-1/2-Syk-PGE2-OX40L through which Th2 immune responses are induced.


Assuntos
Células Dendríticas/imunologia , Dinoprostona/imunologia , Lectinas Tipo C/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Células Th2/imunologia , Animais , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/farmacologia , Comunicação Autócrina , Diferenciação Celular , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/parasitologia , Dinoprostona/metabolismo , Enterotoxinas/farmacologia , Regulação da Expressão Gênica , Humanos , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Sistema de Sinalização das MAP Quinases , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipases A2/genética , Fosfolipases A2/imunologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Esquistossomose mansoni/genética , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/patologia , Quinase Syk/genética , Quinase Syk/imunologia , Células Th2/efeitos dos fármacos , Células Th2/parasitologia , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/imunologia
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