Phospholipase A2 Activity Research Articles

Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8+ T cell.

Increased prevalence of cancer in obese individuals is involved with dyslipidemia- induced chronic inflammation and immune suppression. Although apolipoprotein C-III (ApoC3)-transgenic mice (ApoC3TG mice) or poloxamer 407 (P407)-treated mice had hyperlipidemia, CD8+ T cells with upregulated antitumor activities were observed in ApoC3TG mice, and decreased CD8+ T cell activities were observed in P407-treated mice. Increased ApoC3 expression in hepatocellular carcinoma was associated with increased infiltration of CD8+ T cells and predicted survival. Recombinant ApoC3 had no direct effects on CD8+ T cells. The upregulation of CD8+ T cells in ApoC3TG mice was due to cross-talk with context cells, as indicated by metabolic changes and RNA sequencing results. In contrast to dendritic cells, the macrophages of ApoC3TG mice (macrophagesTG) displayed an activated phenotype and increased IL-1β, TNF-α, and IL-6 production. Coculture with macrophagesTG increased CD8+ T cell function, and the adoptive transfer of macrophagesTG suppressed tumor progression in vivo. Furthermore, spleen tyrosine kinase (Syk) activation induced by TLR2/TLR4 cross-linking after ApoC3 ligation promoted cellular phospholipase A2 (cPLA2) activation, which in turn activated NADPH oxidase 2 (NOX2) to promote an alternative mode of inflammasome activation. Meanwhile, mitochondrial ROS produced by increased oxidative phosphorylation of free fatty acids facilitated the classical inflammasome activation, which exerted an auxiliary effect on inflammasome activation of macrophagesTG. Collectively, the increased antitumor activity of CD8+ T cells was mediated by the ApoC3-stimulated inflammasome activation of macrophages, and the mimetic ApoC3 peptides that can bind TLR2/4 could be a future strategy to target liver cancer.

Capsaicin indirectly regulates TRPA1 via the arachidonic acid cascade, resulting in TJ opening.

Capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. We previously revealed that TRPA1 is involved in the capsaicin-induced Ca2+ influx and TJ permeability increase, although there are no reports that capsaicin directly activates TRPA1. In this study, we investigated the upstream factors of TRPA1 using RNA-seq analysis, and found that the cyclooxygenase 2 (COX2) gene was upregulated by capsaicin. Cyclooxygenase 2 converts arachidonic acid (AA), a metabolite by phospholipase A2 (PLA2), to prostaglandins. Prostaglandin E2 (PGE2) production was stimulated by capsaicin, and capsaicin-induced Ca2+ influx was effectively inhibited by PLA2 and COX2 inhibitors. The AA-induced TJ permeability increase was inhibited by a TRPA1 antagonist, but the capsaicin- and AA-induced TJ permeability increases were hardly inhibited by a COX2 inhibitor. These results suggest that capsaicin-induced PLA2 activation and AA production are the important steps for the TJ permeability increase.

Fucoidan may treat jellyfish dermatitis by inhibiting the inflammatory effect of jellyfish venom

Jellyfish dermatitis is a common medical problem caused by jellyfish stings. However, there are no targeted and effective medications for their treatment. Here, the biological activity of fucoidan for treatment of jellyfish dermatitis was investigated for the first time. 3 mg/mL Fucoidan attenuated the inflammatory effects of Nemopilema nomurai nematocyst venom (NnNV), including dermal toxicity and myotoxicity. Fucoidan may decrease the inflammatory effects of NnNV by downregulating MAPK and NF-κB pathways. This may be attributed to the inhibitory effect of fucoidan on metalloproteinases and phospholipase A2 (PLA2) in NnNV. 3 mg/mL fucoidan reduced the metalloproteinase activity in NnNV from 316.33 ± 20.84 U/mg to 177.33 ± 25.36 U/mg, while the inhibition of PLA2 activity in NnNV by 1 mg/mL fucoidan could reach 37.67 ± 3.42 %. Besides, external application of 3 mg/mL fucoidan can effectively alleviate the symptoms of jellyfish dermatitis. These observations suggest that fucoidan has considerable potential for treatment of jellyfish dermatitis and could be regarded as a novel medicine for jellyfish envenomation. This study provides new ideas for treatment of jellyfish envenomation and suggests evidence for the use of fucoidan in the treatment of jellyfish dermatitis as well as broadens the potential application of fucoidan in clinical practice.

Normolipidic diet containing deep-fried saturated and unsaturated fatty acids rich edible oils promotes metabolic dysregulation and inflammatory microenvironment in Wistar rats.

Deleterious health impacts of high dietary intake of deep-fried edible oils have been reported however, their health impacts at normal dietary levels are yet to be evaluated. This study investigated the influence of prolonged consumption of thermally oxidised long-chain saturated and unsaturated edible oils on metabolic dysregulation and inflammation. The thermally oxidised oils used in the study possess higher p-anisidine values and free fatty acid contents compared to unoxidised oils. Fourier transform infrared spectroscopy and fatty acid methyl ester analysis confirmed the presence of free fatty acids, hydroperoxides, and aldehydes, formed during thermal oxidation. The study analysed the effects of dietary intake of 5% sunflower oil, palm oil, and their thermally-oxidised forms in male Wistar rats for six months. Unoxidised and thermally oxidised palm oil-fed animals experienced metabolic syndrome with obesity-associated inflammatory changes. However, sunflower oil-fed animals exhibited increased inflammation, as evidenced by enhanced C-reactive protein, IL-6, and lipoprotein-associated phospholipase A2 activity and hepatosteatosis condition. This study indicates that prolonged consumption of thermally oxidised oil leads to the dysregulation of carbohydrate and lipid metabolism andpromotes inflammatory microenvironment in Wistar rats that may contribute to metabolic syndrome associated with obesity and hepatic steatosis.

Excessive daytime sleepiness, morning tiredness, and prognostic biomarkers in patients with chronic coronary syndrome

BackgroundSleep-related breathing disorders (SRBD) are related to cardiovascular outcomes in patients with chronic coronary syndrome (CCS). Whether SRBD-related symptoms are associated with prognostic biomarkers in patients with CCS is not established. MethodsAssociations between frequency (never/rarely, sometimes, often, always) of self-reported SRBD-related symptoms (excessive daytime sleepiness [EDS]; morning tiredness [MT]; loud snoring; multiple awakenings/night; gasping, choking, or apnea when asleep) and levels of biomarkers related to cardiovascular prognosis (high-sensitivity C-reactive protein [hs-CRP], interleukin 6 [IL-6], high-sensitivity cardiac troponin T [hs-cTnT], N-terminal pro B-type natriuretic peptide [NT-proBNP], cystatin C, growth differentiation factor 15 [GDF-15] and lipoprotein-associated phospholipase A2 activity) were assessed at baseline in 15,640 patients with CCS on optimal secondary preventive therapy in the STABILITY trial. Cross-sectional associations were assessed by adjusted linear regression models testing for trends with the never/rarely category serving as reference. ResultsEDS was associated (geometric mean ratio, 95% confidence interval) with increased levels of IL-6 (often 1.07 [1.03–1.10], always 1.15 [1.10–1.21]), GDF-15 (often 1.03 [1.01–1.06], always 1.07 [1.03–1.11]), NT-proBNP (always 1.22 [1.12–1.33]), and hs-cTnT (always 1.07 [1.01–1.12]). MT was associated with increased levels of IL-6 (often 1.05 [1.01–1.09], always 1.09 [1.04–1.15]), and GDF-15 (always 1.06 [1.03–1.10]). All symptoms were to some degree associated with higher levels of hs-CRP and loud snoring was also associated with decreased levels of NT-proBNP and hs-cTnT. ConclusionsIn patients with CCS, stepwise increased frequency of SRBD-related symptoms, such as EDS and MT, were associated with gradually higher levels of IL-6 and GDF-15, each reflecting distinct pathophysiological pathways.

Spätzle processing enzyme is required to activate dorsal switch protein 1 induced Toll immune signalling pathway in Tenebrio molitor.

Dorsal switch protein 1 (DSP1) acts as a damage-associated molecular pattern (DAMP) molecule to activate immune responses in Tenebrio molitor. From a previous study in Spodoptera exigua, we found that DSP1 activates Toll immune signalling pathway to induce immune responses by melanisation, PLA2 activity and AMP synthesis. However, the target site of DSP1 in this pathway remains unknown. The objective of this study was to determine the role of spätzle processing enzyme in the DSP1 induced toll immune signalling pathway. To address this, we analyzed spätzle processing enzyme (Tm-SPE) of the three-step serine protease cascade of T. molitor Toll pathway. Tm-SPE expressed in all developmental stages and larval tissues. Upon immune challenge, its expression levels were upregulated but significantly reduced after RNA interference (RNAi). In addition, the induction of immune responses upon immune challenge or recombinant DSP1 injection was significantly increased. Loss of function using RNA interference revealed that the Tm-SPE is involved in connecting DSP1 induced immune responses like hemocyte nodule formation, phenoloxidase (PO) activity, phospholipase A2 (PLA2) activity and antimicrobial peptide (AMP) synthesis. These suggest that Tm-SPE controls the DSP1 induced activation of Toll immune signalling pathway required for both cellular and humoral immune responses. However, to confirm the target molecule of DSP1 in three-step proteolytic cascade, we have to check other upstream serine proteases like Spatzle activating enzyme (SAE) or modular serine protease (MSP).

Identification, structural modeling, gene expression analysis and RNAi effect of putative phospholipase A2 in the lone star tick Amblyomma americanum

Amblyomma americanum, also known as the lone star tick, is a small arachnid that feeds on blood and can spread disease to humans and other animals. Despite the overlapped ecological niche, geographic distribution, and host selection, there is no proof that A. americanum transmits the pathogen Borrelia burgdorferi that causes Lyme disease. Studies have shown that phospholipase A2 (PLA2) may act as a tool to eliminate B. burgdorferi, but particular PLA2 genes in A. americanum have not been identified and functionally characterized. Using the de novo sequencing method, we identified 42 putative A. americanum PLA2 (pAaPLA2) homologs in the present study, of which three pAaPLA2 had calcium binding sites and canonical histidine catalytic sites. Then, we determined phylogenetic relationships, sequence alignments, and conserved protein motifs of these pAaPLA2s. Protein structural analysis demonstrated that pAaPLA2s primarily consisted of α-helices, β-sheets, and random coils. These genes were predicted to be engaged in the phospholipid metabolic process, arachidonic acid secretion, and PLA2 activity by functional annotation analysis. A transcriptional factor (Bgb) was discovered that interacted with pAaPLA2 proteins that may have unrecognized roles in regulating neuronal development. Based on the RNA-seq data, we surveyed expression profiles of key pAaPLA2-related genes to reveal putative modulatory networks of these genes. RNAi knockdown of pAaPLA2_1, a dominant isoform in A. americanum, led to decreased bacterial inhibition ability, suggesting pAaPLA2 may play an important role in mediating immune responses. Collectively, this study provides essential evidence of the identification, gene structure, phylogeny, and expression analysis of pAaPLA2 genes in A. americanum, and offers a deeper understanding of the putative borreliacidal roles in the lone star tick.

Structural and Functional Diversity of the Peroxiredoxin 6 Enzyme Family.

Significance: Peroxiredoxins (Prdxs) with a single peroxidative cysteine (CP) in a conserved motif PXXX(T/S)XXCP within its thioredoxin fold, have been classified as the peroxiredoxin 6 (Prdx6 ) family. All Prdxs can reduce H2O2 and short chain hydroperoxides while Prdx6 in addition, can reduce phospholipid hydroperoxides (PLOOH) due to its ability to interact with peroxidized phospholipid substrate. The single CP of Prdx6 uses various external electron donors including glutathione thioredoxin, and ascorbic acid for resolution of its peroxidized state and, therefore, its peroxidase activity. Prdx6 proteins also exhibit Ca2+-independent phospholipase A2 (PLA2), lysophosphatidylcholine acyltransferase (LPCAT), and chaperone activities that depend on cellular localization and the oxidation and oligomerisation states of the protein. Thus, Prdx6 is a "moonlighting" enzyme. Recent Advance: Physiologically, Prdx6s have been reported to play an important role in protection against oxidative stress, repair of peroxidized cell membranes, mammalian lung surfactant turnover, activation of some NADPH oxidases, the regulation of seed germination in plants, as an indicator of cellular levels of reactive O2 species through Nrf-Klf9 activation, and possibly in male fertility, regulation of cell death through ferroptosis, cancer metastasis, and oxidative stress-related signalling pathways. Critical Issues: This review outlines Prdx6 enzyme unique structural features and explores its wide range of physiological functions. Yet, existing structural data falls short of fully revealing all of human Prdx6 multifunctional roles. Further endeavour is required to bridge this gap in its understanding. Although there are wide variations in both the structure and function of Prdx6 family members in various organisms, all Prdx6 proteins show the unique a long C-terminal extension that is also seen in Prdx1, but not in other Prdxs. Future Directions: As research data continues to accumulate, the potential for detailed insights into the role of C-terminal of Prdx6 in its oligomerisation and activities. There is a need for thorough exploration of structural characteristics of the various biological functions. Additionally, uncovering the interacting partners of Prdx6 and understanding its involvement in signalling pathways will significantly contribute to a more profound comprehension of its role.

Involvement of phospholipase A2 in the neuromuscular blockade caused by coralsnake (Micrurus spp.) venoms in mouse phrenic nerve-diaphragm preparations in vitro

In this work, we examined the neuromuscular blockade caused by venoms from four South-American coralsnakes (Micrurus altirostris – MA, M. corallinus – MC, M. spixii – MS, and M. dumerilii carinicauda – MDC) and the ability of varespladib (VPL), a phospholipase A2 (PLA2) inhibitor, to attenuate this blockade. PLA2 activity was determined using a colorimetric assay and a fixed amount of venom (10 μg). Neurotoxicity was assayed using a single concentration of venom (10 μg/ml) in mouse phrenic nerve-diaphragm (PND) preparations mounted for myographic recordings and then subjected to histological analysis. All venoms showed PLA2 activity, with MS and MA venoms having the highest (15.53 ± 1.9 A425 nm/min) and lowest (0.23 ± 0.14 A425 nm/min) activities, respectively. VPL (292 and 438 μM) inhibited the PLA2 activity of all venoms, although that of MA venom was least affected. All venoms caused neuromuscular blockade, with MS and MDC venoms causing the fastest and slowest 100% blockade [in 40 ± 3 min and 120 ± 6 min (n = 4), respectively]; MA and MC produced complete blockade within 90–100 min. Preincubation of venoms with 292 μM VPL attenuated the blockade to varying degrees: the greatest inhibition was seen with MDC venom and blockade by MS venom was unaffected by this inhibitor. These results indicate that PLA2 has a variable contribution to coralsnake venom-induced neuromuscular blockade in vitro, with the insensitivity of MS venom to VPL suggesting that blockade by this venom is mediated predominantly by post-synaptically-active α-neurotoxins.

Mapping possible interaction sites for crotoxin in CNF, a gamma PLA2 inhibitor from Crotalus durissus terrificus rattle snake, using SPOT synthesis

Phospholipases A2 (PLA2s) are main components of snake venoms. Several snake species possess endogenous PLA2 inhibitors in their circulating blood, which are generally known as sbPLIs (an acronym for snake blood phospholipase A2inhibitors). The sbPLIs are categorized in three classes (alpha, beta or gamma) depending on the existence of distinguishing protein domains in their structure. The Crotalus durrissus terrificus venom has a highly neurotoxic PLA2 - crotoxin (CTX) - in its composition and the self-protection of the snake is mainly ensured by a sbγPLI named CNF (standing for Crotalusneutralizing factor). In an attempt to find smaller molecules able to inhibit the catalytic activity of CTX, in the present study we used linear peptide arrays to identify CNF segments possibly involved in the interaction with the toxin. Five reacting segments were identified as possible interacting regions. The target peptides were synthesized and located in the in silico CNF structure. Although all of them are exposed to the solvent, high concentrations were needed to inhibit the PLA2 activity of the whole venom or CTX. Limitations of the methodology employed and particular characteristics of CTX inhibition by CNF are discussed.

Infection, Inflammation, and Immunity in Sepsis.

Sepsis is triggered by microbial infection, injury, or even major surgery. Both innate and adaptive immune systems are involved in its pathogenesis. Cytoplasmic presence of DNA or RNA of the invading organisms or damaged nuclear material (in the form of micronucleus in the cytoplasm) in the host cell need to be eliminated by various nucleases; failure to do so leads to the triggering of inflammation by the cellular cGAS-STING system, which induces the release of IL-6, TNF-α, and IFNs. These cytokines activate phospholipase A2 (PLA2), leading to the release of polyunsaturated fatty acids (PUFAs), gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which form precursors to various pro- and anti-inflammatory eicosanoids. On the other hand, corticosteroids inhibit PLA2 activity and, thus, suppress the release of GLA, AA, EPA, and DHA. PUFAs and their metabolites have a negative regulatory action on the cGAS-STING pathway and, thus, suppress the inflammatory process and initiate inflammation resolution. Pro-inflammatory cytokines and corticosteroids (corticosteroids > IL-6, TNF-α) suppress desaturases, which results in decreased formation of GLA, AA, and other PUFAs from the dietary essential fatty acids (EFAs). A deficiency of GLA, AA, EPA, and DHA results in decreased production of anti-inflammatory eicosanoids and failure to suppress the cGAS-STING system. This results in the continuation of the inflammatory process. Thus, altered concentrations of PUFAs and their metabolites, and failure to suppress the cGAS-STING system at an appropriate time, leads to the onset of sepsis. Similar abnormalities are also seen in radiation-induced inflammation. These results imply that timely administration of GLA, AA, EPA, and DHA, in combination with corticosteroids and anti-IL-6 and anti-TNF-α antibodies, may be of benefit in mitigating radiation-induced damage and sepsis.

NOX1 and PRDX6 synergistically support migration and invasiveness of hepatocellular carcinoma cells through enhanced NADPH oxidase activity

The NADPH oxidase 1 (NOX1) complex formed by proteins NOX1, p22phox, NOXO1, NOXA1, and RAC1 plays an important role in the generation of superoxide and other reactive oxygen species (ROS) which are involved in normal and pathological cell functions due to their effects on diverse cell signaling pathways. Cell migration and invasiveness are at the origin of tumor metastasis during cancer progression which involves a process of cellular de-differentiation known as the epithelial-mesenchymal transition (EMT). During EMT cells lose their polarized epithelial phenotype and express mesenchymal marker proteins that enable cytoskeletal rearrangements promoting cell migration, expression and activation of matrix metalloproteinases (MMPs), tissue remodeling, and cell invasion during metastasis. In this work, we explored the importance of the peroxiredoxin 6 (PRDX6)-NOX1 enzyme interaction leading to NOXA1 protein stabilization and increased levels of superoxide produced by NOX in hepatocarcinoma cells. This increase was accompanied by higher levels of N-cadherin and MMP2, correlating with a greater capacity for cell migration and invasiveness of SNU475 hepatocarcinoma cells. The increase in superoxide and the associated downstream effects on cancer progression were suppressed when phospholipase A2 or peroxidase activities of PRDX6 were abolished by site-directed mutagenesis, reinforcing the importance of these catalytic activities in supporting NOX1-based superoxide generation. Overall, these results demonstrate a clear functional cooperation between NOX1 and PRDX6 catalytic activities which generate higher levels of ROS production, resulting in a more aggressive tumor phenotype.

Protective effect of exogenous peroxiredoxin 6 and thymic peptide thymulin on BBB conditions in an experimental model of multiple sclerosis

This study aimed to assess the effects of the immunomodulator thymulin, a thymic peptide with anti-inflammatory effects, and peroxiredoxin 6 (Prdx6), an antioxidant enzyme with dual peroxidase and phospholipase A2 activities, on the blood‒brain barrier (BBB) condition and general health status of animals with relapsing-remitting experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis in humans. Both thymulin and Prdx6 significantly improved the condition of the BBB, which was impaired by EAE induction, as measured by Evans blue dye accumulation, tight-junction protein loss in brain tissue, and lymphocyte infiltration through the BBB. The effect was associated with significant amelioration of EAE symptoms. Thymulin treatment was accompanied by a decrease in immune cell activation as judged by interleukin-6, -17, and interferon-gamma cytokine levels in serum and NF-kappaB cascade activation in splenocytes of mice with EAE. Prdx6 did not induce significant immunomodulatory effects but abruptly decreased EAE-induced NOX1 and NOX4 gene expression in brain tissue, which may be one of the possible mechanisms of its beneficial effects on BBB conditions and health status. The simultaneous administration of thymulin and Prdx6 resulted in complete symptomatic restoration of mice with EAE. The results demonstrate prospective strategies for multiple sclerosis treatment.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from “EPR silent” liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured “in vitro” was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of the medically most important Naja spp. African snakes.

Envenomations by African snakes represent a high burden in the sub-Sahara region. The design and fabrication of polyspecific antivenoms with a broader effectiveness, specially tailored for its use in sub-Saharan Africa, require a better understanding of the immunological features of different Naja spp. venoms of highest medical impact in Africa; and to select the most appropriate antigen combinations to generate antivenoms of wider neutralizing scope. Rabbit-derived monospecific antisera were raised against the venoms of five spitting cobras and six non-spitting cobras. The effects of immunization in the animal model were assessed, as well as the development of antibody titers, as proved by immunochemical assays and neutralization of lethal, phospholipase A2 and dermonecrotic activities. By the end of the immunization schedule, the immunized rabbits showed normal values of all hematological parameters, and no muscle tissue damage was evidenced, although alterations in aspartate aminotransferase (AST) and alkaline phosphatase (ALP) suggested a degree of hepatic damage caused mainly by spitting cobra venoms. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within the spitting and no-spitting cobras, yet some antisera showed more extensive cross-reactivity than others. The antisera with the widest coverage were those of anti-Naja ashei and anti-N. nigricollis for the spitting cobras, and anti-N. haje and anti-N. senegalensis for the non-spitting cobras. The methods and study design followed provide a rationale for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against cobra venoms in sub-Saharan Africa. Results suggest that venoms from N. ashei, N. nigricollis within the spitting cobras, and N. haje and N. senegalensis within the non-spitting cobras, generate antisera with a broader cross-reactivity. These experimental results should be translated to larger animal models used in antivenom elaboration to assess whether these predictions are reproduced.

An Increase in Peroxiredoxin 6 Expression Induces Neurotoxic A1 Astrocytes in the Lumbar Spinal Cord of Amyotrophic Lateral Sclerosis Mice Model.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with selective degeneration of motor neurons. It has been reported that an increase in the levels of inflammatory cytokines and glial cells such as reactive astrocytes is closely involved in the pathological progression of ALS. Recently, the levels of neuropathic cytotoxic (A1) astrocytes among reactive astrocytes have reportedly increased in the central nervous system of ALS mice, which induce motor neuron degeneration through the production of inflammatory cytokines and secretion of neuropathic factors. Hence, elucidating the induction mechanism of A1 astrocytes in ALS is important to understand the mechanism of disease progression in ALS. In this study, we observed that the expression of peroxiredoxin 6 (PRDX6), a member of the peroxiredoxin family, was markedly upregulated in astrocytes of the lumbar spinal cord of SOD1G93A mice model for ALS. Additionally, when PRDX6 was transiently transfected into the mouse astrocyte cell line C8-D1A and human astrocytoma cell line U-251 MG, the mRNA expression of complement C3 (a marker for A1 astrocyte phenotype) and inflammatory cytokines was increased. Furthermore, the mRNA expression of C3 and inflammatory cytokine was increased in C8-D1A and U-251 MG cells stably expressing PRDX6, and the increased mRNA expression was significantly suppressed by MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), an inhibitor of the phospholipase A2 activity of PRDX6. Our results suggest that the expression of PRDX6 in astrocytes plays an important role in the induction of A1 astrocytes and expression of inflammatory cytokines in the ALS mice model.

The phospholipase effector Tle1Vc promotes Vibrio cholerae virulence by killing competitors and impacting gene expression

ABSTRACT Vibrio cholerae utilizes the Type VI secretion system (T6SS) to gain an advantage in interbacterial competition by delivering anti-prokaryotic effectors in a contact-dependent manner. However, the impact of T6SS and its secreted effectors on physiological behavior remains poorly understood. In this study, we present Tle1Vc, a phospholipase effector in atypical pathogenic V. cholerae E1 that is secreted by T6SS via its interaction with VgrG1E1. Tle1Vc contains a DUF2235 domain and belongs to the Tle1 (type VI lipase effector) family. Bacterial toxicity assays, lipase activity assays and site-directed mutagenesis revealed that Tle1Vc possessed phospholipase A1 activity and phospholipase A2 activity, and that Tle1Vc-induced toxicity required a serine residue (S356) and two aspartic acid residues (D417 and D496). Cells intoxication with Tle1Vc lead to membrane depolarization and alter membrane permeability. Tli1tox-, a cognate immunity protein, directly interacts with Tle1Vc to neutralize its toxicity. Moreover, Tle1Vc can kill multiple microorganisms by T6SS and promote in vivo fitness of V. cholerae through mediating antibacterial activity. Tle1Vc induces bacterial motility by increasing the expression of flagellar-related genes independently of functional T6SS and the tit-for-tat (TFT) response, where Pseudomonas aeruginosa uses its T6SS-H1 cluster to counterattack other offensive attackers. Our study also demonstrated that the physical puncture of E1 T6SS can induce a moderate TFT response, which is essential to the Tle1Vc-mediated strong TFT response, maximizing effector functions. Overall, our study characterized the antibacterial mechanism of phospholipase effector Tle1Vc and its multiple physiological significance.

Impact of Truncated Oxidized Phosphatidylcholines on Phospholipase A2 Activity in Mono- and Polyunsaturated Biomimetic Vesicles.

The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A2 (sPLA2) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA2 modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA2 activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA2 activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA2 activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA2 activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA2 activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.

Identification of the glycosylphosphatidylinositol-specific phospholipase A2 (GPI-PLA2) that mediates GPI fatty acid remodeling in Trypanosoma brucei

The biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in the parasitic protozoan Trypanosoma brucei involves fatty acid remodeling of the GPI precursor molecules before they are transferred to protein in the endoplasmic reticulum. The genes encoding the requisite phospholipase A2 and A1 activities for this remodeling have thus far been elusive. Here, we identify a gene, Tb927.7.6110, that encodes a protein that is both necessary and sufficient for GPI-phospholipase A2 (GPI-PLA2) activity in the procyclic form of the parasite. The predicted protein product belongs to the alkaline ceramidase, PAQR receptor, Per1, SID-1, and TMEM8 (CREST) superfamily of transmembrane hydrolase proteins and shows sequence similarity to Post-GPI-Attachment to Protein 6 (PGAP6), a GPI-PLA2 that acts after transfer of GPI precursors to protein in mammalian cells. We show the trypanosome Tb927.7.6110 GPI-PLA2 gene resides in a locus with two closely related genes Tb927.7.6150 and Tb927.7.6170, one of which (Tb927.7.6150) most likely encodes a catalytically inactive protein. The absence of GPI-PLA2 in the null mutant procyclic cells not only affected fatty acid remodeling but also reduced GPI anchor sidechain size on mature GPI-anchored procyclin glycoproteins. This reduction in GPI anchor sidechain size was reversed upon the re-addition of Tb927.7.6110 and of Tb927.7.6170, despite the latter not encoding GPI precursor GPI-PLA2 activity. Taken together, we conclude that Tb927.7.6110 encodes the GPI-PLA2 of GPI precursor fatty acid remodeling and that more work is required to assess the roles and essentiality of Tb927.7.6170 and the presumably enzymatically inactive Tb927.7.6150.

Ferrostatin-1 attenuates brain injury in animal model of subarachnoid hemorrhage via phospholipase A2 activity of PRDX6.

Subarachnoid hemorrhage (SAH) is an acute catastrophic neurological disorder with high morbidity and mortality. Ferroptosis is one of the pathophysiological processes during secondary brain injury of SAH, which could be inhibited by ferrostatin-1 (Fer-1) effectively. Peroxiredoxin6 (PRDX6) is an antioxidant protein and is currently proven to be associated with lipid peroxidation in ferroptosis except in GSH/GPX4 and FSP1/CoQ10 antioxidant systems. However, the alteration and function of PRDX6 in SAH are still unknown. In addition, whether PRDX6 is involved in the neuroprotection of Fer-1 in SAH is yet to be investigated. Endovascular perforation was employed to induce the SAH model. Fer-1 and in vivo siRNA aiming to knockdown PRDX6 were administrated intracerebroventricularly to investigate relevant regulation and mechanism. We confirmed the inhibition of ferroptosis and neuroprotection from brain injury by Fer-1 in SAH. The induction of SAH reduced the expression of PRDX6, which could be alleviated by Fer-1. Accordingly, dysregulated lipid peroxidation indicated by GSH and MDA was improved by Fer-1, which was counteracted by si-PRDX6. Similarly, the neuroprotection of Fer-1 in SAH was diminished by the knockdown of PRDX6 and the administration of a calcium-independent phospholipase A2 (iPLA2) inhibitor. PRDX6 is involved in ferroptosis induced by SAH and is associated with Fer-1 neuroprotection from brain injury via its iPLA2 activity.