RESUMEN
Extracellular vesicles (EVs) represent small vesicles secreted from cells, including exosomes (40-150 nm in diameter), which are released via the multivesicular endosomal pathway, and microvesicles and ectosomes (100-1000 nm), which are produced by plasma membrane budding. Broadly, EVs also include vesicles generated from dying cells, such as apoptotic bodies (5-10 µm), as well as exomeres (< 50 nm), which are very small, non-membranous nanoparticles. EVs play important roles in cell-to-cell signaling in various aspects of cancer, immunity, metabolism, and so on by transferring proteins, microRNAs (miRNAs), and metabolites as cargos from donor cells to recipient cells. Although lipids are one of the major components of EVs, they have long been recognized as merely the "wall" that partitions the lumen of the vesicle from the outside. However, it has recently become obvious that lipid composition of EVs influences their properties and functions, that EVs act as a carrier of a variety of lipid mediators, and that lipid mediators are produced in EV membranes by the hydrolytic action of secreted phospholipase A2s (sPLA2s). In this article, we will make an overview of the roles of lipids in EVs, with a particular focus on sPLA2-driven mobilization of lipid mediators from EVs and its biological significance.
Asunto(s)
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Hidrólisis , Fosfolipasas A2 Secretoras/metabolismo , Fosfolipasas A2 Secretoras/genética , Animales , Exosomas/metabolismoRESUMEN
Myocardial reperfusion injury occurs when blood flow is restored after ischemia, an essential process to salvage ischemic tissue. However, this phenomenon is intricate, characterized by various harmful effects. Tissue damage in ischemia-reperfusion injury arises from various factors, including the production of reactive oxygen species, the sequestration of proinflammatory immune cells in ischemic tissues, the induction of endoplasmic reticulum stress, and the occurrence of postischemic capillary no-reflow. Secretory phospholipase A2 (sPLA2) plays a crucial role in the eicosanoid pathway by releasing free arachidonic acid from membrane phospholipids' sn-2 position. This liberated arachidonic acid serves as a substrate for various eicosanoid biosynthetic enzymes, including cyclooxygenases, lipoxygenases, and cytochromes P450, ultimately resulting in inflammation and an elevated risk of reperfusion injury. Therefore, the activation of sPLA2 directly correlates with the heightened and accelerated damage observed in myocardial ischemia-reperfusion injury (MIRI). Presently, clinical trials are in progress for medications aimed at sPLA2, presenting promising avenues for intervention. Cardiolipin (CL) plays a crucial role in maintaining mitochondrial function, and its alteration is closely linked to mitochondrial dysfunction observed in MIRI. This paper provides a critical analysis of CL modifications concerning mitochondrial dysfunction in MIRI, along with its associated molecular mechanisms. Additionally, it delves into various pharmacological approaches to prevent or alleviate MIRI, whether by directly targeting mitochondrial CL or through indirect means.
Asunto(s)
Cardiolipinas , Daño por Reperfusión Miocárdica , Humanos , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Animales , Cardiolipinas/metabolismo , Fosfolipasas A2 Secretoras/metabolismoRESUMEN
Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.
Asunto(s)
Fagocitosis , Proteína Quinasa C-alfa , Transducción de Señal , Animales , Fagocitosis/efectos de los fármacos , Ratones , Transducción de Señal/efectos de los fármacos , Masculino , Proteína Quinasa C-alfa/metabolismo , Macrófagos/efectos de los fármacos , Fosfolipasas A2 Secretoras/metabolismo , Venenos de Serpiente/toxicidad , Rifabutina/análogos & derivados , Rifabutina/farmacologíaRESUMEN
Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.
Asunto(s)
Bombyx , Fosfolipasas A2 Secretoras , Bombyx/genética , Bombyx/enzimología , Animales , Fosfolipasas A2 Secretoras/genética , Fosfolipasas A2 Secretoras/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Larva/genética , Clonación Molecular , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/biosíntesis , Secuencia de Aminoácidos , Perfilación de la Expresión GénicaRESUMEN
The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT-III-induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the ß-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.
Asunto(s)
Bothrops , Venenos de Crotálidos , Macrófagos , Óxido Nítrico , Fagocitosis , Transducción de Señal , Zimosan , Animales , Fagocitosis/efectos de los fármacos , Zimosan/farmacología , Transducción de Señal/efectos de los fármacos , Óxido Nítrico/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Fosfolipasas A2 Secretoras/metabolismoRESUMEN
Previous research suggests that group IIA-secreted phospholipase A2 (sPLA2-IIA) plays a role in and predicts lethal COVID-19 disease. The current study reanalyzed a longitudinal proteomic data set to determine the temporal relationship between levels of several members of a family of sPLA2 isoforms and the severity of COVID-19 in 214 ICU patients. The levels of six secreted PLA2 isoforms, sPLA2-IIA, sPLA2-V, sPLA2-X, sPLA2-IB, sPLA2-IIC, and sPLA2-XVI, increased over the first 7 ICU days in those who succumbed to the disease but attenuated over the same time period in survivors. In contrast, a reversed pattern in sPLA2-IID and sPLA2-XIIB levels over 7 days suggests a protective role of these two isoforms. Furthermore, decision tree models demonstrated that sPLA2-IIA outperformed top-ranked cytokines and chemokines as a predictor of patient outcome. Taken together, proteomic analysis revealed temporal sPLA2 patterns that reflect the critical roles of sPLA2 isoforms in severe COVID-19 disease.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/mortalidad , COVID-19/sangre , Femenino , Masculino , Persona de Mediana Edad , Anciano , Fosfolipasas A2 Secretoras/sangre , Proteómica/métodos , Índice de Severidad de la Enfermedad , Fosfolipasas A2 Grupo II/sangre , Adulto , Isoformas de Proteínas/sangre , Citocinas/sangreRESUMEN
Crotalus neutralizing factor (CNF) is an endogenous glycoprotein from Crotalus durissus terrificus snake blood that inhibits secretory phospholipases A2 (sPLA2) from the Viperid but not from Elapid venoms (subgroups IA and IIA, respectively). In the present study, we demonstrated that CNF can inhibit group III-PLA2 from bee venom by forming a stable enzyme-inhibitor complex. This finding opens up new possibilities for the potential use of CNF and/or CNF-based derivatives in the therapeutics of bee stings.
Asunto(s)
Venenos de Abeja , Crotalus , Serpientes Venenosas , Animales , Venenos de Abeja/farmacología , Inhibidores de Fosfolipasa A2/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Abejas , Fosfolipasas A2 , Glicoproteínas/farmacología , Fosfolipasas A2 Secretoras/antagonistas & inhibidoresRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Hawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. AIM OF THE STUDY: The in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. MATERIALS AND METHODS: The primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE-/-) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 µg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. RESULTS: The chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4â³-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2â³-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE-/- mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1êµ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1ß (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). CONCLUSION: HLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.
Asunto(s)
Aterosclerosis , Crataegus , Fosfolipasas A2 Secretoras , Placa Aterosclerótica , Ratones , Animales , Crataegus/química , Quercetina/uso terapéutico , Fosfolipasas A2 Secretoras/metabolismo , Interleucina-6/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Espectrometría de Masas en Tándem , Aterosclerosis/metabolismo , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/metabolismo , Macrófagos/metabolismo , Flavonoides/uso terapéutico , Lipoproteínas LDL/metabolismo , Transducción de Señal , Colesterol/metabolismo , Ratones Noqueados , Apolipoproteínas E/genéticaRESUMEN
Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of airway obstruction, hyperresponsiveness, remodeling, and eosinophilia. Phospholipase A2 s (PLA2 s), which release fatty acids and lysophospholipids from membrane phospholipids, have been implicated in exacerbating asthma by generating pro-asthmatic lipid mediators, but an understanding of the association between individual PLA2 subtypes and asthma is still incomplete. Here, we show that group III-secreted PLA2 (sPLA2 -III) plays an ameliorating, rather than aggravating, role in asthma pathology. In both mouse and human lungs, sPLA2 -III was expressed in bronchial epithelial cells and decreased during the asthmatic response. In an ovalbumin (OVA)-induced asthma model, Pla2g3-/- mice exhibited enhanced airway hyperresponsiveness, eosinophilia, OVA-specific IgE production, and type 2 cytokine expression as compared to Pla2g3+/+ mice. Lipidomics analysis showed that the pulmonary levels of several lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid (LPA), were decreased in OVA-challenged Pla2g3-/- mice relative to Pla2g3+/+ mice. LPA receptor 2 (LPA2 ) agonists suppressed thymic stromal lymphopoietin (TSLP) expression in bronchial epithelial cells and reversed airway hyperresponsiveness and eosinophilia in Pla2g3-/- mice, suggesting that sPLA2 -III negatively regulates allergen-induced asthma at least by producing LPA. Thus, the activation of the sPLA2 -III-LPA pathway may be a new therapeutic target for allergic asthma.
Asunto(s)
Asma , Eosinofilia , Fosfolipasas A2 Secretoras , Hipersensibilidad Respiratoria , Humanos , Animales , Ratones , Lisofosfolípidos , Fosfolipasas A2 Secretoras/genética , CitocinasRESUMEN
Phospholipase A2 (PLA2 ) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then oxygenated into different eicosanoids, which mediate the diverse physiological processes in insects. Any inhibition of the PLA2 catalysis would give rise to serious malfunctioning in insect growth and development. An onion moth, Acrolepiopsis sapporensis, encodes four different PLA2 genes (As-PLA2 A-As-PLA2 D), in which As-PLA2 A is dominantly expressed at all developmental stages and in different larval tissues. RNA interference of the As-PLA2 A expression significantly reduced the PLA2 activity of A. sapporensis, which suffered from immunosuppression. A recombinant As-PLA2 A protein was purified from a bacterial expression system, which exhibited a typical Michaelis-Menten kinetics and hence susceptible to a specific inhibitor to sPLA2 and dithiothreitol. A total of 19 bacterial metabolites derived from Xenorhabdus and Photorhabdus were screened against the recombinant As-PLA2 A. Five potent metabolites were highly inhibitory and followed a competitive enzyme inhibition. These five inhibitors suppressed the immune responses of A. sapporensis by inhibiting hemocyte-spreading behavior and phenoloxidase activity. However, an addition of AA could significantly rescue the immunosuppression induced by the selected inhibitors. These studies suggest that the recombinant As-PLA2 A protein can be applied for high-throughput screening of insect immunosuppressive compounds.
Asunto(s)
Fosfolipasas A2 Secretoras , Animales , Spodoptera , Fosfolipasas A2 Secretoras/genética , Fosfolipasas A2 Secretoras/metabolismo , Eicosanoides/metabolismo , Larva/metabolismo , Insectos , Ácido Araquidónico/metabolismoRESUMEN
Secreted phospholipase A2s (sPLA2s) are a group of enzymes with 6-8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.
Asunto(s)
Fosfolipasas A2 Secretoras , Saccharomycetales , Sales (Química) , Humanos , Proteínas Recombinantes/química , Pichia/genética , Pichia/metabolismo , Fosfolipasas A2 Secretoras/genética , Fosfolipasas A2 Secretoras/metabolismo , Disulfuros/metabolismoRESUMEN
This study presents a novel approach to synthesizing silver nanoparticles (Ag NPs) using a solution combustion synthesis (SCS) method with Catharanthus roseus (C. roseus) leaf extract. The NPs were thoroughly characterized through X-ray diffraction (XRD), Scanning electron microscopy (SEM), Energy dispersive X-ray (EDX), Transmission electron microscopy (TEM), and Selected area electron diffraction (SAED), elucidating their crystal structure. Notably, the synthesized Ag NPs exhibited a significant dose-dependent decline in viability of the MDA-MB 231 breast cancer cell line, with an IC50 value of 13.3â µg/mL, underscoring their potential as potent anticancer agent. Beyond cytotoxicity, the study pioneers an investigation into the biocompatibility of Ag NPs by blood hemolsysis, providing critical insights into their safety and biomedical applicability. Furthermore, this research uncovers a distinctive facet of Ag NPs, revealing their inhibitory effects on the inflammatory enzyme secretory phospholipase A2 (sPLA2), a recognized biomarker for breast cancer. The demonstrated inâ vitro and inâ vivo inhibition of sPLA2 highlights the multifaceted potential of Ag NPs in not only targeting cancer cells but also modulating inflammatory responses associated with breast cancer, positioning the study at the forefront of advancements in nanomedicine and cancer therapeutics.
Asunto(s)
Neoplasias de la Mama , Nanopartículas del Metal , Fosfolipasas A2 Secretoras , Humanos , Femenino , Plata/farmacología , Plata/química , Nanopartículas del Metal/química , Difracción de Rayos X , Neoplasias de la Mama/tratamiento farmacológico , Inflamación , Extractos Vegetales/química , Antibacterianos/farmacología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs-CoV-2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). METHODS: Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. RESULTS: Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID-19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α-III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12-HETE, exhibited relative specificity for COVID-19 and correlated with sPLA2 and the interleukin-13 receptor to drive lymphopenia, a marker of disease severity. Pro-inflammatory eicosanoids remained correlated with severity in COVID-19 28 days after admission. Amongst non-COVID ICU patients, elevations in 5- and 15-HETE and 9- and 13-HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflected disease severity in COVID-19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non-B non-T cell activation. In COVID-19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids - ChoE 18:3, LPC-O-16:0 and PC-O-30:0 - were altered specifically in COVID. LPC-O-16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi-omic inflammatory pathways and disease severity. CONCLUSIONS: A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12-HETE and the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients and correlate with the inflammatory response to link to disease severity.
Asunto(s)
COVID-19 , Fosfolipasas A2 Secretoras , Sepsis , Humanos , SARS-CoV-2 , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Lipidómica , Leucocitos Mononucleares , Leucotrieno E4 , Prostaglandina D2 , Ciclooxigenasa 2 , EicosanoidesRESUMEN
Epidermal lipids play important roles in skin homeostasis and diseases. Psoriasis is an inflammatory disease characterized by keratinocyte hyperproliferation and Th17 immune responses. We previously reported that ethanolamine-type lysoplasmalogen (P-LPE), preferentially produced by group IIF secreted PLA2 (sPLA2-IIF/PLA2G2F) that is expressed in the suprabasal epidermis, promotes epidermal hyperplasia in psoriatic inflammation. Herein, we show that forcible degradation of epidermal P-LPE by topical application of recombinant lysophospholipase D (LyPls-PLD) from Thermocrispum, a lysoplasmalogen-specific hydrolase, attenuated epidermal hyperplasia and inflammation in imiquimod-induced and K5.Stat3C-transgenic mouse psoriasis models. In humans, P-LPE levels were elevated in the tape-stripped stratum corneum of patients with psoriasis. Moreover, in primary cultured human epidermal keratinocytes, aberrant cell proliferation and activation by psoriatic cytokines were sPLA2-IIF/P-LPE-dependent and were suppressed by the addition of LyPls-PLD with a decrease in P-LPE. These findings confirm that the sPLA2-IIF/P-LPE axis in the epidermis indeed regulates psoriasis, that P-LPE is a lipid biomarker that predicts the severity of psoriasis, and that pharmacological removal of this bioactive lipid is useful to prevent the disease. Thus, our study may lead to the development of drug discovery and diagnostic techniques based on this pathway.
Asunto(s)
Fosfolipasas A2 Secretoras , Psoriasis , Ratones , Animales , Humanos , Hiperplasia/metabolismo , Epidermis/metabolismo , Epidermis/patología , Queratinocitos/metabolismo , Inflamación/metabolismo , Psoriasis/metabolismo , Ratones Transgénicos , Fosfolipasas A2 Secretoras/metabolismo , LípidosRESUMEN
Antivenom is currently the standard-of-care treatment for snakebite envenoming, but its efficacy is limited by treatment delays, availability, and in many cases, species specificity. Many of the rapidly lethal effects of envenoming are caused by venom-derived toxins, such as phospholipase A2 (sPLA2); therefore, small molecule direct toxin inhibitors targeting these toxins may have utility as initial and adjunct therapies after envenoming. Varespladib (intravenous, IV) and varespladib-methyl (oral) have been shown to potently inhibit sPLA2s from snake venoms in murine and porcine models, thus supporting their further study as potential treatments for snakebite envenoming. In this pilot study, we tested the ability of these compounds to reverse neurotoxic effects of venom from the Australian and Papuan taipan (Oxyuranus scutellatus) subspecies in juvenile pigs (Sus domesticus). The mean survival time for control animals receiving Australian taipan venom (0.03 mg/kg, n = 3) was 331 min ± 15 min; for those receiving Papuan taipan venom (0.15 mg/kg, n = 3) it was 178 ± 31 min. Thirteen pigs received Australian taipan venom and treatment with either IV or oral varespladib (or with IV to oral transition) and all 13 survived the duration of the study (≥96 h). Eight pigs received Papuan taipan venom followed by treatment: Briefly: Two animals received antivenom immediately and survived to the end of the study. Two animals received antivenom treatment delayed 45 min from envenoming and died within 4 h. Two animals received similarly delayed antivenom treatment and were rescued by varespladib. Two animals were treated with varespladib alone after a 45-min delay. Treatment with varespladib only was effective but required repeat dosing over the course of the study. Findings highlight both the importance of early treatment and, as well, a half-life for the investigational inhibitors now in Phase II clinical trials for snakebite. Varespladib rapidly reversed weakness even when administered many hours post-envenoming and, overall, our results suggest that varespladib and varespladib-methyl could be efficacious tools in the treatment of sPLA2-induced weakness from Oxyuranus envenoming. Further clinical study as initial therapy and as potential method of rescue from some types of antivenom-resistant envenomings are supported by these data.
Asunto(s)
Fosfolipasas A2 Secretoras , Mordeduras de Serpientes , Animales , Porcinos , Ratones , Antivenenos/farmacología , Antivenenos/uso terapéutico , Mordeduras de Serpientes/tratamiento farmacológico , Proyectos Piloto , Australia , Venenos Elapídicos/toxicidadRESUMEN
We investigated the cell functions of the Ca2+ signaling genes phospholipase C-1 (plc-1), Ca2+/H+ exchanger (cpe-1), and secretory phospholipase A2 (splA2) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA2 mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA2 mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants. Moreover, the ΔsplA2 mutant could not grow on microcrystalline cellulose during ER stress. Furthermore, plc-1, cpe-1, and splA2 synthetically regulate the acquisition of thermotolerance induced by heat shock, responses to alkaline pH and ER stress, and utilization of cellulose and other alternate carbon sources in N. crassa. In addition, expression of the alkaline pH regulator, pac-3, and heat shock proteins, hsp60, and hsp80 was reduced in the Δplc-1, Δcpe-1, and ΔsplA2 single and double mutants. The expression of the unfolded protein response (UPR) markers grp-78 and pdi-1 was also significantly reduced in the mutants showing growth defect during ER stress. The increased cellulolytic activities of the ΔsplA2 and Δcpe-1; ΔsplA2 mutants were due to increased cbh-1, cbh-2, and endo-2 expression in N. crassa. Therefore, plc-1, cpe-1, and splA2 are involved in stress responses and cellulose utilization in N. crassa.
Asunto(s)
Neurospora crassa , Fosfolipasas A2 Secretoras , Neurospora crassa/genética , Metabolismo de los Hidratos de Carbono , Carbono , CelulosaRESUMEN
Breast cancer is the second most cancer worldwide in females. The primary factor responsible for tumor recurrence is the presence of breast cancer stem cells (BCSCs), which escape the chemo-radiotherapy. In this study, we have investigated the role of Secretory phospholipase-A2 Group 2A (sPLA2-IIA) that is overexpressed in BCSCs of MCF7 and MDA-MB-231 breast cancer cell lines. Further, overexpression of sPLA2-IIA revealed an increased EGFR/JNK/c-JUN/c-FOS signaling in BCSCs, while sPLA2-IIA knockdown significantly reduced the percentage of BCSCs and decreased signaling in both the cell lines. Importantly, sPLA2-IIA knockdown showed differentiation of BCSCs. Strikingly, PET imaging showed a decreased metastatic potential of BCSCs. Our study revealed a novel role of sPLA2-IIA in regulating BCSCs, which play a crucial role in regulating the differentiation and metastatic potential of BCSCs.
Asunto(s)
Neoplasias de la Mama , Fosfolipasas A2 Secretoras , Femenino , Humanos , Fosfolipasas A2 Secretoras/genética , Fosfolipasas , Recurrencia Local de Neoplasia , Diferenciación Celular , Células Madre Neoplásicas , Fosfolipasas A2 Grupo II/genéticaRESUMEN
Serum amyloid A (SAA) is named after a life-threatening disease, yet this small evolutionarily conserved protein must have played a vital role in host defense. Most circulating SAA binds plasma lipoproteins and modulates their metabolism. However, this hardly justifies the rapid and dramatic SAA upregulation in inflammation, which is concomitant with upregulation of secretory phospholipase A2 (sPLA2). We proposed that these proteins synergistically clear cell membrane debris from the sites of injury. The present study uses biochemical and biophysical approaches to further explore the beneficial function of SAA and its potential links to amyloid formation. We show that murine and human SAA1 are powerful detergents that solubilize diverse lipids, including mammalian biomembranes, converting them into lipoprotein-size nanoparticles. These nanoparticles provide ligands for cell receptors, such as scavenger receptor CD36 or heparin/heparan sulfate, act as substrates of sPLA2, and sequester toxic products of sPLA2. Together, these functions enable SAA to rapidly clear unprotected lipids. SAA can also adsorb, without remodeling, to lipoprotein-size nanoparticles such as exosomal liposomes, which are proxies for lipoproteins. SAA in complexes with zwitterionic phospholipids stabilizes α-helices, while SAA in complexes containing anionic lipids or micelle-forming sPLA2 products forms metastable ß-sheet-rich species that readily aggregate to form amyloid. Consequently, the synergy between SAA and sPLA2 extends from the beneficial lipid clearance to the pathologic amyloid formation. Furthermore, we show that lipid composition alters SAA conformation and thereby can influence the metabolic fate of SAA-lipid complexes, including their proamyloidogenic and proatherogenic binding to heparan sulfate.
Asunto(s)
Fosfolipasas A2 Secretoras , Proteína Amiloide A Sérica , Humanos , Ratones , Animales , Proteína Amiloide A Sérica/metabolismo , Lipoproteínas , Fosfolípidos , Fosfolipasas A2 Secretoras/metabolismo , Heparitina Sulfato , Mamíferos/metabolismoRESUMEN
Background: Eicosanoids are a group of the oxygenated C20 polyunsaturated fatty acids and play crucial roles in mediating various insect physiological processes. Catalytic activity of phospholipase A2 (PLA2) provides an initial substrate, arachidonic acid (AA), for subsequent eicosanoid biosynthesis. Results: This study identified four different secretory PLA2 (As-PLA2A-As-PLA2D) genes encoded in the Asian onion moth, Acrolepiopsis sapporensis. A phylogenetic analysis indicated that As-PLA2A and As-PLA2D are clustered with Group III PLA2s while As-PLA2B and As-PLA2C are clustered with Group XII and Group X PLA2s, respectively. Expression levels of these PLA2 genes increased along with larval development, especially in the fat body. A bacterial immune challenge upregulated the basal expression levels of the four PLA2 genes, which resulted in significant increases of the PLA2 enzyme activity. The enzyme activity was susceptible to a calcium chelator or reducing agent, suggesting Ca2+ dependency and disulfide linkage required for the catalytic activities of the secretory type of PLA2s. In addition, the PLA2 activity was also susceptible to bromophenacyl bromide (BPB), a specific inhibitor to sPLA2, but not to intracellular PLA2 inhibitors. An addition of BPB to the immune challenge significantly prevented hemocyte-spreading behavior of A. sapporensis. BPB treatment also suppressed a cellular immune response measured by hemocyte nodule formation. However, the immunosuppression was significantly rescued by the AA addition. To determine the PLA2(s) responsible for the immunity, individual RNA interference (RNAi) treatments specific to each of the four PLA2s were performed. Injection of gene-specific double-stranded RNAs caused significant reductions in the transcript level in all four PLA2s. In all four PLA2s, the RNAi treatments prevented the cellular immune response even after the immune challenge. Conclusion: This study reports four secretory PLA2s encoded in A. sapporensis and their function in mediating cellular immunity.
Asunto(s)
Fosfolipasas A2 Secretoras , Animales , Ácido Araquidónico , Inmunidad Celular , Insectos , Fosfolipasas A2 Secretoras/genética , Filogenia , Spodoptera/metabolismoRESUMEN
Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.