Search Results (23)

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones. Full article

Integrins were originally identified as receptors for extracellular matrix (ECM) and cell-surface molecules (e.g., VCAM-1 and ICAM-1). Later, we discovered that many soluble growth factors/cytokines bind to integrins and play a critical role in growth factor/cytokine signaling (growth factor–integrin crosstalk). We performed a virtual screening of protein data bank (PDB) using docking simulations with the integrin headpiece as a target. We showed that several growth factors (e.g., FGF1 and IGF1) induce a integrin-growth factor-cognate receptor ternary complex on the surface. Growth factor/cytokine mutants defective in integrin binding were defective in signaling functions and act as antagonists of growth factor signaling. Unexpectedly, several growth factor/cytokines activated integrins by binding to the allosteric site (site 2) in the integrin headpiece, which is distinct from the classical ligand (RGD)-binding site (site 1). Since 25-hydroxycholesterol, a major inflammatory mediator, binds to site 2, activates integrins, and induces inflammatory signaling (e.g., IL-6 and TNFα secretion), it has been proposed that site 2 is involved in inflammatory signaling. We showed that several inflammatory factors (CX3CL1, CXCL12, CCL5, sPLA2-IIA, and P-selectin) bind to site 2 and activate integrins. We propose that site 2 is involved in the pro-inflammatory action of these proteins and a potential therapeutic target. It has been well-established that platelet integrin αIIbβ3 is activated by signals from the inside of platelets induced by platelet agonists (inside-out signaling). In addition to the canonical inside-out signaling, we showed that αIIbβ3 can be allosterically activated by inflammatory cytokines/chemokines that are stored in platelet granules (e.g., CCL5, CXCL12) in the absence of inside-out signaling (e.g., soluble integrins in cell-free conditions). Thus, the allosteric activation may be involved in αIIbβ3 activation, platelet aggregation, and thrombosis. Inhibitory chemokine PF4 (CXCL4) binds to site 2 but did not activate integrins, Unexpectedly, we found that PF4/anti-PF4 complex was able to activate integrins, indicating that the anti-PF4 antibody changed the phenotype of PF4 from inhibitory to inflammatory. Since autoantibodies to PF4 are detected in vaccine-induced thrombocytopenic thrombosis (VIPP) and autoimmune diseases (e.g., SLE, and rheumatoid arthritis), we propose that this phenomenon is related to the pathogenesis of these diseases. P-selectin is known to bind exclusively to glycans (e.g., sLex) and involved in cell–cell interaction by binding to PSGL-1 (CD62P glycoprotein ligand-1). Unexpectedly, through docking simulation, we discovered that the P-selectin C-type lectin domain functions as an integrin ligand. It is interesting that no one has studied whether P-selectin binds to integrins in the last few decades. The integrin-binding site and glycan-binding site were close but distinct. Also, P-selectin lectin domain bound to site 2 and allosterically activated integrins. Full article

Host molecules with antimicrobial properties belong to a large family of mediators including type-IIA secreted phospholipase A2 (sPLA2-IIA). The latter is a potent bactericidal agent with high selectivity against Gram-positive bacteria, but it may also play a role in modulating the host inflammatory response. However, several pathogen-associated molecular patterns (PAMPs) or toxins produced by pathogenic bacteria can modulate the levels of sPLA2-IIA by either inducing or inhibiting its expression in host cells. Thus, the final sPLA2-IIA concentration during the infection process is determined by the orchestration between the levels of toxins that stimulate and those that downregulate the expression of this enzyme. The stimulation of sPLA2-IIA expression is a process that participates in the clearance of invading bacteria, while inhibition of this expression highlights a mechanism by which certain bacteria can subvert the immune response and invade the host. Here, we will review the major functions of sPLA2-IIA in the airways and the role of bacterial toxins in modulating the expression of this enzyme. We will also summarize the major mechanisms involved in this modulation and the potential consequences for the pulmonary host response to bacterial infection. Full article

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A₂ that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A₂ IIA (sPLA₂-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA₂-IIA levels together with eicosanoids in the sera of COVID patients. sPLA₂ could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A₂, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA₂-IIA in COVID-19-associated coagulopathy (CAC). Full article

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with a very poor prognosis as it has a 2.5 to 5 years mean survival after proper diagnosis. Even nintedanib and pirfenidone cannot halt the progression, though they slow the progression of IPF. Hence, there is a need to understand the novel pathophysiology. Phospholipase A2 (PLA2) could be the ideal candidate to study in IPF, as they have a role in both inflammation and fibrosis. In the present study, we have shown the expression profile of various secretory Phospholipase A2 (PLA2) isoforms by analyzing publicly available transcriptome data of single cells from the lungs of healthy individuals and IPF patients. Among 11 members of sPLA2, PLA2G2A is found to be increased in the fibroblasts and mesothelial cells while PLA2G5 is found to be increased in the fibroblasts of IPF patients. We identified a subset of fibroblasts expressing high PLA2G2A with moderate expression of PLA2G5 and which are specific to IPF only; we named it as PLA2G2A+ IPF fibroblast. Pathway analysis revealed that these PLA2G2A+ IPF fibroblast have upregulation of both inflammatory and fibrosis-related pathways like the TGF-β signaling pathway, IL-17 signaling, the arachidonic acid metabolism pathway and ECM-receptor interaction. In addition to this, we found elevated levels of sPLA2-IIA in plasma samples of IPF patients in our cohort. PLA2G3, PLA2G10 and PLA2G12B are found in to be increased in certain epithelial cells of IPF patients. Thus, these findings indicate that these five isoforms have a disease-dominant role along with innate immune roles as these isoforms are found predominantly in structural cells of IPF patients. Further, we have targeted sPLA2 in mice model of bleomycin-induced lung fibrosis by pBPB, a known sPLA2 inhibitor. pBPB treatment attenuated lung fibrosis induced by bleomycin along with a reduction in TGF-β and deposition of extracellular matrix in lung. Thus, these findings indicate that these sPLA2 isoforms especially PLA2G2A may serve as a therapeutic target in lung fibrosis. Full article

Cancer-related inflammation has recently emerged as an important component of cancer pathogenesis that is able to promote tumor initiation and progression, and the acquisition of the known hallmark capabilities, including evasion from immunosurveillance. Several soluble and cellular mediators participate in tumor microenvironment formation, leading to cancer initiation and progression. In this view, Tumor-Associated Macrophages (TAMs) are pivotal players and, due to their characteristic plasticity, can acquire a variety of distinct phenotypes and contribute in different ways to the different phases of carcinogenesis. Different stimuli have been shown to modulate macrophage polarization. Secreted phospholipase A₂ enzymes (sPLA₂s) exert multiple biological effects on cancer-related inflammation due to their enzymatic activity and ability to activate inflammatory cells by non-enzymatic mechanisms. Among the different sPLA₂ isoforms, several studies have suggested that group IIA and group X are mainly involved in a wide variety of cancer types. A deeper insight into the molecular mechanisms regulating the link between tumor-infiltrating immune cells and cancer could lead to identifying new prognostic/predictive biomarkers and a broader view of cancer immunotherapy. Full article

Human Group IIA secreted phospholipase A₂ (sPLA₂-IIA) enzyme plays a crucial role in several chronic inflammatory diseases such asasthma, atherosclerosis, gout, bronchitis, etc. Several studies showed that the antioxidants exert an anti-inflammatory function by inhibiting the sPLA₂-IIA enzyme. Hence, the present study evaluated an antioxidant molecule, sinapic acid, for sPLA₂-IIA inhibition as an anti-inflammatory function. Initially, the antioxidant efficacy of sinapic acid was evaluated, and it showed greater antioxidant potency. Further, sinapic acid inhibited 94.4 ± 4.83% of sPLA₂-IIA activity with an IC₅₀ value of 4.16 ± 0.13 µM. The mode of sPLA₂-IIA inhibition was examined by increasing the substrate concentration from 30 to 120nM and the calcium concentration from 2.5 to 15 mM, which did not change the level of inhibition. Further, sinapic acid altered the intrinsic fluorescence and distorted the far UltraViolet Circular Dichroism (UV-CD) spectra of the sPLA₂-IIA, indicating the direct enzyme-inhibitor interaction. Sinapic acid reduced the sPLA₂-IIA mediated hemolytic activity from 94 ± 2.19% to 12.35 ± 2.57% and mouse paw edema from 171.75 ± 2.2% to 114.8 ± 1.98%, demonstrating the anti-inflammatory efficiency of sinapic acid by in situ and in vivo methods, respectively. Finally, sinapic acid reduced the hemorrhagic effect of Vipera russelli venom hemorrhagic complex-I (VR-HC-I) as an anti-hemorrhagic function. Thus, the above experimental results revealed the sinapic acid potency to be an antioxidant, anti-inflammatory and anti-hemorrhagic molecule, and therefore, it appears to be a promising therapeutic agent. Full article

Among the phospholipase A₂ (PLA₂) superfamily, the secreted PLA₂ (sPLA₂) family contains 11 mammalian isoforms that exhibit unique tissue or cellular distributions and enzymatic properties. Current studies using sPLA₂-deficient or -overexpressed mouse strains, along with mass spectrometric lipidomics to determine sPLA₂-driven lipid pathways, have revealed the diverse pathophysiological roles of sPLA₂s in various biological events. In general, individual sPLA₂s exert their specific functions within tissue microenvironments, where they are intrinsically expressed through hydrolysis of extracellular phospholipids. Recent studies have uncovered a new aspect of group IIA sPLA₂ (sPLA₂-IIA), a prototypic sPLA₂ with the oldest research history among the mammalian PLA₂s, as a modulator of the gut microbiota. In the intestine, Paneth cell-derived sPLA₂-IIA acts as an antimicrobial protein to shape the gut microbiota, thereby secondarily affecting inflammation, allergy, and cancer in proximal and distal tissues. Knockout of intestinal sPLA₂-IIA in BALB/c mice leads to alterations in skin cancer, psoriasis, and anaphylaxis, while overexpression of sPLA₂-IIA in Pla2g2a-null C57BL/6 mice induces systemic inflammation and exacerbates arthritis. These phenotypes are associated with notable changes in gut microbiota and fecal metabolites, are variable in different animal facilities, and are abrogated after antibiotic treatment, co-housing, or fecal transfer. These studies open a new mechanistic action of this old sPLA₂ and add the sPLA₂ family to the growing list of endogenous factors capable of affecting the microbe–host interaction and thereby systemic homeostasis and diseases. Full article

Phospholipases A₂s (PLA₂s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA₂s (svPLA₂s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA₂s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA₂s are classified into the group IIA secreted PLA₂s, which encompasses mammalian inflammatory sPLA₂s. Thus, research on svPLA₂s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA₂s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA₂s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells. Full article

Phospholipase A₂ (PLA₂) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA₂) enzymes were the first of the five major classes of human PLA₂s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA₂, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA₂ field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function. Full article

Matrix metalloproteinases (MMPs) are proteolytic enzymes that have been associated with the pathogenesis of inflammatory diseases and obesity. Adipose tissue in turn is an active endocrine organ capable of secreting a range of proinflammatory mediators with autocrine and paracrine properties, which contribute to the inflammation of adipose tissue and adjacent tissues. However, the potential inflammatory effects of MMPs in adipose tissue cells are still unknown. This study investigates the effects of BmooMPα-I, a single-domain snake venom metalloproteinase (SVMP), in activating an inflammatory response by 3T3-L1 preadipocytes in culture, focusing on prostaglandins (PGs), cytokines, and adipocytokines biosynthesis and mechanisms involved in prostaglandin E₂ (PGE₂) release. The results show that BmooMPα-I induced the release of PGE₂, prostaglandin I₂ (PGI₂), monocyte chemoattractant protein-1 (MCP-1), and adiponectin by preadipocytes. BmooMPα-I-induced PGE₂ biosynthesis was dependent on group-IIA-secreted phospholipase A₂ (sPLA₂-IIA), cytosolic phospholipase A₂-α (cPLA₂-α), and cyclooxygenase (COX)-1 and -2 pathways. Moreover, BmooMPα-I upregulated COX-2 protein expression but not microsomal prostaglandin E synthase-1 (mPGES-1) expression. In addition, we demonstrate that the enzymatic activity of BmooMPα-I is essential for the activation of prostanoid synthesis pathways in preadipocytes. These data highlight preadipocytes as important targets for metalloproteinases and provide new insights into the contribution of these enzymes to the inflammation of adipose tissue and tissues adjacent to it. Full article

P21 activated kinases (or group I PAKs) are serine/threonine kinases whose expression is altered in prostate and breast cancers. PAK-1 activity is inhibited by the small molecule “Inhibitor targeting PAK-1 activation-3” (IPA-3), which has selectivity for PAK-1 but is metabolically unstable. Secretory Group IIA phospholipase A₂ (sPLA₂) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA_2, called Secretory Phospholipase Responsive liposomes or SPRL-IPA-3, and demonstrated their ability to alter prostate cancer growth. The efficacy of SPRL against other types of cancers is not well understood. We addressed this limitation by determining the ability of SPRL to induce cell death in a diverse panel of cells representing different stages of breast cancer, including the invasive but non-metastatic MCF-7 cells, and metastatic triple-negative breast cancer (TNBC) cells such as MDA-MB-231, MDA-MB-468, and MDA-MB-435. We investigated the role of sPLA₂ in the disposition of these liposomes by comparing the efficacy of SPRL-IPA-3 to IPA-3 encapsulated in sterically stabilized liposomes (SSL-IPA-3), a formulation shown to be less sensitive to sPLA₂. Both SSL-IPA-3 and SPRL-IPA-3 induced time- and dose-dependent decreases in MTT staining in all cell lines tested, but SPRL-IPA-3-induced effects in metastatic TNBC cell lines were superior over SSL-IPA-3. The reduction in MTT staining induced by SPRL-IPA-3 correlated to the expression of Group IIA sPLA₂. sPLA₂ expression also correlated to increased induction of apoptosis in TNBC cell lines by SPRL-IPA-3. These data suggest that SPRL-IPA-3 is selective for metastatic TNBC cells and that the efficacy of SPRL-IPA-3 is mediated, in part, by the expression of Group IIA sPLA₂. Full article

Adipose tissue secretes proinflammatory mediators which promote systemic and adipose tissue inflammation seen in obesity. Group IIA (GIIA)-secreted phospholipase A₂ (sPLA₂) enzymes are found to be elevated in plasma and adipose tissue from obese patients and are active during inflammation, generating proinflammatory mediators, including prostaglandin E₂ (PGE₂). PGE₂ exerts anti-lipolytic actions and increases triacylglycerol levels in adipose tissue. However, the inflammatory actions of GIIA sPLA₂s in adipose tissue cells and mechanisms leading to increased PGE₂ levels in these cells are unclear. This study investigates the ability of a representative GIIA sPLA₂, MT-III, to activate proinflammatory responses in preadipocytes, focusing on the biosynthesis of prostaglandins, adipocytokines and mechanisms involved in these effects. Our results showed that MT-III induced biosynthesis of PGE₂, PGI₂, MCP-1, IL-6 and gene expression of leptin and adiponectin in preadipocytes. The MT-III-induced PGE₂ biosynthesis was dependent on cytosolic PLA₂ (cPLA₂)-α, cyclooxygenases (COX)-1 and COX-2 pathways and regulated by a positive loop via the EP₄ receptor. Moreover, MT-III upregulated COX-2 and microsomal prostaglandin synthase (mPGES)-1 protein expression. MCP-1 biosynthesis induced by MT-III was dependent on the EP4 receptor, while IL-6 biosynthesis was dependent on EP3 receptor engagement by PGE₂. These data highlight preadipocytes as targets for GIIA sPLA₂s and provide insight into the roles played by this group of sPLA₂s in obesity. Full article

Secretory phospholipase-IIA A₂ (sPLA₂-IIA) is expressed in a variety of cell types under inflammatory conditions. Its presence in the bronchoalveolar lavage (BAL) fluid of patients with acute respiratory distress syndrome (ARDS) is associated with the severity of the injury. Exosomal type extracellular vesicles, (EVs), are recognized to perform intercellular communication. They may alter the immune status of recipient target cells through cargo shuttling. In this work, we characterized the exosomal type EVs isolated from BAL fluid of patients with early and late ARDS as compared to control/non-ARDS patients, through morphological (confocal and electron microscopy) and biochemical (dynamic light scattering, qRT-PCR, immunoblotting) approaches. We provide evidence for the presence of an sPLA₂-IIA-carrying EV pool that coprecipitates with exosomes in the BAL fluid of patients with ARDS. PLA2G2A mRNA was present in all the samples, although more prominently expressed in early ARDS. However, the protein was found only in EVs from early phase ARDS. Under both forms, sPLA₂-IIA might be involved in inflammatory responses of recipient lung cells during ARDS. The perception of the association of sPLA₂-IIA to the early diagnosis of ARDS or even with a mechanism of development and propagation of lung inflammation can help in the adoption of appropriate and innovative therapeutic strategies. Full article

Phospholipase A₂s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca²⁺-dependent secreted phospholipase A₂ (sPLA₂) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A₂s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A₂α. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A₂, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response. Full article