Low Molecular Weight Phospholipases A2 in Mammalian Brain and Neural Cells: Roles in Functions and Dysfunctions

Several “low molecular weight” or “secretory” phospholipases A₂ isoforms may be expressed in mammalian neural cells. Indeed, mRNAs for GIB, GIIA, GIIE, GIII, GV, GX, and GXII were detected in brain tissues despite different levels. However, only the presence of GIB, GIIA, and GV proteins has been clearly demonstrated in neural cells or in the nervous tissue. Although the roles of GIB and GV in the nervous tissue are still elusive, there is evidence to support the involvement of GIIA in physiological and pathological events, including neurotransmission, long-term potentiation, and neuritogenesis. The neurotoxic effects of an increase in GIIA may be envisaged under pathological conditions associated with the activation of astrocytes during inflammation or through activation of neurons and enzymes due to the stimulation of the NMDA glutamate receptor. In the past, elevation of GIIA expression in many acute and chronic neurological diseases is well known. Although each neurodegenerative disease has a separate etiology, many share similar neurochemical common processes, such as excitotoxicity, oxidative stress, and mitochondrial dysfunction, phenomena where GIIA play an important role.     

Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum

Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0.8-5.8 microg/L, n = 28, P < 0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA(2) in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA(2)s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA(2)s above the detection limits of the current TR-FIAs.     

NGF Induces the Expression of Group IIA Secretory Phospholipase A2 in PC12 Cells: The Newly Synthesized Enzyme Is Addressed to Growing Neurites

We proposed that group IIA secretory phospholipase A₂ (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168–2174, 2010). The involvement of other secretory PLA₂ isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA₂ (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation–reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.     

Inhibition of secreted phospholipases A₂ by 2-oxoamides based on α-amino acids: Synthesis, in vitro evaluation and molecular docking calculations

Group IIA secreted phospholipase A? (GIIA sPLA?) is a member of the mammalian sPLA? enzyme family and is associated with various inflammatory conditions. In this study, the synthesis of 2-oxoamides based on α-amino acids and the in vitro evaluation against three secreted sPLA?s (GIIA, GV and GX) are described. The long chain 2-oxoamide GK126 based on the amino acid (S)-leucine displayed inhibition of human and mouse GIIA sPLA?s (IC?? 300nM and 180nM, respectively). It also inhibited human GV sPLA? with similar potency, while it did not inhibit human GX sPLA?. The elucidation of the stereoelectronic characteristics that affect the in vitro activity of these compounds was achieved by using a combination of simulated annealing to sample low-energy conformations before the docking procedure, and molecular docking calculations.     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

Groups IV, V, and X phospholipases A2s in human neutrophils: role in eicosanoid production and gram-negative bacterial phospholipid hydrolysis.

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable. GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2). GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.     

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A₂ (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A₂ (sPLA₂), but not a catalytically inactive mutant form of GX sPLA₂, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA₂ inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA₂-overexpressing Y1 cells, ruling out a role for this sPLA₂ receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA₂. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA₂ antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA₂ is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.     

Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations

Inhibition of group IIA secreted phospholipase A₂ (GIIA sPLA₂) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA₂s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC₅₀ = 143 nM and 68 nM against human and mouse GIIA sPLA₂, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity.     

Effect of NGF on the Subcellular Localization of Group IIA Secretory Phospholipase A2 (GIIA) in PC12 Cells: Role in Neuritogenesis

Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.     

Inhibitors of secreted phospholipase A2 suppress the release of PGE2 in renal mesangial cells

The upregulation of PGE₂ by mesangial cells has been observed under chronic inflammation condition. In the present work, renal mesangial cells were stimulated to trigger a huge increase of PGE₂ synthesis and were treated in the absence or presence of known PLA₂ inhibitors. A variety of synthetic inhibitors, mainly developed in our labs, which are known to selectively inhibit each of GIVA cPLA₂, GVIA iPLA₂, and GIIA/GV sPLA₂, were used as tools in this study. Synthetic sPLA₂ inhibitors, such as GK115 (an amide derivative based on the non-natural amino acid (R)-γ-norleucine) as well as GK126 and GK241 (2-oxoamides based on the natural (S)-α-amino acid leucine and valine, respectively) presented an interesting effect on the suppression of PGE₂ formation.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism

Group X secretory phospholipase A₂ (GX sPLA₂) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA₂ is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA₂-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA₂ overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA₂ (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA₂ on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA₂ plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

The Role of Secretory Phospholipase A2 in the Central Nervous System and Neurological Diseases

Secretory phospholipase A₂ (sPLA₂s) are small secreted proteins (14–18 kDa) and require submillimolar levels of Ca²⁺ for liberating arachidonic acid from cell membrane lipids. In addition to the enzymatic function, sPLA₂ can exert various biological responses by binding to specific receptors. Physiologically, sPLA₂s play important roles on the neurotransmission in the central nervous system and the neuritogenesis in the peripheral nervous system. Pathologically, sPLA₂s are involved in the neurodegenerative diseases (e.g., Alzheimer’s disease) and cerebrovascular diseases (e.g., stoke). The common pathology (e.g., neuronal apoptosis) of Alzheimer’s disease and stroke coexists in the mixed dementia, suggesting common pathogenic mechanisms of the two neurological diseases. Among mammalian sPLA₂s, sPLA₂-IB and sPLA₂-IIA induce neuronal apoptosis in rat cortical neurons. The excess influx of calcium into neurons via l-type voltage-dependent Ca²⁺ channels mediates the two sPLA₂-induced apoptosis. The elevated concentration of intracellular calcium activates PKC, MAPK and cytosolic PLA₂. Moreover, it is linked with the production of reactive oxygen species and apoptosis through activation of the superoxide producing enzyme NADPH oxidase. NADPH oxidase is involved in the neurotoxicity of amyloid β peptide, which impairs synaptic plasticity long before its deposition in the form of amyloid plaques of Alzheimer’s disease. In turn, reactive oxygen species from NADPH oxidase can stimulate ERK1/2 phosphorylation and activation of cPLA₂ and result in a release of arachidonic acid. sPLA₂ is up-regulated in both Alzheimer’s disease and cerebrovascular disease, suggesting the involvement of sPLA₂ in the common pathogenic mechanisms of the two diseases. Thus, our review presents evidences for pathophysiological roles of sPLA₂ in the central nervous system and neurological diseases.     

Effects of the propeptide of group X secreted phospholipase A2 on substrate specificity and interfacial activity on phospholipid monolayers

Group X secreted phospholipase A₂ (GX sPLA₂) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA₂ activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA₂ activity of recombinant mouse GX sPLA₂ (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA₂ activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30–35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA₂ activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA₂. This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance.     

Lactadherin Inhibits Secretory Phospholipase A2 Activity on Pre-Apoptotic Leukemia Cells

Secretory phospholipase A₂ (sPLA₂) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA₂ concentrations are associated with autoimmune diseases and septic shock. Many sPLA₂’s do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A₂-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA₂ plateaued at 50–60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA₂ and >70% of human secretory phospholipase A₂-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA₂.     

Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

Background

Previous work has shown that disruption of the gene for group X secreted phospholipase A₂ (sPLA₂-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA₂s in the mammalian genome, the involvement of other sPLA₂s in the asthma model is possible – in particular, the group V sPLA₂ (sPLA₂-V) that like sPLA₂-X is highly active at hydrolyzing membranes of mammalian cells.

Methodology and Principal Findings

The allergen-driven asthma phenotype was significantly reduced in sPLA₂-V-deficient mice but to a lesser extent than observed previously in sPLA₂-X-deficient mice. The most striking difference observed between the sPLA₂-V and sPLA₂-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA₂-V^−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V^−/− mice diminishes Th2 cytokine responses in the airways.

Conclusions

This paper illustrates the diverse roles of sPLA₂s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA₂-V as a potential new therapy to treat asthma and other allergic disorders.     

Exploring the binding mechanism and kinetics of Piperine with snake venom secretory Phospholipase A2

Secreted venom Phospholipase A₂ is highly responsible for pharmacological effects like neurotoxicity, myotoxicity, hemolytic, anti-coagulation, and platelet aggregation. Neutralization of these pharmacological behaviors is one of the challenges existing for many decades and a potent drug compound for this is very much needed to control local effects of venom sPLA₂. In this study, we investigated binding mechanism and kinetics of inhibition of Piperine (major constitute of Piper nigrum) with sPLA₂ using DFT, MD simulation, MM-PBSA, and SPR method. Frontier MO properties were suggested that it procured better chemical reactivity and druglikeness and binding mode of Piperine with EcPLA₂ defined that it occupied well in N-terminal hydrophobic cleft. The persistence of Piperine interactions with and without calcium ion was analyzed and confirmed by MD simulation analysis. The dPCA-based FEL shows the nature of apo- and Piperine-bound conformational behavior of EcPLA₂ including intermediate forms. Further, binding energy of Piperine was calculated by high-throughput MM-PBSA which states that calcium ion presence enhances the Piperine binding by additional electrostatic interactions. Finally, kinetics of inhibition between Piperine and EcPLA₂ implied that it secured better binding affinity (K_D: as 1.708 pM) and the result gives clear evidence for the binding mechanism and binding energy calculated. In conclusion, Piperine was authenticated with better drug ability, entrenched binding interaction, and robust kinetics of inhibition with EcPLA₂ through which it can become an exceeding drug candidate for pharmacological as well as catalytic activity of sPLA₂.     

Functional differences between human and yeast protein disulfide isomerase family proteins

Previously, it has been reported that a mammalian protein disulfide isomerase (PDI), when expressed on a single copy number plasmid, can rescue growth of a PDI1-disrupted yeast. However, here, for the first time we demonstrated by tetrad analysis that human PDI (hPDI) is unable to replace yeast PDI (yPDI) when hPDI cDNA is integrated into the yeast chromosome. This observation indicates that hPDI is not functionally equivalent to yPDI. Estimation of the actual copy number of the plasmid, as well as comparison of isomerase and chaperone activities between human and yeast PDI homologues, indicates that one copy of hPDI cDNA is not sufficient to rescue the PDI1-disrupted strain. Notably, the isomerase activities of yPDI family proteins, Mpd1p, Mpd2p, and Eug1p, were extremely low, although yPDI itself exhibited twice as much isomerase activity as hPDI in vitro. Moreover, with the exception of Mpd1p, all hPDI and yPDI family proteins had chaperone activity, this being particularly strong in the case of yPDI and Mpd2p. These observations indicate that the growth of Saccharomyces cerevisiae is completely dependent on the isomerase activity of yPDI.