PLA2 Isoforms Research Articles

Whole transcriptome sequencing reveals the activity of the PLA2 family members in Androctonus crassicauda (Scorpionida: Buthidae) venom gland.

Phospholipase A2 is the most abundant venom gland enzyme, whose activity leads to the activation of the inflammatory response by accumulating lipid mediators. This study aimed to identify, classify, and investigate the properties of venom PLA2 isoforms. Then, the present findings were confirmed by chemically measuring the activity of PLA2. The sequences representing PLA2 annotation were extracted from the Androctonus crassicauda transcriptome dataset using BLAS searches against the local PLA2 database. We found several cDNA sequences of PLA2 classified and named by conducting multiple searches as platelet-activating factor acetylhydrolases, calcium-dependent PLA2s, calcium-independent PLA2s, and secreted PLA2s. The largest and smallest isoforms of these proteins range between approximately 70.34 kDa (iPLA2) and 17.75 kDa (cPLA2). Among sPLA2 isoforms, sPLA2GXIIA and sPLA2G3 with ORF encoding 169 and 299 amino acids are the smallest and largest secreted PLA2, respectively. These results collectively suggested that A. crassicauda venom has PLA2 activity, and the members of this protein family may have important biological roles in lipid metabolism. This study also revealed the interaction between members of PLA2s in the PPI network. The results of this study would greatly help with the classification, evolutionary relationships, and interactions between PLA2 family proteins in the gene network.

Temporal associations of plasma levels of the secreted phospholipase A2 family and mortality in severe COVID-19.

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.

A Novel Non‐Linear Approach to Characterizing Astrocytic Neurovascular Coupling with Optogenetics and Computational Modeling

The role of astrocytes in neurovascular coupling remains hotly contested with various proposed mechanisms of hemodynamic response. Past findings have proven calcium activity to be the essential component in all astrocytic pathways of vascular control. However, these findings have proven contradictory over the last two decades, suggesting that an additive approach may not be appropriate for the quantification and characterization of astrocytic neurovascular coupling. We hypothesize that these astrocyte‐governed biochemical pathways have an unequal contribution and that they enact hemodynamic change in a dynamic, non‐linear fashion. Using a tetracycline‐based transgenic mice model, expressing ChR2‐EYFP solely in astrocytes, we optogenetically stimulated the production of a calcium wave in mice cortical astrocytes and evaluated hemodynamic change with a Laser Doppler Flowmetry (LDF) modality. Under these conditions, a sustained increase in localized perfusion of around 20% was found within stimulated areas. Subsequent pharmacological inhibition of phospholipase (PLA2) governed pathways facilitated the characterization of PLA2isoforms within cortical astrocytes. Furthermore, we created a computational biophysical model of hemodynamic response aiming to reproduce the experimental data generated from this study and previous ones. This data better elucidates the role of astrocytes in regional resource delivery, suggesting a new perspective in the diagnosis, treatment, and prevention of neurodegenerative diseases with vascular presentations such as Alzheimer’s Disease. We believe our findings provide a new approach by which future studies may revisit the use of pharmacology to characterize the neurovascular coupling phenomenon.

The Influence of Phospholipase A 2 (PLA 2 ) Enzymes on Fatty Acid Release and Oxylipin Formation in Rat Hearts

Polyunsaturated fatty acids (PUFA) released by PLA2 are converted into diverse bioactive lipid mediators called oxylipins. However, little is known about the preference of PLA2 isoforms for PUFA release, and even less is known about how this further impacts the formation of oxylipins. Further, oxylipin levels in tissues do not always mimic the levels of their precursor PUFA, so conclusions about oxylipins cannot be drawn from PUFA content. Therefore, we aimed to determine the impact of PLA2 isoforms on PUFA release and oxylipin formation. We hypothesized that different PLA2 isoforms would impact PUFA release and oxylipin formation differently, and that oxylipin formation would not necessarily reflect changes seen in PUFA. Rat heart homogenates were incubated at 37C for 20 min with or without inhibitors, as the increase of both free PUFA and oxylipins was linear at 20 min incubation. Free PUFA and oxylipins were analyzed by HPLC-MS/MS. Two inhibitors were used: Varespladib (VAR) targeting secreted (sPLA2) isoforms IIA, V and X, and methyl arachidonyl fluorophosphonate (MAFP) targeting cytosolic (cPLA2) and calcium-independent (iPLA2) isoforms. Arachidonic Acid (ARA) release was similarly inhibited by both VAR and MAFP, however, while 15 out of 29 ARA oxylipins were inhibited with MAFP, only 3 out of 29 were inhibited with VAR. Docosahexaenoic acid (DHA) release was more strongly inhibited by VAR than by MAFP, which corresponded with 7 out of 12 DHA oxylipins being more strongly inhibited by VAR than by MAFP. Both alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA) were only inhibited by MAFP, which also inhibited 3 out of 5 ALA and 1 out of 3 EPA oxylipins. Further, 9 out of 10 LA oxylipins were inhibited by MAFP while none were affected by VAR. In conclusion, in the rat heart ARA is released by sPLA2 IIA, V, and/or X as well as by cPLA2 and/or iPLA2, DHA is released more by sPLA2 IIA, V, and/or X than by cPLA2 and/or iPLA2, and EPA and ALA are released primarily by cPLA2 and/or iPLA2. Effects on oxylipin formation largely reflected those on precursor PUFA release, with an exception, as inhibition of sPLA2 IIA, V and X by VAR mostly did not affect ARA oxylipin formation even though it inhibited ARA release. Therefore, careful evaluation of the effects on the oxylipin profile is needed when considering PLA2 inhibitors for potential treatments of diseases.

The Synergy of Membranotropic Effect of the Two Pla2 Fractions of Macrovipera lebetina obtusa Venom on the Model Membranes.

It is known that snake venoms are a complex of enzymes and proteins and the interaction of different venom components with the membranes could be significantly enhanced in course of their action in an orchestra. The aim of the proposed investigation is to obtain detailed information about the mechanism and topology of two snake venom PLA2 isoforms from the Macrovipera lebetina obtusa venom in the membrane-binding process. We investigated the impact of the interaction on the properties of the model membrane (namely, GUVs and erythrocytes ghost) for each of these isoforms, as well as their synergetic action if they act simultaneously. The 6-lauroyl-2-dimethylaminonaphthalene and 6-propionyl-2-dimethylaminonaphthalene fluorescence probes were used to allow us to determine the membrane polarity more accurately via a generalized polarization function. Our results show that two types of PLA2 bring viscosity reduction in GUVs membrane and the effect became more potent when these PLA2 acts together. Intriguingly, we have not observed any significant difference in the case of the erythrocytes ghost membrane.

Geographic variation of individual venom profile of Crotalus durissus snakes.

Background: South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states.Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting.Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar.Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.

Predator-prey interactions and venom composition in a high elevation lizard specialist, Crotalus pricei (Twin-spotted Rattlesnake)

The Twin-spotted Rattlesnake (Crotalus pricei) is a small lizard specialist restricted to higher montane habitat in the Sky Islands of Arizona and México. Though this species is restricted to high elevations and dispersal between mountaintops is impossible, few studies have investigated venom composition or the predator-prey relationship between C. p. pricei and its primary prey source, Yarrow's Spiny Lizard (Sceloporus jarrovii). Because of current isolation of populations in disjunct mountain ranges, it is possible that populations show local adaptation to prey and/or environmental conditions, as reflected by distinct venom phenotypes. Here we characterize venom composition of C. pricei from several different Sky Island mountain ranges and its relationship with S. jarrovii by using various analytical techniques and comparative toxicity tests. Results of venom analyses indicate that there is limited geographic variation in venom composition, occurring primarily in venoms of C. pricei from Durango, México, which had unusual PLA2 isoforms that are lacking from venoms of U.S. populations. Toxicity assays reveal that S. jarrovii has not developed resistance specific to C. p. pricei venom but does display a general tolerance to venom of several snakes in the genus Crotalus. These results provide insight into the evolutionary relationship between a lizard specialist and its natural prey, in addition to novel information on the venom composition of a little-studied species with a narrow range in the United States.

Engineered nanoparticles bind elapid snake venom toxins and inhibit venom-induced dermonecrosis.

Envenomings by snakebites constitute a serious and challenging global health issue. The mainstay in the therapy of snakebite envenomings is the parenteral administration of animal-derived antivenoms. Significantly, antivenoms are only partially effective in the control of local tissue damage. A novel approach to mitigate the progression of local tissue damage that could complement the antivenom therapy of envenomings is proposed. We describe an abiotic hydrogel nanoparticle engineered to bind to and modulate the activity of a diverse array of PLA2 and 3FTX isoforms found in Elapidae snake venoms. These two families of protein toxins share features that are associated with their common (membrane) targets, allowing for nanoparticle sequestration by a mechanism that differs from immunological (epitope) selection. The nanoparticles are non-toxic in mice and inhibit dose-dependently the dermonecrotic activity of Naja nigricollis venom.

Benzo(a)pyrene, an Active Product of Cigarette Smoke, Role in PLA2 Isoforms Activation in Colon Cancer

Background and aim: One of the active combustion product of cigarette smoke, Benzo[a]pyrenes, role in pulmonary cancer is clearly understood. However, its role in gastrointestinal cancer including colon cancer is not clearly understood. Methods: In this study, benzo(a)pyrenes was treated to colon cells to evaluate its role in cell viability, cellular ROS, and gene expression of various PLA2 isoforms was evaluated by FACS and PCR. The identified PLA2 was silenced at the gene level to evaluate its role in cell viability and ROS generation. Results: B(a)P treatment at 1 µg/mL for 48 h to HCT-15 male colon cells significantly reduced the cell viability without affecting HT-29 female colon cells. Higher doses and longer treatment duration with B(a)P showed that female colon cells were highly sensitive than male colon cells. Annexin-V/PI staining for preapoptotic detection showed that B(a)P treatment increased the apoptosis in both the cell types in a concentration and time-dependent manner. The cytosolic ROS (cROS) and superoxide radical (SOR) formation in the female colon cells was significantly higher than male colon cells unlike the mitochondrial ROS (mtROS) production which was significantly higher in male colon cells. Treatment with B(a)P significantly upregulated the IID and IVA PLA2 isoform groups in HCT-15 male colon cells, whereas IB was upregulated in HT-29 female colon cells among the various PLA2 isozyme gene studied (IB, IID, III, IVA, IVB, IVC, VI, X, aiPLA2 and iPLA2). Gene silencing experiments targeting PLA2 IID and IVA in the HCT-15 male colon cells and IB in HT-29 female colon cells showed no effect with B(a)P treatment on the cell proliferation, apoptosis, membrane integrity and free radicals (ROS, mtROS, and SOR) generation. Conclusion: Targeting specific PLA2 isozymes in a cell-specific manner abolished the B(a)P-induced PLA2-mediated oxidative damage–related signaling pathways.

Therapeutic Potential of Cytosolic PLA2 Isoform–Specific Inhibitor Arachidonyl Trifluromethyl Ketone in Cigarette Smoke Condensate–Induced Pathological Conditions in Alveolar Type I and II Epithelial Cells

Background: Cigarette smoking is responsible for various lung pathologies including chronic lung inflammation, emphysema, chronic obstructive pulmonary disease (COPD), cancer, and annually causes almost 10 million deaths globally. During smoke exposure, most affected cells are the alveolar epithelial cells where as a repair mechanism, activation of cytosolic phospholipase A2 enzymes takes place. High free radicals and cPLA2 activity due to continuous exposure of smoke exposure leads to elevated levels of secondary metabolites and various pathophysiologic conditions such as chronic inflammation, oxidative stress and cancer. To reduce the burden of chronic inflammation as well as oxidative stress, and higher levels of secondary metabolites whose role is well defined in progression of cancer, we checked the therapeutic potential of cPLA2 inhibitor arachidonyl trifluromethyl ketone (ATK) by pharmacologically targeting the most expressible cPLA2 during continuous exposure of cigarette smoke. Aim: To check the therapeutic potential of cytosolic PLA2 isoform specific inhibitor arachidonyl trifluromethyl ketone in cigarette smoke condensate–induced pathologic conditions in alveolar type I and II epithelial cells. Methods: Effect of cPLA2 inhibitor on CSC-induced cPLA2 activity were checked using colorimetric assay, cell viability using MTT assay, FDA uptake assay using fluorescence microscopy, ROS levels and apoptosis markers through flow cytometry, and ERK levels using ELISA, in both type of alveolar epithelial cells. Results: ATK significantly mimicked CSC-induced cPLA2 activity, free radicals, primary apoptosis, ratio of apoptotic/apoptotic proteins and levels of ERK whereas protected cells from loss of cell viability and membrane integrity. Conclusion: Current observations revealed cPLA2s as a potential therapeutic target and their inhibitor ATK as a potential therapeutic agent in Cigarette smoke induced pathological conditions in alveolar type I and II epithelial cells.

PO-239 Benzo(a)pyrene, an active product of cigarette smoke, role in PLA2 isoforms activation in colon cancer

IntroductionOne of the active combustion product of cigarette smoke, Benzo[a]pyrenes, role in pulmonary cancer is clearly understood. However, its role in gastrointestinal cancer including colon cancer is not clearly understood.Material and methodsIn this study, benzo(a)pyrene’s was treated to colon cells to evaluate its role in cell viability, cellular ROS, and gene expression of various PLA2 isoforms was evaluated by FACS and PCR. The identified PLA2 was silenced at the gene level to evaluate its role in cell viability and ROS generation.Results and discussionsB(a)P treatment at 1 µg/ml for 48 hour to HCT-15 male colon cells significantly reduced the cell viability without affecting HT-29 female colon cells. Higher doses and longer treatment duration with B(a)P showed that female colon cells were highly sensitive than male colon cells. Annexin-V/PI staining for pre-apoptotic detection showed that B(a)P treatment increased the apoptosis in both the cell types in a concentration and time-dependent manner. The cytosolic ROS (cROS) and superoxide radical (SOR) formation in the female colon cells was significantly higher than male colon cells unlike the mitochondrial ROS (mtROS) production which was significantly higher in male colon cells. Treatment with B(a)P significantly upregulated the IID and IVA PLA2 isoform groups in HCT-15 male colon cells, whereas IB was upregulated in HT-29 female colon cells among the various PLA2 isozyme gene studied (IB, IID, III, IVA, IVB, IVC, VI, X, aiPLA2 and iPLA2). Gene silencing experiments targeting PLA2 IID and IVA in the HCT-15 male colon cells and IB in HT-29 female colon cells showed no effect with B(a)P treatment on the cell proliferation, apoptosis, membrane integrity and free radicals (ROS, mtROS, and SOR) generation.ConclusionTargeting specific PLA2 isozymes in a cell specific manner abolished the B(a)P-induced PLA2 mediated oxidative damage related signalling pathways.

Differential expression of Lp-PLA2 in obesity and type 2 diabetes and the influence of lipids

Aims/hypothesisLipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulatory macrophage-derived factor that increases with obesity and leads to a higher risk of cardiovascular disease (CVD). Despite this, its role in adipose tissue and the adipocyte is unknown. Therefore, the aims of this study were to clarify the expression of Lp-PLA2 in relation to different adipose tissue depots and type 2 diabetes, and ascertain whether markers of obesity and type 2 diabetes correlate with circulating Lp-PLA2. A final aim was to evaluate the effect of cholesterol on cellular Lp-PLA2 in an in vitro adipocyte model.MethodsAnalysis of anthropometric and biochemical variables from a cohort of lean (age 44.4 ± 6.2 years; BMI 22.15 ± 1.8 kg/m2, n = 23), overweight (age 45.4 ± 12.3 years; BMI 26.99 ± 1.5 kg/m2, n = 24), obese (age 49.0 ± 9.1 years; BMI 33.74 ± 3.3 kg/m2, n = 32) and type 2 diabetic women (age 53.0 ± 6.13 years; BMI 35.08 ± 8.6 kg/m2, n = 35), as part of an ethically approved study. Gene and protein expression of PLA2 and its isoforms were assessed in adipose tissue samples, with serum analysis undertaken to assess circulating Lp-PLA2 and its association with cardiometabolic risk markers. A human adipocyte cell model, Chub-S7, was used to address the intracellular change in Lp-PLA2 in adipocytes.ResultsLp-PLA2 and calcium-independent PLA2 (iPLA2) isoforms were altered by adiposity, as shown by microarray analysis (p < 0.05). Type 2 diabetes status was also observed to significantly alter gene and protein levels of Lp-PLA2 in abdominal subcutaneous (AbdSc) (p < 0.01), but not omental, adipose tissue. Furthermore, multivariate stepwise regression analysis of circulating Lp-PLA2 and metabolic markers revealed that the greatest predictor of Lp-PLA2 in non-diabetic individuals was LDL-cholesterol (p = 0.004). Additionally, in people with type 2 diabetes, oxidised LDL (oxLDL), triacylglycerols and HDL-cholesterol appeared important predictors, accounting for 59.7% of the variance (p < 0.001). Subsequent in vitro studies determined human adipocytes to be a source of Lp-PLA2, as confirmed by mRNA expression, protein levels and immunochemistry. Further in vitro experiments revealed that treatment with LDL-cholesterol or oxLDL resulted in significant upregulation of Lp-PLA2, while inhibition of Lp-PLA2 reduced oxLDL production by 19.8% (p < 0.05).Conclusions/interpretationOur study suggests adipose tissue and adipocytes are active sources of Lp-PLA2, with differential regulation by fat depot and metabolic state. Moreover, levels of circulating Lp-PLA2 appear to be influenced by unfavourable lipid profiles in type 2 diabetes, which may occur in part through regulation of LDL-cholesterol and oxLDL metabolism in adipocytes.

“HETE”ing up mitochondria in human heart failure

A decade of research has established the phospholipase iPLA2γ as being involved in cardiomyocyte dysfunction and necrosis leading to heart failure, but the mechanisms by which iPLA2γ acts and its interaction with the mitochondrial permeability transition pore (mPTP) that is critical for cardiac homeostasis are unclear. New investigations by Moon et al. demonstrate that mitochondria in failing hearts undergo dynamic shifts in PLA2 isoform expression, leading to a redistribution of eicosanoid composition that contributes to pathologic mPTP opening.

Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence

It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla−strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification of epidemic process from endemic natural focality with sporadic cases in men to rapidly expanding epizootics followed by human epidemic outbreaks, local epidemics or even pandemics.

CDNA and gene structures of two phospholipase A2 isoforms, acidic PLA2 PA4 and PLA2 PA3A/PA3B/PA5, in Nemopilema nomurai jellyfish venom

We have shown that Nemopilema nomurai jellyfish venom (NnV) contains various kinds of proteolytic enzyme activities, including phospholipase (PLA), metalloproteinase (MP) and hyaluronidase activities. In this study, we reported the full-length cDNA and gene sequences of two PLA2 isoforms: acidic PLA2 PA4 and PLA2 PA3A/PA3B/PA5. The full-length cDNA of acidic PLA2 PA4 contains 483 nucleotides (nt), which encode 160 amino acids (and the stop codon), including a signal peptide, six cysteine residues that form disulfide bonds, and metal-binding and catalytic active sites. The gene sequence of the acidic PLA2 PA4 is 1667 base pairs (bp) long and encodes three exons and two introns. The 5′ donor (GT) and 3′ acceptor (AG) splice sites are highly conserved. The PLA2 PA3A/PA3B/PA5 gene contains 1366 bp, and the 498 nt of the mature mRNA encode 165 amino acids (and the stop codon). The protein includes a signal peptide, six cysteine residues that form disulfide bonds, and metal-binding and catalytic active sites. The three exons and two introns also have highly conserved donor and acceptor splice sites. InterProScan predicted PLA2 activity domains in both isoforms. These results extend our understanding of the PLA2 venom of the N. nomurai jellyfish and will facilitate further research.

Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential

Snake venom is a complex mixture of active compounds consisting of 80–90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications.

Abstract B06: TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism

Abstract Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease characterized by multi-organ proliferation of TSC2-deficient cells with aberrant activation of the mechanistic/mammalian Target of Rapamycin Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Up to 80% of women with TSC develop lymphangioleiomyomatosis (LAM), which consists of diffuse proliferation of TSC2-deficient smooth muscle-like cells with progressive cystic destruction of the lung. LAM can also occur as a sporadic disorder of women. A downstream effector of mTORC1, the Sterol Regulatory Element-Binding Protein (SREBP), regulates the transcription of de novo fatty acid synthesis enzymes in TSC2-deficient cells. The impact of loss of the TSC proteins on the lipidome and how complex lipid species are affected by rapamycin and its analogs are unknown. Here, we report the first systematic study of the TSC lipidome, revealing unexpected findings. Using mass spectrometry, we profiled 131 complex lipid species discovered elevated levels (p&amp;lt;0.05) of 4 lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, C20:4) in the plasma of LAM patients compared with healthy control women. LPC are a class of bioactive lipids generated by phospholipase A (PLA) activity. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro pre-clinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid, including the LPC precursors phosphatidylcholines (PC) and sphingolipids, and neutral lipid species, including triacylglycerols and cholesterol esters. To determine whether TSC2-deficient cells generate LPC using de novo synthesized PC, TSC2-deficient and TSC2-expressing cells were labeled with deuterated choline (choline chloride 1,1,2,2-d4) for 6 hours, and lipids isolated to measure choline incorporation into two saturated LPC (C16:0 and C18:0) and two parent PC (C32:0 and C34:0) species. Incorporation of deuterated choline into PC and LPC was greater than 2 fold higher in TSC2-deficient cells compared to TSC2-expressing controls (p&amp;lt;0.05). Surprisingly, treatment with rapamycin, the mTOR catalytic inhibitor Torin1, or downregulation of the mTORC1-regulated lipogenic transcription factor SREBP (Sterol Regulatory Element-Binding Protein) did not suppress LPC in TSC2-deficient cells, while they suppressed other phospholipid species. Finally, pharmacologic and genetic approaches revealed that inhibition of distinct isoforms of PLA2 may decrease the proliferation of TSC2-deficient but not TSC2-expressing cells. Collectively, these results demonstrate for the first time that TSC2-deficient cells have enhanced choline phospholipid metabolism, revealing a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies. Citation Format: Carmen Priolo, Stéphane J. H. Ricoult, Damir Khabibullin, Harilaos Filippakis, Jane Yu, Brendan Manning, Clary Clish, Elizabeth P. Henske. TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B06.

PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse fibrin clots preventing bacteria dissemination after bites of infected fleas or subcutaneous challenge is believed to be the main Y. pestis factor responsible for generalization of infectious process. Pla-mediated ability of Y. pestis for selective binding with extracellular matrix and basal membranes may promote further hydrolysis of these structures by the host’s plasmin and overcoming tissue barriers by the pathogen. Y. pestis plasminogen activator also hydrolyses C3 complement component, human antimicrobial peptide — cathelicidin LL-37 and such cytokines as tumor necrosis factor α, interferon γ, interleukin 8 and protein 1 of monocyte chemotaxis. The main endogenic TFPI tissue factor pathway inhibitor also highly susceptible to proteolytic action of Pla, and efficiency of TFPI inactivation is much higher than efficacy of plasminogen activation. The review also debates the possibility of using Pla as a molecular target for prophylaxis and treatment of plague.

Ovarian Steroid Regulation of Endometrial Phospholipase A2 Isoforms in Horses

Real-time PCR was used to investigate the role of progesterone (P4) and oestradiol (E2) in regulation of endometrial cytosolic, secretory and calcium-independent phospholipase A2 (PLA2G4A, PLA2G2A and PLA2G6, respectively) gene expression. Ovariectomized mares underwent 6 days of E2 pre-treatment followed by 14 days of P4 supplementation. At the start of P4 treatment (Day 1), mares were assigned in a 2 × 2 factorial design to receive either E2 or vehicle starting on Day 11 and endometrial biopsy collection on either Day 14 when P4 concentrations remained high (>4 ng/ml) or Day 16 when P4 concentrations had declined (0.5-2 ng/ml). Additional biopsies were collected from ovariectomized mares on Day 8, which served as control. Blood samples were collected for P4 determination. PLA2G4A expression was higher (p < 0.05) on Day 14 compared with Day 8. In contrast, PLA2G2A did not change significantly (p < 0.12). PLA2G4A and PLA2G2A gene expression increased (p < 0.05), as P4 concentration dropped, on Day 16. In contrast, PLA2G6 gene expression did not show differences between days. Treatment with oestradiol did not increase PLA2 isoforms expression when compared to treatment with the vehicle. PLA2G4A and PLA2G2A were positively correlated with each other and negatively correlated with P4 concentrations. In conclusion, P4 withdrawal upregulated PLA2G4A and PLA2G2A gene expression, and this was not affected by E2. PLA2G4A and PLA2G2A but not PLA2G6 gene expression may be involved in controlling prostaglandin F2 alpha synthesis and luteolysis.

Phospholipase A2 and its molecular mechanism after spinal cord injury.

Phospholipases A(2) (PLA(2)s) are a diverse family of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor which have been implicated in pathological states of numerous acute and chronic neurological disorders. To date, more than 27 isoforms of PLA(2) have been found in the mammalian system which can be classified into four major categories: secretory PLA(2), cytosolic PLA(2), Ca(2+)-independent PLA(2), and platelet-activating factor acetylhydrolases. Multiple isoforms of PLA(2) are found in the mammalian spinal cord. Under physiological conditions, PLA(2)s are involved in diverse cellular responses, including phospholipid digestion and metabolism, host defense, and signal transduction. However, under pathological situations, increased PLA(2) activity, excessive production of free fatty acids and their metabolites may lead to the loss of membrane integrity, inflammation, oxidative stress, and subsequent neuronal injury. There is emerging evidence that PLA(2) plays a key role in the secondary injury process after traumatic spinal cord injury. This review outlines the current knowledge of the PLA(2) in the spinal cord with an emphasis being placed on the possible roles of PLA(2) in mediating the secondary SCI.