Improvement of a low-cost protocol for a simultaneous comparative evaluation of hydrolytic activity between sessile and planktonic cells: Candida albicans as a study model.

Candida albicans is often implicated in nosocomial infections with fatal consequences. Its virulence is contributed to hydrolytic enzymes and biofilm formation. Previous research focused on studying these virulence factors individually. Therefore, this study aimed to investigate the impact of biofilm formation on the hydrolytic activity using an adapted low-cost method. Eleven strains of C. albicans were used. The biofilms were formed on pre-treated silicone discs using 24-well plates and then deposited on the appropriate agar to test each enzyme, while the planktonic cells were conventionally seeded. Biofilms were analysed using Raman spectroscopy, fluorescent and scanning electron microscopy. The adapted method provided an evaluation of hydrolytic enzymes activity in C. albicans biofilm and showed that sessile cells had a higher phospholipase and proteinase activities compared with planktonic cells. These findings were supported by spectroscopic and microscopic analyses, which provided valuable insights into the virulence mechanisms of C. albicans during biofilm formation.

Comparative analysis of enzymatic profiles and biofilm formation in clinical and environmental Candida kefyr isolates.

The global landscape of Candida infections has seen a significant shift. Previously, Candida albicans was the predominant species. However, there has been an emergence of non-albicans Candida species, which are often less susceptible to antifungal treatment. Candida kefyr, in particular, has been increasingly associated with infections. This study aimed to investigate the profiles of enzymatic activity and biofilm formation in both clinical and non-clinical isolates of C. kefyr. A total of 66 C. kefyr isolates were analysed. The activities of proteinase and phospholipase were assessed using bovine serum albumin and egg yolk agar, respectively. Haemolysin, caseinolytic and esterase activities were evaluated using specific methods. Biofilm formation was investigated using crystal violet staining. The findings indicated that biofilm and proteinase activity were detected in 81.8% and 93.9% of all the isolates, respectively. Haemolysin activity was observed with the highest occurrence (95.5%) among normal microbiota isolates. Esterase activity was predominantly identified in dairy samples and was absent in hospital samples. Caseinase production was found with the highest occurrence (18.2%) in normal microbiota and hospital samples. Phospholipase activity was limited, found in only 3% of all the isolates. These findings reveal variations in enzyme activity between clinical and non-clinical C. kefyr isolates. This sheds light on their pathogenic potential and has implications for therapeutic strategies.

Exploration of the pearl millet phospholipase gene family to identify potential candidates for grain quality traits.

BACKGROUND: Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in plant development and stress responses is well documented. RESULTS: In the present investigation, a thorough genome-wide analysis revealed that the pearl millet genome contains at least 44 phospholipase genes distributed across its 7 chromosomes, with chromosome one harbouring the highest number of these genes. The synteny analysis suggested a close genetic relationship of pearl millet phospholipases with that of foxtail millet and sorghum. All identified genes were examined to unravel their gene structures, protein attributes, cis-regulatory elements, and expression patterns in two pearl millet genotypes contrasting for rancidity. All the phospholipases have a high alpha-helix content and distorted regions within the predicted secondary structures. Moreover, many of these enzymes possess binding sites for both metal and non-metal ligands. Additionally, the putative promoter regions associated with these genes exhibit multiple copies of cis-elements specifically responsive to biotic and abiotic stress factors and signaling molecules. The transcriptional profiling of 44 phospholipase genes in two genotypes contrasting for rancidity across six key tissues during pearl millet growth revealed a predominant expression in grains, followed by seed coat and endosperm. Specifically, the genes PgPLD-alpha1-1, PgPLD-alpha1-5, PgPLD-delta1-7a, PgPLA1-II-1a, and PgPLD-delta1-2a exhibited notable expression in grains of both the genotypes while showing negligible expression in the other five tissues. The sequence alignment of putative promoters revealed several variations including SNPs and InDels. These variations resulted in modifications to the corresponding cis-acting elements, forming distinct transcription factor binding sites suggesting the transcriptional-level regulation for these five genes in pearl millet. CONCLUSIONS: The current study utilized a genome-wide computational analysis to characterize the phospholipase gene family in pearl millet. A comprehensive expression profile of 44 phospholipases led to the identification of five grain-specific candidates. This underscores a potential role for at least these five genes in grain quality traits including the regulation of rancidity in pearl millet. Therefore, this study marks the first exploration highlighting the possible impact of phospholipases towards enhancing agronomic traits in pearl millet.

Measuring Urease and Phospholipase Secretion in Cryptococcus neoformans.

Urease and phospholipase are enzymes that are important virulence factors for Cryptococcus neoformans. These are two of the most studied enzymes involved in how C. neoformans breaches the blood-brain barrier. Additionally, phospholipase secretion also supports dissemination from the lungs. This chapter describes the methods used to measure the secretion of these enzymes, which may be used to characterize strain invasiveness and virulence.

The activation mechanism of Atg15, a vacuolar phospholipase required for the disintegration of organelle membranes.

The disintegration of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies in vacuoles is essential to the Cvt pathway and macroautophagy in yeast. Atg15 is a vacuolar lipase required for the degradation of both Cvt and autophagic bodies. However, the molecular mechanism of their degradation by Atg15 remains poorly understood. In a recent study, we showed that recombinant Chaetomium thermophilum Atg15 (CtAtg15) possesses phospholipase activity, and that this activity is significantly elevated by proteolytic cleavage at a site away from the active center. The proteolytic cleavage of CtAtg15 causes a conformational change around the active center, resulting in the active open state. Interestingly, activated CtAtg15 can degrade not only Cvt and autophagic bodies but also organelle membranes. On the basis of these results, we propose an activation mechanism by which Atg15, as an "organellase," functions only in vacuoles.

Vascular ATGL-dependent lipolysis and the activation of cPLA2-PGI2 pathway protect against postprandial endothelial dysfunction.

Adipose triglyceride lipase (ATGL) is involved in lipolysis and displays a detrimental pathophysiological role in cardio-metabolic diseases. However, the organo-protective effects of ATGL-induced lipolysis were also suggested. The aim of this work was to characterize the function of lipid droplets (LDs) and ATGL-induced lipolysis in the regulation of endothelial function. ATGL-dependent LDs hydrolysis and cytosolic phospholipase A2 (cPLA2)-derived eicosanoids production were studied in the aorta, endothelial and smooth muscle cells exposed to exogenous oleic acid (OA) or arachidonic acid (AA). Functional effects of ATGL-dependent lipolysis and subsequent activation of cPLA2/PGI2 pathway were also studied in vivo in relation to postprandial endothelial dysfunction.The formation of LDs was invariably associated with elevated production of endogenous AA-derived prostacyclin (PGI2). In the presence of the inhibitor of ATGL or the inhibitor of cytosolic phospholipase A2, the production of eicosanoids was reduced, with a concomitant increase in the number of LDs. OA administration impaired endothelial barrier integrity in vitro that was further impaired if OA was given together with ATGL inhibitor. Importantly, in vivo, olive oil induced postprandial endothelial dysfunction that was significantly deteriorated by ATGL inhibition, cPLA2 inhibition or by prostacyclin (IP) receptor blockade.In summary, vascular LDs formation induced by exogenous AA or OA was associated with ATGL- and cPLA2-dependent PGI2 production from endogenous AA. The inhibition of ATGL resulted in an impairment of endothelial barrier function in vitro. The inhibition of ATGL-cPLA2-PGI2 dependent pathway resulted in the deterioration of endothelial function upon exposure to olive oil in vivo. In conclusion, vascular ATGL-cPLA2-PGI2 dependent pathway activated by lipid overload and linked to LDs formation in endothelium and smooth muscle cells has a vasoprotective role by counterbalancing detrimental effects of lipid overload on endothelial function.

Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease.

Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands.

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants-R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low-affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called "high"-affinity biological responses arise from activation by isolated receptor dimers, whereas "low"-affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

PNPLA1 knockdown inhibits esterification of γ-linolenic acid to ceramide 1 in differentiated keratinocytes.

Patatin-like phospholipase domain-containing 1 (PNPLA1) is crucial in the esterification of linoleic acid (LA; 18:2n-6) to ω-hydroxy fatty acids (FA) of ceramide 1 (Cer1), the major barrier lipid of the differentiated epidermis. We previously reported that γ-linolenic acid (GLA; 18:3n-6) as well as LA is esterified to Cer1 subspecies with sphingosine (d18:1) or eicosasphingosine (d20:1) amide-linked to two different ω-hydroxy FA (30wh:0; 32wh:1). Here, we further investigated whether PNPLA1 is also responsible for esterification of GLA to these Cer1 subspecies in normal human keratinocytes (NHK). As late/terminal differentiation was induced in NHK, PNPLA1 and differentiation markers were expressed, and LA-esterified Cer1 subspecies (18:2n-6/C30wh:0 or C32wh:0/d18:1; 18:2n-6/C32wh:0/d20:1) were detected, which were further increased with LA treatment. GLA-esterified Cer1 subspecies (18:3n-6/C30wh:0 or C32wh:0/d18:1; 18:3n-6/C32wh:0/d20:1) were detected only with GLA treatment. Specific small interfering RNA-mediated knockdown of PNPLA1 (KDP) in differentiated NHK decreased levels of these LA-esterified Cer1 subspecies overall and of involucrin (IVL), a terminal differentiation marker. Moreover, KDP resulted in lesser LA/GLA responses as characterized by more significant decreases in IVL and LA/GLA-esterified Cer1 subspecies overall and an accumulation of non-esterified ω-hydroxy ceramides, their putative precursors; the decrease of 18:3n-6/C32wh:0/d18:1, the predominant GLA-esterified Cer1 subspecies, specifically paralleled the increase of C32wh:0/d18:1, its corresponding precursor. PNPLA1 is responsible for NHK terminal differentiation and also for esterification of GLA to the ω-hydroxy FA of Cer1.

Gpcpd1-GPC metabolic pathway is dysfunctional in aging and its deficiency severely perturbs glucose metabolism.

Skeletal muscle plays a central role in the regulation of systemic metabolism during lifespan. With aging, this function is perturbed, initiating multiple chronic diseases. Our knowledge of mechanisms responsible for this decline is limited. Glycerophosphocholine phosphodiesterase 1 (Gpcpd1) is a highly abundant muscle enzyme that hydrolyzes glycerophosphocholine (GPC). The physiological functions of Gpcpd1 remain largely unknown. Here we show, in mice, that the Gpcpd1-GPC metabolic pathway is perturbed in aged muscles. Further, muscle-specific, but not liver- or fat-specific, inactivation of Gpcpd1 resulted in severely impaired glucose metabolism. Western-type diets markedly worsened this condition. Mechanistically, Gpcpd1 muscle deficiency resulted in accumulation of GPC, causing an 'aged-like' transcriptomic signature and impaired insulin signaling in young Gpcpd1-deficient muscles. Finally, we report that the muscle GPC levels are markedly altered in both aged humans and patients with type 2 diabetes, displaying a high positive correlation between GPC levels and chronological age. Our findings reveal that the muscle GPCPD1-GPC metabolic pathway has an important role in the regulation of glucose homeostasis and that it is impaired during aging, which may contribute to glucose intolerance in aging.

Tools and mechanisms of vacuolar escape leading to host egress in Legionella pneumophila infection: Emphasis on bacterial phospholipases.

The phenomenon of host cell escape exhibited by intracellular pathogens is a remarkably versatile occurrence, capable of unfolding through lytic or non-lytic pathways. Among these pathogens, the bacterium Legionella pneumophila stands out, having adopted a diverse spectrum of strategies to disengage from their host cells. A pivotal juncture that predates most of these host cell escape modalities is the initial escape from the intracellular compartment. This critical step is increasingly supported by evidence suggesting the involvement of several secreted pathogen effectors, including lytic proteins. In this intricate landscape, L. pneumophila emerges as a focal point for research, particularly concerning secreted phospholipases. While nestled within its replicative vacuole, the bacterium deftly employs both its type II (Lsp) and type IVB (Dot/Icm) secretion systems to convey phospholipases into either the phagosomal lumen or the host cell cytoplasm. Its repertoire encompasses numerous phospholipases A (PLA), including three enzymes-PlaA, PlaC, and PlaD-bearing the GDSL motif. Additionally, there are 11 patatin-like phospholipases A as well as PlaB. Furthermore, the bacterium harbors three extracellular phospholipases C (PLCs) and one phospholipase D. Within this comprehensive review, we undertake an exploration of the pivotal role played by phospholipases in the broader context of phagosomal and host cell egress. Moreover, we embark on a detailed journey to unravel the established and potential functions of the secreted phospholipases of L. pneumophila in orchestrating this indispensable process.

Protective effect of arctiin against Toxoplasma gondii HSP70-induced allergic acute liver injury by disrupting the TLR4-mediated activation of cytosolic phospholipase A2 and platelet-activating factor.

Toxoplasma gondii (T. gondii)-derived heat shock protein 70 (T.g.HSP70) is a toxic protein that downregulates host defense responses against T. gondii infection. T.g.HSP70 was proven to induce fatal anaphylaxis in T. gondii infected mice through cytosolic phospholipase A2 (cPLA2) activated-platelet-activating factor (PAF) production via Toll-like receptor 4 (TLR4)-mediated signaling. In this study, we investigated the effect of arctiin (ARC; a major lignan compound of Fructus arctii) on allergic liver injury using T.g.HSP70-stimulated murine liver cell line (NCTC 1469) and a mouse model of T. gondii infection. Localized surface plasmon resonance, ELISA, western blotting, co-immunoprecipitation, and immunofluorescence were used to investigate the underlying mechanisms of action of ARC on T. gondii-induced allergic acute liver injury. The results showed that ARC suppressed the T.g.HSP70-induced allergic liver injury in a dose-dependent manner. ARC could directly bind to T.g.HSP70 or TLR4, interfering with the interaction between these two factors, and inhibiting activation of the TLR4/mitogen-activated protein kinase/nuclear factor-kappa B signaling, thereby inhibiting the overproduction of cPLA2, PAF, and interferon-γ. This result suggested that ARC ameliorates T.g.HSP70-induced allergic acute liver injury by disrupting the TLR4-mediated activation of inflammatory mediators, providing a theoretical basis for ARC therapy to improve T.g.HSP70-induced allergic liver injury.

Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

DADLE promotes motor function recovery by inhibiting cytosolic phospholipase A2 mediated lysosomal membrane permeabilization after spinal cord injury.

BACKGROUND AND PURPOSE: Autophagy is a protective factor for controlling neuronal damage, while necroptosis promotes neuroinflammation after spinal cord injury (SCI). DADLE (D-Ala2 , D-Leu5 ]-enkephalin) is a selective agonist for delta (δ) opioid receptor and has been identified as a promising drug for neuroprotection. The aim of this study was to investigate the mechanism/s by which DADLE causes locomotor recovery following SCI. EXPERIMENTAL APPROACH: Spinal cord contusion model was used and DADLE was given by i.p. (16 mg·kg-1 ) in mice for following experiments. Motor function was assessed by footprint and Basso mouse scale (BMS) score analysis. Western blotting used to evaluate related protein expression. Immunofluorescence showed the protein expression in each cell and its distribution. Network pharmacology analysis was used to find the related signalling pathways. KEY RESULTS: DADLE promoted functional recovery after SCI. In SCI model of mice, DADLE significantly increased autophagic flux and inhibited necroptosis. Concurrently, DADLE restored autophagic flux by decreasing lysosomal membrane permeabilization (LMP). Additionally, chloroquine administration reversed the protective effect of DADLE to inhibit necroptosis. Further analysis showed that DADLE decreased phosphorylated cPLA2 , overexpression of cPLA2 partially reversed DADLE inhibitory effect on LMP and necroptosis, as well as the promotion autophagy. Finally, AMPK/SIRT1/p38 pathway regulating cPLA2 is involved in the action DADLE on SCI and naltrindole inhibited DADLE action on δ receptor and on AMPK signalling pathway. CONCLUSION AND IMPLICATION: DADLE causes its neuroprotective effects on SCI by promoting autophagic flux and inhibiting necroptosis by decreasing LMP via activating δ receptor/AMPK/SIRT1/p38/cPLA2 pathway.

Phospholipase Modulation of Synaptic Membrane Landscape: Driving Force Behind Memory Formation?

The synapse is the communication unit of the brain, linking billions of neurons through trillions of synaptic connections. The lipid landscape of the synaptic membrane underpins neurotransmitter release through the exocytic fusion of neurotransmitter-containing vesicles, endocytic recycling of these synaptic vesicles, and the postsynaptic response following binding of the neurotransmitter to specialized receptors. How the connected brain can learn and acquire memories through synaptic plasticity is unresolved. Phospholipases, and especially the phospholipase A1 isoform DDHD2, have recently been shown to play a critical role in memory acquisition through the generation of saturated free fatty acids such as myristic and palmitic acids. This emerging synaptic plasticity pathway suggests that phospholipases cannot only respond to synaptic activity by altering the phospholipid landscape but also contribute to the establishment of long-term memories in our brain.

The patatin-like phospholipase PfPNPLA2 is involved in the mitochondrial degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development.

Plasmodium falciparum is an Apicomplexa responsible for human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Increasing evidences show that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and that these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly divided parasites in the host cell. Parasite membrane homeostasis is achieved by an essential combination of parasite de novo lipid synthesis/recycling and massive host lipid scavenging. Latest data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell, but the molecular actors allowing lipid acquisition are poorly characterized. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possesses an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase PfPNPLA2, for which we generated an glmS-inducible knockdown line and investigated its role during blood stages malaria. Disruption of the mitochondrial PfPNPLA2 in the asexual blood stages affected mitochondrial morphology and further induced a significant defect in parasite replication and survival, in particular under low host lipid availability. Lipidomic analyses revealed that PfPNPLA2 specifically degrades the parasite membrane lipid phosphatidylglycerol to generate lysobisphosphatidic acid. PfPNPLA2 knockdown further resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite phosphatidylglycerol to sustain optimal intraerythrocytic development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

Atg15 is a vacuolar phospholipase that disintegrates organelle membranes.

Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar/intralysosomal autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here, we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity. The activity of CtAtg15(73-475) was markedly elevated by limited digestion. We inserted the human rhinovirus 3C protease recognition sequence and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). Our molecular dynamics simulation suggested that the cleavage facilitated conformational change around the active center of CtAtg15, resulting in an exposed state. We confirmed that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC of S. cerevisiae were disintegrated by CtAtg15. This study suggests Atg15 plays a role in disrupting any organelle membranes delivered to vacuoles by autophagy.

Sperm-Induced Ca2+ Release in Mammalian Eggs: The Roles of PLCζ, InsP3, and ATP.

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca2+ concentration. These Ca2+ oscillations are due to the production of InsP3 within the egg and the subsequent release of Ca2+ from the endoplasmic reticulum into the cytosol. The generation of InsP3 is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion. PLCζ enables a positive feedback loop of InsP3 production and Ca2+ release which then stimulates further InsP3 production. Most cytosolic Ca2+ increases in eggs at fertilization involve a fast Ca2+ wave; however, due to the limited diffusion of InsP3, this means that InsP3 must be generated from an intracellular source rather than at the plasma membrane. All mammalian eggs studied generated Ca2+ oscillations in response to PLCζ, but the sensitivity of eggs to PLCζ and to some other stimuli varies between species. This is illustrated by the finding that incubation in Sr2+ medium stimulates Ca2+ oscillations in mouse and rat eggs but not eggs from other mammalian species. This difference appears to be due to the sensitivity of the type 1 InsP3 receptor (IP3R1). I suggest that ATP production from mitochondria modulates the sensitivity of the IP3R1 in a manner that could account for the differential sensitivity of eggs to stimuli that generate Ca2+ oscillations.