RESUMO
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.
Assuntos
Glicoproteínas de Membrana , Lipofuscinoses Ceroides Neuronais , Camundongos , Animais , Criança , Humanos , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Lisossomos/metabolismo , Fosfolipases/metabolismo , Glicerofosfolipídeos/metabolismo , Fosfolipídeos/metabolismoRESUMO
The outer membrane of Gram-negative bacteria is unique in both structure and function. The surface-exposed outer leaflet is composed of lipopolysaccharide, while the inner leaflet is composed of glycerophospholipids. This lipid asymmetry creates mechanical strength, lowers membrane permeability, and is necessary for virulence in many pathogens. Glycerophospholipids that mislocalize to the outer leaflet are removed by the Mla pathway, which consists of the outer membrane channel MlaA, the periplasmic lipid carrier MlaC, and the inner membrane transporter MlaBDEF. The opportunistic pathogen Pseudomonas aeruginosa has two proteins of the MlaA family: PA2800 and PA3239. Here, we show that PA2800 is part of a canonical Mla pathway, while PA3239 functions with the putative lipase PA3238. While loss of either pathway individually has little to no effect on outer membrane integrity, loss of both pathways weakens the outer membrane permeability barrier and increases production of the secondary metabolite pyocyanin. We propose that mislocalized glycerophospholipids are removed from the outer leaflet by PA3239 (renamed MlaZ), transferred to PA3238 (renamed MlaY), and degraded. This pathway streamlines recycling of glycerophospholipid degradation products by removing glycerophospholipids from the outer leaflet prior to degradation.
Assuntos
Lipídeos de Membrana , Pseudomonas aeruginosa , Lipídeos de Membrana/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transporte Biológico , Fosfolipases/genética , Fosfolipases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Glicerofosfolipídeos/metabolismoRESUMO
Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells under various conditions. They are found in the extracellular milieu in all biological fluids. As the concentrations, contents, and origin of EVs can change during inflammation, the assessment of EVs can be used as a proxy of cellular activation. Here, we review the literature regarding EVs, more particularly those released by platelets and their mother cells, the megakaryocytes. Their cargo includes cytokines, growth factors, organelles (mitochondria and proteasomes), nucleic acids (messenger and non-coding RNA), transcription factors, and autoantigens. EVs may thus contribute to intercellular communication by facilitating exchange of material between cells. EVs also interact with other molecules secreted by cells. In autoimmune diseases, EVs are associated with antibodies secreted by B cells. By definition, EVs necessarily comprise a phospholipid moiety, which is thus the target of secreted phospholipases also abundantly expressed in the extracellular milieu. We discuss how platelet-derived EVs, which represent the majority of the circulating EVs, may contribute to immunity through the activity of their cargo or in combination with the secretory interactome.
Assuntos
Vesículas Extracelulares , Ácidos Nucleicos , Autoantígenos/metabolismo , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Ácidos Nucleicos/metabolismo , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , RNA não Traduzido/metabolismo , Fatores de TranscriçãoRESUMO
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.
Assuntos
Receptores ErbB , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ligantes , Mutação , Fosfolipases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Domínios Proteicos/genética , Células CHO , Animais , Cricetinae , Humanos , Glioblastoma/genéticaRESUMO
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.
Assuntos
Legionella pneumophila , Doença dos Legionários , Humanos , Fosfolipases/metabolismo , Doença dos Legionários/microbiologia , Vacúolos/metabolismo , Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Fosfolipases A/metabolismoRESUMO
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.
Assuntos
Eicosanoides , Lipólise , Lipólise/fisiologia , Azeite de Oliva , Ácido Araquidônico/metabolismo , Eicosanoides/metabolismo , Prostaglandinas I/metabolismo , Fosfolipases/metabolismoRESUMO
Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.
Assuntos
Legionella , Doença dos Legionários , Humanos , Legionella/metabolismo , Vacúolos/metabolismo , Doença dos Legionários/microbiologia , Fosfolipases/metabolismo , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Lisofosfolipídeos/metabolismoRESUMO
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.
Assuntos
Aterosclerose , Células Espumosas , Receptores Purinérgicos P2 , Humanos , Camundongos , Animais , Células Espumosas/metabolismo , Células Espumosas/patologia , Cálcio/metabolismo , Calreticulina/metabolismo , Calreticulina/farmacologia , Proteômica , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/farmacologia , Aterosclerose/genética , Macrófagos/metabolismo , Lipoproteínas LDL/metabolismo , Receptores Depuradores/metabolismo , Camundongos Knockout , Fosfolipases/metabolismo , Fosfolipases/farmacologiaRESUMO
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.
Assuntos
Biflavonoides , Nucleotídeos Cíclicos , Fosfolipases , Humanos , Animais , Camundongos , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/farmacologia , Fosfolipase C gama/metabolismo , Ácido Araquidônico/farmacologia , Ácido Araquidônico/metabolismo , Fosfolipases/metabolismo , Fosfolipases/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Ativação Plaquetária , Plaquetas/metabolismo , Agregação Plaquetária , Proteína Quinase C/metabolismo , Fosforilação , Colágeno/metabolismoRESUMO
Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, we show Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. We explored the light-dependent regulation of Pkc53E and demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCß4/NorpA as degeneration of norpAP24 photoreceptors was enhanced by a reduced Pkc53E activity. We further show that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Proteína Quinase C , Animais , Citoesqueleto de Actina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fosfolipases/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
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.
Assuntos
Grão Comestível , Família Multigênica , Pennisetum , Fosfolipases , Pennisetum/genética , Pennisetum/metabolismo , Fosfolipases/genética , Fosfolipases/metabolismo , Fosfolipases/química , Grão Comestível/genética , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sintenia , Perfilação da Expressão Gênica , Genótipo , Mapeamento CromossômicoRESUMO
The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here, we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to high-resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å-resolution cryo-electron microscopy tri-dimensional structure of the (VgrG)3 -(Tle1)3 complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfolipases/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fosfolipases/genética , Sistemas de Secreção Tipo VI/genéticaRESUMO
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.
Assuntos
Queratinócitos , Ácido gama-Linolênico , Humanos , Ácido gama-Linolênico/metabolismo , Esterificação , Epiderme/metabolismo , Ceramidas/metabolismo , Ácidos Graxos/metabolismo , Ácido Linoleico/metabolismo , Aciltransferases/metabolismo , Fosfolipases/metabolismoRESUMO
Phosphate is a vital macronutrient for plant growth, and its availability in soil is critical for agricultural sustainability and productivity. A substantial amount of cellular phosphate is used to synthesize phospholipids for cell membranes. Here, we identify a key enzyme, nonspecific phospholipase C4 (NPC4) that is involved in phosphosphingolipid hydrolysis and remodeling in Arabidopsis during phosphate starvation. The level of glycosylinositolphosphorylceramide (GIPC), the most abundant sphingolipid in Arabidopsis thaliana, decreased upon phosphate starvation. NPC4 was highly induced by phosphate deficiency, and NPC4 knockouts in Arabidopsis decreased the loss of GIPC and impeded root growth during phosphate starvation. Enzymatic analysis showed that NPC4 hydrolyzed GIPC and displayed a higher activity toward GIPC as a substrate than toward the common glycerophospholipid phosphatidylcholine. NPC4 was associated with the plasma membrane lipid rafts in which GIPC is highly enriched. These results indicate that NPC4 uses GIPC as a substrate in planta and the NPC4-mediated sphingolipid remodeling plays a positive role in root growth in Arabidopsis response to phosphate deficiency.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfolipases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Fosfatos/deficiência , Fosfolipases/genéticaRESUMO
The phospholipase A and acyltransferase (PLAAT) family is composed of three isoforms in mice (PLAAT1, 3, and 5), all of which function as phospholipid-metabolizing enzymes exhibiting phospholipase A1 /A2 and acyltransferase activities. Plaat3-deficient (Plaat3-/- ) mice were previously reported to show lean phenotype and remarkable hepatic fat accumulation under high-fat diet (HFD) feeding, while Plaat1-/- mice have not been analyzed. In the present study, we generated Plaat1-/- mice and investigated the effects of PLAAT1 deficiency on HFD-induced obesity, hepatic lipid accumulation, and insulin resistance. After HFD treatment, PLAAT1 deficiency caused a lower body weight gain compared to wild-type mice. Plaat1-/- mice also showed reduced liver weight with negligible hepatic lipid accumulation. In accordance with these findings, PLAAT1 deficiency improved HFD-induced hepatic dysfunction and lipid metabolism disorders. Lipidomics analysis in the liver revealed that in Plaat1-/- mice, the levels of various glycerophospholipids tended to increase, while all classes of lysophospholipids examined tended to decrease, suggesting that PLAAT1 functions as phospholipase A1 /A2 in the liver. Interestingly, the HFD treatment of wild-type mice significantly increased the mRNA level of PLAAT1 in the liver. Furthermore, the deficiency did not appear to elevate the risk of insulin resistance in contrast to PLAAT3 deficiency. These results suggested that the suppression of PLAAT1 improves HFD-induced overweight and concomitant hepatic lipid accumulation.
Assuntos
Dieta Hiperlipídica , Resistência à Insulina , Animais , Camundongos , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina/genética , Metabolismo dos Lipídeos , Fígado/metabolismo , Fosfolipídeos/metabolismo , Fosfolipases/metabolismo , Fosfolipases/farmacologia , Aciltransferases/genética , Aciltransferases/metabolismo , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Biofilmes , Candida albicans , Plâncton , Candida albicans/fisiologia , Biofilmes/crescimento & desenvolvimento , Hidrólise , Microscopia Eletrônica de Varredura , Fosfolipases/metabolismo , Análise Espectral Raman/métodos , Peptídeo Hidrolases/metabolismoRESUMO
Type VI secretion systems (T6SSs) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce autopermeabilization through unopposed activity of the Tle phospholipase effector. This hyperpermeability phenotype is T6SS dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyperpermeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyperpermeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG ß-spike protein. Together, these findings indicate that Tli has distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export. IMPORTANCE Gram-negative bacteria use type VI secretion systems deliver toxic effector proteins directly into neighboring competitors. Secreting cells also produce specific immunity proteins that neutralize effector activities to prevent autointoxication. Here, we show the Tli immunity protein of Enterobacter cloacae has two distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to block Tle lipase effector activity, while cytoplasmic Tli is required to activate the lipase prior to export. These results indicate Tle interacts transiently with its cognate immunity protein to promote effector protein folding and/or packaging into the secretion apparatus.
Assuntos
Sistemas de Secreção Tipo VI , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Fosfolipases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sinais Direcionadores de Proteínas , Lipase/metabolismoRESUMO
Intracellular lipolysis-the enzymatic breakdown of lipid droplet-associated triacylglycerol (TAG)-depends on the cooperative action of several hydrolytic enzymes and regulatory proteins, together designated as lipolysome. Adipose triglyceride lipase (ATGL) acts as a major cellular TAG hydrolase and core effector of the lipolysome in many peripheral tissues. Neurons initiate lipolysis independently of ATGL via DDHD domain-containing 2 (DDHD2), a multifunctional lipid hydrolase whose dysfunction causes neuronal TAG deposition and hereditary spastic paraplegia. Whether and how DDHD2 cooperates with other lipolytic enzymes is currently unknown. In this study, we further investigated the enzymatic properties and functions of DDHD2 in neuroblastoma cells and primary neurons. We found that DDHD2 hydrolyzes multiple acylglycerols in vitro and substantially contributes to neutral lipid hydrolase activities of neuroblastoma cells and brain tissue. Substrate promiscuity of DDHD2 allowed its engagement at different steps of the lipolytic cascade: In neuroblastoma cells, DDHD2 functioned exclusively downstream of ATGL in the hydrolysis of sn-1,3-diacylglycerol (DAG) isomers but was dispensable for TAG hydrolysis and lipid droplet homeostasis. In primary cortical neurons, DDHD2 exhibited lipolytic control over both, DAG and TAG, and complemented ATGL-dependent TAG hydrolysis. We conclude that neuronal cells use noncanonical configurations of the lipolysome and engage DDHD2 as dual TAG/DAG hydrolase in cooperation with ATGL.
Assuntos
Lipólise , Humanos , Lipase/genética , Lipase/metabolismo , Neurônios/metabolismo , Paraplegia , Fosfolipases/metabolismo , Triglicerídeos/metabolismoRESUMO
Loss-of-function mutations in patatin-like phospholipase domain-containing protein 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, and altered PNPLA1 activity is implicated in the pathogenesis of atopic dermatitis and other common skin diseases. To examine the hypothesis that PNPLA1 catalyzes the synthesis of acylceramides and acyl acids, we expressed and partially purified a soluble, truncated form of PNPLA1 in Escherichia coli, (PNPLA1trun) along with the related protein PNPLA2 (ATGL, adipose triglyceride lipase) and coactivator CGI-58. Liposomal substrates were incubated with recombinant enzymes for 0.5-24 h and products analyzed by HPLC-UV and LC-MS. Using trilinolein or dilinolein substrates, PNPLA1trun, like ATGLtrun, catalyzed lipolysis and acyltransferase reactions with 2-30% conversion into linoleic acid, monolinolein, and trilinolein. CGI-58 enhanced ATGL-catalyzed lipolysis as previously reported, but transacylase activity was not enhanced with ATGL or PNPLA1. In matching the proposed activity in vivo, PNPLA1 catalyzed acyl transfer from trilinolein and dilinolein donors to omega-hydroxy ceramide, omega-hydroxy glucosylceramide, and omega-hydroxy acid acceptors to form acylceramide, glucosyl-acylceramide, and acyl acid, respectively, albeit with only â¼0.05% conversion of the substrates. Notably, in experiments comparing dilinolein vs. diolein acyl donors, PNPLA1 transferred linoleate with 3:1 selectivity over oleate into acylceramide. These results support the role for PNPLA1 in the synthesis of acylceramides and acyl acids in epidermis and suggest that the enrichment of these lipids with linoleic acid could result from the substrate selectivity of PNPLA1.
Assuntos
Ictiose Lamelar , Pele , Humanos , Pele/metabolismo , Ácido Linoleico/metabolismo , Lipase/genética , Lipase/metabolismo , Epiderme/metabolismo , Ictiose Lamelar/genética , Ictiose Lamelar/metabolismo , Ceramidas/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Fosfolipases/metabolismoRESUMO
Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2-/- mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA2 enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2-/- photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2-/- and Pnpla2+/- mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.