Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

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.

Genome-wide screening and characterization of phospholipase A (PLA)-like genes in sorghum (Sorghum bicolor L.).

MAIN CONCLUSION: The characterisation of PLA genes in the sorghum genome using in-silico methods revealed their essential roles in cellular processes, providing a foundation for further detailed studies. Sorghum bicolor (L.) Moench is the fifth most cultivated crop worldwide, and it is used in many ways, but it has always gained less popularity due to the yield, pest, and environmental constraints. Improving genetic background and developing better varieties is crucial for better sorghum production in semi-arid tropical regions. This study focuses on the phospholipase A (PLA) family within sorghum, comprehensively characterising PLA genes and their expression across different tissues. The investigation identified 32 PLA genes in the sorghum genome, offering insights into their chromosomal localization, molecular weight, isoelectric point, and subcellular distribution through bioinformatics tools. PLA-like family genes are classified into three groups, namely patatin-related phospholipase A (pPLA), phospholipase A1 (PLA1), and phospholipase A2 (PLA2). In-silico chromosome localization studies revealed that these genes are unevenly distributed in the sorghum genome. Cis-motif analysis revealed the presence of several developmental, tissue and hormone-specific elements in the promoter regions of the PLA genes. Expression studies in different tissues such as leaf, root, seedling, mature seed, immature seed, anther, and pollen showed differential expression patterns. Taken together, genome-wide analysis studies of PLA genes provide a better understanding and critical role of this gene family considering the metabolic processes involved in plant growth, defence and stress response.

A Complex of Type I Platelet-Activating Factor Acetylhydrolase (PAF-AH) Catalytic Subunits Switches from α1/α2 Heterodimer to α2/α2 Homodimer during Adipocyte Differentiation of 3T3-L1 Cells.

Platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes an acetyl ester at the sn-2 position of platelet-activating factor (PAF), thereby mediating a variety of biological functions. PAF-AH is found in three isoforms: Type I PAF-AH (PAF-AH I) and Type II PAF-AH (PAF-AH II) are intracellular enzymes whereas plasma PAF-AH is characterized by association with lipoprotein in plasma. PAF-AH I forms a tetramer constituted by two catalytic subunits (α1 and α2) with ß regulatory subunits. We recently showed that a deficiency of PAF-AH I catalytic subunits in male mice caused an increase of body weight, food intake, and white adipose tissue (WAT) weight. In this study, we examined whether the expression of this enzyme was altered in the differentiation of 3T3-L1 preadipocytes into adipocytes. The amount of PAF-AH I α1 subunit protein was significantly reduced in 3T3-L1 differentiation, while the amount of the PAF-AH I α2 subunit was not changed. Immunoprecipitation analysis of 3T3-L1 differentiation showed that the complex of PAF-AH I catalytic subunits was changed from α1/α2 heterodimer to α2/α2 homodimer. Our findings suggest that changes in PAF-AH I catalytic subunits are involved in adipocyte differentiation of 3T3-L1 and obesity in mice.

PLAAT1 Exhibits Phosphatidylcholine:Monolysocardiolipin Transacylase Activity.

Tissue-specific cardiolipin fatty acyl profiles are achieved by remodeling of de novo synthesized cardiolipin, and four remodeling enzymes have thus far been identified. We studied the enzyme phospholipase A and acyltransferase 1 (PLAAT1), and we report the discovery that it has phosphatidylcholine (PC):monolysocardiolipin (MLCL) transacylase activity. Subcellular localization was analyzed by differential centrifugation and immunoblotting. Total levels of major phospholipids, and the fatty acyl profile of cardiolipin, were analyzed in HEK293 cells expressing murine PLAAT1 using gas chromatography. Apparent enzyme kinetics of affinity-purified PLAAT1 were calculated using radiochemical enzyme assays. This enzyme was found to localize predominantly to the endoplasmic reticulum (ER) but was detected at low levels in the mitochondria-associated ER matrix. Cells expressing PLAAT1 had higher levels of total cardiolipin, but not other phospholipids, and it was primarily enriched in the saturated fatty acids myristate, palmitate, and stearate, with quantitatively smaller increases in the n-3 polyunsaturated fatty acids linolenate, eicosatrienoate, and eicosapentanoate and the monounsaturated fatty acid erucate. Affinity-purified PLAAT1 did not catalyze the transacylation of MLCL using 1-palmitoyl-2-[14C]-linoleoyl-PC as an acyl donor. However, PLAAT1 had an apparent Vmax of 1.61 µmol/min/mg protein and Km of 126 µM using [9,10-3H]-distearoyl-PC as an acyl donor, and 0.61 µmol/min/mg protein and Km of 16 µM using [9,10-3H]-dioleoyl-PC. PLAAT1 is therefore a novel PC:MLCL transacylase.

The Simple Method of Preparation of Highly Carboxylated Bacterial Cellulose with Ni- and Mg-Ferrite-Based Versatile Magnetic Carrier for Enzyme Immobilization.

The bacterial cellulose (BC) is a versatile biopolymer of microbial origin characterized by high purity and unusual water and material properties. However, the native BC contains a low number of functional groups, which significantly limits its further application. The main goal of its effective modification is to use methods that allow the unusual properties of BC to be retained and the desired functional group to be efficiently introduced. In the present study, the new magnetic carrier based on functionalized citric acid (CA) bacterial cellulose was developed and tested to support critical industrial enzymes such as lipase B from Candida antarctica and phospholipase A from Aspergillus oryzae. The applied method allowed BC to be effectively modified by citric acid and a sufficient number of carboxylic groups to be introduced, up to 3.6 mmol of COOH per gram of dry mass of the prepared carrier. The DSC and TGA analyses revealed carrier stability at operational temperatures in the range of 20 °C to 100 °C and substantially influenced the amount of the introduced carboxyl groups on carrier properties. Both enzymes' immobilization significantly improves their thermal stability at 60 °C without a significant thermal and pH optima effect. The analyzed enzymes showed good operational stability with a significant residual activity after ten cycles of repeated uses. The new magnetic carrier based on highly carboxylated bacterial cellulose has a high application capability as matrix for immobilization the various enzymes of industrial interest.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain.

Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.

Quercetin Potentiates Docosahexaenoic Acid to Suppress Lipopolysaccharide-induced Oxidative/Inflammatory Responses, Alter Lipid Peroxidation Products, and Enhance the Adaptive Stress Pathways in BV-2 Microglial Cells.

High levels of docosahexaenoic acid (DHA) in the phospholipids of mammalian brain have generated increasing interest in the search for its role in regulating brain functions. Recent studies have provided evidence for enhanced protective effects when DHA is administered in combination with phytochemicals, such as quercetin. DHA and quercetin can individually suppress lipopolysaccharide (LPS)⁻induced oxidative/inflammatory responses and enhance the antioxidative stress pathway involving nuclear factor erythroid-2 related factor 2 (Nrf2). However, studies with BV-2 microglial cells indicated rather high concentrations of DHA (IC50 in the range of 60⁻80 µM) were needed to produce protective effects. To determine whether quercetin combined with DHA can lower the levels of DHA needed to produce protective effects in these cells is the goal for this study. Results showed that low concentrations of quercetin (2.5 µM), in combination with DHA (10 µM), could more effectively enhance the expression of Nrf2 and heme oxygenase 1 (HO-1), and suppress LPS⁻induced nitric oxide, tumor necrosis factor-α, phospho-cytosolic phospholipase A2, reactive oxygen species, and 4-hydroxynonenal, as compared to the same levels of DHA or quercetin alone. These results provide evidence for the beneficial effects of quercetin in combination with DHA, and further suggest their potential as nutraceuticals for improving health.

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin.

The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general.

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.

In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase.

Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes.

AIMS: We investigated whether Listeria monocytogenes strains differ in their ability to escape from the primary phagosome after internalization into human intestinal epithelial cells. METHODS AND RESULTS: Food and clinical strains were used to study specific alleles; the activities of listeriolysin O (LLO) and phospholipases PlcA and PlcB, which promote rupture of the phagocytic vacuole; and initial intracellular bacterial growth in Caco-2 cells. Results showed no difference in LLO activities between food and clinical strains or among serotypes. In contrast, the LLO truncation mutant lacked detectable haemolytic activity and intracellular growth. PlcA and PlcB produced by the strains of serotypes 4b/4e and 1/2b exhibited significantly lower activities than those of serotypes 1/2a and 1/2c. In contrast, the strains of serotype 1/2b grew significantly faster than those of serotypes 4b/4e and 1/2a. Moreover, the PrfA truncation mutants lacked LLO and phospholipases activities and did not show intracellular growth. CONCLUSIONS: We determined that LLO and PrfA mutants exert a significant effect on intracellular growth, although it was unclear from this study whether PlcA and PlcB alleles affect escape from vacuoles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study estimates that low-virulence L. monocytogenes strains associated with escape ability from the primary vacuoles are not widely distributed among food strains.

Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) are ethanolamides of long-chain fatty acids, including palmitoylethanolamide, oleoylethanolamide and anandamide. In animal tissues, NAEs are biosynthesized from membrane phospholipids. The classical "transacylation-phosphodiesterase" pathway proceeds via N-acyl-phosphatidylethanolamine (NAPE), which involves the actions of two enzymes, NAPE-generating Ca2+-dependent N-acyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD). Recent identification of Ca-NAT as Ɛ isoform of cytosolic phospholipase A2 enabled the further molecular biological approaches toward this enzyme. In addition, Ca2+-independent NAPE formation was shown to occur by N-acyltransferase activity of a group of proteins named phospholipase A/acyltransferases (PLAAT)-1-5. The analysis of NAPE-PLD-deficient mice confirmed that NAEs can be produced through multi-step pathways bypassing NAPE-PLD. The NAPE-PLD-independent pathways involved three members of the glycerophosphodiesterase (GDE) family (GDE1, GDE4 and GDE7) as well as α/ß-hydrolase domain-containing protein (ABHD)4. In this review article, we will focus on recent progress made and latest insights in the enzymes involved in NAE synthesis and their further characterization.

Improved recombinant Api m 1- and Ves v 5-based IgE testing to dissect bee and yellow jacket allergy and their correlation with the severity of the sting reaction.

BACKGROUND: No study has assessed the diagnostic sensitivity of rApi m 1 and rVes v 5 on Immulite testing system. OBJECTIVE: To compare the diagnostic sensitivity of commercially available venom recombinant allergens between the currently available immunoassays [ImmunoCAP (CAP) and Immulite (LITE)] and establish their correlation with the severity of the sting reaction. METHODS: This study evaluated 95 bee venom and 110 yellow jacket venom-allergic subjects. We measured the levels of sIgE to rApi m 1, rVes v 5 (LITE and CAP), rApi m 2 (LITE), rVes v 1 (CAP) and total IgE (CAP). Forty-nine healthy subjects served as controls. RESULTS: The diagnostic sensitivity of rApi m 1 and rVes v 5 was significantly higher with the LITE than with the CAP system (71% vs. 88% and 82% vs. 93%). The specificity of both assays for both allergens was between 94% and 98%. Twenty-nine patients that tested negative for rApi m 1 or rVes v 5 with CAP were positive with LITE, but none of the patients that tested negative with LITE were positive with CAP. The positive values of rApi m 1 and rVes v 5 were on average 2.7 and 2.3 times higher, with the LITE than with the CAP system. The combination of rApi m 1 and rApi m 2 (LITE) and the combination of rVes v 5 (LITE) and rVes v 1 (CAP) almost matched the sensitivity of native venoms (95% and 97%, respectively), whereas the diagnostic sensitivity of the combination of rVes v 5 and rVes v 1 (CAP) did not reach the sensitivity of rVes v 5 (LITE) alone (90% vs. 93%). IgE levels to venom recombinants and total IgE did not correlate with the severity of sting reaction. CONCLUSIONS & CLINICAL RELEVANCE: The use of rApi m 1 and rVes v 5 with the LITE system significantly enhanced diagnostic utility of venom recombinants and should improve the dissection of bee and yellow jacket venom allergy.

The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.

Neutral lipid storage disease comprises a heterogeneous group of autosomal recessive disorders characterized by systemic accumulation of triglycerides in cytoplasmic droplets. Here we report a neutral lipid storage disease subgroup characterized by mild myopathy, absence of ichthyosis and mutations in both alleles of adipose triglyceride lipase (PNPLA2, also known as ATGL). Three of these mutations are predicted to lead to a truncated ATGL protein with an intact patatin domain containing the active site, but with defects in the hydrophobic domain. The block in triglyceride degradation was mimicked by short interfering RNA directed against ATGL. NLSDM is distinct from Chanarin-Dorfman syndrome, which is characterized by neutral lipid storage disease with ichthyosis, mild myopathy and hepatomegaly due to mutations in ABHD5 (also known as CGI-58).

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Overexpression of patatin-related phospholipase AIIIδ altered plant growth and increased seed oil content in camelina.

Camelina sativa is a Brassicaceae oilseed species being explored as a biofuel and industrial oil crop. A growing number of studies have indicated that the turnover of phosphatidylcholine plays an important role in the synthesis and modification of triacylglycerols. This study manipulated the expression of a patatin-related phospholipase AIIIδ (pPLAIIIδ) in camelina to determine its effect on seed oil content and plant growth. Constitutive overexpression of pPLAIIIδ under the control of the constitutive cauliflower mosaic 35S promoter resulted in a significant increase in seed oil content and a decrease in cellulose content. In addition, the content of major membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, in 35S::pPLAIIIδ plants was increased. However, these changes in 35S::pPLAIIIδ camelina were associated with shorter cell length, leaves, stems, and seed pods and a decrease in overall seed production. When pPLAIIIδ was expressed under the control of the seed specific, ß-conglycinin promoter, the seed oil content was increased without compromising plant growth. The results suggest that pPLAIIIδ alters the carbon partitioning by decreasing cellulose content and increasing oil content in camelina.

Patatin-related phospholipase A, pPLAIIIα, modulates the longitudinal growth of vegetative tissues and seeds in rice.

Patatin-related phospholipase A (pPLA) hydrolyses glycerolipids to produce fatty acids and lysoglycerolipids. The Oryza sativa genome has 21 putative pPLAs that are grouped into five subfamilies. Overexpression of OspPLAIIIα resulted in a dwarf phenotype with decreased length of rice stems, roots, leaves, seeds, panicles, and seeds, whereas OspPLAIIIα-knockout plants had longer panicles and seeds. OspPLAIIIα-overexpressing plants were less sensitive than wild-type and knockout plants to gibberellin-promoted seedling elongation. OspPLAIIIα overexpression and knockout had an opposite effect on the expression of the growth repressor SLENDER1 in the gibberellin signalling process. OspPLAIIIα-overexpressing plants had decreased mechanical strength and cellulose content, but exhibited increases in the expression of several cellulose synthase genes. These results indicate that OspPLAIIIα plays a role in rice vegetative and reproductive growth and that the constitutive, high activity of OspPLAIIIα suppresses cell elongation. The decreased gibberellin response in overexpressing plants is probably a result of the decreased ability to make cellulose for anisotropic cell expansion.

The HRASLS (PLA/AT) subfamily of enzymes.

The H-RAS-like suppressor (HRASLS) subfamily consists of five enzymes (1-5) in humans and three (1, 3, and 5) in mice and rats that share sequence homology with lecithin:retinol acyltransferase (LRAT). All HRASLS family members possess in vitro phospholipid metabolizing abilities including phospholipase A1/2 (PLA1/2) activities and O-acyltransferase activities for the remodeling of glycerophospholipid acyl chains, as well as N-acyltransferase activities for the production of N-acylphosphatidylethanolamines. The in vivo biological activities of the HRASLS enzymes have not yet been fully investigated. Research to date indicates involvement of this subfamily in a wide array of biological processes and, as a consequence, these five enzymes have undergone extensive rediscovery and renaming within different fields of research. This review briefly describes the discovery of each of the HRASLS enzymes and their role in cancer, and discusses the biochemical function of each enzyme, as well as the biological role, if known. Gaps in current understanding are highlighted and suggestions for future research directions are discussed.