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BACKGROUND AND AIM: The aim of this study was to investigate the comprehensive genetic effects of exploratory variants of LYPLAL1, GCKR, HSD17B13, TRIB1, APOC3, MBOAT7, and PARVB on pediatric nonalcoholic fatty liver disease in addition to the previously reported variants of TM6SF2, PNPLA3, and SAMM50 in Korean children. METHODS: A prospective case-control study was conducted involving 309 patients diagnosed using ultrasound and 339 controls. Anthropometric measurements, liver function tests, and metabolic marker analysis were conducted, and fibrosis scores were calculated. Transient elastography was performed in 69 some patients with nonalcoholic fatty liver disease. TaqMan allelic discrimination assays were used for genotyping. The genetic risk scores were calculated using significant variants, namely, HSD17B13, PARVB, PNPLA3, SAMM50, and TM6SF2, to evaluate the additive effect. RESULTS: Risk allele carriers of the PARVB variant showed significantly higher levels of aminotransferases, gamma-glutamyl transferase, alkaline phosphatase, pediatric nonalcoholic fatty liver disease fibrosis score, and aspartate aminotransferase/platelet ratio index. Individuals with a homozygous variant of HSD17B13 showed significantly lower levels of aminotransferase, gamma-glutamyl transferase, liver stiffness measurement, and aspartate aminotransferase/platelet ratio index than those with other genotypes. These parameters did not significantly differ among other variants of LYPLAL1, GCKR, TRIB1, APOC3, and MBOAT7. The genetic risk scores was identified as an independent risk factor for nonalcoholic fatty liver disease and had a positive association with severity. CONCLUSION: HSD17B13 has protective effects on the severity of pediatric nonalcoholic fatty liver disease. Variants of HSD17B13, PARVB, PNPLA3, SAMM50, and TM6SF2 had an additive effect on nonalcoholic fatty liver disease.
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17-Hidroxiesteroide Deshidrogenasas , Aciltransferasas , Proteínas de la Membrana , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/genética , Masculino , Femenino , Niño , 17-Hidroxiesteroide Deshidrogenasas/genética , Estudios de Casos y Controles , Aciltransferasas/genética , Estudios Prospectivos , Proteínas de la Membrana/genética , Adolescente , Lipasa/genética , Predisposición Genética a la Enfermedad , Péptidos y Proteínas de Señalización Intracelular/genética , Variación Genética , Proteínas Adaptadoras Transductoras de Señales/genética , Diagnóstico por Imagen de Elasticidad , Alelos , Lisofosfolipasa , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fosfolipasas A2 Calcio-IndependienteRESUMEN
To understand the lifespan of higher organisms, including humans, it is important to understand lifespan at the cellular level as a prerequisite. So, fission yeast is a good model organism for the study of lifespan. To identify the novel factors involved in longevity, we are conducting a large-scale screening of long-lived mutant strains that extend chronological lifespan (cell survival in the stationary phase) using fission yeast. One of the newly acquired long-lived mutant strains (No.98 mutant) was selected for analysis and found that the long-lived phenotype was due to a missense mutation (92Phe â Ile) in the plb1+ gene. plb1+ gene in fission yeast is a nonessential gene encoding a homolog of phospholipase B, but its functions under normal growth conditions, as well as phospholipase B activity, remain unresolved. Our analysis of the No.98 mutant revealed that the plb1 mutation reduces the integrity of the cellular membrane and cell wall and activates Sty1 via phosphorylation.
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Lisofosfolipasa , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Regulación Fúngica de la Expresión Génica , Longevidad/genética , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Mutación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismoRESUMEN
The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.
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Malaria Falciparum , Parásitos , Animales , Humanos , Plasmodium falciparum , Lisofosfatidilcolinas/metabolismo , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Malaria Falciparum/parasitología , Eritrocitos/metabolismo , Parásitos/metabolismo , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
Genome sequence of Pyrococcus abyssi DSM25543 contains a coding sequence (PAB_RS01410) for α/ß hydrolase (WP_010867387.1). Structural analysis revealed the presence of a consensus motif GXSXG and a highly conserved catalytic triad in the amino acid sequence of α/ß hydrolase that were characteristic features of lysophospholipases. A putative lysophospholipase from P. abyssi with its potential applications in oil degumming and starch processing was heterologously produced in E. coli Rosetta (DE3) pLysS in soluble form followed by its purification and characterization. The recombinant enzyme was found to be active at temperature of 40-90 °C and pH 5.5-7.0. However, the enzyme exhibited its optimum activity at 65 °C and pH 6.5. None of the metal ions (Mn2+, Mg2+, Ni2+, Cu2+, Fe2+, Co2+, Zn2+ and Ca2+) being tested had stimulatory effect on lysophospholipase activity. Km and Vmax for hydrolysis of 4-nitrophenyl butyrate were calculated to be 1 ± 0.089 mM and 1637 ± 24.434 U/mg, respectively. It is the first report on the soluble production and characterization of recombinant lysophospholipase from P. abyssi which exhibits its lipolytic activity in the absence of divalent metal ions. Broad substrate specificity, activity and stability at elevated temperatures make recombinant lysophospholipase an ideal candidate for potential industrial applications.
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Lisofosfolipasa , Pyrococcus abyssi , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , Lisofosfolipasa/química , Escherichia coli/genética , Archaea/metabolismo , Metales/farmacología , Metales/metabolismo , Iones/metabolismo , Especificidad por Sustrato , Proteínas Recombinantes/química , Clonación MolecularRESUMEN
Legionella pneumophila, the causative agent of a life-threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella-containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB-secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/ß-hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate-binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.
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Legionella pneumophila , Lisofosfolipasa , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Proteínas Bacterianas/metabolismo , Disulfuros/metabolismo , Vacuolas/metabolismo , Ácidos Grasos/metabolismo , Relación Estructura-ActividadRESUMEN
INTRODUCTION: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity. METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB). RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB. CONCLUSION: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.
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Queratitis por Acanthamoeba , Acanthamoeba , Amebiasis , Encefalitis , Lectina de Unión a Manosa , Animales , Lisofosfolipasa/genética , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Queratitis por Acanthamoeba/parasitología , Amebiasis/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa , Expresión Génica , Péptido HidrolasasRESUMEN
Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.
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Antifúngicos , Biopelículas , Cryptococcus neoformans , Proteínas Fúngicas , Lisofosfolipasa , Macrófagos Alveolares , Própolis , Humanos , Biopelículas/efectos de los fármacos , Línea Celular Tumoral , Criptococosis/prevención & control , Criptococosis/terapia , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/patogenicidad , Etanol/química , Proteínas Fúngicas/antagonistas & inhibidores , Liposomas , Enfermedades Pulmonares Fúngicas/prevención & control , Enfermedades Pulmonares Fúngicas/terapia , Lisofosfolipasa/antagonistas & inhibidores , Macrófagos Alveolares/microbiología , Própolis/química , Própolis/farmacología , Virulencia/efectos de los fármacos , Factores de Virulencia/antagonistas & inhibidores , Antifúngicos/química , Antifúngicos/farmacologíaRESUMEN
The microaerotolarent amitochondriate protozoan Giardia lamblia causes Giardiasis and produces a unique enzyme called Phospholipase B (PLB) in contrast to higher eukaryotes. The enzyme is produced upon induction with oxidative (H2O2) stress, thus leading to prostaglandin E2 (PGE2) production. It exists in dimeric form, and its molecular weight is 56 kDa. This PLB was extracellularly cloned in the pET21d vector. The ORF is 1620 bp (Genbank accession no. -OM939681) long and codes for a protein 539 amino acid long, with a 15 amino acid long amino-terminal signal peptide. The highest enzyme activity of PLB was identified at pH 7.5 and 35 °C. This specific enzyme was also active at 50 °C pH 10, but activity was low. We also analyzed the expression of PLB protein in G. lamblia, which was significantly induced under increased oxidative stress.
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Giardia lamblia , Giardiasis , Humanos , Lisofosfolipasa , Giardia lamblia/genética , Peróxido de Hidrógeno , AminoácidosRESUMEN
Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis-and-ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant-specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP-producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome-localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity-related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over-accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP-producing microbial pathogens.
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Lisofosfolipasa , Verticillium , Lisofosfolipasa/genética , Gossypium/genética , Peroxisomas , Fitomejoramiento , Enfermedades de las Plantas/microbiología , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Phospholipid metabolism is crucial for membrane biogenesis and homeostasis of Plasmodium falciparum. To generate such phospholipids, the parasite extensively scavenges, recycles, and reassembles host lipids. P. falciparum possesses an unusually large number of lysophospholipases, whose roles and importance remain to be elucidated. Here, we functionally characterize one P. falciparum lysophospholipase, PfLPL3, to reveal its key role in parasite propagation during asexual blood stages. PfLPL3 displays a dynamic localization throughout asexual stages, mainly localizing in the host-parasite interface. Inducible knockdown of PfLPL3 disrupts parasite development from trophozoites to schizont, inducing a drastic reduction in merozoite progenies. Detailed lipidomic analyses show that PfLPL3 generates fatty acids from scavenged host lipids to generate neutral lipids. These are then timely mobilized to allow schizogony and merozoite formation. We then identify inhibitors of PfLPL3 from Medicine for Malaria Venture (MMV) with potent antimalarial activity, which could also serve as pertinent chemical tools to study parasite lipid synthesis.
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Malaria Falciparum , Parásitos , Animales , Plasmodium falciparum , Parásitos/metabolismo , Ácidos Grasos/metabolismo , Lisofosfolipasa/metabolismo , Malaria Falciparum/parasitología , Eritrocitos/parasitología , Proteínas Protozoarias/metabolismoRESUMEN
The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.
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Colina , Lisofosfolipasa , Animales , Humanos , Ratones , Colina/metabolismo , Glicerilfosforilcolina/metabolismo , Hígado/metabolismo , Lisofosfolipasa/metabolismo , Metionina/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Mitophagy, which selectively eliminates the dysfunctional and excess mitochondria by autophagy, is crucial for cellular homeostasis under stresses such as hypoxia. Dysregulation of mitophagy has been increasingly linked to many disorders including neurodegenerative disease and cancer. Triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype, is reported to be characterized by hypoxia. However, the role of mitophagy in hypoxic TNBC as well as the underlying molecular mechanism is largely unexplored. Here, we identified GPCPD1 (glycerophosphocholine phosphodiesterase 1), a key enzyme in choline metabolism, as an essential mediator in hypoxia-induced mitophagy. Under the hypoxic condition, we found that GPCPD1 was depalmitoylated by LYPLA1, which facilitated the relocating of GPCPD1 to the outer mitochondrial membrane (OMM). Mitochondria-localized GPCPD1 could bind to VDAC1, the substrate for PRKN/PARKIN-dependent ubiquitination, thus interfering with the oligomerization of VDAC1. The increased monomer of VDAC1 provided more anchor sites to recruit PRKN-mediated polyubiquitination, which consequently triggered mitophagy. In addition, we found that GPCPD1-mediated mitophagy exerted a promotive effect on tumor growth and metastasis in TNBC both in vitro and in vivo. We further determined that GPCPD1 could serve as an independent prognostic indicator in TNBC. In conclusion, our study provides important insights into a mechanistic understanding of hypoxia-induced mitophagy and elucidates that GPCPD1 could act as a potential target for the future development of novel therapy for TNBC patients.Abbreviations: ACTB: actin beta; 5-aza: 5-azacytidine; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; ChIP: chromatin immunoprecipitation; co-IP: co-immunoprecipitation; CQ: chloroquine; CsA: cyclosporine; DOX: doxorubicin; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GPCPD1: glycerophosphocholine phosphodiesterase 1; HAM: hydroxylamine; HIF1A: hypoxia inducible factor 1 subunit alpha; HRE: hypoxia response element; IF: immunofluorescence; LB: lysis buffer; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; LC-MS: liquid chromatography-mass spectrometry; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; MDA231: MDA-MB-231; MDA468: MDA-MB-468; MFN1: mitofusin 1; MFN2: mitofusin 2; MKI67: marker of proliferation Ki-67; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; OS: overall survival; PalmB: palmostatin B; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SDS: sodium dodecyl sulfate; TOMM20: translocase of outer mitochondrial membrane 20; TNBC: triple-negative breast cancer; VBIT-4: VDAC inhibitor; VDAC1: voltage dependent anion channel 1; WT: wild type.
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Enfermedades Neurodegenerativas , Neoplasias de la Mama Triple Negativas , Humanos , Autofagia , Lisofosfolipasa/metabolismo , Lisofosfolipasa/farmacología , Mitofagia , Fosfolipasas/metabolismo , Fosfolipasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Cryptococcus neoformans (Cn) is an opportunistic, encapsulated, yeast-like fungus that causes severe meningoencephalitis, especially in countries with high HIV prevalence. In addition to its well-known polysaccharide capsule, Cn has other virulence factors such as phospholipases, a heterogeneous group of enzymes that hydrolyze ester linkages in glycerophospholipids. Phospholipase B (PLB1) has been demonstrated to play a key role in Cn pathogenicity. In this study, we used a PLB1 mutant (plb1) and its reconstituted strain (Rec1) to assess the importance of this enzyme on Cn brain infection in vivo and in vitro. Mice infected with the plb1 strain survive significantly longer, have lower peripheral and central nervous system (CNS) fungal loads, and have fewer and smaller cryptococcomas or biofilm-like brain lesions compared to H99- and Rec1-infected animals. PLB1 causes extensive brain tissue damage and changes microglia morphology during cryptococcal disease, observations which can have important implications in patients with altered mental status or dementia as these manifestations are related to poorer survival outcomes. plb1 cryptococci are significantly more phagocytosed and killed by NR-9460 microglia-like cells. plb1 cells have altered capsular polysaccharide biophysical properties which impair their ability to stimulate glial cell responses or morphological changes. Here, we provide significant evidence demonstrating that Cn PLB1 is an important virulence factor for fungal colonization of and survival in the CNS as well as in the progression of cryptococcal meningoencephalitis. These findings may potentially help fill in a gap of knowledge in our understanding of cerebral cryptococcosis and provide novel research avenues in Cn pathogenesis. IMPORTANCE Cryptococcal meningoencephalitis (CME) is a serious disease caused by infection by the neurotropic fungal pathogen Cryptococcus neoformans. Due to the increasing number of cases in HIV-infected individuals, as well as the limited therapies available, investigation into potential targets for new therapeutics has become critical. Phospholipase B is an enzyme synthesized by Cn that confers virulence to the fungus through capsular enlargement, immunomodulation, and intracellular replication. In this study, we examined the properties of PLB1 by comparing infection of a Cn PLB1 mutant strain with both the wild-type and a PLB1-reconstituted strain. We show that PLB1 augments the survival and proliferation of the fungus in the CNS and strengthens virulence by modulating the immune response and enhancing specific biophysical properties of the fungus. PLB1 expression causes brain tissue damage and impacts glial cell functions, which may be responsible for the dementia observed in patients which may persist even after resolving from CME. The implications of PLB1 inhibition reveal its involvement in Cn infection and suggest that it may be a possible molecular target in the development of antifungal therapies. The results of this study support additional investigation into the mechanism of PLB1 to further understand the intricacies of cerebral Cn infection.
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Criptococosis , Cryptococcus neoformans , Demencia , Infecciones por VIH , Meningoencefalitis , Animales , Ratones , Cryptococcus neoformans/metabolismo , Lisofosfolipasa/metabolismo , Criptococosis/microbiología , Sistema Nervioso Central/patología , Meningoencefalitis/microbiología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Infecciones por VIH/complicacionesRESUMEN
Human genome-wide association studies found single-nucleotide polymorphisms (SNPs) near LYPLAL1 (Lysophospholipase-like protein 1) that have sex-specific effects on fat distribution and metabolic traits. To determine whether altering LYPLAL1 affects obesity and metabolic disease, we created and characterized a mouse knockout (KO) of Lyplal1. We fed the experimental group of mice a high-fat, high-sucrose (HFHS) diet for 23 weeks, and the controls were fed regular chow diet. Here, we show that CRISPR-Cas9 whole-body Lyplal1 KO mice fed an HFHS diet showed sex-specific differences in weight gain and fat accumulation as compared to chow diet. Female, not male, KO mice weighed less than WT mice, had reduced body fat percentage, had white fat mass, and had adipocyte diameter not accounted for by changes in the metabolic rate. Female, but not male, KO mice had increased serum triglycerides, decreased aspartate, and decreased alanine aminotransferase. Lyplal1 KO mice of both sexes have reduced liver triglycerides and steatosis. These diet-specific effects resemble the effects of SNPs near LYPLAL1 in humans, suggesting that LYPLAL1 has an evolutionary conserved sex-specific effect on adiposity. This murine model can be used to study this novel gene-by-sex-by-diet interaction to elucidate the metabolic effects of LYPLAL1 on human obesity.
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Estudio de Asociación del Genoma Completo , Lisofosfolipasa , Obesidad , Animales , Femenino , Humanos , Masculino , Ratones , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo , Triglicéridos , Lisofosfolipasa/genéticaRESUMEN
BACKGROUND: Trichosporon asahii is an opportunistic pathogenic yeast-like fungus. Phospholipase B1 (PLB1) is an important virulence factor of pathogenic fungi such as Candida albicans and Cryptococcus neoformans, and there are few studies on the role of PLB1 in the pathogenicity of T. asahii. OBJECTIVES: To investigate the role of PLB1 in the pathogenicity of T. asahii. METHODS: A strain with low secretion of PLB1 (4848) was screened, a PLB1 overexpression strain (PLB1OX ) was constructed, and the differences in histopathology, fungal load of organ, survival time of mice, the levels of IL-6, IL-10, TNF-α, and GM-GSF in the serum and organs caused by the two strains were compared. RESULTS: Histopathology showed that spores and hyphae were observed in both groups, and PLB1OX led to more fungal invasion. The fungal loads in the kidney, lung, spleen and liver in the PLB1OX group were significantly higher than those in the 4848 group, and the survival time of mice was significantly lower than that in the 4848 group. The levels of TNF-α in the serum, liver, spleen, lung and kidney of the PLB1OX group were lower than those of the 4848 group, while the level of IL-10 in the serum was higher than that of the 4848 group. CONCLUSIONS: These results suggest that PLB1 can enhance the invasive function of T. asahii and affect the secretion of TNF-α and IL-10 which may affect the host antifungal immune response, providing evidence that PLB1 plays a role in the pathogenic infection of T. asahii.
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Interleucina-10 , Trichosporon , Animales , Ratones , Fosfolipasas , Trichosporon/genética , Factor de Necrosis Tumoral alfa , Virulencia , Lisofosfolipasa/metabolismoRESUMEN
Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.
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Hígado , Lisofosfolipasa , Metionina , Fosfatidilcolinas , Animales , Ratones , Colina/metabolismo , Glicerilfosforilcolina/metabolismo , Hígado/metabolismo , Metionina/metabolismo , Racemetionina/metabolismo , S-Adenosilmetionina/metabolismo , Triglicéridos/metabolismo , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Fosfatidilcolinas/metabolismoRESUMEN
Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01â¯mg/kg and the yield of oil reached to 98.80â¯% after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.
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Geobacillus , Aceite de Soja , Aceite de Soja/química , Lisofosfolipasa/metabolismo , Monoacilglicerol Lipasas , Lisofosfatidilcolinas , Glycine max/metabolismoRESUMEN
Neuropathy target esterase (NTE) has been proven to act as a lysophospholipase (LysoPLA) and phospholipase B (PLB) in mammalian cells. In this study, we took human neuroblastoma SK-N-SH cells as the research object and explored the effect of NTE on phospholipid homeostasis. The results showed that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels significantly increased (> 40%), while glycerophosphocholine (GPC) decreased (below 60%) after NTE gene was knockdown in the cells (NTE < 30% of control), which were prepared by gene silencing with dsRNA-NTE. However, in the NTE-overexpressed cells (NTE > 50% of control), which were prepared by expressing recombinant catalytic domain of NTE, LPC remarkably decreased (below 80%) and GPC enhanced (> 40%). Mipafox, a neuropathic organophosphorus compound (OP), significantly inhibited NTE-LysoPLA and NTE-PLB activities (> 95-99% inhibition at 50 µM), which was accompanied with a decreased GPC level (below 40%) although no change of the PC and LPC levels was observed; while paraoxon, a non-neuropathic OP, suppresses neither the activities of NTE-phospholipases nor the levels of PC, LPC, and GPC. Thus, we concluded that both the stable up- or down-regulated expression of NTE gene and the loss of NTE-LysoPLA/PLB activities disrupts phospholipid homeostasis in the cells although the inhibition of NTE activity only decreased GPC content without altering PC and LPC levels.
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Neuroblastoma , Fosfolípidos , Humanos , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Homeostasis , Lisofosfatidilcolinas/farmacología , Lisofosfatidilcolinas/metabolismo , Lisofosfolipasa/metabolismo , Lisofosfolipasa/farmacología , Mamíferos/metabolismo , Compuestos Organofosforados/farmacología , Fosfatidilcolinas/farmacologíaRESUMEN
Lysophosphatidylcholine (LPC) is a bioactive lipid that modulates macrophage polarization during immune responses, inflammation, and tissue remodeling. Patatin-like phospholipase domain containing protein 7 (PNPLA7) is a lysophospholipase with a preference for LPC. However, the role of PNPLA7 in macrophage polarization as an LPC hydrolase has not been explored. In the current study, we found that PNPLA7 is highly expressed in naïve macrophages and downregulated upon lipopolysaccharide (LPS)-induced polarization towards the classically activated (M1) phenotype. Consistently, overexpression of PNPLA7 suppressed the expression of proinflammatory M1 marker genes, including interleukin 1ß (IL-1ß), IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor α (TNF-α), whereas knockdown of PNPLA7 augmented the inflammatory gene expression in LPS-challenged macrophages. PNPLA7 overexpression and knockdown increased and decreased Sirtuin1 (SIRT1) mRNA and protein levels, respectively, and affected the acetylation of the nuclear factor-kappa B (NF-κB) p65 subunit, a key transcription factor in M1 polarization. In addition, the levels of phosphorylated p38 mitogen-activated protein kinase (MAPK) were suppressed and enhanced by PNPLA7 overexpression and knockdown, respectively. Taken together, these findings suggest that PNPLA7 suppresses M1 polarization of LPS-challenged macrophages by modulating SIRT1/NF-κB- and p38 MAPK-dependent pathways.
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Lisofosfolipasa , Activación de Macrófagos , FN-kappa B , Sirtuina 1 , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Inflamación/metabolismo , Lipopolisacáridos , Macrófagos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Sirtuina 1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Lisofosfolipasa/metabolismoRESUMEN
Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase-mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold KD reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels.Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden-Fletcher-Goldfarb-Shanno; CHO, Chinese Hamster Ovary; KD, Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab'), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.