LIPA Gene Research Articles

Genetic Heterogeneity of Familial Hypercholesterolemia: Repercussions for Molecular Diagnosis.

Genetics of Familial Hypercholesterolemia (FH) is ascribable to pathogenic variants in genes encoding proteins leading to an impaired LDL uptake by the LDL receptor (LDLR). Two forms of the disease are possible, heterozygous (HeFH) and homozygous (HoFH), caused by one or two pathogenic variants, respectively, in the three main genes that are responsible for the autosomal dominant disease: LDLR, APOB and PCSK9 genes. The HeFH is the most common genetic disease in humans, being the prevalence about 1:300. Variants in the LDLRAP1 gene causes FH with a recessive inheritance and a specific APOE variant was described as causative of FH, contributing to increase FH genetic heterogeneity. In addition, variants in genes causing other dyslipidemias showing phenotypes overlapping with FH may mimic FH in patients without causative variants (FH-phenocopies; ABCG5, ABCG8, CYP27A1 and LIPA genes) or act as phenotype modifiers in patients with a pathogenic variant in a causative gene. The presence of several common variants was also considered a genetic basis of FH and several polygenic risk scores (PRS) have been described. The presence of a variant in modifier genes or high PRS in HeFH further exacerbates the phenotype, partially justifying its variability among patients. This review aims to report the updates on the genetic and molecular bases of FH with their implication for molecular diagnosis.

Y12F mutation in Pseudomonas plecoglossicida S7 lipase enhances its thermal and pH stability for industrial applications: a combination of in silico and in vitro study.

Appropriate amino acid substitutions are critical for protein engineering to redesign catalytic properties of industrially important enzymes like lipases. The present study aimed for improving the environmental stability of lipase from Pseudomonas plecoglossicida S7 through site-directed mutagenesis driven by computational studies. lipA gene was amplified and sequenced. Both wild type (WT) and mutant type (MT) lipase genes were expressed into the pET SUMO system. The expressed proteins were purified and characterized for pH and thermostability. The lipase gene belonged to subfamily I.1 lipase. Molecular dynamics revealed that Y12F-palmitic acid complex had a greater binding affinity (-6.3 Kcal/mol) than WT (-6.0 Kcal/mol) complex. Interestingly, MDS showed that the binding affinity of WT-complex (-130.314 ± 15.11 KJ/mol) was more than mutant complex (-108.405 ± 69.376 KJ/mol) with a marked increase in the electrostatic energy of mutant (-26.969 ± 12.646 KJ/mol) as compared to WT (-15.082 ± 13.802 KJ/mol). Y12F mutant yielded 1.27 folds increase in lipase activity at 55°C as compared to the purified WT protein. Also, Y12F mutant showed increased activity (~ 1.2 folds each) at both pH 6 and 10. P. plecoglossicida S7. Y12F mutation altered the kinetic parameters of MT (Km- 1.38 mM, Vmax- 22.32 µM/min) as compared to WT (Km- 1.52 mM, Vmax- 29.76 µM/min) thus increasing the binding affinity of mutant lipase. Y12F mutant lipase with better pH and thermal stability can be used in biocatalysis.

A Field Study of Some Bacterial Causes of Mass Mortality Syndrome in Nile Tilapia Fish Farms with a Treatment Trial

The goal of this study was to evaluate some of the bacterial microorganisms that contribute to the summer mass mortality syndrome in Nile tilapia fish (Oreochromis niloticus) kept in captivity in the Kafr El-Sheikh governorate. The fish samples were from five private fish farms with high mortality. Hundred diseased Nile tilapia fish (weighing 100–150 g) were collected from these farms (20 samples from each farm). The fish were examined at the farm to detect the clinical signs. The collected samples were sent to the lab for postmortem examination, bacteriological examination, and an antibiogram (sensitivity test) of isolated bacteria. The examined fish farms suffered from mass mortalities; the affected fish displayed respiratory symptoms, dark coloration of the skin, hemorrhagic skin lesions, ulceration, exophthalmia, and ascetics. PM examination showed congestion with enlargement and adhesion of the internal organs (liver, spleen) with abdominal fluid. The isolated bacteria were Aeromonas spp. (28%), Pseudomonas spp. (16%), and Edwardsiella tarda (5%). Polymerase chain reaction (PCR) was used to identify primers targeting gyrB as well as the detection of some virulence genes of Aeromonas hydrophila using one set of primers targeting the detection of the virulent genes AST gene (331bp), Lip gene (382 bp) and Alt gene (442bp). All nine PCR examined isolates were positive for the gyrB-conserved gene, and five examined samples were positive for three genes except sample number 2, which was negative for the Lip gene and the Alt gene. The result of the sensitivity test revealed that isolated bacteria were resistant to Erythromycin, Sulfamethazole Trimethoprim, and Lincomycin, while they were sensitive to Doxycycline and Enrofloxacin. Treatment of farm number 3 with doxycycline for 10 days and farm number 4 with Florfenicol for 10 days resulted in a significant decrease in mortality rates and an improvement in the health status of the fish.

Lysosomal Acid Lipase Deficiency: Genetics, Screening, and Preclinical Study.

Lysosomal acid lipase (LAL) is a lysosomal enzyme essential for the degradation of cholesteryl esters through the endocytic pathway. Deficiency of the LAL enzyme encoded by the LIPA gene leads to LAL deficiency (LAL-D) (OMIM 278000), one of the lysosomal storage disorders involving 50-60 genes. Among the two disease subtypes, the severe disease subtype of LAL-D is known as Wolman disease, with typical manifestations involving hepatomegaly, splenomegaly, vomiting, diarrhea, and hematopoietic abnormalities, such as anemia. In contrast, the mild disease subtype of this disorder is known as cholesteryl ester storage disease, with hypercholesterolemia, hypertriglyceridemia, and high-density lipoprotein disappearance. The prevalence of LAL-D is rare, but several treatment options, including enzyme replacement therapy, are available. Accordingly, a number of screening methodologies have been developed for this disorder. This review summarizes the current discussion on LAL-D, covering genetics, screening, and the tertiary structure of human LAL enzyme and preclinical study for the future development of a novel therapy.

Deacylation of galactolipids decomposes photosystem II dimers to enhance degradation of damaged D1 protein.

Photosystem II (PSII) contains many lipid molecules that are essential for the function and maintenance of PSII. Under strong light conditions, PSII complexes are dynamically modified during the repair process; however, the molecular mechanism of the dynamic changes in the PSII structure is still unclear. In the present study, we investigated the role of a lipase in the repair of PSII in Synechocystis sp. PCC 6803. We identified a protein encoded by the sll1969 gene, previously named lipase A (lipA), in the Synechocystis sp. PCC 6803 genome as a candidate for the lipase involved in PSII repair. Recombinant protein expressed in Escherichia coli cells hydrolyzed fatty acids at the sn-1 position of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol as well as triacylglycerol esterified with stearic acids. PSII repair in a disrupted mutant of the lipA gene was suppressed by the slow degradation of damaged D1 protein under strong light. The level of the PSII dimer remained higher in lipA mutant cells than wild-type (WT) cells under strong light. LipA protein was associated with the PSII dimer in vivo, and recombinant LipA protein decomposed PSII dimers purified from WT cells to monomers by reducing MGDG content in the PSII complex. These results indicate that LipA reacts with PSII dimers, dissociates them into monomers by digesting MGDG, and enhances D1 degradation during PSII repair.

Lysosomal acid lipase deficiency: A rare inherited dyslipidemia but potential ubiquitous factor in the development of atherosclerosis and fatty liver disease.

Lysosomal acid lipase (LAL), encoded by the gene LIPA, is the sole neutral lipid hydrolase in lysosomes, responsible for cleavage of cholesteryl esters and triglycerides into their component parts. Inherited forms of complete (Wolman Disease, WD) or partial LAL deficiency (cholesteryl ester storage disease, CESD) are fortunately rare. Recently, LAL has been identified as a cardiovascular risk gene in genome-wide association studies, though the directionality of risk conferred remains controversial. It has also been proposed that the low expression and activity of LAL in arterial smooth muscle cells (SMCs) that occurs inherently in nature is a likely determinant of the propensity of SMCs to form the majority of foam cells in atherosclerotic plaque. LAL also likely plays a potential role in fatty liver disease. This review highlights the nature of LAL gene mutations in WD and CESD, the association of LAL with prediction of cardiovascular risk from genome-wide association studies, the importance of relative LAL deficiency in SMC foam cells, and the need to further interrogate the pathophysiological impact and cell type-specific role of enhancing LAL activity as a novel treatment strategy to reduce the development and induce the regression of ischemic cardiovascular disease and fatty liver.

Genetic characterization and antimicrobial profiling of bacterial isolates collected from Nile tilapia (Oreochromis niloticus) affected by summer mortality syndrome.

In recent years, Egyptian tilapia aquaculture has experienced mortality episodes during the summer months. The causative agents responsible for such mortalities have not been clearly identified. A total of 400 fish specimens were collected from affected tilapia farms within five Egyptian governorates. A total of 344 bacterial isolates were identified from the examined fish specimens. Bacterial isolates were grouped into seven genera based on API 20E results. The most prevalent pathogens were Aeromonas spp. (42%), Vibrio spp. (21%), and Streptococcus agalactiae (14.5%). Other emerging infections like, Plesiomonas shigelloides (10%), Staphyloccocus spp. (8%), Pseudomonas oryzihabitans, and Acinetobacter lwoffii (2.3%) were also detected. Sequence analysis of the 16S ribosomal RNA bacterial gene of some isolates, confirmed the phenotypic identification results. The analysis of antibiotic resistance genes revealed the presence of aac(6')-Ib-cr (35.7%), blaCTX gene (23.8%), qnrS (19%), ampC (16.7%), floR (14.3%), sul1, tetA, and van.C1 (2.4%) genes in some isolates. The antimicrobia resistance gene, qac was reported in 46% of screened isolates. Bacterial strains showed variable virulence genes profiles. Aeromonas spp. harboured (act, gcat, aerA, lip, fla, and ser) genes. All Vibrio spp. possessed the hlyA gene, while cylE, hylB, and lmb genes, were detected in S. agalactiae strains. Our findings point to the possible role of the identified bacterial pathogens in tilapia summer mortality syndrome and highlight the risk of the irresponsible use of antibiotics on antimicrobial resistance in aquaculture.

Screening, lipA Gene Amplification and Molecular Identification of Lipolytic Fungi from Contaminated Soils

Lipase is a glycerol ester hydrolase that hydrolyzes esters of triglycerides. This study aimed to isolate, screen and characterize lipolytic fungi with lipA gene from soil environments. Fungi [molds and yeasts] were isolated from contaminated soil samples randomly collected from Palm oil mills, Abattoirs and Automobile servicing workshops in Eleme and Obio Akpor local governments in Rivers State, Nigeria using standard microbiological techniques. Qualitative Lipase screening assay was carried out on the fungal isolates through gel diffusion assay using Tributryin, Tween 80 and Olive oil respectively. Isolates with maximum zones of clearance were selected and screened for Lipase gene for hydrolysis of triacylglyceride [lipA gene] through Lipase gene amplification and were identified by Internal Transcribed Spacer characterization.
 A total of 96 fungal isolates were obtained, lipolytic screening showed 57 lipolytic strains in which 21 lipolytic fungal strains were screened from contaminated soil from palm oil mills, 17 lipolytic strains from abattoirs and 19 lipolytic strains from automobile workshops respectively. Seven isolates with maximum zones of clearance showed lipA gene in their genomic DNA and were identified as Candida orthopsilosis PMS3 [Accession No: OL546803], Fusarium proliferatum PMS4 [Accession No: OL546804], Saccharomyces cerevisiae PMS8 [Accession No: OL546805], Penicillium paxilli ABS8 [Accession No: OL546799], Pichia kudriavzevii ENS1 [Accession No: OL546800] Aspergillus niger ENS3 [Accession No: OL546801] and Trichoderma viride ENS5 [Accession No: OL546802].
 This study showed that soil fungi are potential producers of biotechnologically important lipase with diverse industrial applications.

Optimization of Submerged Fermentation Parameters for Enhanced Lipase Production from Newly Isolated Lipolytic Yeasts using Agro Waste Substrates

Fungi are currently recognized as the most preferred sources of lipase due to their ability to produce copious amount of extracellular lipase with lower production’s and extraction’s costs. Lipase production was carried out in agro wastes formulated-liquid fermentation media at 300C, at 130rpm, at initial pH of 7 for 96 hours using newly isolated LipA gene identified yeasts [Pichia kudriavzevii ENS1 [Accession No: OL546800], Candida orthopsilosis PMS3 [Accession No: OL546803] and Saccharomyces cerevisiae PMS8 [Accession No: OL546805] at inoculum size of 2%. Agrowastes such as Mango kernel and orange peel were used as alternative carbon sources; beans hull and groundnut shell as nitrogen sources while lipidic sources such as olive oil, soyabeans oil, palm oil, groundnut oil and coconut oil were used as inducers. Single factor optimization process was carried out to determine the effects of carbon sources, nitrogen sources, inducers, incubation time, inoculum size, pH and carbon: nitrogen ratio on lipase production and cell growth. Extraction by filtration, centrifugation and acetone precipitation; Quantitative lipase assay using titrimetric method and Cell growth by measuring optic density at 600nm were done. Proximate analysis of the agro-waste substrates showed higher percentage of carbohydrate and crude fat in mango kernel powder at 68.05±0.56% and 19.54±0.62% while groundnut shell recorded higher crude protein at 11.63±0.05%. Single factor optimization study showed optimum lipase production and cell growth at 50.83±1.18u/ml/min and 0.69±0.00[OD] with Candida orthopsilosis PMS3[Accession No: OL546803] using hot water treated mango kernel hydrolysate as carbon source. The results from this study showed that mango kernel would be an economical alternative carbon source for industrial-scale lipase production.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

ABSTRACTVaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 μg/mL) as well as minimum fungicidal concentrations (2 to 32 μg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 μg/mL and 32 μg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis.IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Uncommon presentation of Cholesteryl Ester Storage Disease (CESD): Description of a case and genetic characterization by next generation sequencing

Background and Aims : The LIPA gene encodes the lysosomal acidic lipase (LAL), an enzyme which hydrolyzes cholesterol esters (CE) and triglycerides (TG). Cholesteryl Ester Storage Disease (CESD) is a rare recessive disease caused by mutations in LIPA gene which result in residual LAL activity. Complete LAL deficiency is associated with a more severe form of disease known as Wolman’s disease. Hyperlipidemia and liver steatosis are common clinical features of CESD.

Early Discovery of Children With Lysosomal Acid Lipase Deficiency With the Universal Familial Hypercholesterolemia Screening Program.

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive lysosomal storage disorder, caused by homozygous or compound heterozygous pathogenic variants in the LIPA gene. Clinically, LAL-D is under- and misdiagnosed, due to similar clinical and laboratory findings with other cholesterol or liver misfunctions. As a part of the Slovenian universal familial hypercholesterolemia (FH) screening, LAL-D is screened as a secondary condition among other rare dyslipidemias manifesting with hypercholesterolemia. Out of 669 children included, three were positive for a homozygous disease-causing splicing variant NM_000235.4: c.894G > A (NP_000226.2:p. Gln298Gln) in the LIPA gene (NG_008194.1). The mean age by the diagnosis of LAL-D was 9.8 ± 0.9 years. Moreover, all three LAL-D-positive children had an important elevation of transaminases and decreased activity of the lysosomal acid lipase enzyme. Abdominal MRI in all children detected an enlarged liver but a normal-sized spleen. In conclusion, universal FH screening algorithms with the confirmatory genetic analysis in the pediatric population enable also rare dyslipidemia detection at an early age. An important clinical criterion for differentiation between FH and the LAL-D-positive children has elevated transaminase levels (AST and ALT). In all three LAL-D positive children, an improvement in cholesterol and transaminase levels and steatosis of the liver has been seen after early treatment initiation.

Metagenomic analysis displays the potential predictive biodegradation pathways of the persistent pesticides in agricultural soil with a long record of pesticide usage

Microbes are crucial in removing various xenobiotics, including pesticides, from the environment, specifically by mineralizing these hazardous pollutants. However, the specific procedure of microbe-mediated pesticide degradation and its consequence on the environment remain elusive owing to limitations in culturing techniques. Therefore, in this study, we have investigated i) the physicochemical and elemental compositions of PCAS (pesticide-contaminated agricultural soils) and NS (natural soils); ii) the bacterial communities and degradation pathways, as well as some novel biodegradation genes (BDGs) and pesticide degradation genes (PDGs) across two different landscapes (PCAS and NS) by applying high-throughput sequencing. The chemical and elemental composition analyses showed that all nutrients (P, K, N, S, Mn, B, and Zn) were significantly higher in PCAS than in NS (p ≤ 0.05). The results of the 16S rRNA amplicon sequencing analysis of pesticide-contaminated (PCAS-1, PCAS-2, PCAS-3, PCAS-4) samples showed that the relative abundance of the phylum Proteobacteria (30-36%) > Actinobacteria (15-20%) > Firmicutes (13-14%) > Bacteroidetes (7-13%), were higher compared to the natural soil (NS-1, NS2). Consistent with this, a phylogenetic shift was observed with (alpha, beta, and gamma Proteobacteria) being abundant in PCAS, whereas delta and epsilon groups were more prevalent in NS. The functional characterization of the PCAS and NS by PICRUSt2 revealed that bacterial communities play a significant role in pesticide metabolism. Predictive metagenome analysis of contaminated soils showed the role of core degrading genes in membrane transport, stress response, regulatory genes, resource transport, and environmental sensing. Furthermore, 14 BDGs and 30 PDGs were examined, with a relative abundance of 0.081−1.029 % and 0.107–0.8903 % in each PCAS, respectively. The major BDGs and PDGs, with the compounds they hydrolyze, include ppo (polyphenol oxidase and laccase), CYP (cytochrome p450 protein), lip gene (lignin peroxidase), similarly, among the PDGs mhel (carbendazim), opd (organophosphate), mpd (methyl parathion), atzA, atzB, atzD, atzF and trzN (atrazine), chd (chlorothalonil), hdx (metamitron), hdl-1 (isoproturon) and fmo (nicosulfuron). Overall, our findings demonstrated the significance of utilizing metagenomic methods to predict microbial aided degradation in the ecology of contaminated environments.

Morroniside, a novel GATA3 binding molecule, inhibits hepatic stellate cells activation by enhancing lysosomal acid lipase expression

BackgroundLiver fibrosis can be easily developed into irreversible liver cirrhosis or even liver cancer. Lysosomal acid lipase (LAL), encoded by the lipase A (Lipa) gene, is a critical enzyme involved in liver fibrosis development. Morroniside, an iridoid glycoside isolated from Cornus officinalis Sieb. et Zucc., exerts hepatic protective effects. However, the mechanism of action underling the anti-liver fibrosis effects of morroniside have not been fully elucidated. PurposeTo explore whether Lipa served as a biomarker for liver fibrosis and investigate the anti-liver fibrosis effects of morroniside and the underlying action mechanism in liver fibrosis cell models. MethodsLAL expression was examined in the liver tissues of CCl4 and high-fat diet (HFD)-induced liver fibrosis animal models. α-smooth muscle actin (α-SMA) level, collagen and GATA family expressions were analyzed by Real-time PCR and Western blot. Putative transcription factor binding sites in the DNA sequences of Lipa was identified by PROMO-ALGGEN v8.3 online software and ENCODE ChIP-Seq Significance Tool. MD simulation was performed to explore the protein-ligand interaction. ResultsWe found that the expression of hepatic LAL is lower in the liver fibrosis animal models than the control models. The reduced LAL expression is associated with HSCs activation, suggesting LAL is novel liver fibrosis biomarker. More importantly, our data showed that morroniside exerts anti-liver fibrosis effects in vitro. Mechanistic studies reveal that it binds to the hydrophobic sites of GATA3 and also reduces GATA3 expression, which increases LAL expression. ConclusionsThis study, for the first time, suggests LAL is a novel biomarker for liver fibrosis. Besides, morroniside exerts its anti-liver fibrosis effects by targeting GATA3 and LAL and hence inhibits HSC activation. These findings provide strong scientific evidence to support the development of morroniside as novel alternative or complementary therapeutics for liver injury prevention and treatment.

The lip gene contributes to the virulence of Aeromonas veronii strain TH0426

Aeromonas veronii (A. veronii) is a pathogen that can infect aquatic organisms and mammals and has caused irrecoverable economic losses to the aquaculture industry. The results of an epidemiological investigation showed that the number of cases of A. veronii have increased gradually in recent years, and its drug resistance and virulence has shown an upward trend. In this study, we constructed an A. veronii mutant strain Δlip, by homologous recombination and studied its function. The results showed that there was no significant difference in the biofilm formation ability between the Δlip and the wild-type strain, but the toxicity of the Δlip to EPC cells and its ability to adhere to EPC cells were significantly reduced. The LD50 value of the Δlip to zebrafish was 7.40-fold higher than that of the wild-type strain. In addition, after 24 h and 72 h, the bacterial loads of the Δlip in the organs of crucian carp were significantly lower than those in the wild-type strain. In conclusion, the mutant strain Δlip led to a decrease in the adhesion and virulence of the wild-type strain, which lays a foundation to further understand lip gene function and the pathogenic mechanism of A. veronii.

Characterization of Polymer Degrading Lipases, LIP1 and LIP2 From Pseudomonas chlororaphis PA23.

The outstanding metabolic and bioprotective properties of the bacterial genus Pseudomonas make these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacterium Pseudomonas chlororaphis PA23 and subsequently performed cloning and expression in Escherichia coli. The lip1 gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas the lip2 consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant α/β-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyanion pocket, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with an oxyanion pocket of GGX-type, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. Our three-dimensional models of LIP1 and LIP2 complexed with a 3-hydroxyoctanoate dimer revealed the core α/β hydrolase-type domain with an exposed substrate binding pocket in LIP1 and an active-site capped with a closing lid domain in LIP2. The recombinant LIP1 was optimally active at 45°C and pH 9.0, and the activity improved in the presence of Ca2+. LIP2 exhibited maximum activity at 40°C and pH 8.0, and was unaffected by Ca2+. Despite different properties, the enzymes exhibited broadsubstrate specificity and were able to hydrolyze short chain length and medium chain length polyhydroxyalkanoates (PHAs), polylactic acid (PLA), and para-nitrophenyl (pNP) alkanoates. Gel Permeation Chromatography (GPC) analysis showed a decrease in the molecular weight of the polymers after incubation with LIP1 and LIP2. The enzymes also manifested some polymer-degrading activity on petroleum-based polymers such as poly(ε-caprolactone) (PCL) and polyethylene succinate (PES), suggesting that these enzymes could be useful for biodegradation of synthetic polyester plastics. The study will be the first report of the complete characterization of intracellular lipases from bacterial and/or Pseudomonas species. The lipases, LIP1 and LIP2 are different from other bacterial lipases/esterases in having broad substrate specificity for polyesters.

Lysosomal acid lipase deficiency – an underestimated cause of hypercholesterolemia in children

Lysosomal acid lipase deficiency (LAL-D) is a rare, progressive, autosomal recessive disease, which develops due to impaired degradation and subsequent intra-lysosomal accumulation of triglycerides and cholesterol esters causing dyslipidemia. The clinical manifestations of the disease presumably depend on the residual activity of the enzyme, lysosomal acid lipase. A profound deficiency of the enzyme known as Wolman’s disease has an onset in the first 6 months of life. The disease reveals itself by dyspeptic disorders in the form of vomiting and diarrhea, lack of weight gain, hepatosplenomegaly, and adrenal calcification. If the Wolman’s disease is not treated, children die within the first 6 months as a result of exhaustion caused by malabsorption syndrome combined with progressive deterioration of liver and adrenal glands. Partial deficiency of lysosomal acid lipase manifests itself at a later age and is called cholesterol ester storage disease. Its clinical presentations include hepatosplenomegaly, elevated transaminases, hypercholesterolemia, and, in some cases, hypertriglyceridemia. Liver failure is the main cause of death in the natural course of cholesterol ester storage disease. Delayed diagnosis of the disease leads to its progression causing irreversible liver damage. The implementation of mass screening programs with the determination of cholesterol levels in childhood is critical to identifying asymptomatic patients.The article presents a clinical case of a patient aged 3 years. The molecular genetic testing showed a mutation in exon 8 of the LIPA gene: NM_000235.3:c.894G>A synonymous variant in the homozygous state. It was also found that both parents of the girl had this type of mutation in the heterozygous state. The patient was prescribed sebelipase alfa in a dose of 1 mg/kg once every 14 days. The treatment was well tolerated. Due to the early verification of the diagnosis and timely pathogenetic therapy, the prognosis of the course of LAL-D, the duration and quality of life of the child were considered to be favourable.Raising the awareness of doctors along with the introduction of effective screening programs for the timely detection of dyslipidemia in children contributes to timely diagnosis and early initiation of pathogenetic therapy, which can increase the life expectancy of patients with lysosomal acid lipase deficiency and improve their quality of life.

Interpreting coronary artery disease GWAS results: A functional genomics approach assessing biological significance.

Genome-wide association studies (GWAS) have implicated 58 loci in coronary artery disease (CAD). However, the biological basis for these associations, the relevant genes, and causative variants often remain uncertain. Since the vast majority of GWAS loci reside outside coding regions, most exert regulatory functions. Here we explore the complexity of each of these loci, using tissue specific RNA sequencing data from GTEx to identify genes that exhibit altered expression patterns in the context of GWAS-significant loci, expanding the list of candidate genes from the 75 currently annotated by GWAS to 245, with almost half of these transcripts being non-coding. Tissue specific allelic expression imbalance data, also from GTEx, allows us to uncover GWAS variants that mark functional variation in a locus, e.g., rs7528419 residing in the SORT1 locus, in liver specifically, and rs72689147 in the GUYC1A1 locus, across a variety of tissues. We consider the GWAS variant rs1412444 in the LIPA locus in more detail as an example, probing tissue and transcript specific effects of genetic variation in the region. By evaluating linkage disequilibrium (LD) between tissue specific eQTLs, we reveal evidence for multiple functional variants within loci. We identify 3 variants (rs1412444, rs1051338, rs2250781) that when considered together, each improve the ability to account for LIPA gene expression, suggesting multiple interacting factors. These results refine the assignment of 58 GWAS loci to likely causative variants in a handful of cases and for the remainder help to re-prioritize associated genes and RNA isoforms, suggesting that ncRNAs maybe a relevant transcript in almost half of CAD GWAS results. Our findings support a multi-factorial system where a single variant can influence multiple genes and each genes is regulated by multiple variants.

Drosophila Lipase 3 Mediates the Metabolic Response to Starvation and Aging

The human LIPA gene encodes for the enzyme lysosomal acid lipase, which hydrolyzes cholesteryl ester and triacylglycerol. Lysosomal acid lipase deficiency results in Wolman disease and cholesteryl ester storage disease. The Drosophila genome encodes for two LIPA orthologs, Magro and Lipase 3. Magro is a gut lipase that hydrolyzes triacylglycerides, while Lipase 3 lacks characterization based on mutant phenotypes. We found previously that Lipase 3 transcription is highly induced in mutants with defects in peroxisome biogenesis, but the conditions that allow a similar induction in wildtypic flies are not known. Here we show that Lipase 3 is drastically upregulated in starved larvae and starved female flies, as well as in aged male flies. We generated a lipase 3 mutant that shows sex-specific starvation resistance and a trend to lifespan extension. Using lipidomics, we demonstrate that Lipase 3 mutants accumulate phosphatidylinositol, but neither triacylglycerol nor diacylglycerol. Our study suggests that, in contrast to its mammalian homolog LIPA, Lipase 3 is a putative phospholipase that is upregulated under extreme conditions like prolonged nutrient deprivation and aging.

Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad

BackgroundMannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment.ResultsM. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species.ConclusionsSequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.