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1.
PLoS Biol ; 18(1): e3000595, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961851

RESUMO

Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. Here, we use Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. Our analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. We find that loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although we show that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, we identify an additional role for bmm function in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, we demonstrate that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, our data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male-female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/genética , Drosophila/metabolismo , Lipase/fisiologia , Metabolismo dos Lipídeos/genética , Lipólise/genética , Caracteres Sexuais , Animais , Animais Geneticamente Modificados , Metabolismo Energético/genética , Feminino , Lipase/genética , Lipase/metabolismo , Masculino , Micronutrientes/metabolismo , Triglicerídeos/metabolismo
2.
PLoS One ; 15(1): e0226838, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990908

RESUMO

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.


Assuntos
Bactérias/enzimologia , Lipase/química , Lipase/metabolismo , Mutação , Pergelissolo/microbiologia , Motivos de Aminoácidos , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Lipase/genética , Metagenoma , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Serina/metabolismo , Sibéria , Especificidade por Substrato
3.
Lett Appl Microbiol ; 70(3): 221-229, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31854000

RESUMO

Hard-shelled mussel (Mytilus coruscus) is a popular seafood in Korea. This study aimed to determine the virulence markers and antimicrobial resistance patterns of 33 Aeromonas strains isolated from mussels. The isolates were identified as A. salmonicida (n = 14), A. veronii (n = 9), A. enteropelogenes (n = 4), A. caviae (n = 3), A. allosaccharophila (n = 2) and A. bivalvium (n = 1) by gyrB gene sequencing. The sequence divergence between and within the species ranged from 3·70 to 10·40% and 0-1·50% respectively. Every species formed a distinct group in a neighbour-joining phylogenetic tree. The DNase, gelatinase, caseinase, ß-haemolysis, biofilm and lipase activities were observed in 33 (100·00%), 31 (93·93%), 30 (90·90%), 27 (81·81%), 21 (63·63%) and 17 (51·51%) isolates respectively. The virulence genes were detected by PCR in the following frequencies: fla (90·09%), aer (87·88%), hlyA (87·88%), ahyB (81·19%), gcaT (75·76%), ser (69·70%), lip (66·67%), alt (57·58%), ast (51·51%) and act (21·21%). Every isolate was resistant to at least three of 18 antimicrobials in the disk diffusion test. The multiple antimicrobial resistance index values ranged from 0·11 to 0·44 among the isolates. Our study suggests that mussels can be a potential reservoir of virulent and multidrug-resistant Aeromonas sp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas sp. are known as common pathogenic bacteria isolated from seafood. The virulence factors and antimicrobial resistance profiles of mussel-borne Aeromonas sp. are poorly understood. This study demonstrated for the first time the existence of virulence markers and antimicrobial resistance of Aeromonas sp. from mussels in Korea. Majority of the isolates were positive for phenotypic virulence characteristics and harboured several virulence genes which reveal the potential virulence of mussel-borne Aeromonas sp. Multiple antimicrobial resistance was also observed among the isolates. Our study highlights the importance of food safety standards in mussel consumption.


Assuntos
Aeromonas/classificação , Aeromonas/isolamento & purificação , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Mytilus/microbiologia , Aciltransferases/genética , Aeromonas/genética , Aeromonas/patogenicidade , Animais , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções por Bactérias Gram-Negativas/microbiologia , Lipase/genética , Filogenia , Reação em Cadeia da Polimerase , República da Coreia , Alimentos Marinhos/microbiologia , Virulência/genética , Fatores de Virulência/genética
4.
Enzyme Microb Technol ; 132: 109416, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731975

RESUMO

A thermostable lipase from Bacillus thermocatenulatus was glycosylated by forming the consensus sequence (-NXS/T-) for N-linked glycosylation by site-directed mutagenesis. Among the eight BTL2 mutants including the consensus sequence, six BTL2 mutants, A277 N, A290 N, Y200 N, T236 N, T238 N, and P261 N, were glycosylated. Among the six mutants, glycosylated A277 N and T236 N showed higher stability in the presence of 25% (v/v) DMSO (74.3 and 72.8% of initial activity was remained after incubation at 45 °C for 20 h, respectively) than deglycosylated A277 N and T236 N (57.2 and 45.1% of initial activity was remained, respectively). These glycosylated mutants also showed higher remaining activity than wild-type BTL2 (56.0% of the initial activity were remained). Furthermore, the glycosylated mutant T236 N showed longer half-lives in the presence of 25% (v/v) ethylene glycol, DMSO, and DMF (161, 133, and 56.7 h at 45 °C, respectively) than deglycosylated mutant T236 N (107, 91.9, and 42.8 h, respectively). N-linked glycosylation may be a promising approach for preparing enzymes to retain their activity in the presence of organic solvents.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Lipase/metabolismo , Compostos Orgânicos/química , Solventes/química , Bacillus/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosilação , Lipase/genética , Mutagênese Sítio-Dirigida , Mutação
5.
J Agric Food Chem ; 67(51): 14048-14055, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31791125

RESUMO

Chemical residues in the environment are considered to be important factors that cause obesity. Bifenthrin is one of the pyrethroid pesticides and is widely used worldwide. However, its effect on adipose tissue is ill-defined. Here, we administered bifenthrin/corn oil to adult C57BL/6 mice by gavage. After 6 weeks, the bifenthrin treatment significantly increased their body weight (P = 0.015) and fat mass (P < 0.001). Then we identified 246 differently expressed proteins by proteomic analysis, and they were highly involved in fatty acid uptake and lipid metabolism processes. Interestingly, protein hormone-sensitive lipase and adipose triacylglyceride lipase were downregulated while lipoprotein lipase is upregulated after bifenthrin treatment. Similar effects in 3T3-L1 cells treated with bifenthrin validated the in vivo results. Thus, this study suggests that long-term exposure to low-dose bifenthrin induces fat deposition in mice by improving fatty acid uptake and inhibiting lipolysis, and it may cause obesity in humans.


Assuntos
Ácidos Graxos/metabolismo , Lipólise/efeitos dos fármacos , Obesidade/metabolismo , Praguicidas/efeitos adversos , Piretrinas/efeitos adversos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Feminino , Humanos , Lipase/genética , Lipase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/fisiopatologia , Praguicidas/metabolismo , Piretrinas/metabolismo , Esterol Esterase/genética , Esterol Esterase/metabolismo
6.
Adv Exp Med Biol ; 1185: 377-382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884641

RESUMO

Oxidative stress-mediated injury of the retinal pigment epithelium (RPE) can precede progressive retinal degeneration and ultimately lead to blindness (e.g., age-related macular degeneration (AMD)). The RPE expresses the PNPLA2 gene and produces its protein product PEDF-R that exhibits lipase activity. We have shown that transient PNPLA2 overexpression decreases dead-cell proteolytic activity and that synthetic peptides derived from a central region of PEDF-R efficiently protect ARPE-19 and pig primary RPE cells from oxidative stress. This study aims to evaluate the effect of loss of PNPLA2 in RPE cells undergoing oxidative stress. Loss of PNPLA2 conferred increased resistance to cells when subjected to oxidative stress.


Assuntos
Lipase/genética , Estresse Oxidativo , Epitélio Pigmentado da Retina/patologia , Animais , Epitélio Pigmentado da Retina/enzimologia , Suínos
7.
Int J Mol Sci ; 21(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881781

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). The aim of this retrospective study was to determine the risk factors for progression of CKD in patients with biopsy-proven NAFLD including patatin-like phospholipase domain containing 3 (PNPLA3) polymorphism. A total of 344 patients with biopsy-proven NAFLD were enrolled consecutively in this study. Multivariate analysis identified males (odds ratio (OR) 5.46), age (per 1 year, OR 1.07), and FIB-4 index (≥1.30, OR 3.85) as factors associated with CKD. Of the 154 patients with a baseline estimated glomerular filtration rate (eGFR) ≥60 mL/min, 30 had a deterioration in CKD stage and 15 developed CKD after 3 years. Multivariate analysis identified diabetes mellitus (OR 2.44) as a risk factor for deterioration in CKD stage, while diabetes mellitus (OR 21.54) and baseline eGFR (per 1 mL/min OR 0.88) were risk factors for development of CKD. PNPLA3 did not affect the change in eGFR. In NAFLD patients, a high FIB-4 index was associated with CKD to increases in the index linked to reductions in eGFR. In order to prevent development of CKD, an appropriate therapy focusing on renal function is needed for NAFLD patients, especially those with diabetes.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Insuficiência Renal Crônica/patologia , Índice de Gravidade de Doença , Adulto , Idoso , Idoso de 80 Anos ou mais , Diabetes Mellitus Tipo 2/complicações , Feminino , Taxa de Filtração Glomerular , Humanos , Japão/epidemiologia , Lipase/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Análise Multivariada , Hepatopatia Gordurosa não Alcoólica/etiologia , Razão de Chances , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/etiologia , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
8.
J Agric Food Chem ; 67(47): 13176-13184, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31690075

RESUMO

Lipases or triacylglycerol (TAG) lipases belong to the α/ß-hydrolases superfamily, which are enzymes capable of catalyzing the hydrolysis of the ester bond between fatty acids and glycerol. Interestingly, some lipases have been found to not only possess hydrolysis activity but also acyltransferase activity in yeasts and microalgae. Our present study reported a novel dual-functional Mucor circinelloides lipase Lip10 with a slight lipolysis activity but a noteworthy phospholipid/diacylglycerol acyltransferase (PDAT) activity. The purified Lip10 mutants prefer to utilize phosphatidyl serine to form TAG over phosphatidyl ethanolamine and phosphatidylcholine. Site-directed mutagenesis indicated that the histidine residue in the acyltransferase motif H-(X)4-D is indispensable for the PDAT activity of Lip10. Overexpression of the acyltransferase motif of Lip10 promoted cell growth by 12% and increased lipid production by 14% compared to the control, whilst overexpression of the lipase motif induced lipid degradation in M. circinelloides.


Assuntos
Aciltransferases/metabolismo , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Mucor/enzimologia , Aciltransferases/química , Aciltransferases/genética , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Lipase/química , Lipase/genética , Metabolismo dos Lipídeos , Mucor/química , Mucor/genética , Mutagênese Sítio-Dirigida , Alinhamento de Sequência
9.
Curr Med Sci ; 39(5): 766-777, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31612395

RESUMO

The activation of hepatic stellate cells (HSCs) is a major event during hepatic fibrogenesis. Restoration of intracellular lipid droplet (LD) formation turns the activated HSC back to a quiescent state. Our previous studies have shown that curcumin suppresses HSC activation through increasing peroxisome proliferator-activated receptor, gamma (PPARγ) and 5' adenosine monophosphate-activated protein kinase (AMPK) activities. This study aims at evaluating the effect of curcumin on lipid accumulation in HSCs and hepatocytes, and further elucidating the underlying mechanisms. Now we showed that curcumin increased LD formation in activated HSCs and stimulated the expression of sterol regulatory element-binding protein and fatty acid synthase, and reduced the expression of adipose triglyceride lipase. Exogenous perilin5 expression in primary HSCs promoted LD formation. Perilipin 5 siRNA eliminated curcumin-induced LD formation in HSCs. These results suggest that curcumin recovers LD formation and lipid accumulation in activated HSCs by increasing perilipin 5 gene expression. Furthermore, inhibition of AMPK or PPARγ activity blocked curcumin's effect on Plin5 gene expression and LD formation. Our results provide a novel evidence in vitro for curcumin as a safe, effective candidate to treat liver fibrosis.


Assuntos
Curcumina/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Gotículas Lipídicas/efeitos dos fármacos , Perilipina-1/genética , Perilipina-5/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Regulação da Expressão Gênica , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lipase/genética , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Camundongos , Especificidade de Órgãos , PPAR gama/genética , PPAR gama/metabolismo , Perilipina-1/agonistas , Perilipina-1/metabolismo , Perilipina-5/agonistas , Perilipina-5/metabolismo , Cultura Primária de Células , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismo
10.
Microb Cell Fact ; 18(1): 167, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601223

RESUMO

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. RESULTS: The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL-1) within 24 h as compared to the mono-copy strain. CONCLUSIONS: The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.


Assuntos
Proteínas Fúngicas , Lipase , Microrganismos Geneticamente Modificados , Proteínas Recombinantes , Yarrowia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica/genética , Lipase/genética , Lipase/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
11.
J Basic Microbiol ; 59(12): 1173-1184, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31621083

RESUMO

Lipids are hydrocarbons comprised of long-chain fatty acids and are found in all living things. In the environment, microorganisms degrade them to obtain energy using esterases and lipases. These enzymes are nowadays used in different industrial applications. We report isolation of 24 bacteria with esteresic and lipolytic activity from Lake Magadi, Kenya. The isolates were characterised using morphological, biochemical, and molecular methods. Isolates grew at an optimum salt concentration of 5-8% (w/v), pH range of 8.0-9.0, and temperature range of 35-40°C. The isolates were positive for esterase and lipase assay as well as other extracellular enzymes. Phylogenetic analysis of the 16S ribosomal RNA gene showed that the isolates were affiliated to the genus Bacillus, Alkalibacterium, Staphylococcus, Micrococcus, Halomonas, and Alkalilimnicola. None of the bacterial isolates produced antimicrobial agents, and all of them were resistant to trimethoprim and nalidixic acid but susceptible to streptomycin, amoxillin, chloramphenicol, and cefotaxime. Growth at elevated pH, salt, and temperature is an indicator that the enzymes from these organisms could function well under haloalkaline conditions. Therefore, Lake Magadi could be a good source of isolates with the potential to produce unique biocatalysts for the biotechnology industry.


Assuntos
Bactérias/classificação , Bactérias/enzimologia , Biodiversidade , Esterases/metabolismo , Lagos/microbiologia , Lipase/metabolismo , Microbiologia da Água , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , DNA Bacteriano/genética , Esterases/genética , Concentração de Íons de Hidrogênio , Quênia , Lagos/química , Lipase/genética , Testes de Sensibilidade Microbiana , Filogenia , RNA Ribossômico 16S/genética , Tolerância ao Sal , Análise de Sequência de DNA , Temperatura
12.
Infect Immun ; 88(1)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31636137

RESUMO

Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. However, its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. We overexpressed and purified the recombinant LipE (rLipE) protein and demonstrated that LipE has a lipase/esterase activity. rLipE prefers medium-chain ester substrates, with the maximal activity on hexanoate. Its activity is the highest at 40°C and pH 9. We determined that rLipE hydrolyzes trioctanoate. Using site-directed mutagenesis, we confirmed that the predicted putative activity triad residues Ser97, Gly342, and His363 are essential for the lipase activity of rLipE. The expression of the lipE gene was induced under stressed conditions mimicking M. tuberculosis' intracellular niche. The gene-disrupting mutation of lipE led to significantly reduced bacterial growth inside THP-1 cells and human peripheral blood mononuclear cell-derived macrophages and attenuated M. tuberculosis infection in mice (with ∼8-fold bacterial load reduction in mouse lungs). Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection.


Assuntos
Esterases/metabolismo , Lipase/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/microbiologia , Fatores de Virulência/metabolismo , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Estabilidade Enzimática , Esterases/deficiência , Esterases/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lipase/deficiência , Lipase/genética , Camundongos , Modelos Teóricos , Mutagênese Sítio-Dirigida , Células THP-1 , Temperatura , Fatores de Virulência/genética
13.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505904

RESUMO

: Nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of kidney disease in adults and children. However, it is uncertain whether this association is influenced by major NAFLD susceptibility genes. In a sample of 230 overweight/obese children, 105 with NAFLD (hepatic fat fraction ≥5% by magnetic resonance imaging) and 125 without NAFLD, rs738409 in PNPLA3, rs58542926 in TM6SF2, rs1260326 in GCKR, and rs641738 in MBOAT7 were genotyped. Abnormal kidney function was defined as estimated glomerular filtration rate (eGFR) < 90 mL/min/1.73 m2 and/or the presence of microalbuminuria (24 h urinary albumin excretion between 30 and 300 mg). In comparison with children without NAFLD, those with NAFLD showed increased prevalence of reduced eGFR (13.3% vs. 1.6%; p < 0.001) and microalbuminuria (8.6% vs. 3.4%, p = 0.025). TM6SF2, GCKR, and MBOAT7 risk alleles did not show any impact on kidney function, while the PNPLA3 G allele was associated with lower eGFR, but only in children with NAFLD (p = 0.003). After adjustment for confounders, NAFLD (OR, 4.7; 95% CI, 1.5-14.8; padj = 0.007), but not the PNPLA3 gene variant, emerged as the main independent predictor of renal dysfunction. Overall, our findings suggest that NAFLD remains the main determinant of decline in kidney function in overweight/obese children, while the PNPLA3 rs738409 prosteatogenic variant has a small impact, if any.


Assuntos
Albuminúria , Variação Genética , Nefropatias , Lipase/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica , Obesidade Pediátrica , Adolescente , Albuminúria/genética , Albuminúria/urina , Criança , Estudos Transversais , Feminino , Taxa de Filtração Glomerular , Humanos , Nefropatias/genética , Nefropatias/urina , Masculino , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/urina , Obesidade Pediátrica/genética , Obesidade Pediátrica/urina
14.
Molecules ; 24(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480403

RESUMO

Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus epidermidis AT2 (rT-M386) was markedly enhanced by directed evolution. The newly evolved mutant, G210C, demonstrated an optimal temperature shift from 25 to 45 °C and stability up to 50 °C. Interestingly, a cysteine residue was randomly introduced on the loop connecting the two lids and accounted for the only cysteine found in the lipase. We further investigated the structural and mechanistic insights that could possibly cause the significant temperature shift. Both rT-M386 and G210C were modeled and simulated at 25 °C and 50 °C. The results clearly portrayed the effect of cysteine substitution primarily on the lid stability. Comparative molecular dynamics simulation analysis revealed that G210C exhibited greater stability than the wild-type at high temperature simulation. The compactness of the G210C lipase structure increased at 50 °C and resulted in enhanced rigidity hence stability. This observation is supported by the improved and stronger non-covalent interactions formed in the protein structure. Our findings suggest that the introduction of a single cysteine residue at the lid region of cold-adapted lipase may result in unexpected increased in thermostability, thus this approach could serve as one of the thermostabilization strategies in engineering lipase stability.


Assuntos
Temperatura Baixa , Cisteína/genética , Glicina/genética , Lipase/genética , Mutação/genética , Staphylococcus/enzimologia , Cátions , Estabilidade Enzimática , Ligação de Hidrogênio , Lipase/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/metabolismo , Sais/química , Solventes
15.
J Biochem Mol Toxicol ; 33(11): e22391, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476254

RESUMO

Abnormal metabolism, including abnormal lipid metabolism, is a hallmark of cancer cells. Some studies have demonstrated that the lipogenic pathway might promote the development of hepatocellular carcinoma (HCC). However, the role of adipose triglyceride lipase (ATGL) in hepatocellular carcinoma cells has not been elucidated. We evaluated the function of ATGL in hepatocellular carcinoma using methyl azazolyl blue and migration assay through overexpression of ATGL in HepG2 cells. Quantitative reverse-transcription polymerase chain reaction and Western blot analyses were used to assess the mechanisms of ATGL in hepatocellular carcinoma. In the current study, we first constructed and transiently transfected ATGL into hepatocellular carcinoma cells. Secondly, we found that ATGL promoted the proliferation of hepatoma cell lines via upregulating the phosphorylation of AKT, but did not affect the metastatic ability of HCC cells. Moreover, the p-AKT inhibitor significantly eliminated the effect of ATGL on the proliferation of hepatoma carcinoma cells. Taken together, our results indicated that ATGL promotes hepatocellular carcinoma cells proliferation through upregulation of the AKT signaling pathway.


Assuntos
Carcinoma Hepatocelular/patologia , Proliferação de Células , Lipase/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Células Hep G2 , Humanos , Lipase/genética , Neoplasias Hepáticas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Regulação para Cima
16.
Protein Eng Des Sel ; 32(1): 13-24, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31403166

RESUMO

An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.


Assuntos
Evolução Molecular Direcionada , Proteínas de Drosophila/química , Lipase/química , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Estabilidade Enzimática/genética , Lipase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
17.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140261, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31401312

RESUMO

Bacillus lipases are industrially attractive enzymes due to their broad substrate specificity and optimum alkaline pH. However, narrow temperature range of action and low thermostability restrain their optimal use and thus, necessitate attention. Several laboratories are engaged in protein engineering of Bacillus lipases to generate variants with improved attributes for decades using techniques such as directed evolution or rational design. This review summarizes the effect of mutations on the conformational changes through in silico modeling and their manifestation with respect to various biochemical parameters. Various studies have been put together to develop a perspective on the molecular basis of biocatalysis of lipases holding industrial importance.


Assuntos
Substituição de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/química , Temperatura Alta , Lipase/química , Bacillus/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática/genética , Lipase/genética , Mutação de Sentido Incorreto , Relação Estrutura-Atividade
18.
Int J Biol Macromol ; 137: 1190-1198, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31299254

RESUMO

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 °C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 °C, which was about 5.0 °C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5 U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.


Assuntos
Lipase/química , Lipase/genética , Engenharia de Proteínas , Temperatura , Yarrowia/enzimologia , Biocatálise , Estabilidade Enzimática/genética , Temperatura Alta , Lipase/metabolismo , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica
19.
BMC Med ; 17(1): 135, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31311600

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver illness with a genetically heterogeneous background that can be accompanied by considerable morbidity and attendant health care costs. The pathogenesis and progression of NAFLD is complex with many unanswered questions. We conducted genome-wide association studies (GWASs) using both adult and pediatric participants from the Electronic Medical Records and Genomics (eMERGE) Network to identify novel genetic contributors to this condition. METHODS: First, a natural language processing (NLP) algorithm was developed, tested, and deployed at each site to identify 1106 NAFLD cases and 8571 controls and histological data from liver tissue in 235 available participants. These include 1242 pediatric participants (396 cases, 846 controls). The algorithm included billing codes, text queries, laboratory values, and medication records. Next, GWASs were performed on NAFLD cases and controls and case-only analyses using histologic scores and liver function tests adjusting for age, sex, site, ancestry, PC, and body mass index (BMI). RESULTS: Consistent with previous results, a robust association was detected for the PNPLA3 gene cluster in participants with European ancestry. At the PNPLA3-SAMM50 region, three SNPs, rs738409, rs738408, and rs3747207, showed strongest association (best SNP rs738409 p = 1.70 × 10- 20). This effect was consistent in both pediatric (p = 9.92 × 10- 6) and adult (p = 9.73 × 10- 15) cohorts. Additionally, this variant was also associated with disease severity and NAFLD Activity Score (NAS) (p = 3.94 × 10- 8, beta = 0.85). PheWAS analysis link this locus to a spectrum of liver diseases beyond NAFLD with a novel negative correlation with gout (p = 1.09 × 10- 4). We also identified novel loci for NAFLD disease severity, including one novel locus for NAS score near IL17RA (rs5748926, p = 3.80 × 10- 8), and another near ZFP90-CDH1 for fibrosis (rs698718, p = 2.74 × 10- 11). Post-GWAS and gene-based analyses identified more than 300 genes that were used for functional and pathway enrichment analyses. CONCLUSIONS: In summary, this study demonstrates clear confirmation of a previously described NAFLD risk locus and several novel associations. Further collaborative studies including an ethnically diverse population with well-characterized liver histologic features of NAFLD are needed to further validate the novel findings.


Assuntos
Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Idoso , Índice de Massa Corporal , Estudos de Casos e Controles , Redes Comunitárias/organização & administração , Redes Comunitárias/estatística & dados numéricos , Progressão da Doença , Registros Eletrônicos de Saúde/organização & administração , Registros Eletrônicos de Saúde/estatística & dados numéricos , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genômica/organização & administração , Genômica/estatística & dados numéricos , Humanos , Lipase/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Morbidade , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Transdução de Sinais/genética
20.
Bioprocess Biosyst Eng ; 42(11): 1739-1746, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31321527

RESUMO

A rational enhancement of kinetic resolution process for producing (S)-N-(2-ethyl-6-methylphenyl) alanine from racemic methyl ester using lipase B from Candida antarctica (CalB) was investigated. With the benefit results that lipase CalB-catalyzed reactions can be effectively regulated using amino acids (such as histidine and lysine) as additives, CalBs modified (mCalBs) by n-histidines at the N terminal and n-lysines at the C terminal were constructed and expressed. The results show that both soluble and precipitated mCalBs can effectively catalyze the hydrolysis reaction without adding any extra additives. The enantioselective ratio (E value) of soluble and precipitated mCalBs could be improved from 12.1 to 20.3, which were higher than that (E value was only 10.2) of commercial Novozym 435 (immobilized CalB). The study indicated that the amino acid-rich molecules introduced on lipase CalB can produce positive effects on enantioselectivity of enzyme. It provides unusual ideas for reasonable regulation of enzyme-catalyzed reactions.


Assuntos
Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lipase/química , Catálise , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipase/genética , Lipase/metabolismo , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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