RESUMO
AIMS: Beyond the pivotal roles of the gut microbiome in initiating physiological processes and modulating genetic factors, a query persists: Can a single gene mutation alter the abundance of the gut microbiome community? Not only this, but the intricate impact of gut microbiome composition on skin pigmentation has been largely unexplored. METHODS AND RESULTS: Based on these premises, our study examines the abundance of lipase-producing gut microbes about differential gene expression associated with bile acid synthesis and lipid metabolism-related blood metabolites in red (whole wild) and white (whole white wild and SCARB1-/- mutant) Oujiang colour common carp. Following the disruption of the SCARB1 gene in the resulting mutant fish with white body colour (SCARB1-/-), there is a notable decrease in the abundance of gut microbiomes (Bacillus, Staphylococcus, Pseudomonas, and Serratia) associated with lipase production. This reduction parallels the downregulation seen in wild-type white body colour fish (WW), as contrasting to the wild-type red body colour fish (WR). Meanwhile, in SCARB1-/- fish, there was a downregulation noted not only at the genetic and metabolic levels but also a decrease in lipase-producing bacteria. This consistency with WW contrasts significantly with WR. Similarly, genes involved in the bile acid synthesis pathway, along with blood metabolites related to lipid metabolism, exhibited downregulation in SCARB1-/- fish. CONCLUSIONS: The SCARB1 knockout gene blockage led to significant alterations in the gut microbiome, potentially influencing the observed reduction in carotenoid-associated skin pigmentation. Our study emphasizes that skin pigmentation is not only impacted by genetic factors but also by the gut microbiome. Meanwhile, the gut microbiome's adaptability can be rapidly shaped and may be driven by specific single-gene variations.
Assuntos
Carpas , Microbioma Gastrointestinal , Pigmentação da Pele , Animais , Carpas/microbiologia , Pigmentação da Pele/genética , Lipase/genética , Lipase/metabolismo , Mutação , Metabolismo dos Lipídeos , Ácidos e Sais Biliares/metabolismo , Bactérias/genética , Bactérias/metabolismo , Bactérias/isolamento & purificação , Bactérias/classificaçãoRESUMO
BACKGROUND: Recent studies have suggested an association between H. pylori and metabolic-disfunction associated fatty liver disease (MASLD). However, epidemiologic studies have yielded inconsistent results. We aim to evaluate the association of H. pylori and G-allele PNPLA3 in MASLD diagnosis, and markers of severity. METHODS: A multi-center cross-sectional study was conducted. A total 224 functional dyspepsia (FD) patients cohort who underwent gastroscopy was selected. Biochemical, clinical parameters, ultrasound, FIB-4 score, LSM by VCTE, gastric biopsies, H. pylori status, and rs738409 PNPLA3 were evaluated. A second retrospective cohort of 86 patients with biopsy-proven MASLD who underwent gastroscopy with gastric biopsies was analyzed. RESULTS: In the FD cohort MASLD was observed in 52%, and H. pylori-positive in 51%. H. pylori infection was associated with MASLD prevalence, but in multivariate analyses adjusted for G-allele PNPLA3, it became not significant. Then in MASLD-only dyspeptic cohort, H. pylori infection was significantly linked to elevated serum AST levels and increased liver stiffness measurements, suggesting a potential role in liver injury and fibrosis. Histopathological analysis in biopsy-proven MASLD patients further supported these findings, showing a significant association between H. pylori infection and increased NAS score, fibrosis stage, and prevalence of MASH. Notably, the combination of H. pylori infection and G-allele PNPLA3 appeared to exacerbate MASLD severity beyond individual effects. CONCLUSIONS: Our results suggest that H. pylori infection may play a role in the progression of liver injury and fibrosis in patients with MASLD, especially in those with specific genetic predispositions.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Lipase , Proteínas de Membrana , Humanos , Masculino , Feminino , Lipase/genética , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Infecções por Helicobacter/complicações , Infecções por Helicobacter/genética , Adulto , Estudos Transversais , Estudos Retrospectivos , Fígado Gorduroso/genética , Fígado Gorduroso/complicações , Alelos , Polimorfismo de Nucleotídeo Único , Dispepsia/microbiologia , Dispepsia/genética , Dispepsia/complicações , Fígado/patologia , Fígado/metabolismo , Aciltransferases , Fosfolipases A2 Independentes de CálcioRESUMO
Sirtuin 1 (SIRT1) is a key upstream regulator of lipid metabolism; however, the molecular mechanisms by which SIRT1 regulates milk fat synthesis in dairy goats remain unclear. This study aimed to investigate the regulatory roles of SIRT1 in modulating lipid metabolism in goat mammary epithelial cells (GMECs) and its impact on the adipose triglyceride lipase (ATGL) promoter activity using RNA interference (RNAi) and gene overexpression techniques. The results showed that SIRT1 is significantly upregulated during lactation compared to the dry period. Additionally, SIRT1 knockdown notably increased the expressions of genes related to fatty acid synthesis (SREBP1, SCD1, FASN, ELOVL6), triacylglycerol (TAG) production (DGAT2, AGPAT6), and lipid droplet formation (PLIN2). Consistent with the transcriptional changes, SIRT1 knockdown significantly increased the intracellular contents of TAG and cholesterol and the lipid droplet abundance in the GMECs, while SIRT1 overexpression had the opposite effects. Furthermore, the co-overexpression of SIRT1 and Forkhead box protein O1 (FOXO1) led to a more pronounced increase in ATGL promoter activity, and the ability of SIRT1 to enhance ATGL promoter activity was nearly abolished when the FOXO1 binding sites (FKH1 and FKH2) were mutated, indicating that SIRT1 enhances the transcriptional activity of ATGL via the FKH element in the ATGL promoter. Collectively, our data reveal that SIRT1 enhances the transcriptional activity of ATGL through the FOXO1 binding sites located in the ATGL promoter, thereby regulating lipid metabolism. These findings provide novel insights into the role of SIRT1 in fatty acid metabolism in dairy goats.
Assuntos
Células Epiteliais , Ácidos Graxos , Proteína Forkhead Box O1 , Cabras , Lipase , Glândulas Mamárias Animais , Regiões Promotoras Genéticas , Sirtuína 1 , Animais , Sirtuína 1/metabolismo , Sirtuína 1/genética , Lipase/metabolismo , Lipase/genética , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/citologia , Feminino , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Metabolismo dos Lipídeos , Lactação , Triglicerídeos/metabolismo , Triglicerídeos/biossíntese , Regulação da Expressão GênicaRESUMO
Arecoline (ACL), an active constituent derived from Areca catechu L., exerts various pharmacological effects and serves as a potential plant-based insecticide. However, the effects of ACL on Spodoptera litura, an important and widely distributed agricultural pest, remain unknown. This study aimed to elucidate the mechanism underlying ACL-induced toxicity and its inhibitory effects on larval growth and development through intestinal pathology observations, intestinal transcriptome sequencing, intestinal digestive enzyme activity analysis. The results indicated that ACL exposure leads to pathological alterations in the S. litura midgut. Furthermore, the detection of digestive enzyme activity revealed that ACL inhibits the activities of acetyl CoA carboxylase, lipase, α-amylase, and trypsin. Simultaneously, upregulation of superoxide dismutase activity and downregulation of malondialdehyde levels were observed after ACL exposure. Transcriptome analysis identified 1118 genes that were significantly differentially expressed in the midgut after ACL exposure, potentially related to ACL toxic effects. Notably, ACL treatment downregulated key enzymes involved in lipid metabolism, such as fatty acid binding protein 2-like, pancreatic triacylglycerol lipase-like, pancreatic lipid-related protein 2-like, and fatty acid binding protein 1-like. Taken together, these results suggest that ACL induces midgut damage and impedes larval growth by suppressing digestive enzyme activity in the intestine. These findings can aid in the development of environmentally friendly plant-derived insecticides, utilizing ACL to effectively combat S. litura proliferation.
Assuntos
Intestinos , Larva , Spodoptera , Animais , Spodoptera/efeitos dos fármacos , Spodoptera/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Inseticidas/toxicidade , Inseticidas/farmacologia , Lipase/metabolismo , Lipase/genéticaRESUMO
Lung parenchyma destruction represents a severe condition commonly found in chronic obstructive pulmonary disease (COPD), a leading cause of morbidity and mortality worldwide. Promoting lung regeneration is crucial for achieving clinical improvement. However, no therapeutic drugs are approved to improve the regeneration capacity due to incomplete understanding of the underlying pathogenic mechanisms. Here, we identify a positive feedback loop formed between adipose triglyceride lipase (ATGL)-mediated lipolysis and overexpression of CD36 specific to lung epithelial cells, contributing to disease progression. Genetic deletion of CD36 in lung epithelial cells and pharmacological inhibition of either ATGL or CD36 effectively reduce COPD pathogenesis and promote lung regeneration in mice. Mechanistically, disruption of the ATGL-CD36 loop rescues Z-DNA binding protein 1 (ZBP1)-induced cell necroptosis and restores WNT/ß-catenin signaling. Thus, we uncover a crosstalk between lipolysis and lung epithelial cells, suggesting the regenerative potential for therapeutic intervention by targeting the ATGL-CD36-ZBP1 axis in COPD.
Assuntos
Antígenos CD36 , Lipase , Lipólise , Pulmão , Necroptose , Doença Pulmonar Obstrutiva Crônica , Regeneração , Animais , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Antígenos CD36/metabolismo , Antígenos CD36/genética , Necroptose/genética , Regeneração/fisiologia , Camundongos , Lipase/metabolismo , Lipase/genética , Humanos , Pulmão/patologia , Pulmão/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Camundongos Endogâmicos C57BL , Masculino , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Via de Sinalização Wnt , Camundongos Knockout , AciltransferasesRESUMO
Many factors are associated with the development and progression of metabolic dysfunction-associated steatotic liver disease (MASLD); however, genetics and gut microbiota are representative factors. Recent studies have highlighted the link between host genes and the gut microbiota. Although there have been many studies on the separate effects of single nucleotide polymorphisms (SNPs) and gut bacteria on MASLD, few epidemiological studies have examined how SNPs and gut bacteria interact in the development and progression of MASLD. This study aimed to investigate the association between PNPLA3 rs738409, a representative MASLD-related SNP, and gut bacteria in MASLD using a cross-sectional study of the general population. The 526 participants (318 normal and 208 MASLD groups) were grouped into the PNPLA3 rs738409 SNP, CC, CG, and GG genotypes, and the differences in the gut microbiota were investigated in each group. The PNPLA3 rs738409 CC and CG genotypes were associated with decreased Blautia and Ruminococcaceae in the MASLD group. They were negatively correlated with controlled attenuation parameter levels, body mass index, serum blood glucose, and triglycerides. In contrast, there was no association between the normal and MASLD groups and the gut bacteria in the PNPLA3 rs738409, the GG genotype group. This finding implies that dietary interventions and probiotics may be more effective in preventing and treating MASLD in individuals with the PNPLA3 rs738409 CC and CG genotypes. In contrast, their efficacy may be limited in those with the GG genotype.
Assuntos
Microbioma Gastrointestinal , Lipase , Proteínas de Membrana , Polimorfismo de Nucleotídeo Único , Humanos , Microbioma Gastrointestinal/genética , Proteínas de Membrana/genética , Masculino , Feminino , Pessoa de Meia-Idade , Lipase/genética , Estudos Transversais , Adulto , Idoso , Fígado Gorduroso/genética , Fígado Gorduroso/microbiologia , Fígado Gorduroso/patologia , Genótipo , Aciltransferases , Fosfolipases A2 Independentes de CálcioRESUMO
Agropyron mongolicum Keng is a diploid perennial grass of triticeae in gramineae. It has strong drought resistance and developed roots that can effectively fix the soil and prevent soil erosion. GDSL lipase or esterases/lipase has a variety of functions, mainly focusing on plant abiotic stress response. In this study, a GDSL gene from A. mongolicum, designated as AmGDSL1, was successfully cloned and isolated. The subcellular localization of the AmGDSL1 gene (pCAMBIA1302-AmGDSL1-EGFP) results showed that the AmGDSL1 protein of A. mongolicum was only localized in the cytoplasm. When transferred into tobacco (Nicotiana benthamiana), the heterologous expression of AmGDSL1 led to enhanced drought tolerance. Under drought stress, AmGDSL1 overexpressing plants showed fewer wilting leaves, longer roots, and larger root surface area. These overexpression lines possessed higher superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and proline (PRO) activities. At the same time, the malondialdehyde (MDA) content was lower than that in wild-type (WT) tobacco. These findings shed light on the molecular mechanisms involved in the GDSL gene's role in drought resistance, contributing to the discovery and utilization of drought-resistant genes in A. mongolicum for enhancing crop drought resistance.
Assuntos
Agropyron , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Nicotiana , Proteínas de Plantas , Agropyron/genética , Agropyron/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Secas , Estresse Fisiológico/genética , Plantas Geneticamente Modificadas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Lipase/metabolismo , Lipase/genéticaRESUMO
Stearoyl-CoA desaturase (SCD) is a lipogenic enzyme that catalyzes formation of the first double bond in the carbon chain of saturated fatty acids. Four isoforms of SCD have been identified in mice, the most poorly characterized of which is SCD4, which is cardiac-specific. In the present study, we investigated the role of SCD4 in systemic and cardiac metabolism. We used WT and global SCD4 KO mice that were fed standard laboratory chow or a high-fat diet (HFD). SCD4 deficiency reduced body adiposity and decreased hyperinsulinemia and hypercholesterolemia in HFD-fed mice. The loss of SCD4 preserved heart morphology in the HFD condition. Lipid accumulation decreased in the myocardium in SCD4-deficient mice and in HL-1 cardiomyocytes with knocked out Scd4 expression. This was associated with an increase in the rate of lipolysis and, more specifically, adipose triglyceride lipase (ATGL) activity. Possible mechanisms of ATGL activation by SCD4 deficiency include lower protein levels of the ATGL inhibitor G0/G1 switch protein 2 and greater activation by protein kinase A under lipid overload conditions. Moreover, we observed higher intracellular Ca2+ levels in HL-1 cells with silenced Scd4 expression. This may explain the activation of protein kinase A in response to higher Ca2+ levels. Additionally, the loss of SCD4 inhibited mitochondrial enlargement, NADH overactivation, and reactive oxygen species overproduction in the heart in HFD-fed mice. In conclusion, SCD4 deficiency activated lipolysis, resulting in a reduction of cardiac steatosis, prevented the induction of left ventricular hypertrophy, and reduced reactive oxygen species levels in the heart in HFD-fed mice.
Assuntos
Dieta Hiperlipídica , Estearoil-CoA Dessaturase , Animais , Dieta Hiperlipídica/efeitos adversos , Camundongos , Estearoil-CoA Dessaturase/deficiência , Estearoil-CoA Dessaturase/metabolismo , Estearoil-CoA Dessaturase/genética , Camundongos Knockout , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Lipase/metabolismo , Lipase/deficiência , Lipase/genética , Remodelação Ventricular , Miocárdio/metabolismo , Miocárdio/patologia , Camundongos Endogâmicos C57BL , AciltransferasesRESUMO
BACKGROUND: Free fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study. RESULTS: This study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling. CONCLUSIONS: The differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.
Assuntos
Encéfalo , Ácidos Graxos não Esterificados , Homeostase , Animais , Encéfalo/metabolismo , Encéfalo/crescimento & desenvolvimento , Ácidos Graxos não Esterificados/metabolismo , Lipase/metabolismo , Lipase/genética , Mariposas/crescimento & desenvolvimento , Mariposas/fisiologia , Mariposas/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Hormônios Juvenis/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Metamorfose Biológica/fisiologia , Ecdisterona/metabolismoRESUMO
Lipid droplets (LDs) are organelles specialized in the storage of neutral lipids, cholesterol esters and triglycerides, thereby protecting cells from the toxicity of excess lipids while allowing for the mobilization of lipids in times of nutrient deprivation. Defects in LD function are associated with many diseases. S-acylation mediated by zDHHC acyltransferases modifies thousands of proteins, yet the physiological impact of this post-translational modification on individual proteins is poorly understood. Here, we show that zDHHC11 regulates LD catabolism by modifying adipose triacylglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, both in hepatocyte cultures and in mice. zDHHC11 S-acylates ATGL at cysteine 15. Preventing the S-acylation of ATGL renders it catalytically inactive despite proper localization. Overexpression of zDHHC11 reduces LD size, whereas its elimination enlarges LDs. Mutating ATGL cysteine 15 phenocopies zDHHC11 loss, causing LD accumulation, defective lipolysis and lipophagy. Our results reveal S-acylation as a mode of regulation of ATGL function and LD homoeostasis. Modulating this pathway may offer therapeutic potential for treating diseases linked to defective lipolysis, such as fatty liver disease.
Assuntos
Aciltransferases , Hepatócitos , Homeostase , Lipase , Gotículas Lipídicas , Lipólise , Gotículas Lipídicas/metabolismo , Animais , Hepatócitos/metabolismo , Camundongos , Acilação , Aciltransferases/metabolismo , Aciltransferases/genética , Lipase/metabolismo , Lipase/genética , Humanos , Metabolismo dos LipídeosRESUMO
Frataxin (FXN) is required for iron-sulfur cluster biogenesis, and its loss causes the early-onset neurodegenerative disease Friedreich ataxia (FRDA). Loss of FXN is a susceptibility factor in the development of diabetes, a common metabolic complication after myocardial hypertrophy in patients with FRDA. The underlying mechanism of FXN deficient-induced hyperglycemia in FRDA is, however, poorly understood. In this study, we confirmed that the FXN deficiency mouse model YG8R develops insulin resistance in elder individuals by disturbing lipid metabolic homeostasis in adipose tissues. Evaluation of lipolysis, lipogenesis, and fatty acid ß-oxidation showed that lipolysis is most severely affected in white adipose tissues. Consistently, FXN deficiency significantly decreased expression of lipolytic genes encoding adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) resulting in adipocyte enlargement and inflammation. Lipolysis induction by fasting or cold exposure remarkably upregulated FXN expression, though FXN deficiency lessened the competency of lipolysis compared with the control or wild type mice. Moreover, we found that the impairment of lipolysis was present at a young age, a few months earlier than hyperglycemia and insulin resistance. Forskolin, an activator of lipolysis, or pioglitazone, an agonist of PPARγ, improved insulin sensitivity in FXN-deficient adipocytes or mice. We uncovered the interplay between FXN expression and lipolysis and found that impairment of lipolysis, particularly the white adipocytes, is an early event, likely, as a primary cause for insulin resistance in FRDA patients at later age.
Assuntos
Adipócitos Brancos , Modelos Animais de Doenças , Frataxina , Ataxia de Friedreich , Resistência à Insulina , Proteínas de Ligação ao Ferro , Lipólise , Animais , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Camundongos , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Adipócitos Brancos/metabolismo , Adipócitos Brancos/patologia , Masculino , Lipase/metabolismo , Lipase/genética , HumanosRESUMO
Quorum sensing (QS) is a cellular communication mechanism in which bacteria secrete and recognize signaling molecules to regulate group behavior. Lipases provide energy for bacterial cell growth but it is unknown whether they influence nutrient-dependent QS by hydrolyzing substrate. A high-yield lipase-producing strain, Burkholderia pyrrocinia WZ10-3, was previously identified in our laboratory, but the composition of its crude enzymes was not elucidated. Here, we identified a key extracellular lipase, Lip1728, in WZ10-3, which accounts for 99 % of the extracellular lipase activity. Lip1728 prefers to hydrolyze triglycerides at sn-1,3 positions, with pNP-C16 being its optimal substrate. Lip1728 exhibited activity at pH 5.0-10.0 and regardless of the presence of metal ions. It had strong resistance to sodium dodecyl sulfate and short-chain alcohols and was activated by phenylmethanesulfonylfluoride (PMSF). Lip1728 knockout significantly affected lipid metabolism and biofilm formation in the presence of olive oil. Finally, oleic acid, a hydrolysate of Lip1728, influenced the production of the signal molecule N-acyl homoserine lactone (AHL) and biofilm formation by downregulating the AHL synthetase gene pyrI. In conclusion, Lip1728, as a key extracellular lipase in B. pyrrocinia WZ10-3, exhibits superior properties that make it suitable for biodiesel production and plays a crucial role in QS.
Assuntos
Burkholderia , Lipase , Percepção de Quorum , Lipase/metabolismo , Lipase/genética , Percepção de Quorum/genética , Burkholderia/genética , Burkholderia/enzimologia , Burkholderia/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Especificidade por Substrato , Metabolismo dos Lipídeos , Nutrientes/metabolismo , Acil-Butirolactonas/metabolismoRESUMO
The acidic lipase from Rasamsonia emersonii named LIPR has great potential for biodiesel synthesis due to its strong methanol tolerance. Nonetheless, the limited thermostability of LIPR and low expression level in Escherichia coli remain major obstacles to its use in biodiesel synthesis. To enhance the thermostability, the mutant LIPR harboring mutations A126C-P238C for the formation of a new disulfide bond and amino acid substitution D214L was obtained through rational design. To our delight, the thermostability of LIPR mutant was greatly improved. Moreover, a comprehensive optimization strategy, such as employing the Mss signal peptide, co-expressing the molecular chaperone protein disulfide isomerase (PDI), knocking out the vacuolar sorting receptor gene VPS10-01, and overexpressing the dihydroxyacetone synthase gene DAS2, was adopted to obtain the combination-optimized mutant Pichia pastoris strain GS54. Furthermore, the biodiesel synthetic capability with the mutant GS54-LIPR was verified and the production yield was 52.2 % after 24 h in a shake flask. Subsequently, a continuous flow system was adopted to increase the biodiesel yield to 73.6 % within 3 h, demonstrating its efficacy in enhancing enzyme biocatalysis. The engineered GS54-LIPR mutant lipase is an efficient and reusable biocatalyst for the sustained production of biodiesel in a continuous flow reaction.
Assuntos
Biocombustíveis , Estabilidade Enzimática , Lipase , Lipase/genética , Lipase/química , Lipase/metabolismo , Lipase/biossíntese , Reatores Biológicos , Saccharomycetales/genética , Saccharomycetales/enzimologia , Temperatura , Mutação , Expressão GênicaRESUMO
The improvement of enzyme thermostability often accompanies the decreased activity due to the loss of the key regions' flexibility. As a representative structure, unlocking the potential of loop dynamics will not only provide new ideas for stabilization strategies, but also help to deepen the understanding of the relationship between enzyme structural dynamics and function. In this study, a creative "hook loop dynamics engineering" (HLoD) strategy was successfully proposed for simultaneously improving the thermostability and maintaining activity of the model enzyme, Candida Antarctica lipase B. A small and smart mutant library involving five key residues located at the "hook loop" was meticulously identified and systematically investigated and thus yielded a five-point multiple mutant M1 (L147S/T244P/S250P/T256D/N292D), demonstrating a remarkable 7.0-fold increase in thermostability at 60 °C compared to the wild-type (WT). Furthermore, the activity of M1 remained comparable to that of WT, effectively transcending the barrier of activity-stability trade-off. Molecular dynamics simulations revealed that the precise regulation of hook loop dynamics via intermolecular interactions, such as salt bridges and hydrogen bonding, curbed the excessive flexibility of the pivotal regions α5 and α10 at high temperatures, thus driving the substantial enhancement of the thermostability of M1. Refining the dynamics of the flexible region via HLoD, which transcended the barrier of activity-stability trade-off, exhibited to be a robust and potentially universal strategy for designing enzymes with outstanding thermostability and activity.
Assuntos
Estabilidade Enzimática , Proteínas Fúngicas , Lipase , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Lipase/química , Lipase/genética , Lipase/metabolismo , Engenharia de Proteínas/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Temperatura , Mutação , Conformação ProteicaRESUMO
In adipose tissue, reduced expression of the glycerol channel aquaporin 7 (AQP7) has been associated with increased accumulation of triglyceride. The present study determines the relative protein abundances of lipolytic enzymes, AQP7, and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in paired mesenteric and omental visceral adipose tissue (VAT) and abdominal and femoral subcutaneous adipose tissue (SAT) in women with either normal weight or upper-body obesity. No differences in the expression of hormone-sensitive lipase (HSL) or AQP7 were found between the two groups in the four depots. The expression of adipocyte triglyceride lipase (ATGL) and HSL were higher in omental VAT and femoral SAT than in mesenteric VAT in both groups of women. Similarly, AQP7 expression was higher in omental VAT than in mesenteric VAT. The expression of PEPCK-C was lower in omental VAT than in femoral SAT. No correlation between the expression of AQP7 and the mean adipocyte size was observed; however, the expression of PEPCK-C positively correlated with the mean adipocyte size. In conclusion, a depot-specific protein expression pattern was found for ATGL, HSL, AQP7, and PEPCK-C. The expression pattern supports that the regulation of AQP7 protein expression is at least in part linked to the lipolytic rate. Furthermore, the results support that the synthesis of glycerol-3-phosphate via glyceroneogenesis contributes to regulating triglyceride accumulation in white adipose tissue in women.
Assuntos
Aquaporinas , Glicerol , Gordura Intra-Abdominal , Obesidade , Gordura Subcutânea , Humanos , Feminino , Gordura Subcutânea/metabolismo , Aquaporinas/metabolismo , Aquaporinas/genética , Glicerol/metabolismo , Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Adulto , Pessoa de Meia-Idade , Lipólise , Esterol Esterase/metabolismo , Esterol Esterase/genética , Lipase/metabolismo , Lipase/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Adipócitos/metabolismo , Triglicerídeos/metabolismo , AciltransferasesRESUMO
BACKGROUND: Neutral Lipid Storage Disease with Myopathy (NLSDM) is a rare lipid metabolism disorder caused by PNPLA2 gene mutations. Clinical manifestations are heterogeneous, and diagnosis is often delayed, usually gaining patients' attention due to the increased risk of cardiomyopathy. CASE PRESENTATION: We herein report a 36-year-old Asian male presenting with progressive limb weakness, muscle atrophy of limbs and trunk, dysarthria, and heart failure. Electromyography indicated myogenic changes, and muscle biopsy results revealed characteristics of lipid storage myopathy. Genetic analysis of PNPLA2 revealed two heterozygous mutations: c.757 + 1G > T (chr11-823588, splice-5) on intron 6 and c.919delG (chr11-823854, p.A307Pfs*13) on exon 7. The patient improved limb strength, and dysarthria disappeared after the Medium Chain Fatty Acids diet. CONCLUSIONS: In conclusion, we report for the first time that the two heterozygous mutations PNPLA2 c.919delG and c.757 + 1G > T together induced NLSDM, which was confirmed by muscle biopsy.
Assuntos
Heterozigoto , Lipase , Erros Inatos do Metabolismo Lipídico , Doenças Musculares , Mutação , Humanos , Masculino , Lipase/genética , Adulto , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Doenças Musculares/genética , Doenças Musculares/diagnóstico , Músculo Esquelético/patologia , AciltransferasesRESUMO
Chronic stress-induced epinephrine (EPI) accelerates breast cancer progression and metastasis, but the molecular mechanisms remain unclear. Herein, we found a strong positive correlation between circulating EPI levels and the tumoral expression of ubiquitin-specific peptidase 22 (USP22) in patients with breast cancer. USP22 facilitated EPI-induced breast cancer progression and metastasis by enhancing adipose triglyceride lipase (ATGL)-mediated lipolysis. Targeted USP22 deletion decreased ATGL expression and lipolysis, subsequently inhibiting EPI-mediated breast cancer lung metastasis. USP22 acts as a bona fide deubiquitinase for the Atgl gene transcription factor FOXO1, and EPI architects a lipolysis signaling pathway to stabilize USP22 through AKT-mediated phosphorylation. Notably, USP22 phosphorylation levels are positively associated with EPI and with downstream pathways involving both FOXO1 and ATGL in breast cancers. Pharmacological USP22 inhibition synergized with ß-blockers in treating preclinical xenograft breast cancer models. This study reveals a molecular pathway behind EPI's tumor-promoting effects and provides a strong rationale for combining USP22 inhibition with ß-blockers to treat aggressive breast cancer.
Assuntos
Neoplasias da Mama , Epinefrina , Lipólise , Ubiquitina Tiolesterase , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Lipólise/efeitos dos fármacos , Feminino , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Epinefrina/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Lipase/metabolismo , Lipase/genética , Transdução de Sinais/efeitos dos fármacos , Metástase Neoplásica , Fosforilação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , AciltransferasesRESUMO
Developing reusable and easy-to-operate biocatalysts is of significant interest in biodiesel production. Here, magnetic whole-cell catalysts constructed through immobilizing recombinant Escherichia coli cells (containing MAS1 lipase) into Fe3O4-chitosan magnetic microspheres (termed MWCC@MAS1) were used for fatty acid methyl ester (FAME) production from waste cooking oil (WCO). During the preparation process of immobilized cells, the effects of chitosan concentration and cell concentration on their activity and activity recovery were investigated. Optimal immobilization was achieved with 3% (w/v) chitosan solution and 10 mg wet cell/mL cell suspension. Magnetic immobilization endowed the whole-cell catalysts with superparamagnetism and improved their methanol tolerance, enhancing the recyclability of the biocatalysts. Additionally, we studied the effects of catalyst loading, water content, methanol content, and reaction temperature on FAME yield, optimizing these parameters using response surface methodology and Box-Behnken design. An experimental FAME yield of 89.19% was gained under the optimized conditions (3.9 wt% catalyst loading, 22.3% (v/w) water content, 23.0% (v/w) methanol content, and 32 °C) for 48 h. MWCC@MAS1 demonstrated superior recyclability compared to its whole-cell form, maintaining about 86% of its initial productivity after 10 cycles, whereas the whole-cell form lost nearly half after just five cycles. These results suggest that MWCC@MAS1 has great potential for the industrial production of biodiesel.
Assuntos
Biocombustíveis , Quitosana , Escherichia coli , Microesferas , Escherichia coli/genética , Escherichia coli/metabolismo , Quitosana/química , Células Imobilizadas/metabolismo , Óleos de Plantas/química , Lipase/metabolismo , Lipase/genética , Metanol/química , CulináriaRESUMO
Lipase with unique regioselectivity is an attractive biocatalyst for elaborate lipid modification. However, the excavation of novel sn-2 regioselective lipases is difficult due to their scarcity in nature, with Candida antarctica lipase A (CALA) being the pronouncedly reported one. Here, we identified a novel CALA-like lipase from Cordyceps militaris (CACML7) via in silico mining. Through chiral-phase high-performance liquid chromatography, we determined that CACML7 displays sn-2 regioselectivity (>68 %) as does CALA, but exhibits distinctive chain length selectivity and bias against unsaturated fats. Notably, the curvature of the acyl-binding tunnel was expected to contribute to the 2.2-fold higher preference for cis-fatty acid (C18:1, cis-Δ9) over trans-fatty acid (C18:1, trans-Δ9) unlike trans-active CALA. Random pose docking of trioleoylglycerol (TOG) into the active site of a lid-truncated mutant of CACML7 revealed that TOG accepts a tuning fork conformation, of which the precise positioning of the reactive ester group towards the catalytic center was only favorable via sn-2 binding mode. The unique active site morphology, which we refer to as an "acyl-binding tunnel with a narrow entrance," may contribute to the sn-2 regioselectivity of CACML7. Our data provide an attractive model to better understand the mechanism underlying sn-2 regioselectivity.
Assuntos
Cordyceps , Ácidos Graxos , Lipase , Lipase/química , Lipase/metabolismo , Lipase/genética , Cordyceps/enzimologia , Cordyceps/química , Cordyceps/metabolismo , Especificidade por Substrato , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Estereoisomerismo , Simulação de Acoplamento Molecular , Domínio Catalítico , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMO
The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3-/- and PNPLA3I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells.