Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

Single-cell RNA sequencing of preadipocytes reveals the cell fate heterogeneity induced by melatonin.

Obesity is a global epidemic health disorder and associated with several diseases. Body weight-reducing effects of melatonin have been reported; however, no investigation toward examining whether the beneficial effects of melatonin are associated with preadipocyte heterogeneity has been reported. In this study, we profiled 25 071 transcriptomes of normal and melatonin-treated preadipocytes using scRNA-seq. By tSNE analysis, we present a cellular-state landscape for melatonin-treated preadipocytes that covers multiple-cell subpopulations, defined as cluster 0 to cluster 13. Cluster 0 and cluster 1 were the largest components of normal and melatonin-treated preadipocytes, respectively. G0S2, an inhibitor of adipose triglyceride lipase (ATGL), was significantly upregulated in cluster 0 and downregulated in cluster 1. We redefined cluster 0 as the G0S2-positive cluster (G0S2+ ) and cluster 1 as the G0S2-negative cluster (G0S2- ). Through pseudotime analysis, the G0S2- cluster cell differentiation trajectory was divided into three major structures, that is, the prebranch, the lipid catabolism branch, and the cell fate 2 branch. In vitro, G0S2 knockdown enhanced the expression levels of ATGL, BAT markers and fatty acid oxidation-related genes, but inhibited C/EBPα and PPARγ expression. In vivo, knockdown of G0S2 reduced the body weight gain in high-fat-fed mice. The beneficial effects of the G0S2- cell cluster in promoting lipolysis and inhibiting adipogenesis are dependent on two major aspects: first, downregulation of the G0S2 gene in the G0S2- cluster, resulting in activation of ATGL, which is responsible for the bulk of triacylglycerol hydrolase activity; and second, upregulation of FABP4 in the G0S2- cluster, resulting in inhibition of PPARγ and further reducing adipogenesis.

Genetic Programs Driving Oncogenic Transformation: Lessons from in Vitro Models.

Cancer complexity relies on the intracellular pleiotropy of oncogenes/tumor suppressors and in the strong interplay between tumors and micro- and macro-environments. Here we followed a reductionist approach, by analyzing the transcriptional adaptations induced by three oncogenes (RAS, MYC, and HDAC4) in an isogenic transformation process. Common pathways, in place of common genes became dysregulated. From our analysis it emerges that, during the process of transformation, tumor cells cultured in vitro prime some signaling pathways suitable for coping with the blood supply restriction, metabolic adaptations, infiltration of immune cells, and for acquiring the morphological plasticity needed during the metastatic phase. Finally, we identified two signatures of genes commonly regulated by the three oncogenes that successfully predict the outcome of patients affected by different cancer types. These results emphasize that, in spite of the heterogeneous mutational burden among different cancers and even within the same tumor, some common hubs do exist. Their location, at the intersection of the various signaling pathways, makes a therapeutic approach exploitable.

Assessment of the Main Compounds of the Lipolytic System in Treadmill Running Rats: Different Response Patterns between the Right and Left Ventricle.

The aim of the present study was to investigate the time and intensity dependent effects of exercise on the heart components of the lipolytic complex. Wistar rats ran on a treadmill with the speed of 18 m/min for 30 min (M30) or 120 min (M120) or with the speed of 28 m/min for 30 min (F30). The mRNA and protein expressions of the compounds adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58), G0/G1 switch gene 2 (G0S2), hormone sensitive lipase (HSL) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were examined by real-time PCR and Western blot, respectively. Lipid content of free fatty acids (FFA), diacylglycerols (DG) and triacylglycerols (TG) were estimated by gas liquid chromatography. We observed virtually no changes in the left ventricle lipid contents and only minor fluctuations in its ATGL mRNA levels. This was in contrast with its right counterpart i.e., the content of TG and DG decreased in response to both increased duration and intensity of a run. This occurred in tandem with increased mRNA expression for ATGL, CGI-58 and decreased expression of G0S2. It is concluded that exercise affects behavior of the components of the lipolytic system and the lipid content in the heart ventricles. However, changes observed in the left ventricle did not mirror those in the right one.

G0S2: A small giant controller of lipolysis and adipose-liver fatty acid flux.

The discovery of adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) provided a major paradigm shift in the understanding of intracellular lipolysis in both adipocytes and nonadipocyte cells. The subsequent discovery of G0/G1 switch gene 2 (G0S2) as a potent endogenous inhibitor of ATGL revealed a unique mechanism governing lipolysis and fatty acid (FA) availability. G0S2 is highly conserved in vertebrates, and exhibits cyclical expression pattern between adipose tissue and liver that is critical to lipid flux and energy homeostasis in these two tissues. Biochemical and cell biological studies have demonstrated that a direct interaction with ATGL mediates G0S2's inhibitory effects on lipolysis and lipid droplet degradation. In this review we examine evidence obtained from recent in vitro and in vivo studies that lends support to the proof-of-principle concept that G0S2 functions as a master regulator of tissue-specific balance of TG storage vs. mobilization, partitioning of metabolic fuels between adipose and liver, and the whole-body adaptive energy response. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

G0/G1 Switch Gene 2 Regulates Cardiac Lipolysis.

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids to prevent lipotoxicity, whereas TAG hydrolysis (lipolysis) remobilizes fatty acids from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G0/G1 switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild-type and mutant mice, we show the following: (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2 overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.

Increases in skeletal muscle ATGL and its inhibitor G0S2 following 8 weeks of endurance training in metabolically different rat skeletal muscles.

Adipose triglyceride lipase (ATGL) catalyzes the rate-limiting removal of the first fatty acid from a triglyceride. ATGL is activated by comparative gene identification-58 and inhibited by G(0)/G(1) switch gene-2 protein (G0S2). Research in other tissues and cell culture indicates that inhibition is dependent on relative G0S2-to-ATGL protein content. G0S2 may also have several roles within mitochondria; however, this has yet to be observed in skeletal muscle. The purpose of this study was to determine if muscle G0S2 relative to ATGL content would decrease to facilitate intramuscular lipolysis following endurance training. Male Sprague-Dawley rats (n = 10; age 51-53 days old) were progressively treadmill trained at a 10% incline for 8 wk ending with 25 m/min for 1 h compared with control. Sciatic nerve stimulation for hind-limb muscle contraction (and lipolysis) was administered for 30 min to one leg, leaving the opposing leg as a resting control. Soleus (SOL), red gastrocnemius (RG), and white gastrocnemius were excised from both legs following stimulation or control. ATGL protein increased in all trained muscles. Unexpectedly, G0S2 protein was greater in the trained SOL and RG. In RG-isolated mitochondria, G0S2 also increased with training, yet mitochondrial G0S2 content was unaltered with acute contraction; therefore, any role of G0S2 in the mitochondria does not appear to be acutely mediated by content alone. In summary, G0S2 increased with training in oxidative muscles and mitochondria but not following acute contraction, suggesting that inhibition is not through relative G0S2-to-ATGL content but through more complicated intracellular mechanisms.

Upregulation of Pnpla2 and Abhd5 and downregulation of G0s2 gene expression in mesenteric white adipose tissue as a potential reason for elevated concentration of circulating NEFA after removal of retroperitoneal, epididymal, and inguinal adipose tissue.

Elevated concentrations of circulating non-esterified fatty acids (NEFA) were reported in (a) humans with lipodystrophy, (b) humans following bariatric surgery, and (c) transgenic mice with reduced amounts of adipose tissue. Paradoxically, these findings suggest that the reduction of adipose tissue mass is associated with elevated circulating NEFA concentrations. To explain a molecular background of this phenomenon, we analyzed the effects of surgical removal of inguinal, epididymal, and retroperitoneal white adipose tissue (WAT) on (a) circulating NEFA concentrations, (b) expression of Pnpla2, a gene that encodes adipose triglyceride lipase (ATGL), genes encoding abhydrolase domain containing 5 (ABHD5) and G0/G1 switch 2 (G0S2), i.e., a coactivator and inhibitor of ATGL, respectively, and (c) expression of Lipe gene coding hormone-sensitive lipase (HSL) in mesenteric WAT. Reduction of adipose tissue mass resulted in an increase in circulating NEFA concentration, which was associated with (a) an increase in the expressions of Pnpla2 and Abhd5, (b) decrease in G0s2 expression, and (c) upregulation of Lipe expression, all measured on both mRNA and protein levels in mesenteric WAT of male rats. The rate of lipolysis in mesenteric WAT explants and isolated adipocytes from lipectomized rats was significantly higher than that from the controls. In conclusion, upregulation of Pnpla2 expression and activation of ATGL (due to an increase in ABHD5 and decrease in G0S2 levels), as well as a coordinated interplay of these genes with Lipe in mesenteric WAT, contribute, at least in part, to an increase in the concentration of circulating NEFA in rats with reduced fat mass.

Lipolytic inhibitor G0/G1 switch gene 2 inhibits reactive oxygen species production and apoptosis in endothelial cells.

G0/G1 switch gene 2 (G0S2), a novel target gene of peroxisome proliferator-activated receptor, is highly expressed in fat tissues. G0S2 acts as proapoptotic factor toward human cancer cells. Endothelial cell (EC) apoptosis may be an initiating event in the development of atherosclerosis. However, the expression and function of G0S2 in vascular ECs remain unknown. Here, we reported for the first time that G0S2 is expressed in arterial ECs. Ectopic expression of G0S2 increased neutral lipid accumulation in cultured ECs. However, G0S2 prevented ECs from serum-free starvation stress- and hydrogen peroxide (H2O2)-induced apoptosis. G0S2 blocked the H2O2-induced dissipation of mitochondrial membrane potential. G0S2 decreased the release of cytochrome c from mitochondria into the cytosol, followed by activation of caspase-9 and caspase-3. The anti-apoptotic effect of G0S2 was Bcl-2 and adipose triglyceride lipase independent. In contrast, gene silence of G0S2 increased serum-free starvation stress-induced EC apoptosis and decreased the formation of capillary-like structures. We further found that G0S2 couples with the F0F1-ATP synthase in ECs. Levels of ATP were elevated, whereas reactive oxygen species levels were reduced in G0S2-expressing ECs. G0S2 can inhibit endothelial denudation secondary to H2O2-induced injury to ECs in vivo. These results indicate that G0S2 acts as a prosurvival molecule in ECs. Taken together, our results indicate that G0S2 has a protective function in ECs and may be a potential target for the treatment of cardiovascular diseases associated with reactive oxygen species-induced EC injury, such as atherosclerosis and restenosis.

Defective adipose lipolysis and altered global energy metabolism in mice with adipose overexpression of the lipolytic inhibitor G0/G1 switch gene 2 (G0S2).

Biochemical and cell-based studies have identified the G0S2 (G0/G1 switch gene 2) as a selective inhibitor of the key intracellular triacylglycerol hydrolase, adipose triglyceride lipase. To better understand the physiological role of G0S2, we constructed an adipose tissue-specific G0S2 transgenic mouse model. In comparison with wild type animals, the transgenic mice exhibited a significant increase in overall fat mass and a decrease in peripheral triglyceride accumulation. Basal and adrenergically stimulated lipolysis was attenuated in adipose explants isolated from the transgenic mice. Following fasting or injection of a ß3-adrenergic agonist, in vivo lipolysis and ketogenesis were decreased in G0S2 transgenic mice when compared with wild type animals. Consequently, adipose overexpression of G0S2 prevented the "switch" of energy substrate from carbohydrates to fatty acids during fasting. Moreover, G0S2 overexpression promoted accumulation of more and larger lipid droplets in brown adipocytes without impacting either mitochondrial morphology or expression of oxidative genes. This phenotypic change was accompanied by defective cold adaptation. Furthermore, feeding with a high fat diet caused a greater gain of both body weight and adiposity in the transgenic mice. The transgenic mice also displayed a decrease in fasting plasma levels of free fatty acid, triglyceride, and insulin as well as improved glucose and insulin tolerance. Cumulatively, these results indicate that fat-specific G0S2 overexpression uncouples adiposity from insulin sensitivity and overall metabolic health through inhibiting adipose lipolysis and decreasing circulating fatty acids.

A peptide derived from G0/G1 switch gene 2 acts as noncompetitive inhibitor of adipose triglyceride lipase.

The protein G0/G1 switch gene 2 (G0S2) is a small basic protein that functions as an endogenous inhibitor of adipose triglyceride lipase (ATGL), a key enzyme in intracellular lipolysis. In this study, we identified a short sequence covering residues Lys-20 to Ala-52 in G0S2 that is still fully capable of inhibiting mouse and human ATGL. We found that a synthetic peptide corresponding to this region inhibits ATGL in a noncompetitive manner in the nanomolar range. This peptide is highly selective for ATGL and does not inhibit other lipases, including hormone-sensitive lipase, monoacylglycerol lipase, lipoprotein lipase, and patatin domain-containing phospholipases 6 and 7. Because increased lipolysis is linked to the development of metabolic disorders, the inhibition of ATGL by G0S2-derived peptides may represent a novel therapeutic tool to modulate lipolysis.

TNF-α reduces g0s2 expression and stimulates lipolysis through PPAR-γ inhibition in 3T3-L1 adipocytes.

Tumor necrosis factor-α (TNF-α) is a multifunctional cytokine that acts as a mediator of obesity-linked insulin resistance (IR). It is commonly accepted that macrophage-derived TNF-α acts in a paracrine manner on adjacent adipocytes, induces lipolysis, which contributes to obesity-linked hyperglycemia. Several studies suggested that G0/G1 switch gene 2 (g0s2) was up-regulated during adipogenesis, and its protein could be degraded in response to TNF-α stimulation. The aim of the present work was to investigate the transcriptional regulation of g0s2 by TNF-α stimulation. In this study, 3T3-L1 pre-adipocytes were differentiated, and treated with TNF-α for 24h. The effects of TNF-α on lipolysis and lipase expression were then examined. Our results revealed that TNF-α exerted dose- and time-dependent lipolytic effects, which could be partially reversed by overexpression of g0s2 and peroxisome proliferator-activated receptor-γ (ppar-γ). In addition, TNF-α treatment significantly reduced the expression of adiponectin, ppar-γ, hormone-sensitive Lipase (hsl), adipose triglyceride lipase (atgl) as well as ATGL co-factors. Interestingly, TNF-α significantly decreased adiponectin and PPAR-γ protein levels, while treatment with the proteasomal inhibitor MG-132 maintained PPAR-γ levels. Degradation of PPAR-γ almost completely abolished the binding of PPAR-γ to the g0s2 promoter in adipocytes treated with TNF-α. We propose that proteasomal degradation of PPAR-γ and the reduction of g0s2 content are permissive for prolonged TNF-α induced lipolysis.

G0S2 promotes antiestrogenic and pro-migratory responses in ER+ and ER- breast cancer cells.

G0/G1 switch gene 2 (G0S2) is known to inhibit lipolysis by inhibiting adipose triglyceride lipase (ATGL). In this report, we dissect the role of G0S2 in ER+ versus ER- breast cancer. Overexpression of G0S2 in ER- cells increased cell proliferation, while G0S2 overexpression in ER+ cells decreased cell proliferation. Transcriptome analysis revealed that G0S2 mediated distinct but overlapping transcriptional responses in ER- and ER+ cells. G0S2 reduced genes associated with an epithelial phenotype, especially in ER- cells, including CDH1, ELF3, STEAP4 and TACSTD2, suggesting promotion of the epithelial-mesenchymal transition (EMT). G0S2 also repressed estrogen signaling and estrogen receptor target gene signatures, especially in ER+ cells, including TFF1 and TFF3. In addition, G0S2 overexpression increased cell migration in ER- cells and increased estrogen deprivation sensitivity in ER+ cells. Interestingly, two genes downstream of ATGL in fat utilization and very important in steroid hormone biosynthesis, HMGCS1 and HMGCS2, were downregulated in G0S2 overexpressing ER+ cells. In addition, HSD17B11, a gene that converts estradiol to its less estrogenic derivative, estrone, was highly upregulated in G0S2 overexpressing ER+ cells, suggesting G0S2 overexpression has a negative effect on estradiol production and maintenance. High expression of G0S2 and HSD17B11 was associated with improved relapse-free survival in breast cancer patients while high expression of HMGSC1 was associated with poor survival. Finally, we deleted G0S2 in breast cancer-prone MMTV-PyMT mice. Our data indicates a complex role for G0S2 in breast cancer, dependent on ER status, that may be partially mediated by suppression of the estrogen signaling pathway.

Gastric Cancer Growth Modulated by circSNTB2/miR-6938-5p/G0S2 and PDCD4.

BACKGROUND: Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Increasing studies have indicated that circular RNAs (circRNAs) play critical roles in cancer progression. However, the precise mechanism and functions of most circRNAs are still unknown in gastric cancer. METHODS: In the present study, we aim to uncover the mechanism by which circRNAs regulate gastric cancer tumorigenesis. By analyzing the microarray data, we screened differential expressed circRNAs in the gastric cancer group and identified a down-regulated circRNA, hsa_circ_0040039 (circSNTB2). Mechanically, circSNTB2 served as a sponge for the miR-6938-5p and up-regulated its expression. RESULTS: Meanwhile, G0/G1 switch gene 2 (G0S2) and programmed cell death gene 4 (PDCD4) were identified to be the aim genes of miR-6938-5p, constructing circSNTB2/miR-6938-5p/G0S2 and PDCD4 pathways. CONCLUSION: Taken together, our findings demonstrated that circSNTB2 plays an essential role in gastric cancer by regulating miR-6938-5p through G0S2 and PDCD4 genes. CircSNTB2 could be a promising biomarker for GC diagnosis and targeted therapy.

The effect of G0S2 on insulin sensitivity: A proteomic analysis in a G0S2-overexpressed high-fat diet mouse model.

Background: Previous research has shown a tight relationship between the G0/G1 switch gene 2 (G0S2) and metabolic diseases such as non-alcoholic fatty liver disease (NAFLD) and obesity and diabetes, and insulin resistance has been shown as the major risk factor for both NAFLD and T2DM. However, the mechanisms underlying the relationship between G0S2 and insulin resistance remain incompletely understood. Our study aimed to confirm the effect of G0S2 on insulin resistance, and determine whether the insulin resistance in mice fed a high-fat diet (HFD) results from G0S2 elevation. Methods: In this study, we extracted livers from mice that consumed HFD and received tail vein injections of AD-G0S2/Ad-LacZ, and performed a proteomics analysis. Results: Proteomic analysis revealed that there was a total of 125 differentially expressed proteins (DEPs) (56 increased and 69 decreased proteins) among the identified 3583 proteins. Functional enrichment analysis revealed that four insulin signaling pathway-associated proteins were significantly upregulated and five insulin signaling pathway -associated proteins were significantly downregulated. Conclusion: These findings show that the DEPs, which were associated with insulin resistance, are generally consistent with enhanced insulin resistance in G0S2 overexpression mice. Collectively, this study demonstrates that G0S2 may be a potential target gene for the treatment of obesity, NAFLD, and diabetes.

Acidosis-induced regulation of adipocyte G0S2 promotes crosstalk between adipocytes and breast cancer cells as well as tumor progression.

Bidirectional interactions between cancer cells and their microenvironment govern tumor progression. Among the stromal cells in this microenvironment, adipocytes have been reported to upregulate cancer cell migration and invasion by producing fatty acids. Conversely, cancer cells alter adipocyte phenotype notably via increased lipolysis. We aimed to identify the mechanisms through which cancer cells trigger adipocyte lipolysis and evaluate the functional consequences on cancer progression. Here, we show that cancer cell-induced acidification of the extracellular medium strongly promotes preadipocyte lipolysis through a mechanism that does not involve lipophagy but requires adipose triglyceride lipase (ATGL) activity. This increased lipolysis is triggered mainly by attenuation of the G0/G1 switch gene 2 (G0S2)-induced inhibition of ATGL. G0S2-mediated regulation in preadipocytes affects their communication with breast cancer cells, modifying the phenotype of the cancer cells and increasing their resistance to chemotherapeutic agents in vitro. Furthermore, we demonstrate that the adipocyte-specific overexpression of G0S2 impairs mammary tumor growth and lung metastasis formation in vivo. Our results highlight the importance of acidosis in cancer cell-adipocyte crosstalk and identify G0S2 as the main regulator of cancer-induced lipolysis, regulating tumor establishment and spreading.

Residues of the minimal sequence of G0S2 collectively contribute to ATGL inhibition while C-and N-terminal extensions promote binding to ATGL.

The protein encoded by the G0/G1 switch gene 2 (G0S2) is a potent inhibitor of adipose triglyceride lipase (ATGL) and thus an important regulator of intracellular lipolysis. Since dysfunction of lipolysis is associated with metabolic diseases including diabetes and obesity, inhibition of ATGL is considered a therapeutic strategy. G0S2 interacts with ATGL's patatin-domain to mediate non-competitive inhibition, however atomic details of the inhibition mechanism are incompletely understood. Sequences of G0S2 from higher organisms show a highly conserved N-terminal part, including a hydrophobic region covering amino acids 27 to 42. We show that predicted G0S2 orthologs from platypus, chicken and Japanese rice-fish are able to inhibit human and mouse ATGL, emphasizing the contribution of conserved amino acid to ATGL inhibition. Our site directed mutagenesis and truncation studies give insights in the protein-protein interaction on a per-residue level. We determine that the minimal sequence required for ATGL inhibition ranges from amino acids 20 to 44. Residues Y27, V28, G30, A34 G37, V39 or L42 within this sequence play a substantial role in ATGL inhibition. Furthermore, we show that unspecific interactions of the N-terminal part (amino acids 20-27) of the minimal sequence facilitate the interaction to ATGL. Our studies also demonstrate that full-length G0S2 shows higher tolerance to specific single amino acid exchanges in the hydrophobic region due to the stronger contributions of unspecific interactions. However, exchanges of more than one amino-acid in the hydrophobic region also result in the loss of function as ATGL inhibitor even in the full-length protein.

G0S2 Gene Polymorphism and Its Relationship with Carcass Traits in Chicken.

Gene single nucleotide polymorphisms can be used as auxiliary markers in molecular breeding and are an effective method to improve production performance. G0S2 is a key gene involved in regulating fat metabolism, but little research has been conducted on this gene regarding its role in poultry. In this study, the specialized commercial partridge chicken strain G0S2 gene was cloned and sequenced, and the relationship between the SNP sites on G0S2 and the carcass traits of chickens was investigated. The results showed that a total of seven SNPs were detected on G0S2 (g.102G > A, g.255G > A, g.349C > T, g.384A > G, g.386G > A, g.444G > A, g.556G > A). Two sites are located in the coding region and five sites are located in the 3'-UTR. SNPs located in the coding region are synonymous mutations. g.444G > A has a significant correlation with abdominal fat weight. The chickens with AG and GG genotypes have the highest abdominal fat weight, while the AA genotype is lower. The g.102G > A genotype has a significant correlation with live and abdominal fat weight. The live weight and abdominal fat weight of the chickens with AA and AG genotypes are at a higher level and have a larger gap than the GG genotype. Chickens with the AA genotype in g.556G > A had the lowest fat weight. The results of present study can provide practical information for molecular marker-assisted breeding of chicken carcass traits.

G0S2 ameliorates oxidized low-density lipoprotein-induced vascular endothelial cell injury by regulating mitochondrial apoptosis.

Background: Oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell damage is a major risk factor for atherosclerosis and its related cardiovascular diseases. The G0/G1 switch gene 2 (G0S2) is a multifunctional protein which has been poorly studied in atherosclerosis. Methods: In this study, ox-LDL was utilized to construct a human aortic endothelial cell (HAEC) injury model. Results: It was found that ox-LDL impaired cell viability, augmented lactate dehydrogenase (LDH) release, and reduced G0S2 levels in HAECs in a dose-dependent manner. Further, G0S2 overexpression improved the viability and restrained apoptosis of HAECs treated by ox-LDL. Conversely, G0S2 depletion decreased the viability and aggravated apoptosis of HAECs treated by ox-LDL. At the molecular level, G0S2 overexpression significantly increased the secretion of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPH-Px), promoted intracellular reactive oxygen species (ROS) production and malondialdehyde (MDA) content in HAECs under either normal or ox-LDL conditions. Meanwhile, the ox-LDL-induced mitochondrial dysfunction, as demonstrated by decreased mitochondrial membrane potential, translocation of mitochondrial cytochrome c (Cyt-c) to the cytoplasm, and activation of caspase-3 and caspase-9, was significantly reversed by G0S2 overexpression. In addition, G0S2 overexpression promoted the activation of AMP-activated protein kinase (AMPK) and increased the expression of nuclear factor erythroid-2-related factor-2 (Nrf2), sirtuin 1 (SIRT1) and heme oxygenase 1 (HO-1) under normal and ox-LDL conditions. Conclusions: This study demonstrated that G0S2 protects against ox-LDL-induced vascular endothelial cell injury by regulating oxidative damage and mitochondrial homeostasis and may be a promising target for the treatment of atherosclerosis.

Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism.

Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.