Stearic acid promotes lipid synthesis through CD36/Fyn/FAK/mTORC1 axis in bovine mammary epithelial cells.

Stearic acid (C18:0, SA) is a saturated long-chain fatty acid (LCFA) that has a prominent function in lactating dairy cows. It is obtained primarily from the diet and is stored in the form of triacylglycerol (TAG) molecules. The transmembrane glycoprotein cluster of differentiation 36 (CD36) is also known as fatty acid translocase, but whether SA promotes lipid synthesis through CD36 and FAK/mTORC1 signaling is unknown. In this study, we examined the function and mechanism of CD36-mediated SA-induced lipid synthesis in bovine mammary epithelial cells (BMECs). SA-enriched supplements enhanced lipid synthesis and the FAK/mTORC1 pathway in BMECs. SA-induced lipid synthesis, FAK/mTORC1 signaling, and the expression of lipogenic genes were impaired by anti-CD36 and the CD36-specific inhibitor SSO, whereas overexpression of CD36 effected the opposite results. Inhibition of FAK/mTORC1 by TAE226/Rapamycin attenuated SA-induced TAG synthesis, inactivated FAK/mTORC1 signaling, and downregulated the lipogenic genes PPARG, CD36, ACSL1, SCD, GPAT4, LIPIN1, and DGAT1 at the mRNA and protein levels in BMECs. By coimmunoprecipitation and yeast two-hybrid screen, CD36 interacted directly with Fyn but not Lyn, and Fyn bound directly to FAK; FAK also interacted directly with TSC2. CD36 linked FAK through Fyn, and FAK coupled mTORC1 through TSC2 to form the CD36/Fyn/FAK/mTORC1 signaling axis. Thus, stearic acid promotes lipogenesis through CD36 and Fyn/FAK/mTORC1 signaling in BMECs. Our findings provide novel insights into the underlying molecular mechanisms by which LCFA supplements promote lipid synthesis in BMECs.

Chlorogenic acid enhances PPARγ-mediated lipogenesis through preventing Lipin 1 nuclear translocation in Staphylococcus aureus-exposed bovine mammary epithelial cells.

Chlorogenic acid (CGA) as one of the most ubiquitously dietary polyphenolic compounds, has been reported to have various antimicrobial effects and exhibit strong anti-inflammatory ability. Staphylococcus aureus is a gram-positive bacterium that can induce mastitis. However, the mechanism through which S. aureus infection affects lipid synthesis and whether CGA have protective effect on S. aureus reduced lipid synthesis is not fully understood. In this study, the internalization of S. aureus reduced intracellular lipid droplet formation, decreased the levels of intracellular triacylglycerol, total cholesterol and 7 types of fatty acid and downregulated the expression of lipogenic genes FAS, ACC, and DGAT1 in bovine mammary epithelial cells (BMECs). In addition, we found that S. aureus intracellular infection attenuated mTORC1 activation resulting in Lipin 1 nuclear localization. Remarkablely, S. aureus infection-mediated repression of lipid synthesis related to the mTORC1 signaling and Lipin 1 nuclear localization can be alleviated by CGA. Thus, our findings provide a novel mechanism by which lipid synthesis is regulated under S. aureus infection and the protective effects of CGA on lipid synthesis in BMECs.

Therapeutic drug combinations against COVID-19 obtained by employing a collaborative filtering method.

The outbreak of coronavirus disease 2019 (COVID-19) has severely harmed human society and health. Because there is currently no specific drug for the treatment and prevention of COVID-19, we used a collaborative filtering algorithm to predict which traditional Chinese medicines (TCMs) would be effective in combination for the prevention and treatment of COVID-19. First, we performed drug screening based on the receptor structure prediction method, molecular docking using q-vina to measure the binding ability of TCMs, TCM formulas, and neo-coronavirus proteins, and then performed synergistic filtering based on Laplace matrix calculations to predict potentially effective TCM formulas. Combining the results of molecular docking and synergistic filtering, the new recommended formulas were analyzed by reviewing data platforms or tools such as PubMed, Herbnet, the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, the Guide to the Dispensing of Medicines for Clinical Evidence, and the Dictionary of Chinese Medicine Formulas, as well as medical experts' treatment consensus in terms of herbal efficacy, modern pharmacological studies, and clinical identification and typing of COVID-19 pneumonia, to determine the recommended solutions. We found that the therapeutic effect of a combination of six TCM formulas on the COVID-19 virus is the result of the overall effect of the formula rather than that of specific components of the formula. Based on this, we recommend a formula similar to that of Jinhua Qinggan Granules for the treatment of COVID-19 pneumonia. This study may provide new ideas and new methods for future clinical research. Classification: Biological Science.

Signaling repurposable drug combinations against COVID-19 by developing the heterogeneous deep herb-graph method.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has spurred a boom in uncovering repurposable existing drugs. Drug repurposing is a strategy for identifying new uses for approved or investigational drugs that are outside the scope of the original medical indication. MOTIVATION: Current works of drug repurposing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are mostly limited to only focusing on chemical medicines, analysis of single drug targeting single SARS-CoV-2 protein, one-size-fits-all strategy using the same treatment (same drug) for different infected stages of SARS-CoV-2. To dilute these issues, we initially set the research focusing on herbal medicines. We then proposed a heterogeneous graph embedding method to signaled candidate repurposing herbs for each SARS-CoV-2 protein, and employed the variational graph convolutional network approach to recommend the precision herb combinations as the potential candidate treatments against the specific infected stage. METHOD: We initially employed the virtual screening method to construct the 'Herb-Compound' and 'Compound-Protein' docking graph based on 480 herbal medicines, 12,735 associated chemical compounds and 24 SARS-CoV-2 proteins. Sequentially, the 'Herb-Compound-Protein' heterogeneous network was constructed by means of the metapath-based embedding approach. We then proposed the heterogeneous-information-network-based graph embedding method to generate the candidate ranking lists of herbs that target structural, nonstructural and accessory SARS-CoV-2 proteins, individually. To obtain precision synthetic effective treatments forvarious COVID-19 infected stages, we employed the variational graph convolutional network method to generate candidate herb combinations as the recommended therapeutic therapies. RESULTS: There were 24 ranking lists, each containing top-10 herbs, targeting 24 SARS-CoV-2 proteins correspondingly, and 20 herb combinations were generated as the candidate-specific treatment to target the four infected stages. The code and supplementary materials are freely available at https://github.com/fanyang-AI/TCM-COVID19.

Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells.

Staphylococcus aureus (S. aureus) is one of the main pathogens in cow mastitis, colonizing mammary tissues and being internalized into mammary epithelial cells, causing intracellular infection in the udder. Milk that is produced by cows that suffer from mastitis due to S. aureus is associated with decreased production and changes in protein composition. However, there is limited information on how mastitis-inducing bacteria affect raw milk, particularly with regard to protein content and protein composition. The main purpose of this work was to examine how S. aureus infection affects milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were infected with S. aureus, and milk protein and amino acid levels were determined by ELISA after S. aureus invasion. The activity of mTORC1 signaling and the transcription factors NF-κB and STAT5 and the expression of the amino acid transporters SLC1A3 and SLC7A5 were measured by western blot or immunofluorescence and RT-qPCR. S. aureus was internalized by BMECs in vitro, and the internalized bacteria underwent intracellular proliferation. Eight hours after S. aureus invasion, milk proteins were downregulated, and the level of BMECs that absorbed Glu, Asp, and Leu from the culture medium and the exogenous amino acids induced ß-casein synthesis declined. Further, the activity of mTORC1 signaling, NF-κB, and STAT5 was impaired, and SLC1A3 and SLC7A5 were downregulated. Eight hours of treatment with 100 nM rapamycin inhibited NF-κB and STAT5 activity, SLC1A3 and SLC7A5 expression, and milk protein synthesis in BMECs. Thus mTORC1 regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5. These findings constitute a model by which S. aureus infection suppresses milk protein synthesis by decreasing amino acids uptake in BMECs.

Phosphoproteomics Analysis Reveals a Pivotal Mechanism Related to Amino Acid Signals in Goat Fetal Fibroblast.

In addition to serving as the building blocks for protein synthesis, amino acids serve as critical signaling molecules in cells. However, the mechanism through which amino acid signals are sensed in cells is not yet fully understood. This study examined differences in the phosphorylation levels of proteins in response to amino acid signals in Cashmere goat fetal fibroblasts (GFb). Amino acid deficiency was found to induce autophagy and attenuate mammalian/mechanistic target of rapamycin complex (mTORC1)/Unc-51-like autophagy activating kinase 1 (ULK1) signaling in GFb cells. A total of 144 phosphosites on 102 proteins positively associated with amino acid signaling were screened using phosphorylation-based proteomics analysis. The mitogen-activated protein kinase (MAPK) signaling pathway was found to play a potentially important role in the interaction network involved in the response to amino acid signals, according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and MAPK1/3 may serve as a central hub for the entire network. Motif analysis identified three master motifs, xxx_S_Pxx, xxx_S_xxE, and xxx_S_xDx, which were centered on those phosphosites at which phosphorylation was positively regulated by amino acid signaling. Additionally, the phosphorylation levels of three membrane proteins, the zinc transporter SLC39A7, the sodium-dependent neutral amino acid transporters SLC1A5 and SLC38A7, and three translation initiation factors, eukaryotic initiation factor (eIF)5B, eIF4G, and eIF3C, were positively regulated by amino acid signals. These pivotal proteins were added to currently known signaling pathways to generate a novel model of the network pathways associated with amino acid signals. Finally, the phosphorylation levels of threonine 203 and tyrosine 205 on MAPK3 in response to amino acid signals were examined by western blot analysis, and the results were consistent with the data from the phosphoproteomics analysis. The findings of this study provide new evidence and insights into the precise mechanism through which amino acid signals are sensed and conducted in Cashmere goat fetal fibroblasts.

Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

BACKGROUND: Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established. METHODS: Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used to examine the protein changes. IFA was used to identify typical budding vesicles of pyroptosis and the STAT3 nuclear localization. SEM was used to observe the morphological characteristics of pyroptosis. ELISA was used to detect the level of inflammatory cytokines. Pyroptosis was filmed in macrophages by LSCM. RESULTS: S. aureus was internalized by human macrophages. Intracellular S. aureus induced macrophage death. S. aureus invasion increased the expression of NLRP3, Caspase1 (Casp-1 p20) and the accumulation of GSDMD-NT, GSDMD-NT pore structures, and the release of IL-1ß and IL-18 in macrophages. Macrophages pyroptosis induced by S. aureus can be abrogated by blockage of S. aureus phagocytosis. The pyroptosic effect by S. aureus infection was promoted by either rapamycin or Stattic, a specific inhibitor for mTORC1 or STAT3. Inhibition of mTORC1 or STAT3 induced pyroptosis. mTORC1 regulated the pyroptosic gene expression through governing the nuclear localization of STAT3. mTORC1/STAT3 axis may play a regulatory role in pyroptosis within macrophages. CONCLUSIONS: S. aureus infection induces human macrophage pyroptosis, inhibition of mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. mTORC1 and STAT3 are associated with pyroptosis. Our findings demonstrate a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages. Video Abstract Macrophages were infected with S. aureus for 3 h (MOI 25:1), and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell.

mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells.

The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.

The mTORC1/4EBP1/PPARγ Axis Mediates Insulin-Induced Lipogenesis by Regulating Lipogenic Gene Expression in Bovine Mammary Epithelial Cells.

4EBP1 is a chief downstream factor of mTORC1, and PPARγ is a key lipogenesis-related transcription factor. mTORC1 and PPARγ are associated with lipid metabolism. However, it is unknown which effector protein connects mTORC1 and PPARγ. This study investigated the interaction between 4EBP1 with PPARγ as part of the underlying mechanism by which insulin-induced lipid synthesis and secretion are regulated by mTORC1 in primary bovine mammary epithelial cells (pBMECs). Rapamycin, a specific inhibitor of mTORC1, downregulated 4EBP1 phosphorylation and the expression of PPARγ and the following lipogenic genes: lipin 1, DGAT1, ACC, and FAS. Rapamycin also decreased the levels of intracellular triacylglycerol (TAG); 10 types of fatty acid; and the accumulation of TAG, palmitic acid (PA), and stearic acid (SA) in the cell culture medium. Inactivation of mTORC1 by shRaptor or shRheb attenuated the synthesis and secretion of TAG and PA. In contrast, activation of mTORC1 by Rheb overexpression promoted 4EBP1 phosphorylation and PPARγ expression and upregulated the mRNA and protein levels of lipin 1, DGAT1, ACC, and FAS, whereas the levels of intracellular and extracellular TAG, PA, and SA also rose. Further, 4EBP1 interacted directly with PPARγ. Inactivation of mTORC1 by shRaptor prevented the nuclear location of PPARγ. These results demonstrate that mTORC1 regulates lipid synthesis and secretion by inducing the expression of lipin 1, DGAT1, ACC, and FAS, which is likely mediated by the 4EBP1/PPARγ axis. This finding constitutes a novel mechanism by which lipid synthesis and secretion are regulated in pBMECs.

Current models of mammalian target of rapamycin complex 1 (mTORC1) activation by growth factors and amino acids.

Mammalian target of rapamycin (mTOR), which is now referred to as mechanistic target of rapamycin, integrates many signals, including those from growth factors, energy status, stress, and amino acids, to regulate cell growth and proliferation, protein synthesis, protein degradation, and other physiological and biochemical processes. The mTOR-Rheb-TSC-TBC complex co-localizes to the lysosome and the phosphorylation of TSC-TBC effects the dissociation of the complex from the lysosome and activates Rheb. GTP-bound Rheb potentiates the catalytic activity of mTORC1. Under conditions with growth factors and amino acids, v-ATPase, Ragulator, Rag GTPase, Rheb, hVps34, PLD1, and PA have important but disparate effects on mTORC1 activation. In this review, we introduce five models of mTORC1 activation by growth factors and amino acids to provide a comprehensive theoretical foundation for future research.