Paeoniflorin inhibits PRAS40 interaction with Raptor to activate mTORC1 to reverse excessive autophagy in airway epithelial cells for asthma.

BACKGROUND: Bronchial asthma is a chronic condition characterized by airway inflammation and remodeling, which pose complex pathophysiological challenges. Autophagy has been identified as a practical strategy to regulate inflammation and remodeling processes in chronic inflammatory diseases with pathological characteristics, such as asthma. PF (Paeoniflorin) is a potential new autophagy regulatory compound. Previous studies have reported that PF can inhibit airway inflammation to alleviate allergic asthma, but whether this is mediated through the regulation of autophagy and the molecular mechanism of action remains unclear. PURPOSE: The aim of this study was to evaluate the inhibitory effect of natural small molecule PF on asthma by regulating epithelial autophagy. METHODS: The rat asthma model was established through intraperitoneal injection of OVA and aluminum hydroxide suspension, followed by atomized inhalation of OVA for a period of two weeks. Following treatment with PF, histopathology was observed using Masson and H&E staining, while airway Max Rrs was evaluated using a pulmonary function apparatus. Levels of inflammatory cells in BALF were detected using a blood cell analyzer, and levels of inflammatory factors in BALF were detected through Elisa. Expressions of p-PRAS40 and p-Raptor were observed through immunohistochemistry, and levels of Beclin1 and LC3B were observed through immunofluorescence. The structure and quantity of autophagosomes and autophagolysosomal were observed through TEM. An autophagy model of 16HBE cells was established after treatment with 10ng/mL IL13 for 30 minutes. PRAS40 (AKT1S1) overexpression and mutation of PF and Raptor binding site (K207M& L302I& Q417H) were introduced in 16HBE cells. Autophagy in cells was measured by mFRP-GFP-LC3 ADV fluorescent tracer. The binding sites of PF and Raptor were analyzed using the Autodock Tool. The p-mTOR, p-Raptor, p-PRAS40, LC3II/LC3I were detected through Western blot, and interaction between PRAS40-Raptor and Raptor-mTOR was detected through Co-IP. RESULTS: The results showed that PF effectively reduced airway inflammation, improved airway pathological changes and remodeling, and maintained lung function. Additionally, PF was found to reverse excessive autophagy in airway epithelial cells. Interestingly, PF activated the mTORC1 subunit PRAS40 and Raptor in airway epithelial cells by regulating their phosphorylation. PRAS40 is an endogenous mTOR inhibitor that promotes autophagy. PF competitively binds Raptor to PRAS40, promoting Raptor-mTOR interactions to activate mTORC1, an outcome that can be reversed by PRAS40 overexpression and site-specific amino acid codon mutations in Raptor. CONCLUSION: These findings suggest that PF intervention and inhibition of PRAS40-Raptor interaction are effective treatments for bronchial asthma. By activating mTORC1, PF effectively reverses excessive autophagy in airway epithelial cells, leading to improved airway function and reduced inflammation.

A useful mTORC1 signaling-related RiskScore model for the personalized treatment of osteosarcoma patients by using the bulk RNA-seq analysis.

AIMS: This research developed a prognostic model for OS patients based on the Mechanistic Target of Rapamycin Complex 1 (mTORC1) signature. BACKGROUND: The mTORC1 signaling pathway has a critical role in the maintenance of cellular homeostasis and tumorigenesis and development through the regulation of cell growth, metabolism and autophagy. However, the mechanism of action of this signaling pathway in Osteosarcoma (OS) remains unclear. OBJECTIVE: The datasets including the TARGET-OS and GSE39058, and 200 mTORC1 genes were collected. METHODS: The mTORC1 signaling-related genes were obtained based on the Molecular Signatures Database (MSigDB) database, and the single sample gene set enrichment analysis (ssGSEA) algorithm was utilized in order to calculate the mTORC1 score. Then, the WGCNA were performed for the mTORC1-correlated gene module, the un/multivariate and lasso Cox regression analysis were conducted for the RiskScore model. The immune infiltration analysis was performed by using the ssGSEA method, ESTIMATE tool and MCP-Count algorithm. KM survival and Receiver Operating Characteristic (ROC) Curve analysis were performed by using the survival and timeROC package. RESULTS: The mTORC1 score and WGCNA with ß = 5 screened the mTORC1 positively correlated skyblue2 module that included 67 genes, which are also associated with the metabolism and hypoxia pathways. Further narrowing of candidate genes and calculating the regression coefficient, we developed a useful and reliable RiskScore model, which can classify the patients in the training and validation set into high and low-risk groups based on the median value of RiskScore as an independent and robust prognostic factor. High-risk patients had a significantly poor prognosis, lower immune infiltration level of multiple immune cells and prone to cancer metastasis. Finally, we a nomogram model incorporating the metastasis features and RiskScore showed excellent prediction accuracy and clinical practicability. CONCLUSION: We developed a useful and reliable risk prognosis model based on the mTORC1 signaling signature.

EspH utilizes phosphoinositide and Rab binding domains to interact with plasma membrane infection sites and Rab GTPases.

Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen's benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

Targeting Asparagine Metabolism in Well-Differentiated/Dedifferentiated Liposarcoma.

BACKGROUND: mTORC1 activity is dependent on the presence of micronutrients, including Asparagine (Asn), to promote anabolic cell signaling in many cancers. We hypothesized that targeting Asn metabolism would inhibit tumor growth by reducing mTORC1 activity in well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS). METHODS: Human tumor metabolomic analysis was utilized to compare abundance of Asn in WD vs. DD LPS. Gene set enrichment analysis (GSEA) compared relative expression among metabolic pathways upregulated in DD vs. WD LPS. Proliferation assays were performed for LPS cell lines and organoid models by using the combination treatment of electron transport chain (ETC) inhibitors with Asn-free media. 13C-Glucose-labeling metabolomics evaluated the effects of combination treatment on nucleotide synthesis. Murine xenograft models were used to assess the effects of ETC inhibition combined with PEGylated L-Asparaginase (PEG-Asnase) on tumor growth and mTORC1 signaling. RESULTS: Asn was enriched in DD LPS compared to WD LPS. GSEA indicated that mTORC1 signaling was upregulated in DD LPS. Within available LPS cell lines and organoid models, the combination of ETC inhibition with Asn-free media resulted in reduced cell proliferation. Combination treatment inhibited nucleotide synthesis and promoted cell cycle arrest. In vivo, the combination of ETC inhibition with PEG-Asnase restricted tumor growth. CONCLUSIONS: Asn enrichment and mTORC1 upregulation are important factors contributing to WD/DD LPS tumor progression. Effective targeting strategies require limiting access to extracellular Asn and inhibition of de novo synthesis mechanisms. The combination of PEG-Asnase with ETC inhibition is an effective therapy to restrict tumor growth in WD/DD LPS.

Nonalcoholic Fatty Liver Disease and Staging of Hepatic Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is in parallel with the obesity epidemic, and it is the most common cause of liver diseases. The patients with severe insulin-resistant diabetes having high body mass index (BMI), high-grade adipose tissue insulin resistance, and high hepatocellular triacylglycerols (triglycerides; TAG) content develop hepatic fibrosis within a 5-year follow-up. Insulin resistance with the deficiency of insulin receptor substrate-2 (IRS-2)-associated phosphatidylinositol 3-kinase (PI3K) activity causes an increase in intracellular fatty acid-derived metabolites such as diacylglycerol (DAG), fatty acyl CoA, or ceramides. Lipotoxicity-related mechanism of NAFLD could be explained still best by the "double-hit" hypothesis. Insulin resistance is the major mechanism in the development and progression of NAFLD/nonalcoholic steatohepatitis (NASH). Metabolic oxidative stress, autophagy, and inflammation induce NASH progression. In the "first hit" the hepatic concentrations of diacylglycerol increase with an increase in saturated liver fat content in human NAFLD. Activities of mitochondrial respiratory chain complexes are decreased in the liver tissue of patients with NASH. Hepatocyte lipoapoptosis is a critical feature of NASH. In the "second hit," reduced glutathione levels due to oxidative stress lead to the overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling that induces cell death in the steatotic liver. Accumulation of toxic levels of reactive oxygen species (ROS) is caused at least by two ineffectual cyclical pathways. First is the endoplasmic reticulum (ER) oxidoreductin (Ero1)-protein disulfide isomerase oxidation cycle through the downstream of the inner membrane mitochondrial oxidative metabolism and the second is the Kelch like-ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. In clinical practice, on ultrasonographic examination, the elevation of transaminases, γ-glutamyltransferase, and the aspartate transaminase to platelet ratio index indicates NAFLD. Fibrosis-4 index, NAFLD fibrosis score, and cytokeratin18 are used for grading steatosis, staging fibrosis, and discriminating the NASH from simple steatosis, respectively. In addition to ultrasonography, "controlled attenuation parameter," "magnetic resonance imaging proton-density fat fraction," "ultrasound-based elastography," "magnetic resonance elastography," "acoustic radiation force impulse elastography imaging," "two-dimensional shear-wave elastography with supersonic imagine," and "vibration-controlled transient elastography" are recommended as combined tests with serum markers in the clinical evaluation of NAFLD. However, to confirm the diagnosis of NAFLD, a liver biopsy is the gold standard. Insulin resistance-associated hyperinsulinemia directly accelerates fibrogenesis during NAFLD development. Although hepatocyte lipoapoptosis is a key driving force of fibrosis progression, hepatic stellate cells and extracellular matrix cells are major fibrogenic effectors. Thereby, these are pharmacological targets of therapies in developing hepatic fibrosis. Nonpharmacological management of NAFLD mainly consists of two alternatives: lifestyle modification and metabolic surgery. Many pharmacological agents that are thought to be effective in the treatment of NAFLD have been tried, but due to lack of ability to attenuate NAFLD, or adverse effects during the phase trials, the vast majority could not be licensed.

Senescence in the ageing skin: a new focus on mTORC1 and the lysosome.

Ageing is defined as the progressive loss of tissue function and regenerative capacity and is caused by both intrinsic factors i.e. the natural accumulation of damage, and extrinsic factors i.e. damage from environmental stressors. Cellular senescence, in brief, is an irreversible exit from the cell cycle that occurs primarily in response to excessive cellular damage, such as from ultraviolet (UV) exposure and oxidative stress, and it has been comprehensively demonstrated to contribute to tissue and organismal ageing. In this review, we will focus on the skin, an organ which acts as an essential protective barrier against injury, insults, and infection. We will explore the evidence for the existence and contribution of cellular senescence to skin ageing. We discuss the known molecular mechanisms driving senescence in the skin, with a focus on the dysregulation of the master growth regulator, mechanistic Target of Rapamycin Complex 1 (mTORC1). We explore the interplay of dysregulated mTORC1 with lysosomes and how they contribute to senescence phenotypes.

Locked in Structure: Sestrin and GATOR-A Billion-Year Marriage.

Sestrins are a conserved family of stress-responsive proteins that play a crucial role in cellular metabolism, stress response, and ageing. Vertebrates have three Sestrin genes (SESN1, SESN2, and SESN3), while invertebrates encode only one. Initially identified as antioxidant proteins that regulate cell viability, Sestrins are now recognised as crucial inhibitors of the mechanistic target of rapamycin complex 1 kinase (mTORC1), a central regulator of anabolism, cell growth, and autophagy. Sestrins suppress mTORC1 through an inhibitory interaction with the GATOR2 protein complex, which, in concert with GATOR1, signals to inhibit the lysosomal docking of mTORC1. A leucine-binding pocket (LBP) is found in most vertebrate Sestrins, and when bound with leucine, Sestrins do not bind GATOR2, prompting mTORC1 activation. This review examines the evolutionary conservation of Sestrins and their functional motifs, focusing on their origins and development. We highlight that the most conserved regions of Sestrins are those involved in GATOR2 binding, and while analogues of Sestrins exist in prokaryotes, the unique feature of eukaryotic Sestrins is their structural presentation of GATOR2-binding motifs.

RRAGD variants cause cardiac dysfunction in a zebrafish model.

The Ras-related GTP-binding protein D (RRAGD) gene plays a crucial role in cellular processes. Recently, RRAGD variants found in patients have been implicated in a novel disorder with kidney tubulopathy and dilated cardiomyopathy. Currently, the consequences of RRAGD variants at organismal level is unknown. Therefore, this study investigated the impact of RRAGD variants on cardiac function using zebrafish embryo model. Furthermore, the potential usage of rapamycin, an mTOR inhibitor, as a therapy was assessed in this model. Zebrafish embryos were injected with RRAGD p.S76L and p.P119R cRNA and the resulting heart phenotypes were studied. Our findings reveal that overexpression of RRAGD mutants resulted in decreased ventricular fractional shortening, ejection fraction, and pericardial swelling. In RRAGD S76L-injected embryos, lower survival and heartbeat were observed, while survival was unaffected in RRAGD P119R embryos. These observations were reversible following therapy with the mTOR inhibitor rapamycin. Moreover, no effects on electrolyte homeostasis were observed. Together, these findings indicate a crucial role of RRAGD for cardiac function. In the future, the molecular mechanisms by which RRAGD variants result in cardiac dysfunction, and if the effects of rapamycin are specific for RRAGD-dependent cardiomyopathy should be studied in clinical studies.

TRIM35 triggers cardiac remodeling by regulating SLC7A5-mediated amino acid transport and mTORC1 activation in fibroblasts.

BACKGROUND: Cardiac maladaptive remodeling is one of the leading causes of heart failure with highly complicated pathogeneses. The E3 ligase tripartite motif containing 35 (TRIM35) has been identified as a crucial regulator governing cellular growth, immune responses, and metabolism. Nonetheless, the role of TRIM35 in fibroblasts in cardiac remodeling remains elusive. METHODS: Heart tissues from human donors were used to verify tissue-specific expression of TRIM35. Fibroblast-specific Trim35 gene knockout mice (Trim35cKO) were used to investigate the function of TRIM35 in fibroblasts. Cardiac function, morphology, and molecular changes in the heart tissues were analyzed after transverse aortic constriction (TAC) surgery. The mechanisms by which TRIM35 regulates fibroblast phenotypes were elucidated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA sequencing (RNA-Seq). These findings were further validated through the use of adenoviral and adeno-associated viral transfection systems, as well as the mTORC1 inhibitor Rapamycin. RESULTS: TRIM35 expression is primarily up-regulated in cardiac fibroblasts in both murine and human fibrotic hearts, and responds to TGF-ß1 stimulation. Specific deletion of TRIM35 in cardiac fibroblasts significantly improves cardiac fibrosis and hypertrophy. Consistently, the overexpression of TRIM35 promotes fibroblast proliferation, migration, and differentiation. Through paracrine signaling, it induces hypertrophic growth of cardiomyocytes. Mechanistically, we found that TRIM35 interacts with, ubiquitinates, and up-regulates the amino acid transporter SLC7A5, which enhances amino acid transport and activates the mTORC1 signaling pathway. Furthermore, overexpression of SLC7A5 significantly reverses the reduced cardiac fibrosis and hypertrophy caused by conditional knockout of TRIM35. CONCLUSION: Our findings demonstrate a novel role of fibroblast-TRIM35 in cardiac remodeling and uncover the mechanism underlying SLC7A5-mediated amino acid transport and mTORC1 activation. These results provide a potential novel therapeutic target for treating cardiac remodeling.

Identification and Verification of a Prognostic Risk Signature in Oral Squamous Cell Carcinoma.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a prevalent malignant condition. This study aimed to investigate the role of mTORC1 signaling and develop a prognostic model for OSCC. MATERIALS AND METHODS: The single-sample gene set enrichment analysis (ssGSEA) algorithm was utilized to calculate the Z-Score of Hallmarks in OSCC, followed by univariate Cox regression analysis to identify processes associated with prognosis. Weighted gene co-expression network analysis (WGCNA) was performed using transcriptomic data from the cancer genome atlas (TCGA) cohort to identify genes correlated with mTORC1 signaling. A six-gene prognostic model was constructed using multifactorial Cox regression analysis and validated using an external dataset. RESULTS: The study uncovered a strong linkage between mTORC1, glycolysis, hypoxia, and the prognosis of OSCC. mTORC1 signaling emerged as the most significant risk factor, negatively impacting patient survival. Additionally, a six-gene prognostic risk score model was developed which provided a quantitative measure of patients' survival probabilities. Interestingly, within the context of these findings, TP53 gene mutations were predominantly observed in the high-risk group, potentially underlining the genetic complexity of this patient subgroup. Additionally, differential immune cell infiltration and an integrated nomogram were also reported. CONCLUSION: This study highlights the importance of mTORC1 signaling in OSCC prognosis and presents a robust prognostic model for predicting patient outcomes.

mTORC1 pathway activity biases cell fate choice.

Pluripotent stem cells can differentiate into distinct cell types but the intracellular pathways controlling cell fate choice are not well understood. The social amoeba Dictyostelium discoideum is a simplified system to study choice preference as proliferating amoebae enter a developmental cycle upon starvation and differentiate into two major cell types, stalk and spores, organised in a multicellular fruiting body. Factors such as acidic vesicle pH predispose amoebae to one fate. Here we show that the mechanistic target of rapamycin complex 1 (mTORC1) pathway has a role in cell fate bias in Dictyostelium. Inhibiting the mTORC1 pathway activity by disruption of Rheb (activator Ras homolog enriched in brain), or treatment with the mTORC1 inhibitor rapamycin prior to development, biases cells to a spore cell fate. Conversely activation of the pathway favours stalk cell differentiation. The Set1 histone methyltransferase, responsible for histone H3 lysine4 methylation, in Dictyostelium cells regulates transcription at the onset of development. Disruption of Set1 leads to high mTORC1 pathway activity and stalk cell predisposition. The ability of the mTORC1 pathway to regulate cell fate bias of cells undergoing differentiation offers a potential target to increase the efficiency of stem cell differentiation into a particular cell type.

Beneficial effects of rapamycin on endothelial function in systemic lupus erythematosus.

Introduction: Endothelial function is significantly impaired in patients with SLE compared to healthy controls. Elevated activation of the mammalian target of rapamycin complex 1 (mTORC1) is reported in humans and mice with SLE. However, it is unclear if elevated mTORC1 in SLE contributes to impaired mitophagy and endothelial dysfunction. Therefore, we tested the hypothesis that inhibiting mTORC1 with rapamycin would increase mitophagy and attenuate endothelial dysfunction and inflammatory responses in SLE. Methods: Nine-week-old female lupus-prone (MRL/lpr) and healthy control (MRL/MpJ) mice were randomly assigned into rapamycin treatment (lpr_Rapamycin and MpJ_Rapamycin) or control (lpr_Control and MpJ_Control) groups. Rapamycin was injected i.p. 3 days per week for 8 weeks. After 8 weeks, endothelium-dependent vasorelaxation to acetylcholine (ACh) and endothelium-independent vasorelaxation to sodium nitroprusside (SNP) were measured in thoracic aortas using a wire myograph. Results: MTORC1 activity was increased in aorta from lpr mice as demonstrated by increased phosphorylation of s6rp and p70s6k and significantly inhibited by rapamycin (s6rp, p < 0.0001, p70s6k, p = 0.04, respectively). Maximal responses to Ach were significantly impaired in lpr_Control (51.7% ± 6.6%) compared to MpJ_Control (86.7% ± 3.6%) (p < 0.0001). Rapamycin prevented endothelial dysfunction in the thoracic aorta from lupus mice (lpr_Rapamycin) (79.6% ± 4.2%) compared to lpr_Control (p = 0.002). Maximal responses to SNP were not different across groups. Phosphorylation of endothelial nitric oxide synthase also was 42% lower in lpr_Control than MpJ_Control and 46% higher in lpr_Rapamycin than lpr_Control. The inflammatory marker, vascular cell adhesion protein 1 (Vcam 1), was elevated in aorta from lupus mice compared with healthy mice (p = 0.001), and significantly reduced with Rapamycin treatment (p = 0.0021). Mitophagy markers were higher in lupus mice and reduced by rapamycin treatment, suggesting altered mitophagy in lpr mice. Conclusion: Collectively, these results demonstrate the beneficial effects of inhibiting mTORC1 on endothelial function in SLE mice and suggest inflammation and altered mitophagy contribute to endothelial dysfunction in SLE.

BCAA-producing Clostridium symbiosum promotes colorectal tumorigenesis through the modulation of host cholesterol metabolism.

Identification of potential bacterial players in colorectal tumorigenesis has been a focus of intense research. Herein, we find that Clostridium symbiosum (C. symbiosum) is selectively enriched in tumor tissues of patients with colorectal cancer (CRC) and associated with higher colorectal adenoma recurrence after endoscopic polypectomy. The tumorigenic effect of C. symbiosum is observed in multiple murine models. Single-cell transcriptome profiling along with functional assays demonstrates that C. symbiosum promotes the proliferation of colonic stem cells and enhances cancer stemness. Mechanistically, C. symbiosum intensifies cellular cholesterol synthesis by producing branched-chain amino acids (BCAAs), which sequentially activates Sonic hedgehog signaling. Low dietary BCAA intake or blockade of cholesterol synthesis by statins could partially abrogate the C. symbiosum-induced cell proliferation in vivo and in vitro. Collectively, we reveal C. symbiosum as a bacterial driver of colorectal tumorigenesis, thus identifying a potential target in CRC prediction, prevention, and treatment.

Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.

Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.

Arf1 promotes porcine intestinal epithelial cell proliferation via the mTORC1 signaling pathway.

The promotion of gut health, a pervasive problem in modern animal husbandry, positively affects organismal health, productivity, and economics. Porcine intestinal epithelial cells (IPEC-J2) continuously proliferate to maintain intestinal homeostasis, including barrier, immune, and absorptive functions. Gut homeostasis is fundamental to organismal health. ADP-ribosylation factor 1 (Arf1), a small GTPase, plays a crucial role in coordinating mTORC1 in response to nutrients, especially amino acid availability in the gut. mTORC1 is the central hub of proliferation. Thus, it seems likely that Arf1 promotes IPEC-J2 cell proliferation. However, the exact role of Arf1 in the porcine gut remains unclear. Therefore, we evaluated the functional role and possible mechanisms of Arf1 in the porcine intestine through Arf1 overexpression and knockdown in IPEC-J2 cells. Arf1 overexpression and knockdown significantly enhanced and inhibited, respectively, IPEC-J2 cell viability, and PCNA expression varied with Arf1 expression. Moreover, the proportion of Ki67-positive cells was significantly greater in the Arf1-overexpressing group than in the control group. These results suggest that Arf1 improves IPEC-J2 cell proliferation. The underlying mechanism was explored by Western blotting. Arf1 overexpression and knockdown significantly enhanced and suppressed, respectively, the levels of p-S6K1 and p-RPS6, which are key downstream targets of the mTORC1 signaling pathway. Collectively, our findings reveal the role of the Arf1-mTORC1 axis in IPEC-J2 cell proliferation and its potential function in regulating intestinal homeostasis and health.

Morin overcomes doxorubicin resistance in human breast cancer by inducing DNA damage and modulating the LKB1/AMPK/mTORC1 signaling pathway.

Breast cancer chemoresistance hampers chemotherapy efficacy; researchers investigate the pharmacological activities of natural products for potential solutions. This study aimed to determine the effect of morin, a bioflavonoid isolated from Maclura pomifera, on two Dox-resistant human breast cancer cell lines MDA-MB-231 (MDA-DR) and MCF-7 (MCF-DR). Sulforhodamine B and colony-forming assays demonstrated the cytotoxic effect of morin on both cell lines. Morin induced DNA damage and reduced the DNA repair mechanism, a feature of chemoresistance. In addition, morin reduced the protein expressions of cell cycle regulators, such as cyclin D1, CDK4, cyclin E1, cyclin B1, and p-Rb, thereby halting cell cycle progression. Moreover, morin slightly reduced PARP and Bcl-xL expressions but left LC3-II and RIPK3 expressions unchanged. Annexin-V/7-AAD analysis showed morin increased 7-AAD positive cells and annexin-V positive cells among MDA-DR and MCF-DR cells, respectively. In addition, morin increased p-AMPK and p-LKB1 levels; and, thus, inhibited phosphorylation of the mTOR pathway, but decreased t-AMPK levels by inducing lysosomal degradation, and AICAR, an AMPK activator, reduced Raptor, cyclin D1, CDK4, cyclin E1 and phosphorylated, and total mTOR levels, indicating AMPK is a key player in inducing cell death. Also, morin modulated MAPK phosphorylation and attenuated p-Akt and p-GSK3αß levels; and thus, inhibited cell survival. In addition, morin suppressed tumor growth in our MDA-DR xenografted mouse model. These findings indicate that morin is a potential treatment for Dox-resistant breast cancer and that it does so by inducing DNA damage and modulating the LKB1/AMPK/mTORC1 pathway, along with regulating the MAPK, and Akt/GSK3αß signaling pathways.

tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance.

About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.

Diet restriction enhances the effect of immune checkpoint block by inhibiting the intratumoral mTORC1/B7-H3 axis.

Immune checkpoint blockade therapy has demonstrated significant therapeutic efficacy in certain cancer types; however, the impact of dietary restriction remains scarcely reported in this context. This study aimed to investigate the influence of dietary restriction on anti-PDL-1 therapy and the interplay of immune cells within this context. Using an anti-PDL-1 regimen combined with dietary restrictions, tumor progression was assessed in LLC-bearing mice. Flow cytometry was employed to analyze immune cell infiltration and differentiation levels within the tumor microenvironment. The expression of mTORC1/B7-H3 in tumors subjected to dietary restriction was also examined. LLC tumors with elevated B7-H3 expression were validated in mice to determine its inhibitory effect on immune cell proliferation and differentiation. A CD3/B7-H3 chimeric antibody was developed for therapeutic intervention in B7-H3 overexpressing tumors, with subsequent T cell responses assessed through flow cytometry. Dietary restriction potentiated the effect of anti-PDL1 therapy by suppressing the intratumorally mTORC1/B7-H3 axis. In vivo experiments demonstrated that elevated B7-H3 expression in tumors reduced infiltration and activation of CD8 + T cells within the tumor, while it did not affect tumor-infiltrating Tregs. In vitro studies revealed that high B7-H3 expression influenced the proliferation and activation of CD8 + T cells within a Coculture system. The constructed CD3/B7-H3 chimeric antibody prominently activated TCR within B7-H3 overexpressing tumors and impeded tumor progression. The findings suggest that dietary restriction enhances the efficacy of immune checkpoint blockade by modulating the intratumoral mTORC1/B7-H3 axis.

Nutrient Sensing of mTORC1 signaling in cancer and aging.

The mechanistic target of rapamycin complex 1 (mTORC1) is indispensable for preserving cellular and organismal homeostasis by balancing the anabolic and catabolic processes in response to various environmental cues, such as nutrients, growth factors, energy status, oxygen levels, and stress. Dysregulation of mTORC1 signaling is associated with the progression of many types of human disorders including cancer, age-related diseases, neurodegenerative disorders, and metabolic diseases. The way mTORC1 senses various upstream signals and converts them into specific downstream responses remains a crucial question with significant impacts for our perception of the related physiological and pathological process. In this review, we discuss the recent molecular and functional insights into the nutrient sensing of the mTORC1 signaling pathway, along with the emerging role of deregulating nutrient-mTORC1 signaling in cancer and age-related disorders.

Accumulation of branched-chain amino acids deteriorates the neuroinflammatory response of Müller cells in diabetic retinopathy via leucine/Sestrin2-mediated sensing of mTOR signaling.

AIMS: This study aimed to investigate branched-chain amino acid (BCAA) catabolism in diabetic retinopathy (DR). METHODS: Wild-type and db/db mice were fed BCAAs (5 or 10 mg/kg/day) for 12 weeks, and hyperglycemia-exposed Müller cells were treated with BCAAs (2 or 5 mmol/L) for 24 and 48 h. BCAA levels were measured using MS/MS. Western blotting was performed to detect proteins. Flow cytometry, oxygen consumption rate, and Cell Counting Kit-8 assays were used to evaluate Müller cell viability. Each experiment was conducted at least thrice. RESULTS: BCAAs and branched-chain α-keto acids (BCKAs) were increased in the retina and systemic tissues of diabetic mice, and these changes were further enhanced to approximately 2-fold by extra BCAAs compared to wild-type group. In vitro, BCAAs and BCKAs were induced in hyperglycemic Müller cells, and augmented by BCAA supplementation. The aberrant BCAA catabolism was accompanied by mTORC1 activation and subsequently induced TNF-ɑ, VEGFA, GS, and GFAP in retinas and Müller cells under diabetic conditions. The cell apoptosis rate increased by approximately 50%, and mitochondrial respiration was inhibited by hyperglycemia and BCAA in Müller cells. Additionally, mTORC1 signaling was activated by leucine in Müller cells. Knockdown of Sestrin2 or LeuRS significantly abolished the leucine-induced mTORC1 phosphorylation and protected Müller cell viability under diabetic conditions. CONCLUSIONS: We found that BCAA catabolism is hindered in DR through mTORC1 activation. Leucine plays a key role in inducing mTORC1 by sensing Sestrin2 in Müller cells. Targeting Sestrin2 may ameliorate the toxic effects of BCAA accumulation on Müller cells in DR.