Parkinson's disease (PD), characterized by the selective loss of nigral dopaminergic neurons, is a common neurodegenerative disorder for which effective disease-modifying therapies remain unavailable. Rapamycin, a clinical immunosuppressant used for decades, has demonstrated neuroprotective effects in various animal models of neurological diseases, including PD. These effects are believed to be mediated through the inhibition of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling, with rapamycin binding to FKBP12. However, recent studies have suggested that mTOR activation can be neuroprotective in degenerating dopaminergic neurons, presenting a paradox to the neuroprotective mechanism of rapamycin via mTORC1 inhibition. In this study, we showed that mTORC1 signaling was inactivated in nigral dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Notably, the optimal neuroprotective dose of rapamycin did not inhibit mTORC1 signaling nor restore autophagy defects in nigral dopaminergic neurons of MPTP-treated male C57BL/6 mice. Furthermore, acute Raptor knockout in dopaminergic neurons, which abolishes mTORC1 activity, did not diminish rapamycin's neuroprotective effects, suggesting that its protection is independent of mTORC1 inhibition. Importantly, rapamycin is also a potent inhibitor of FKBP12, a peptidyl-prolyl cis-trans isomerase highly expressed in the brain. Selective knockdown of FKBP12 in nigral dopaminergic neurons confers neuroprotective effects comparable to that of rapamycin, with no synergism observed when the two are combined. Collectively, our results indicate that rapamycin exerts neuroprotective effects in parkinsonian mice through inhibition of FKBP12 rather than mTORC1 signaling. These findings suggest that FKBP12 may serve as a novel target for disease-modifying therapies in PD.

Autophagy disruption is important in Alzheimer's disease (AD) as it prevents misfolded proteins from being removed, which leads to the accumulation of amyloid plaques and neurofibrillary tangles (NFTs). Restoring autophagy improves neuronal survival and cognitive function, according to experimental models. In AD models, mTOR inhibition and AMPK activation enhance synaptic plasticity and lessen learning deficits. Inhibitors of phosphodiesterase-4 (PDE4) improve cognition and reduce neuroinflammation via altering cyclic adenosine monophosphate (cAMP) transmission. Furthermore, autophagic-lysosomal clearance is encouraged by upregulating transcription factor EB (TFEB), which lessens the pathogenic damage linked to AD. These results point to autophagy modification as a promising therapeutic approach, with the mTOR, AMPK, cAMP, and TFEB pathways being possible targets for drugs. Though much evidence is based on animal studies, these findings provide valuable insights into autophagy's role in AD pathology, offering promising directions for future research and drug development.

Cells need to reprogram their metabolism to adapt to extracellular nutrient changes. The yeast histone acetyltransferase SAGA (Spt-Ada-Gcn5-acetyltransferase) has been reported to acetylate its subunit Ada3 and form homo-dimers to enhance its ability to acetylate nucleosomes and facilitate metabolic gene transcription. How cells transduce extracellular nutrient changes to SAGA structure and function changes remains unclear. Here, we found that SAGA is deacetylated by Rpd3L complex and uncover how its deacetylase activity is repressed by nutrient sensor protein kinase A (PKA). When sucrose is used as the sole carbon source, PKA catalytic subunit Tpk2 is activated, which phosphorylates Rpd3L catalytic subunit Rpd3 to inhibit its ability to deacetylate Ada3. Moreover, Tpk2 phosphorylates Rpd3L subunit Ash1, which specifically reduces the interaction between Rpd3L and SAGA. By phosphorylating both Rpd3 and Ash1, Tpk2 inhibits Rpd3L-mediated Ada3 deacetylation, which promotes SAGA dimerization, nucleosome acetylation and transcription of genes involved in sucrose utilization and tricarboxylate (TCA) cycle, resulting in metabolic shift from glycolysis to TCA cycle. Most importantly, PKA phosphorylates HDAC1, the Rpd3 homolog in mammals to repress its deacetylase activity, promote TCA cycle gene transcription and facilitate cell growth. Our work hence reveals a conserved role of PKA in regulating Rpd3/HDAC1 and metabolic adaptation.

Light and CO2 assimilation activate the target of rapamycin (TOR) kinase in photosynthetic cells, but how these signals are transmitted to TOR is unknown. Using the green alga Chlamydomonas reinhardtii as a model system, we identified dihydroxyacetone phosphate (DHAP) as the key metabolite regulating TOR in response to carbon and light cues. Metabolomic analyses of synchronized cells revealed that DHAP levels change more than any other metabolite between dark- and light-grown cells and that the addition of the DHAP precursor, dihydroxyacetone (DHA), was sufficient to activate TOR in the dark. We also demonstrated that TOR was insensitive to light or inorganic carbon but not to exogenous DHA in a Chlamydomonas mutant defective in the export of DHAP from the chloroplast. Our results provide a metabolic basis for the mode of TOR control by light and inorganic carbon and indicate that cytoplasmic DHAP is an important metabolic regulator of TOR.

The haploid, olegenious yeast Rhodosporidium toruloides accumulates intracellular lipids and carotenoids upon metabolic stress. Target of Rapamycin (TOR) signaling, essential for cell proliferation, is known to affect cellular lipid accumulation. In contrast to the conventional surrugate cell model S. cerevisiae, which harbours two TOR kinases within its TOR complex, R. toruloides only harbours one TOR kinase, mimicking mammalian systems. We used a proteomics centered approach to probe the cellular response, of the two R. toruloides haplotypes, IFO0559 and IFO0880 upon treatment with the TOR inhibitor rapamycin, with an original focus on difference in carotenoid and lipid accumulation. Unexpectedly, IFO0880 displayed severe growth arrest in response to rapamycin, while IFO0559 did not. Proteomic anaysis revealed differential expression of several proteins involved in cell cycle control, lipogensis, amino acid metabolism and autophagy between the two haplotypes. Among those we identified several proteins previously described in both mammalian oncogenic and aging contexts. This differential haplotype response to rapamycin treatment positions R. toruloides as a promising cell surrugate model to study cellular mechanisms underlying rapamycin response especially for systems with high lipid contents, an emerging hallmark of different forms of mammalian cancer and age related disease.

Gastric cancer (GC) is one of the primary causes of cancer-related fatalities, which requires novel treatment including traditional Chinese medicine (TCM) to prolong survival. Protocatechualdehyde (PCA), a monomer from Chinese herbs, exhibits an anti-cancer effect by inhibiting proliferation and migration, or inducing apoptosis in various types of tumors. However, the anti-cancer effect and underlying mechanism of PCA in gastric cancer are still unclear.

The cell proliferation ability was detected by the cell counting kit-8 (CCK-8) and colony formation. The occurrence of autophagy was observed by TEM (Tansmission electron microscopy) and immunofluorescence. The expression of proteins involved in AMPK/mTOC1 signaling pathway was detected by western blotting. Apoptosis and cell cycle analysis were determined through flow cytometry. A xenograft mouse model was employed to validate the anticancer effect of PCA in vivo.

PCA was first identified as a specific inhibitor to gastric cancer cells that significantly inhibited the proliferation of human gastric cancer cells MKN45 and AGS in a dose- and time-dependent manner, but not that of human gastric epithelial cells. Furthermore, PCA induced tumor suppressive autophagy in both gastric cancer cells, and blockage of the autophagy by silencing ATG5 can partially reverse the proliferation inhibition of PCA. Mechanistically, PCA induced-autophagy was largely dependent on the activation of the AMPK/ULK1 signaling pathway, and blockage of the pathway through AMPK specific inhibitor Compound C (Com C) or siRNAs targeting ULK1 prevented the occurrence of autophagy and partially reversed the proliferation inhibition induced by PCA. In addition, PCA significantly suppressed the growth of gastric cancer in the gastric cancer xenograft mouse model by activating key proteins related to the AMPK/ULK1 signaling pathway of autophagy.

These findings demonstrated that PCA inhibited gastric cancer by inducing tumor suppressive autophagy through the AMPK/ULK1 signaling pathway. PCA may serve as a novel candidate for the treatment of gastric cancer.

The mammalian target of rapamycin (mTOR) is a crucial enzyme in regulating multiple signaling pathways in the body, including autophagy, proliferation and apoptosis. Disruption of these mTOR signaling pathways can lead to an array of abnormalities and trigger disease processes, examples being neurodegenerative conditions, cancer, obesity and diabetes. Under conditions of oxidative stress, mTOR can regulate apoptosis and autophagy, with tissue repair being favored under such circumstances. Moreover, the correlation between mTOR and other signaling pathways could play a pivotal role in the pathophysiology of numerous disorders. mTOR has a tight connection with NF-κB, Akt, PI3K, MAPK, GSK-3β, Nrf2/HO-1, JAK/STAT, CREB/BDNF, and ERK1/2 pathways, which together could play significant roles in the regulation of inflammation, apoptosis, cell survival, and oxidative stress in different body organs. Research suggests that inhibiting mTOR could be beneficial in treating metabolic, neurological and cardiovascular conditions, as well as potentially extending life expectancy. Therefore, identifying new chemicals and agents that can modulate the mTOR signaling pathway holds promise for treating and preventing these disorders. Curcumin is one such agent that has demonstrated regulatory effects on the mTOR pathway, making it an exciting alternative for reducing complications associated with complex diseases by targeting mTOR. This review aims to examine the potential of curcumin in modulating the mTOR signaling pathway and its therapeutic implications.

This study aimed to investigate the role of CKMT1B in the growth, migration, invasion, and apoptosis of colorectal cancer (CRC) cells, focusing on its regulation of the AKT/mTOR/STAT3 signaling pathway. Bioinformatics analysis indicated that the CK family expression exhibited heterogeneity and correlation across various cancers, with CKMT1B significantly underexpressed in CRC, suggesting its potential role as a tumor suppressor gene. Additionally, reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot were employed to evaluate CKMT1B expression in CRC cell lines. To assess the effects of CKMT1B knockdown and overexpression in LOVO cells, CCK-8, plate cloning, scratch, Transwell chamber, and Muse assays were used to measure cell proliferation, migration, invasion, and apoptosis. The regulatory mechanism of CKMT1B in CRC was further explored through the AKT/mTOR/STAT3 pathway and related proteins. Downregulation of CKMT1B enhanced the proliferation, migration, and invasion of LOVO cells while inhibiting apoptosis, while CKMT1B overexpression had the opposite effect. Moreover, CKMT1B overexpression led to a decrease in the anti-apoptotic protein BCL2 and an increase in the tumor suppressor protein p53, suggesting its role in modulating apoptotic pathways. The expression levels of the pathway-related proteins P-AKT, P-mTOR, and P-STAT3 were significantly reduced, suggesting that CKMT1B regulates the AKT/mTOR/STAT3 signaling pathway, thereby modulating CRC progression. Taken together, these findings suggest that CKMT1B could serve as a promising molecular target for CRC treatment.

Amino acids and ammonia serve as sources of nitrogen for cell growth and were previously thought to have similar effects on yeast. Consistent with this idea, depletion of either of these two nitrogen sources inhibits the target of rapamycin complex 1 (TORC1), leading to induction of macroautophagy/autophagy and inhibition of cell growth. In this study, we show that Whi2 and the haloacid dehalogenase (HAD)-type phosphatases Psr1 and Psr2 distinguish between these two nitrogen sources in Saccharomyces cerevisiae, as the Whi2-Psr1-Psr2 complex inhibits TORC1 in response to low leucine but not in the absence of nitrogen. In contrast, a parallel pathway controlled by Npr2 and Npr3, components of the Seh1-associated complex inhibiting TORC1 (SEACIT), suppress TORC1 under both low leucine- and nitrogen-depletion conditions. Co-immunoprecipitations with mutants of Whi2, Psr1, Psr2 and fragments of Tor1 support the model that Whi2 recruits Psr1 and Psr2 to TORC1. In accordance, the interaction between Whi2 and Tor1 appears to increase under low leucine but decreases under nitrogen-depletion conditions. Although the targets of Psr1 and Psr2 phosphatases are not known, mutation of their active sites abolishes their inhibitory effects on TORC1. Consistent with the conservation of HAD phosphatases across species, human HAD phosphatases CTDSP1 (CTD small phosphatase 1), CTDSP2, and CTDSPL can functionally replace Psr1 and Psr2 in yeast, restoring TORC1 inhibition and autophagy activation in response to low leucine conditions.

Ustilaginoidea virens is an economically important plant pathogen that causes rice false smut, which causes yield reduction and produces mycotoxins in infected grains that pose a serious threat to human and animal health. The target of rapamycin (TOR) signaling pathway acts as a master regular in regulating cell growth and secondary metabolism in fungi. However, little is known about the function of the TOR pathway in regulating fungal development, pathogenicity and mycotoxin biosynthesis in U. virens. Here, we demonstrate that the TOR signaling pathway positively regulates the cell growth, conidiation and pathogenicity in U. virens through the biochemical inhibition of TOR kinases. The inhibition of TOR in U. virens (UvTOR) by rapamycin significantly induces the expression of genes related to mycotoxin biosynthesis, especially that of ustiloxins. Transcriptome analysis under TOR inhibition revealed that the TOR signaling pathway is a regulatory hub that governs U. virens growth and metabolism. A total of 275 differentially expressed genes (DEGs), consisting of 109 up-regulated DEGs and 166 down-regulated DEGs, were identified after rapamycin treatment. The up-regulated DEGs were enriched in amino acid- and acetyl-CoA-related metabolism pathways and the down-regulated DEGs were enriched in carbohydrate- and fatty acid-related metabolism pathways. Collectively, our results provide the first in-depth insight into the TOR signaling pathway in regulating vegetable growth, virulence and mycotoxin biosynthesis in U. virens.

One of the biggest challenges to eukaryotic gene expression is coordinating transcription in the nucleus and protein synthesis in the cytoplasm. However, little is known about how these major steps in gene expression are connected. The Target of Rapamycin (TOR) signaling pathway is crucial in connecting these critical phases of gene expression. Highly conserved among eukaryotic cells, TOR regulates growth, metabolism, and cellular equilibrium in response to changes in nutrients, energy levels, and stress conditions. This review examines the extensive role of TOR in gene expression regulation. We highlight how TOR is involved in phosphorylation, remodeling chromatin structure, and managing the factors that facilitate transcription and translation. Furthermore, the critical functions of TOR extend to processing RNA, assembling RNA-protein complexes, and managing their export from the nucleus, demonstrating its wide-reaching impact throughout the cell. Our discussion emphasizes the integral roles of TOR in bridging the processes of transcription and translation and explores how it orchestrates these complex cellular processes.

Oleaginous microorganisms can produce polyunsaturated fatty acids beneficial to human health through adjusting the nitrogen content in the medium. The target of rapamycin complex 1 (TORC1) is important for nitrogen sensing and then regulates lipid metabolism. However, the function of Kog1, a subunit of TORC1, in TORC1-regulated lipid metabolism in oleaginous microorganisms remains unclear. In this study, the gene kog1 was knocked out to explore the mechanism of lipid accumulation in the oleaginous fungus M. circinelloides under nitrogen-limited and nitrogen-rich conditions. The results showed that the cell dry weight (CDW) of the kog1 deletion mutant was obviously decreased from 22.2 to 15.4 g/L under nitrogen-limited conditions; however, the lipid content markedly increased by 43.2% compared to the control, from 20.8% of CDW to 29.9%. A similar trend was observed under nitrogen-rich conditions; the cell growth was significantly inhibited, the CDW was decreased from 28.6 to 23.0 g/L, and the lipid content increased by 79.6% compared to the control strain, reaching 9.7% of CDW. The addition of rapamycin further enhanced lipid accumulation in the kog1 knockout mutant but not in the tor knockout mutant, indicating that Kog1 is the upstream target of rapamycin (TOR) in regulating lipid regulation. Transcriptional analysis under both nitrogen-limited and nitrogen-rich conditions notably suggested that nitrogen stress may activate Snf1/AMPK to inhibit Kog1, facilitating SREBP-1c nuclear translocation and activating fatty acid biosynthesis genes.

Macroautophagy/autophagy is an evolutionarily conserved intracellular degradation pathway that relies on vacuoles or lysosomes. Over 40 ATG genes have been identified in yeast cells as participants in various types of autophagy, although these genes are non-essential. While some essential genes involved in autophagy have been identified using temperature-sensitive yeast strains, systematic research on essential genes in autophagy remains lacking. To address this, we established an essential protein conditional degradation library using the auxin-inducible degron (AID) system. By introducing the GFP-Atg8 plasmid, we identified 29 essential yeast genes involved in autophagy, 19 of which had not been previously recognized. In summary, the yeast essential protein conditional degradation library we constructed will serve as a valuable resource for systematically investigating the roles of essential genes in autophagy and other biological functions.Abbreviation: AID: auxin-inducible degron; ALP: alkaline phosphatase; ATG: autophagy related; CSG: constitutive slow growth; DAmP: Decreased Abundance by mRNA Perturbation; GFP: green fluorescent protein; MMS: methyl methanesulfonate; ORF: open reading frame; PAS: phagophore assembly site; PCR: polymerase chain reaction; SD-G: glucose starvation medium; SD-N: nitrogen starvation medium; TOR: target of rapamycin kinase; YGRC: yeast genetic resource center; YPD: yeast extract peptone dextrose.

Autophagy is a genetically regulated, eukaryotic catabolic pathway that responds to internal and external cellular signals. In plants, it plays crucial roles in development, and responses to abiotic and biotic stresses. Due to its role in limiting the hypersensitive response, research on the molecular mechanisms of autophagic signalling pathways in plant-microbe interactions has primarily focused on plant-pathogen responses. Although there is substantially less information on the role of autophagy signalling in symbiotic plant-microbe interactions, there is accumulating evidence that it is also a key regulator of mutualistic plant-microbe interactions. Here, we review recent progress on the roles of autophagy in symbiotic plant interactions and discuss potential future research directions. Once understood, the central role that autophagy plays within pathogenic and symbiotic plant-microbe interactions has significant potential application for crop improvement. Manipulating autophagy in legume crops could help support crop growth with reduced levels of fertiliser application while maintaining yields with increased protein content in the harvest.

Mammalian target of rapamycin complex 2 (mTORC2) is essential for hearing by regulating auditory hair cell structure and function. However, mechanistic details of how mTORC2 regulates intracellular processes in sensory hair cells have not yet been clarified. To further elucidate the role of mTORC2 in auditory cells, we generated a Rictor knockout cell line from HEI-OC1 auditory cells. mTORC2-deficient auditory cells exhibited significant alterations in actin cytoskeleton morphology and decreased proliferation rates. Additionally, we observed a reduction in phosphorylation of protein kinase C alpha (PKCα) and disrupted actin polymerization in mTORC2-deficient cells. Using proteomics, we found that mTORC2 disruption altered expression of cytoskeleton-related proteins in auditory cells. These findings provide valuable mechanistic insights into the functional role of mTORC2 in auditory cells, potentially opening new perspectives to address sensorineural hearing loss.

Under salt stress, autophagy regulates ionic balance, scavenges ROS, and supports nutrient remobilization, thereby alleviating osmotic and oxidative damage. Salt stress is a major environmental challenge that significantly impacts plant growth and agricultural productivity by disrupting nutrient balance, inducing osmotic stress, and causing the accumulation of toxic ions like Na+. Autophagy, a key cellular degradation and recycling pathway, plays a critical role in enhancing plant salt tolerance by maintaining cellular homeostasis and mitigating stress-induced damage. While autophagy has traditionally been viewed as a response to nutrient starvation, recent research has highlighted its importance under various environmental stresses, particularly salt stress. Under such conditions, plants activate autophagy through distinct signaling pathways involving autophagy-related genes (ATGs), Target of Rapamycin (TOR) proteins, and reactive oxygen species (ROS). Salt stress induces the expression of ATG genes and promotes the formation of autophagosomes, which facilitate the degradation of damaged organelles, denatured proteins, and the sequestration of Na+ into vacuoles, thereby improving stress tolerance. Recent studies have also suggested that autophagy may play a direct role in salt stress signaling, linking it to the regulation of metabolic processes. This review discusses the molecular mechanisms underlying autophagy induction in plants under salt stress, including the roles of ATGs and TOR, as well as the physiological significance of autophagy in mitigating oxidative damage, maintaining ion balance, and enhancing overall salt tolerance. In addition, we discussed the metabolic changes related to autophagy in stressed plants and examined the broader implications for managing plant stress and improving crops.

Historically, plant derived natural products and their crude extracts have been used to treat a wide range of ailments across the world. Biogerontology research aims to explore the molecular basis of aging and discover new anti-aging therapeutic compounds or formulations to combat the detrimental effects of aging and promote a healthy life span. The budding yeast Saccharomyces cerevisiae has been, and continues to be, an indispensable model organism in the field of biomedical research for discovering the molecular basis of aging S. cerevisiae has preserved nutritional signaling pathways (such as the target of rapamycin (TOR)-Sch9 and the Ras-AC-PKA (cAMP-dependent protein kinase) pathways, and shows two distinct aging paradigms chronological life span (CLS) and replicative life span (RLS). This review explores the anti-aging properties of natural products, predominantly derived from plants, and phytoextracts using S. cerevisiae as a model organism.

Historically, plant derived natural products and their crude extracts have been used to treat a wide range of ailments across the world. Biogerontology research aims to explore the molecular basis of aging and discover new anti-aging therapeutic compounds or formulations to combat the detrimental effects of aging and promote a healthy life span. The budding yeast Saccharomyces cerevisiae has been, and continues to be, an indispensable model organism in the field of biomedical research for discovering the molecular basis of aging S. cerevisiae has preserved nutritional signaling pathways (such as the target of rapamycin (TOR)-Sch9 and the Ras-AC-PKA (cAMP-dependent protein kinase) pathways, and shows two distinct aging paradigms chronological life span (CLS) and replicative life span (RLS). This review explores the anti-aging properties of natural products, predominantly derived from plants, and phytoextracts using S. cerevisiae as a model organism.

Tor kinases play diverse and essential roles in control of nutrient signaling and cell growth. These kinases are assembled into two multiprotein complexes known as TORC1 and TORC2. In budding yeast, TORC2 relays nutrient-dependent signals that strongly influence growth rate and cell size. However, the mechanisms that control TORC2 signaling are poorly understood. Activation of TORC2 requires Mss4, a phosphatidylinositol 4-phosphate 5-kinase that recruits and activates downstream targets of TORC2. Localization of Mss4 to the plasma membrane is thought to be controlled by phosphorylation, and previous work has suggested that yeast homologs of casein kinase 1, Yck1 and Yck2 (referred to here collectively as Yck1/2), Control phosphorylation of Mss4. Here, we generated a new analog-sensitive allele of YCK2 and used it to test whether Yck1/2 influence localization of Mss4 or signaling in the TORC2 network. We found that Yck1/2 strongly influence Mss4 phosphorylation and localization, as well as influencing regulation of multiple components of the TORC2 network. However, inhibition of Yck1/2 causes mild effects on the best-characterized signaling axis in the TORC2 pathway, suggesting that Yck1/2 might play a larger role in influencing less well-understood aspects of TORC2 signaling.

Ferroptosis is a novel form of cell death characterized by unlimited accumulation of iron-dependent lipid peroxides. It is often accompanied by disease, and the relationship between ferroptosis of immune cells and immune regulation has been attracting increasing attention. Initially, it was found in cancer research that the inhibition of regulatory T cell (Treg) ferroptosis and the promotion of CD8+ T cell ferroptosis jointly promoted the formation of an immune-tolerant environment in tumors. T-cell ferroptosis has subsequently been found to have immunoregulatory effects in other diseases. As an autoimmune disease characterized by immune imbalance, T-cell ferroptosis has attracted attention for its potential in regulating immune balance in lupus nephritis. This article reviews the metabolic processes within different T-cell subsets in lupus nephritis (LN), including T follicular helper (TFH) cells, T helper (Th)17 cells, Th1 cells, Th2 cells, and Treg cells, and reveals that these cellular metabolisms not only facilitate the formation of a T-cell immune imbalance but are also closely associated with the occurrence of ferroptosis. Consequently, we hypothesize that targeting the metabolic pathways of ferroptosis could become a novel research direction for effectively treating the immune imbalance in lupus nephritis by altering T-cell differentiation and the incidence of ferroptosis.

Background/Objectives: The mammalian target of the rapamycin (mTOR) signaling pathway is a central regulator of cell growth, proliferation, metabolism, and survival. Dysregulation of mTOR signaling contributes to many human diseases, including cancer, diabetes, and obesity. Therefore, inhibitors against mTOR's catalytic kinase domain (KD) have been developed and have shown significant antitumor activities, making it a promising therapeutic target. The ATP-KD interaction is particularly important for mTOR to exert its cellular functions, and such inhibitors have demonstrated efficient attenuation of overall mTOR activity. Methods: In this study, we screened the Traditional Chinese Medicine (TCM) database, which enlists natural products that capture the relationships between drugs targets and diseases. Our aim was to identify potential ATP-competitive agonists that target the mTOR-KD and compete with ATP to bind the mTOR-KD serving as potential potent mTOR inhibitors. Results: We identified two compounds that demonstrated interatomic interactions similar to those of ATP-mTOR. The conformational stability and dynamic features of the mTOR-KD bound to the selected compounds were tested by subjecting each complex to 200 ns molecular dynamic (MD) simulations and molecular mechanics/generalized Born surface area (MM/GBSA) to extract free binding energies. We show the effectiveness of both compounds in forming stable complexes with the mTOR-KD, which is more effective than the mTOR-KD-ATP complex with more robust binding affinities. Conclusions: This study implies that both compounds could serve as potential therapeutic inhibitors of mTOR, regulating its function and, therefore, mitigating human disease progression.

Oral leukoplakia (OLK) is the most emblematic oral potentially malignant disorder that may precede the diagnosis of oral squamous cell carcinoma (OSCC) and has an overall malignant transformation rate of 9.8 %. Early intervention is crucial to reduce the malignant transformation rate from OLK to OSCC but the lack of effective local pharmaceutical preparations poses a challenge to clinical management. Rapamycin is speculated to prevent OLK from carcinogenesis and its inherent lipophilicity facilitates its penetration into stratum corneum. Nevertheless, hydrophilic hydrogels frequently encounter challenges when attempting to deliver lipophilic drugs. Furthermore, the oral cavity presents a complex environment defined by oral motor functions, saliva secretion cycles, dynamic fluctuations, and protective barriers comprising mucus and lipid layers. Consequently, addressing issues of muco-penetration and muco-adhesion is imperative for developing an effective drug delivery system aiming at delivering rapamycin to target oral potentially malignant disorders. Here, a dual-function hydrogel drug delivery system integrating adhesion and lipophilicity was successfully developed based on polyvinyl alcohol (PVA) and dioleoyl phosphatidylglycerol (DOPG) via dynamic boronic ester bonds. Rheological experiments based on orthogonal design revealed that PVA-DOPG hydrogels exhibited ideal adhesive strength (around 6 kPa) and could adhere to various surfaces in both dry and wet conditions. PVA-DOPG hydrogels also significantly promoted lipophilic molecules' penetration into stratum corneum (integrated fluorescence density of 6.95 ± 0.52 × 106 and mean fluorescence depth of 0.96 ± 0.07 mm) of ex-vivo porcine buccal mucosa (p < 0.001). Furthermore, PVA-DOPG hydrogels incorporating rapamycin inhibited malignant transformation of OLK mouse model induced by 4-Nitroquinoline N-oxide (4-NQO), distinct improvements in survival (the neoplasm incidence density at the 40th day is 0.0091) (p < 0.05), decrease in neoplasm incidence density of 36.36 % and inhibition rate in neoplasm volume of 75.04 ± 33.67 % have been demonstrated, suggesting the hydrogels were valuable candidates for potential applications in the management of OLK.

The target of rapamycin (TOR) protein, renowned for its highly conserved nature across species, plays a pivotal role in modulating signaling pathways via its multiprotein complexes, TORC1 and TORC2. The relationship between TOR and its inhibitor, rapamycin, especially in the context of lifespan extension, has earned significant attention. Unlike mammals, which have a single TOR gene, the budding yeast Saccharomyces cerevisiae features two TOR paralogs: TOR1 and TOR2. Non-essential TOR1 gene has been the focus of extensive research, whereas the essential TOR2 gene has received relatively little attention in lifespan studies. In our research, we engineered a point mutation (Ser-1975-Ile) within the FKBP12-rapamycin-binding (FRB) domain of Tor2p to block rapamycin binding. Remarkably, this mutation negated the lifespan-extending benefits of rapamycin, irrespective of the TOR1 gene status. Our findings indicate that the TOR2 gene likely serves as the primary mammalian ortholog, playing a crucial role in mediating the effects of rapamycin on lifespan extension. This discovery opens a new avenue for the development of innovative anti-aging agents targeting the TOR. complex.

Combinational therapies provoking cell death are of major interest in oncology. Combining TORC2 kinase inhibition with the radiomimetic drug Zeocin results in a rapid accumulation of double-strand breaks (DSB) in the budding yeast genome. This lethal Yeast Chromosome Shattering (YCS) requires conserved enzymes of base excision repair. YCS can be attenuated by eliminating three N-glycosylases or endonucleases Apn1/Apn2 and Rad1, which act to convert oxidized bases into abasic sites and single-strand nicks. Adjacent lesions must be repaired in a step-wise fashion to avoid generating DSBs. Artificially increasing nuclear actin by destabilizing cytoplasmic actin filaments or by expressing a nuclear export-deficient actin interferes with this step-wise repair and generates DSBs, while mutants that impair DNA polymerase processivity reduce them. Repair factors that bind actin include Apn1, RFA and the actin-dependent chromatin remodeler INO80C. During YCS, increased INO80C activity could enhance both DNA polymerase processivity and repair factor access to convert clustered lesions into DSBs.

High-density lipoprotein (HDL) protects against myocardial ischemia-reperfusion (I/R) injury. Mammalian target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) play opposing roles in protecting against I/R injury, whereby mTORC1 appears to be detrimental while mTORC2 is protective. However, the role of HDL and mTORC signaling in protecting against I/R in hypertensive rodents is not clearly understood. In this study, we investigated the involvement of mTORC1 and mTORC2 in HDL-mediated protection against myocardial I/R injury in normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).

Hearts from WKY and SHR were subjected to I/R injury using a modified Langendorff system. Hemodynamics data were collected, and infarct size was measured. Rapamycin and JR-AB2-011 were used to test the role of mTORC1 and mTORC2, respectively. MK-2206 was used to test the role of Akt in HDL-mediated cardiac protection. The expression levels and the activation states of mediators of mTORC1 and mTORC2 signaling and myocardial apoptosis were measured by immunoblotting and/or enzyme-linked immunosorbent assay (ELISA).

HDL protected hearts from WKY and SHR against I/R injury as indicated by significant improvements in cardiac hemodynamics and reduction in infarct size. HDL induced greater protection in WKY compared to SHR. HDL treatment attenuated mTORC1 signaling in WKY by reducing the phosphorylation of P70S6K (mTORC1 substrate). In SHR however, HDL attenuated mTORC1 signaling by reducing the levels of phospho-mTORC1, Rag C (mTORC1 activator), and phospho-PRAS40 (mTORC1 inhibitor). HDL increased the phosphorylation of mTORC2 substrate Akt, specifically the Akt2 isoform in SHR and to a greater extent in WKY. HDL-induced protection was abolished in the presence of Akt antagonist and involved attenuation of GSK, caspases 7 and 8 activation, and cytochrome C release.

HDL mediates cardiac protection via attenuation of mTORC1, activation of mTORC2-Akt2, and inhibition of myocardial apoptosis. HDL regulates mTORC1 and mTORC2 signaling via distinct mechanisms in normotensive and hypertensive rats. HDL attenuation of mTORC1 and activation of mTORC2-Akt2 signaling could be a mechanism by which HDL protects against myocardial I/R injury in hypertension.

Domestication can be understood as a symbiotic relationship that benefits both domesticator and domesticated species, involving multiple genetic changes that configure the phenotype of the domesticated species. One of the most important domesticated species is the yeast Saccharomyces cerevisiae, with both domesticated strains used for different fermentations processes for thousands of years and wild strains existing only in environments without human intervention; however, little is known about the phenotypic effects associated with its domestication. In the present work, we studied the effect of domestication on yeast TORC1 activation, a pleiotropic signalling pathway conserved across the eukaryotic domain. To achieve this goal, we improved a previously generated methodology to assess TORC1 activation, which turned out to be as effective as the original one but also presents several practical advantages for its application (such as facilitating confirmation of transformants and putting the Luc reporter gene under the control of the same PRPL26A promoter for each transformed strain). We then generated a mapping population, the so-called TOMAN-G population, derived from the "1002 Yeast Genomes Project" population, the most comprehensive catalogue of the genetic variation in yeasts. Finally, strains belonging to the TOMAN-G population were phenotyped for TORC1 activation, and then we compared the results obtained between yeast strains with different ecological origins, finding differences in TORC1 activation between wild and domesticated strains, particularly wine strains. These results are indicative of the effect of domestication on TORC1 activation, specifically that the different evolutionary trajectories of wild and domesticated strains have in fact caused differences in the activation of this pathway; furthermore, the phenotypic data obtained in this work could be used to continue underlying the genetic bases of TORC1 activation, a process that is still not fully understood, using techniques such as GWAS to search for specific genetic variants underlying the observed phenotypic variability and phylogenetic tree inferences to gain insight into the evolutionary relationships between these genetic variants.

Target of rapamycin complex 1 (TORC1) is a key regulator of metabolism in eukaryotes across multiple pathways. Although TORC1 has been extensively studied in vertebrates and some invertebrates, research on this complex in scallops is limited. In this study, we identified the genes encoding TORC1 complex subunits in the scallop Argopecten irradians irradians through genome-wide in silico scanning. Five genes, including TOR, RAPTOR, LST8, DEPTOR, and PRAS40, that encode the subunits of TORC1 complex were identified in the bay scallop. We then conducted structural characterization and phylogenetic analysis of the A. i. irradians TORC1 (AiTORC1) subunits to determine their structural features and evolutionary relationships. Next, we analyzed the spatiotemporal expressions of AiTORC1-coding genes during various embryo/larvae developmental stages and across different tissues in healthy adult scallops. The results revealed stage- and tissue-specific expression patterns, suggesting diverse roles in development and growth. Furthermore, the regulation of AiTORC1-coding genes was examined in temperature-sensitive tissues (the mantle, gill, hemocyte, and heart) of bay scallops exposed to high-temperature (32 °C) stress over different durations (0 h, 6 h, 12 h, 24 h, 3 d, 6 d, and 10 d). The expression of AiTORC1-coding genes was predominantly suppressed in the hemocyte but was generally activated in the mantle, gill, and heart, indicating a tissue-specific response to heat stress. Finally, functional validation was performed using the TOR inhibitor rapamycin to suppress AiTORC1, leading to an enhanced catabolism, a decreased antioxidant capacity, and a significant reduction in thermotolerance in bay scallops. Collectively, this study elucidates the presence, structural features, evolutional relationships, expression profiles, and roles in antioxidant capacity and metabolism regulation of AiTORC1 in the bay scallop, providing a preliminary understanding of its versatile functions in response to high-temperature challenges in marine mollusks.

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.

The mammalian target of Rapamycin complex 1 (mTORC1) is a serine/threonine kinase that couples nutrient and growth factor signaling to the cellular control of metabolism and plays a fundamental role in aberrant proliferation in cancer. mTORC1 has previously been considered an "on/off" switch, capable of phosphorylating the entire pool of its substrates when activated. However, recent studies have indicated that mTORC1 may be active toward its canonical substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase (S6K), involved in mRNA translation and protein synthesis, and inactive toward TFEB and TFE3, transcription factors involved in the regulation of lysosome biogenesis, in several pathological contexts. Among these conditions are Birt-Hogg-Dubé syndrome (BHD) and, recently, tuberous sclerosis complex (TSC). Furthermore, increased TFEB and TFE3 nuclear localization in these syndromes, and in translocation renal cell carcinomas (tRCC), drives mTORC1 activity toward the canonical substrates, through the transcriptional activation of the Rag GTPases, thereby positioning TFEB and TFE3 upstream of mTORC1 activity toward 4EBP1 and S6K. The expanding importance of TFEB and TFE3 in the pathogenesis of these renal diseases warrants a novel clinical grouping that we term "TFEopathies." Currently, there are no therapeutic options directly targeting TFEB and TFE3, which represents a challenging and critically required avenue for cancer research.

Hydrogen peroxide (H2O2) is naturally produced by plant cells during normal development and serves as a messenger that regulates cell metabolism. Despite its importance, the relationship between hydrogen peroxide and the target of rapamycin (TOR) pathway, as well as its impact on cell division, has been poorly analyzed. In this study, we explore the interaction of H2O2 with TOR, a serine/threonine protein kinase that plays a central role in controlling cell growth, size, and metabolism in Arabidopsis thaliana. By applying two concentrations of H2O2 exogenously (0.5 and 1 mM), we could correlate developmental traits, such as primary root growth, lateral root formation, and fresh weight, with the expression of the cell cycle gene CYCB1;1, as well as TOR expression. When assessing the expression of the ribosome biogenesis-related gene RPS27B, an increase of 94.34% was noted following exposure to 1 mM H2O2 treatment. This increase was suppressed by the TOR inhibitor torin 2. The elimination of H2O2 accumulation with ascorbic acid (AA) resulted in decreased cell division as well as TOR expression. The potential molecular mechanisms associated with the effects of H2O2 on the cell cycle and TOR expression in roots are discussed in the context of the results.

Lung disease development involves multiple cellular processes, including inflammation, cell death, and proliferation. Research increasingly indicates that autophagy and its regulatory proteins can influence inflammation, programmed cell death, cell proliferation, and innate immune responses. Autophagy plays a vital role in the maintenance of homeostasis and the adaptation of eukaryotic cells to stress by enabling the chelation, transport, and degradation of subcellular components, including proteins and organelles. This process is essential for sustaining cellular balance and ensuring the health of the mitochondrial population. Recent studies have begun to explore the connection between autophagy and the development of different lung diseases. This article reviews the latest findings on the molecular regulatory mechanisms of autophagy in lung diseases, with an emphasis on potential targeted therapies for autophagy.

PPI analysis deepens our knowledge in critical processes like carbon fixation and nutrient sensing. Moreover, signaling networks, including pathways like MAPK/ERK and TOR, provide valuable information in how microalgae respond to environmental changes and stress. Additionally, species-species interaction networks for microalgae provide a comprehensive understanding of how different species interact within their environments. This review examines recent advancements in the study of biological networks within microalgae, with a focus on the intricate interactions that define these organisms. It emphasizes how network biology, an interdisciplinary field, offers valuable insights into microalgae functions through various methodologies. Crucial approaches, such as protein-protein interaction (PPI) mapping utilizing yeast two-hybrid screening and mass spectrometry, are essential for comprehending cellular processes and optimizing functions, such as photosynthesis and fatty acid biosynthesis. The application of advanced computational methods and information mining has significantly improved PPI analysis, revealing networks involved in critical processes like carbon fixation and nutrient sensing. The review also encompasses transcriptional networks, which play a role in gene regulation and stress responses, as well as metabolic networks represented by genome-scale metabolic models (GEMs), which aid in strain optimization and the prediction of metabolic outcomes. Furthermore, signaling networks, including pathways like MAPK/ERK and TOR, are crucial for understanding how microalgae respond to environmental changes and stress. Additionally, species-species interaction networks for microalgae provide a comprehensive understanding of how different species interact within their environments. The integration of these network biology approaches has deepened our understanding of microalgal interactions, paving the way for more efficient cultivation and new industrial applications.

Diabetic kidney disease (DKD) represents the most lethal complication in both type 1 and type 2 diabetes. The disease progresses without obvious symptoms and is often refractory when apparent symptoms have emerged. Although the molecular mechanisms underlying the onset/progression of DKD have been extensively studied, only a few effective therapies are currently available. Pathogenesis of DKD involves multifaced events caused by diabetes, which include alterations of metabolisms, signals, and hemodynamics. While the considerable efficacy of sodium/glucose cotransporter-2 (SGLT2) inhibitors or angiotensin II receptor blockers (ARBs) for DKD has been recognized, the ever-increasing number of patients with diabetes and DKD warrants additional practical therapeutic approaches that prevent DKD from diabetes. One plausible but promising target is the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway, which senses cellular nutrients to control various anabolic and catabolic processes. This review introduces the current understanding of the mTOR signaling pathway and its roles in the development of DKD and other chronic kidney diseases (CKDs), and discusses potential therapeutic approaches targeting this pathway for the future treatment of DKD.

Cancer often involves the overactivation of RAS/RAF/MEK/ERK (MAPK) and PI3K-Akt-mTOR pathways due to mutations in genes like RAS, RAF, PTEN, and PIK3CA. Various strategies are employed to address the overactivation of these pathways, among which targeted therapy emerges as a promising approach. Directly targeting specific proteins, leads to encouraging results in cancer treatment. For instance, RTK inhibitors such as imatinib and afatinib selectively target these receptors, hindering ligand binding and reducing signaling initiation. These inhibitors have shown potent efficacy against Non-Small Cell Lung Cancer. Other inhibitors, like lonafarnib targeting Farnesyltransferase and GGTI 2418 targeting geranylgeranyl Transferase, disrupt post-translational modifications of proteins. Additionally, inhibition of proteins like SOS, SH2 domain, and Ras demonstrate promising anti-tumor activity both in vivo and in vitro. Targeting downstream components with RAF inhibitors such as vemurafenib, dabrafenib, and sorafenib, along with MEK inhibitors like trametinib and binimetinib, has shown promising outcomes in treating cancers with BRAF-V600E mutations, including myeloma, colorectal, and thyroid cancers. Furthermore, inhibitors of PI3K (e.g., apitolisib, copanlisib), AKT (e.g., ipatasertib, perifosine), and mTOR (e.g., sirolimus, temsirolimus) exhibit promising efficacy against various cancers such as Invasive Breast Cancer, Lymphoma, Neoplasms, and Hematological malignancies. This review offers an overview of small molecule inhibitors targeting specific proteins within the RAS upstream and downstream signaling pathways in cancer.

Lipases are used in many food, energy, and pharmaceutical processes. Thus, new systems have been sought to synthesize alternative lipases with potential biotechnological applications. Kluyveromyces marxianus is a yeast with recognized lipase activity; at least ten putative lipases/esterases in its genome have been detected, and two of them possess a signal peptide for extracellular secretion. The study of extracellular lipases becomes more relevant since they usually have higher activity rates than intracellular lipases and simpler purification mechanisms. For these reasons, this study aimed to characterize the production and lipase activity of the putative extracellular lipases of the K. marxianus L-2029 strain, encoded in the genes LIP3 and YJR107W. Both genes were heterologously expressed in Saccharomyces cerevisiae BY4742 (yeast strain without extracellular lipase activity) using a pYES2.1/V5-His-TOPO® plasmid. Herein, we show evidence that the strain transformed with the LIP3 gene did not show lipase activity during flask galactose induction. On the other hand, the strain transformed with the YJR107W gene showed a specific activity of 0.397 U/mg, with an optimum temperature of 37 °C and pH 6. For maximum cell production, glucose and yeast extract concentrations were evaluated by a 22 factorial design, followed by the validation of the best concentrations predicted by a statistical model; a 22 factorial design was also carried out to evaluate the concentration of the inducer galactose on the transformed strains, and the intracellular and extracellular lipase specific activities were quantified. Finally, the biomass and lipase production were determined for each strain, which was grown in a stirred tank bioreactor with a working volume of 1.5 L. The specific activities of the transformed strains obtained in the bioreactor were 1.36 U/mg for the LIP3 transformant and 1.25 U/mg for the YJR107W transformant, respectively.

Despite advances in cancer treatment, pancreatic cancer (PC) remains a disease with high mortality rates and poor survival outcomes. The B7 homolog 3 (B7-H3) checkpoint molecule is overexpressed among many malignant tumors, including PC, with low or absent expression in healthy tissues. By modulating various immunological and nonimmunological molecular mechanisms, B7-H3 may influence the progression of PC. However, the impact of B7-H3 on the survival of patients with PC remains a subject of debate. Still, most available scientific data recognize this molecule as a suppressive factor to antitumor immunity in PC. Furthermore, it has been demonstrated that B7-H3 stimulates the migration, invasion, and metastasis of PC cells, and enhances resistance to chemotherapy. In preclinical models of PC, B7-H3-targeting monoclonal antibodies have exerted profound antitumor effects by increasing natural killer cell-mediated antibody-dependent cellular cytotoxicity and delivering radioisotopes and cytotoxic drugs to the tumor site. Finally, PC treatment with B7-H3-targeting antibody-drug conjugates and chimeric antigen receptor T cells is being tested in clinical studies. This review provides a comprehensive analysis of all PC-related studies in the context of B7-H3 and points to deficiencies in the current data that should be overcome by future research.

TORC1 (target of rapamycin complex 1) is a highly conserved protein kinase that plays a central role in regulating cell growth. Given the role of mammalian TORC1 (mTORC1) in metabolism and disease, understanding mTORC1 downstream signaling and feedback loops is important. mTORC1 recognizes some of its substrates via a five amino acid binding sequence called the TOR signaling (TOS) motif. mTORC1 binding to a TOS motif facilitates phosphorylation of a distinct, distal site. Here, we show that LST2, also known as ZFYVE28, contains a TOS motif (amino acids 401 to 405) and is directly phosphorylated by mTORC1 at serine 670 (S670). mTORC1-mediated S670 phosphorylation promotes LST2 monoubiquitination on lysine 87 (K87). Monoubiquitinated LST2 is stable and displays a broad reticular distribution. When mTORC1 is inactive, unphosphorylated LST2 is degraded by the proteasome. The absence of LST2 enhances EGFR (epidermal growth factor receptor) signaling. We propose that mTORC1 negatively feeds back on its upstream receptor EGFR via LST2.

The Phosphatidylinositol-3-kinase (PI3K) family is well-known to comprise three classes of intracellular enzymes. Class I PI3Ks primarily function in signaling by responding to cell surface receptor stimulation, while class II and III are more involved in membrane transport. Under normal physiological conditions, the PI3K signaling network orchestrates cell growth, division, migration and survival. Aberrant activation of the PI3K signaling pathway disrupts cellular activity and metabolism, often marking the onset of cancer. Currently, the Food and Drug Administration (FDA) has approved the clinical use of five class I PI3K inhibitors. These small-molecule inhibitors, which exhibit varying selectivity for different class I PI3K family members, are primarily used in the treatment of breast cancer and hematologic malignancies. Therefore, the development of novel class I PI3K inhibitors has been a prominent research focus in the field of oncology, aiming to enhance potential therapeutic selectivity and effectiveness. In this review, we summarize the specific structures of PI3Ks and their functional roles in cancer progression. Additionally, we critically evaluate small molecule inhibitors that target class I PI3K, with a particular focus on their clinical applications in cancer treatment. Moreover, we aim to analyze therapeutic approaches for different types of cancers marked by aberrant PI3K activation and to identify potential molecular targets amenable to intervention with small-molecule inhibitors. Ultimately, we propose future directions for the development of therapeutic strategies that optimize cancer treatment outcomes by modulating the PI3K family.

Breast cancer (BRCA) has become the most common type of cancer in women. Improving the therapeutic response remains a challenge. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a classic tumour suppressor with emerging new functions discovered in recent years, and myeloid PTEN loss has been reported to impair antitumour immunity. In this study, we revealed a novel mechanism by which myeloid PTEN potentially affects antitumour immunity in BRCA. We detected accelerated stress granule (SG) assembly under oxidative stress in PTEN-deficient bone marrow-derived macrophages (BMDMs) through the EGR1-promoted upregulation of TIAL1 transcription. PI3K/AKT/mTOR (PAM) pathway activation also promoted SG formation. ATP consumption during SG assembly in BMDMs impaired the phagocytic ability of 4T1 cells, potentially contributing to the disruption of antitumour immunity. In a BRCA neoadjuvant cohort, we observed a poorer response in myeloid PTENlow patients with G3BP1 aggregating as SGs in CD68+ cells, a finding that was consistent with the observation in our study that PTEN-deficient macrophages tended to more readily assemble SGs with impaired phagocytosis. Our results revealed the unconventional impact of SGs on BMDMs and might provide new perspectives on drug resistance and therapeutic strategies for the treatment of BRCA patients.

TBK1 positively regulates the growth factor-mediated mTOR signaling pathway by phosphorylating mTOR. However, it remains unclear how the TBK1-mTOR signaling pathway is regulated. Considering that STING not only interacts with TBK1 but also with MARCH1, we speculated that MARCH1 might regulate the mTOR signaling pathway by targeting TBK1. The aim of this study was to determine whether MARCH1 regulates the mTOR signaling pathway by targeting TBK1.

The co-immunoprecipitation (Co-IP) assay was used to verify the interaction between MARCH1 with STING or TBK1. The ubiquitination of STING or TBK1 was analyzed using denatured co-immunoprecipitation. The level of proteins detected in the co-immunoprecipitation or denatured co-immunoprecipitation samples were determined by Western blotting. Stable knocked-down cells were constructed by infecting lentivirus bearing the related shRNA sequences. Scratch wound healing and clonogenic cell survival assays were used to detect the migration and proliferation of breast cancer cells.

We showed that MARCH1 played an important role in growth factor-induced the TBK1- mTOR signaling pathway. MARCH1 overexpression attenuated the growth factor-induced activation of mTOR signaling pathway, whereas its deficiency resulted in the opposite effect. Mechanistically, MARCH1 interacted with and promoted the K63-linked ubiquitination of TBK1. This ubiquitination of TBK1 then attenuated its interaction with mTOR, thereby inhibiting the growth factor-induced mTOR signaling pathway. Importantly, faster proliferation induced by MARCH1 deficiency was weakened by mTOR, STING, or TBK1 inhibition.

MARCH1 suppressed growth factors mediated the mTOR signaling pathway by targeting the STING-TBK1-mTOR axis.