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.

The muscle-building and strengthening effects of the active form of vitamin D in humans remain unclear.

In this ancillary study of the Diabetes Prevention with active Vitamin D trial, we examined clinical and experimental aspects to investigate the effects and mechanisms of eldecalcitol, an active form of vitamin D, in preventing sarcopenia. We examined changes in molecules involved in muscle synthesis and degradation pathways in muscle samples from 32 participants before and after 1 year of eldecalcitol or placebo treatment. The protein levels of molecules involved in muscle synthesis and degradation pathways were examined using western blotting. Additionally, the skeletal muscle and body fat volumes were measured using bioelectrical impedance analysis with a body composition analyzer.

We found that eldecalcitol treatment for 1 year resulted in higher phosphorylation levels of mTOR and FOXO1 signaling pathways, which are associated with increased muscle mass and strength than those with placebo treatment. Body composition measurements at 1 year showed that the eldecalcitol group had significantly higher skeletal muscle mass (1.9 % vs. -3.4 %, p = 3.26E-9) and muscle strength (4.1 % vs. -0.7 %, p = 2.57E-17), and lower fat mass (-3.2 % vs. 1.8 %, p = 1.73E-12) than those in the placebo group.

This study suggested that the active form of vitamin D regulates the protein synthesis and degradation pathways in human skeletal muscle and may help prevent sarcopenia. This study was registered at UMIN clinical trials registry, UMIN 000005394.

Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12-Rapamycin binding site of mTOR (mTORFRB). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTORFRB through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTORFRB. Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTORFRB. Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.

Heat stress (HS) impacts broilers by reducing feed intake which impairs nutrient availability and energy levels, subsequently affecting protein biosynthesis. We hypothesize that an exogenous supply of glucose could provide extra energy resources that enhance protein biosynthesis in broilers reared under HS. Our experimental design involved two levels of temperature (25 °C [thermoneutral, TN]); 35 °C (8.00 AM to 8.00 PM, [Heat Stress, HS]), and two glucose levels (0 % and 6 %). We randomly assigned a total of 456 four-week-old Cobb500 broilers to four different treatment groups (TN0, TN6, HS0, and HS6), respectively. After 7 days post-HS, we observed an inverse relationship between the avian target of rapamycin (avTOR) and autophagy-related genes. The phosphorylation of mTOR and S6K1 at Ser2448 and Thr421/Ser424 respectively was higher (p < 0.05) in the TN0 group than in the HS groups. Additionally, the phosphorylation of Foxo3a at Ser253 was higher (p < 0.05) in the HS0 group than in the HS6 groups, indicating an adaptive response to HS. Thus, the combined effect of HS and glucose could influence the phosphorylation status of key signaling genes in the mTOR pathway. The expression levels of mRNA genes in the mTOR pathway were more pronounced (p < 0.05) in HS6 birds than in HS0 birds except for avTOR, Akt1, and S6K1.

Mutations in the TSC (tuberous sclerosis complex) genes result in the hyperactivation of the mTORC1 (mechanistic/mammalian target of rapamycin 1) growth pathway in mesenchymal pulmonary cells. Rapamycin (sirolimus), a naturally occurring macrolide, is the only therapeutic approved for women with lymphangioleiomyomatosis (LAM), a progressive, destructive lung disease caused by TSC gene mutations and mTORC1 hyperactivation. However, on cessation of the drug, lung function decline continues. We demonstrated here that pulmonary LAM cancer stem-like state (SLS) cells most highly expressed the eIF4E (eukaryotic translation initiation factor 4E)-dependent translation initiation genes. We also showed that the 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) gene has the lowest expression in these cells, indicating that the 4E-BP1/eIF4E ratio in LAM SLS cells favors unrestrained eIF4E oncogenic mRNA translation. The bi-steric mTORC1-selective compound RMC-5552 prevented growth of LAM-associated fibroblasts and phosphorylation of proteins in the ribosomal protein S6K1/ribosomal protein S6 (S6K1/S6) and 4E-BP1/eIF4E translation mTORC1-driven pathways, whereas rapamycin only blocked the S6K/S6 axis. Rapamycin inhibition of LAM-associated fibroblast growth was rapidly reversed, but RMC-5552 inhibition was more durable. RMC-5552, through its potential to eradicate LAM cancer SLS cells, may have therapeutic benefit in LAM and other diseases with mTORC1 hyperactivity.

Senescent foamy macrophages are key drivers of atherosclerosis and plaque instability. N6-methyladenosine (m6A) modification of RNA plays an important role in the development of various diseases including aging. Here, we aim to investigate the role of m6A modification of RNA in the formation of senescent foamy macrophages in atherosclerosis.

To assess m6A methylation, macrophages were isolated from the atherosclerotic plaques of patients with atherosclerosis, and Apoe-/- mice were fed a high-fat diet using flow cytometry. An ALKBH5 (alkB homolog 5)f/f, Lyz2 (lysozyme 2)Cre, Apoe-/- mouse model was generated to determine the infiltration of senescent foamy macrophages into plaques and atherosclerosis progression. Methylated RNA immunoprecipitation, RNA immunoprecipitation sequencing, and dual-luciferase assays were performed to explore the mechanisms underlying the ALKBH5-mediated formation of senescent foamy macrophages.

Decreased m6A methylation and increased ALKBH5 expression were observed in arterial plaques and infiltrating macrophages from patients and mice with atherosclerosis. Compared with control mice, ALKBH5f/f, Lyz2Cre, Apoe-/- mice exhibited fewer atherosclerosis plaques with greater stability, which was attributed to the suppression of senescent foamy macrophage formation and senescence-associated secretory phenotype. In addition, ALKBH5 deletion reduced the mRNA expression level of CCL5 (CC chemokine ligand 5) by increasing m6A methylation in macrophages, which disrupts the stability of CCL5 mRNA. Mechanistically, ALKBH5 promoted senescent foamy macrophage formation through the CCL5/CCR5 (CC chemokine receptor 5)/autophagy signaling pathway. CCL5 also recruited CD8+ IFN (interferon)γ+ T cells via the CCL5-CCR5 axis. The ALKBH5 inhibitor IOX1 (inhibitor of 2OG oxygenases) and the CCR5 antagonist maraviroc were identified as potential clinical interventions for inhibiting senescent foamy macrophage formation and atherosclerosis progression.

Myeloid ALKBH5 deletion attenuates atherosclerosis progression by suppressing the formation of senescent foamy macrophages and the recruitment of CD8+IFNγ+ T cells. These findings identify ALKBH5, CCL5, and CCR5 as novel therapeutic targets for atherosclerosis.

Accumulating clinical evidence suggests an intricate relationship between severe COVID-19 and preexisting metabolic complications, which share some metabolic dysregulations, including hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. However, the potential role of these metabolic risk factors in SARS-CoV-2 infection and entry factor expression remains unknown. Here we report the implication of hyperglycaemia in SARS-CoV-2 infection and therapy. Hyperglycaemia, instead of hyperinsulinaemia and hyperlipidaemia, can significantly induce the expression of SARS-CoV-2 entry factors (Ace2, Tmprss2, Tmprss4, Furin and Nrp1) in liver cells, but not in lung and pancreatic cells, which is attenuated by mTOR inhibition. Correspondingly, pathological glucose levels promote SARS-CoV-2 entry into cultured hepatocytes in pseudovirus cell systems. Conversely, representative glucose-lowering drugs (metformin, dapagliflozin, sitagliptin and exenatide) are able to diminish the enhancement of entry factor expression and SARS-CoV-2 infection in cultured hepatocytes under pathological glucose conditions. Intriguingly, SARS-CoV-2 entry factors are increased in the livers of nonalcoholic fatty liver disease and diabetes patients. These results define hyperglycaemia as a key susceptibility factor for hepatic SARS-CoV-2 infection, and provide insights into the clinical application of glucose-lowering therapies in COVID-19 patients under comorbid hyperglycaemia conditions.

Diamine oxidase (DAO), a well-established biomarker for intestinal damage, histamine intolerance or tumorigenesis, has rarely been reported in immune regulation. This study aimed to identify DAO as a critical enhancer of abnormal inflammation by promoting interferon-gamma (IFN-γ) production from natural killer (NK) cells.

Clinical bioinformatics analyzed aoc1 (DAO-coding gene) expression in PBMCs from patients with inflammatory diseases. Murine models (LPS-induced systemic inflammation, sepsis, DSS-induced colitis) using DAO-/- mice, alongside DAO-/- NK92 cells and DAO inhibitor DIZE, were employed for phenotypic validation. Cellular profiling, bone marrow chimeras, reciprocal transplantation, RNA-sequence, non-targeted metabolomics, and flow cytometry were utilized to dissect DAO's mechanisms in NK cells.

DAO deficiency protected mice from inflammatory pathology by suppressing IFN-γ production. NK cells were identified as the primary target cells during the process, with DAO acting intracellularly to promote IFN-γ via a reactive oxygen species (ROS)-autophagy axis. DAO-derived ROS, distinct from mitochondrial or NOX2 sources, enhanced autophagic flux during NK activation, enabling IFN-γ biosynthesis. DAO did not affect NK homeostasis, including maturation, proliferation, or receptor expressions.

DAO is a novel risk factor in inflammatory diseases, driving IFN-γ production through ROS-autophagy signaling in NK cells. Targeting DAO may offer therapeutic strategies for conditions involving dysregulated IFN-γ responses, including sepsis, colitis, and autoimmune disorders.

Mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates key cellular processes including cell growth, autophagy and metabolism. Hyperactivation of the mTOR pathway causes a group of rare and ultrarare genetic diseases. mTOR pathway diseases have diverse clinical manifestations that are managed by distinct medical disciplines but share a common underlying molecular basis. There is a now a deep understanding of the molecular underpinning that regulates the mTOR pathway but effective treatments for most mTOR pathway diseases are lacking. Translating scientific knowledge into clinical applications to benefit the unmet clinical needs of patients is a major challenge common to many rare diseases. In this article we expound how mTOR pathway diseases provide an opportunity to coordinate basic and translational disease research across the group, together with industry, medical research foundations, charities and patient groups, by pooling expertise and driving progress to benefit patients. We outline the germline and somatic mutations in the mTOR pathway that cause rare diseases and summarise the prevalence, genetic basis, clinical manifestations, pathophysiology and current treatments for each disease in this group. We describe the challenges and opportunities for progress in elucidating the underlying mechanisms, improving diagnosis and prognosis, as well as the development and approval of new therapies for mTOR pathway diseases. We illustrate the crucial role of patient public involvement and engagement in rare disease and mTOR pathway disease research. Finally, we explain how the mTOR Pathway Diseases node, part of the Research Disease Research UK Platform, will address these challenges to improve the understanding, diagnosis and treatment of mTOR pathway diseases.

Venous malformations (VMs) are vascular anomalies lacking curative treatments, often caused by somatic PIK3CA mutations that hyperactivate the PI3Kα-AKT-mTOR signaling pathway. Here, we identify a venous-specific signaling circuit driving disease progression, where excessive PI3Kα activity amplifies upstream TIE2 receptor signaling through autocrine and paracrine mechanisms. In Pik3caH1047R-driven VM mouse models, single-cell transcriptomics and lineage tracking revealed clonal expansion of mutant endothelial cells with a post-capillary venous phenotype, characterized by suppression of the AKT-inhibited FOXO1 and its target genes, including the TIE2 antagonist ANGPT2. An imbalance in TIE2 ligands, likely exacerbated by aberrant recruitment of smooth muscle cells producing the agonist ANGPT1, increased TIE2 activity in both mouse and human VMs. While mTOR blockade had limited effects on advanced VMs in mice, inhibiting TIE2 or ANGPT effectively suppressed their growth. These findings uncover a PI3K-FOXO1-ANGPT-TIE2 circuit as a core driver of PIK3CA-related VMs and highlight TIE2 as a promising therapeutic target.

Parkinson's disease (PD) imposes a significant health burden among older adults and may be related to zinc and ring finger 2 (ZNRF2)-a member of the ubiquitination family. To investigate the role and mechanism of action of ZNRF2 in the regulation of mammalian target of rapamycin (mTOR)-mediated neuroinflammation in a mouse model of PD. Healthy mice were injected intraperitoneally with either saline (control) or MPTP 30 mg/kg. Mouse behavior was tested using rotarod and open field tests. The distribution and expression of tyrosine hydroxylase (TH) were determined by immunoblotting and immunohistochemistry. Inflammatory factors were evaluated using immunoblotting, enzyme-linked immunosorbent assay, and immunofluorescence assay. Compared with mice injected with saline, MPTP-treated mice showed significantly impaired locomotor activity, a significant decrease in the number of TH neurons, and a markedly altered morphology. ZNRF2 expression was significantly increased in the mesencephalon of MPTP-treated mice compared to that in control mice. ZNRF2 knockdown exacerbated motor dysfunction, accelerated dopamine neuron degeneration and death, increased the levels of pro-inflammatory factors (e.g., interleukin (IL)-1β, IL-6), and suppressed the expression of anti-inflammatory factors (e.g., IL-4, IL-10) in the central nervous system of MPTP-treated mice, with more pronounced activation of microglia and astrocytes. ZNRF2 knockdown significantly elevated phosphorylated mTOR protein levels after MPTP treatment; subsequently, phosphorylated mTOR protein levels were inhibited; dyskinesia and dopamine neuronal damage were significantly ameliorated, and neuroinflammation was suppressed in PD mice. ZNRF2 regulates the pathogenesis of MPTP-induced PD in mice via mechanisms related to mTOR-mediated neuroinflammation.

Lung cancer remains the leading cause of cancer-related mortality globally, with lung adenocarcinoma (LUAD) being the most prevalent subtype. Despite extensive research efforts, the role of transcription factors in LUAD progression remains largely uncharacterized. In this study, we focused on ZNF266, a transcription factor whose impacts on LUAD have not been investigated.

Using high-throughput sequencing data, we observed a significant downregulation of ZNF266 expression in LUAD tissues. To validate this finding, we conducted a retrospective analysis of nearly three thousand LUAD patients' data from public databases and our institution. Functional studies were performed using cell lines, organoids, and xenograft models to assess the role of ZNF266 in LUAD progression. RNA sequencing, chromatin immunoprecipitation, DNA pull-down assays, and dual-luciferase reporter assays were employed to elucidate the underlying mechanism. Additionally, adeno-associated virus (AAV)-mediated overexpression of ZNF266 was used to evaluate its therapeutic potential.

Patients with low ZNF266 expression had poorer prognosis compared to those with high expression. ZNF266 inhibits the malignant phenotypes of LUAD, including proliferation, migration, and invasion. Mechanistically, ZNF266 binds to the promoter region of CA9, suppressing its transcription. This leads to a reduction in intracellular pH and subsequent inhibition of the mTOR signaling pathway, which is crucial for cancer cell growth and survival. Furthermore, AAV-mediated overexpression of ZNF266 significantly inhibited tumor growth in patient-derived xenograft models.

Our study demonstrated that ZNF266 inhibits LUAD progression in a pH-dependent manner via modulating CA9 expression, uncovering its therapeutic significance for LUAD treatment.

The lymphatic vasculature comprises lymphatic capillaries and collecting vessels. To support lymphatic development, lymphatic endothelial cells (LECs) utilize nutrients to fuel lymphangiogenic processes. Meanwhile, LECs maintain constant prospero homeobox 1 (PROX1) expression critical for lymphatic specification. However, molecular mechanisms orchestrating nutrient metabolism while sustaining PROX1 levels in LECs remain unclear. Here, we show that loss of RAPTOR, an indispensable mechanistic target of rapamycin complex 1 (mTORC1) component, downregulates PROX1 and impairs lymphatic capillary growth and differentiation of collecting lymphatics in mice. Mechanistically, mTORC1 inhibition in mouse and human LECs causes Myc reduction, which decreases hexokinase 2 (HK2) and glutaminase (GLS), inhibiting glycolysis and glutaminolysis. Myc or HK2/GLS ablation impedes lymphatic capillary and collecting vessel formation. Interestingly, mTORC1 regulation of PROX1 is independent of Myc-HK2/GLS signaling. Moreover, genetic interaction analysis indicates that Myc and PROX1 play crucial roles in mTORC1-regulated lymphatic development. Collectively, our findings identify mTORC1 as a key regulator of metabolic programs and PROX1 expression during lymphangiogenesis.

Metabolic reprogramming is a hallmark of cancer and de novo lipogenesis (DNL) accelerates the progression of ovarian cancer. In this study, we investigated the effects of cardamonin, a natural compound potential to suppress various malignancies, on the lipid anabolism in ovarian cancer. Cell proliferation was assessed using CCK-8 and clone formation assay. Cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI staining and mitochondrial membrane potential (MMP) was measured with JC-10 probe. Free fatty acids (FFA) was measured by fluorescence using acyl-CoA oxidation and carnitine palmitoyl transferase-1 (CPT-1) activity was analyzed by spectrophotometric assay using palmitoyl-CoA and DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) reaction. mRNA expression was measured by Quantitative Real-Time PCR. Protein expression was analyzed through western blotting and immunofluorescence. Raptor was knocked down by shRNA and Raptor was overexpressed by lentiviral transfection. The antitumor effect of cardamonin was evaluated using a xenotransplantation tumor bearing mouse model. Cardamonin suppressed the cell proliferation, induced cell apoptosis and triggered mitochondrial damage in ovarian cancer cells. Cardamonin inhibited the protein expression of sterol regulatory element binding protein 1 (SREBP1) and its downstream lipogenic enzymes and decreased FFA content and CPT-1 activity. Additionally, cardamonin inhibited the activation of mechanistic target of rapamycin complex 1 (mTORC1) and expression of regulatory-associated protein of mTOR (Raptor). Raptor knockdown abolished the inhibitory effect of cardamonin on mTORC1 and SREBP1. Furthermore, cardamonin inhibited mTORC1 activation and lipogenic proteins expression induced by Raptor overexpression. Cardamonin reduced the tumor growth and fatty acid synthase of the tumors, as evidenced by decreased expression of Ki-67 and FASN. It suggests that cardamonin suppresses mTORC1/SREBP1 through reducing the protein level of Raptor and inhibits DNL of ovarian cancer.

Syndecans (SDCs) are glycosaminoglycan-containing cell surface proteins with diverse functions in the immune system with SDC1 (CD138) and SDC4 expressed in B-lineage cells. Here, we show that stem cells lacking either molecule generate fewer B-cell progenitors but give rise to mature B cells in vivo. Deletion of the plasma cell "marker" CD138 has no effect on homeostatic or antigen-induced plasma cell formation. Naive B cells express high SDC4 and encounter with cognate antigen results in transient CD138 upregulation and SDC4 loss, both further modulated by IL-4, IL-21, and CD40 ligation. SDC4 is downregulated on germinal center B cells and absent on most memory B cells. Glycosaminoglycans such as those attached to SDCs, and heparin, a commonly used therapeutic, regulate survival and activation of naive B cells by limiting responsiveness to cognate antigen. Conversely, ablation of SDC4 results in increased baseline and antigen-induced B-cell activation. Collectively, our data reveal B-cell activation- and subset-dependent SDC expression and show that SDC4 and GAGs can limit antigen-induced activation to promote B-cell survival and expansion.

The tuberous sclerosis complex (TSC1/TSC2/TBC1D7) primarily functions to inhibit the mechanistic target of rapamycin complex 1 (mTORC1), a crucial regulator of cell growth. Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder marked by benign tumors in multiple organs that rarely progress to malignancy. Traditionally, TSC proteins are considered tumor suppressive due to their inhibition of mTORC1 and other mechanisms. However, more recent studies have shown that TSC proteins can also promote tumorigenesis in certain cancer types. In this review, we explore the composition and function of the TSC protein complex, the roles of its individual components in cancer biology, and potential future therapeutic targeting strategies.

Ependymal cells are multiciliated glial cells lining the ventricles of the mammalian brain. Their differentiation from progenitor cells involves cell enlargement and progresses through centriole amplification phases and ciliogenesis. These phases are accompanied by the sharp up-regulation of mTOR Complex 1 activity (mTORC1), a master regulator of macromolecule biosynthesis and cell growth, whose function in ependymal cell differentiation is unknown. We demonstrate that mTORC1 inhibition by rapamycin preserves the progenitor pool by reinforcing quiescence and preventing alternative cell cycle progression for centriole amplification. Overexpressing E2F4 and MCIDAS circumvents mTORC1-regulated processes, enabling centriole amplification despite rapamycin, and enhancing mTORC1 activity through positive feedback. Acute rapamycin treatment in multicentriolar cells during the late phases of differentiation causes centriole regrouping, indicating a direct role of mTORC1 in centriole dynamics. By phosphoproteomic and phosphomutant analysis, we reveal that the mTORC1-mediated phosphorylation of GAS2L1, a centrosomal protein that links actin and microtubule cytoskeletons, participates in centriole disengagement. This multilayered and sequential control of ependymal development by mTORC1, from the progenitor pool to centriolar function, has implications for pathophysiological conditions like aging and hydrocephalus-prone genetic diseases.

Insect fat body serves as a central hub for energy mobilization and protein synthesis. During larval metamorphosis, fat body undergoes programmed cell death and tissue disassembly. Following adult eclosion, fat body reconstructs with cell proliferation and becomes competent for large-scale vitellogenin (Vg) synthesis required for the maturation of dozens of eggs.

This study aims to uncover the molecular mechanisms underlying the remodeling of fat body in acquisition of competence for massive Vg production.

RNA-seq and metabolomics were used for identification of differentially expressed genes and metabolites. RNAi was applied for gene knockdown. Transmission electron microscope, MitoTracker staining, mitochondrial DNA quantification, ATP and citrate synthase assays were employed for examining mitochondrial biogenesis. Dual-luciferase reporter assay and EMSA were performed for transcriptional regulation. qRT-PCR and western blot were performed for measuring Vg synthesis.

Transcriptomic and metabolomic analyses revealed significant upregulation of genes and metabolites involved in mitochondrial biogenesis in the fat body of adult locusts. PGC-1α was highly expressed in adult fat body. Knockdown of PGC-1α reduced mitochondrial biogenesis, fat body cell number, Vg synthesis and ovarian development. CREBB bound to PGC-1α promoter and activated its transcription. CREBB depletion impaired mitochondrial biogenesis and fat body remodeling. Moreover, loss of TORC1 function suppressed CREBB function and PGC-1α expression, subsequently disrupting mitochondrial biogenesis and fat body remodeling. Juvenile hormone (JH) deprivation also decreased CREBB function and PGC-1α expression, which was reversible with JH treatment. Our results suggest that TORC1 and JH coordinate CREBB-upregulated PGC-1α expression, which promotes mitochondrial biogenesis and fat body remodeling for Vg synthesis and egg production.

The findings provide new insights into the molecular mechanisms of post-metamorphic fat body development, and highlight the role of JH/TORC1/CREBB/PGC-1α/mitochondrial biogenesis axis in insect reproduction. The data also offer potential targets for insect pest control.

Aberrant activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome increases the release of mature pro-inflammatory cytokines interleukin (IL)-1β and IL-18, and enhances pyroptosis; thereby necessitating tight regulation of the NLRP3 inflammasome. Dysfunctional glutamine metabolism contributes to the pathogenesis of multiple inflammatory disorders, and the precise mechanism remains to be elucidated. Here, we provide evidence that glutamine deprivation enhances NLRP3 inflammasome activation in macrophages. Indeed, the absence of exogenous glutamine specifically enhanced NLRP3 inflammasome assembly, thereby accelerating pyroptosis and promoting the maturation of IL-1β and IL-18. Inhibition of glutaminolysis exhibited a similar effect to glutamine deprivation, whereas this effect was reversed by α-ketoglutarate (α-KG), a tricarboxylic acid (TCA)-cycle intermediate that can be replenished by glutamine supply. We further observed reduced generation of endogenous itaconate by glutamine deprivation and verified that both exogenous supplementation of itaconate derivative and increased endogenous itaconate production by overexpressing immune-responsive gene 1 [IRG1; also known as aconitate decarboxylase 1 (ACOD1)] could replace glutamine to inhibit the NLRP3 inflammasome. Mechanistically, glutamine deprivation decreased the source of substrate and inhibited transcription factor EB (TFEB)-dependent transcriptional upregulation of IRG1, thereby impairing the IRG1/itaconate axis that suppresses the NLRP3 inflammasome. Furthermore, glutamine deficiency was detected in a murine sepsis model, whereas extrinsic glutamine supplementation conferred protection against intestinal inflammation and tissue damage in septic mice. Taken together, our findings provide a novel insight into the link between glutamine metabolism and NLRP3 inflammasome activation, highlighting the target of glutamine metabolism, which holds as a potential therapeutic strategy for inflammatory diseases.

The transmembrane and coiled-coil domains 3 (TMCO3) are highly expressed in many tumors. However, the underlying mechanisms governing the way in which TMCO3 affects the progression of hepatocellular carcinoma (HCC) remain unclear. This study screens out the molecule TMCO3 with high N6-methyladenosine (m6A) modification level in tumor samples compared to the adjacent non-cancerous tissues of three pairs of HCC patients through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and RNA sequencing (RNA-seq). Subsequently, the oncogenic effect of TMCO3 in HCC is verified through in vivo and in vitro experiments. AlkB Homolog 5 (ALKBH5), an m6A demethylase of TMCO3 is then screened out. The following experiments demonstrate that TMCO3 can activate AKT directly through the Phosphatidylinositol-3-Kinase (PI3K) pathway, thus promoting the progression of HCC. Meanwhile, the phosphorylation site on TMCO3: the 85th amino acid-serine, and mutation of this site can directly impair the activity and membrane translocation of AKT is found. Finally, the carcinogenic effect of TMCO3 is further elucidated in HCC through the orthotopic treatment model and the hydrodynamic tail vein injection treatment model. The findings can provide a potential target for targeted AKT treatment in patients with HCC and verify a possible prognostic marker in HCC.

Colorectal cancer is the second most common cause of cancer mortality worldwide, and liver metastasis is the major cause of death of patients with colorectal cancer. Dysfunctional E3 ligase activity has recently been shown to be associated with colorectal cancer. However, the key E3 ligases affecting colorectal cancer liver metastasis remain unknown. Therefore, an shRNA library targeting 156 E3 ubiquitin ligases has been used to perform an in vivo loss-of-function screen of a human colorectal cancer cell line in a mouse model of liver metastasis. The screen reveals that neural precursor cell expressed developmentally down-regulated gene 4-like (NEDD4L) knockdown promotes colorectal cancer liver metastasis. Mechanistic studies reveal that NEDD4L binds to the PPNAY motif in protein arginine methyltransferase 5 (PRMT5) and ubiquitinates PRMT5 to promote its degradation. PRMT5 degradation attenuates the arginine methylation of AKT1 to inhibit the AKT/mTOR signaling pathway. The effect of NEDD4L decreases colorectal cancer cell proliferation to suppress colonization. This study is the first to show that PRMT5 is a substrate of NEDD4L and reveals not only the metastasis-inhibiting function of NEDD4L but also a novel mechanism by which NEDD4L prevents colorectal cancer liver metastasis. These findings may provide a new preventive strategy for liver metastasis.

Human cytomegalovirus (HCMV) and LINE-1 (L1) can co-inhabit a common host and closely interact with each other within a single cell. We have previously shown that HCMV exploits this opportunistic interaction by upregulating L1 expression that promotes its own productive life cycle by facilitating HCMV DNA replication. However, the mechanism by which HCMV increases L1 expression remains unknown. Here, we report that HCMV infection functionally inactivates KRAB-associated protein 1 (KAP1), a key epigenetic repressor of L1, through phosphorylation. HCMV infection of cells activates mTOR kinase that phosphorylates S824 residue of KAP1 and reduces its epigenetic repressive function, leading to increased chromatin accessibility of L1 promoter region. Treatment of potent mTOR inhibitor to the HCMV-infected cells was sufficient to reduce KAP1 phosphorylation and block L1 expression. Furthermore, cells infected with a mutant virus lacking UL38, an HCMV mTOR pathway activator, showed reduced KAP1 S824 phosphorylation and abolished L1 expression. Our results highlight the synergistic interaction between HCMV and L1 where HCMV UL38 serves as a primary viral regulator of L1 expression by upregulating the mTOR-KAP1 pathway.

Oncogenic mutations in EGFR often result in EGF-independent constitutive activation and aberrant trafficking and are associated with several human malignancies, including non-small cell lung cancer. A major consequence of EGFR mutations is the activation of the mechanistic target of rapamycin complex 1 (mTORC1), which requires EGFR kinase activity and downstream PI3K/AKT signaling, resulting in increased cell proliferation. However, recent studies have elucidated kinase-independent roles of EGFR in cell survival and cancer progression. Here, we report a cis mTORC1 activation function of EGFR that is independent of its kinase activity. Our results reveal that lysosomal localization of EGFR is critical to mTORC1 activation, where EGFR physically binds Rheb, acting as a guanine exchange factor (GEF) for Rheb, with its Glu804 serving as a potential glutamic finger. Genetic knock-in of EGFR-E804K in cells reduces the level of GTP-bound Rheb, and significantly suppresses mTORC1 activation, cell proliferation and tumor growth. Different tyrosine kinase inhibitors exhibit distinct effects on EGFR-induced mTORC1 activation, with afatinib, which additionally blocks EGFR's GEF activity, causing a much greater suppression of mTORC1 activation and cell growth, and erlotinib, which targets only kinase activity, resulting in only a slight decrease. Moreover, a novel small molecule, BIEGi-1, was designed to target both the Rheb-GEF and kinase activities of EGFR, and shows a strong inhibitory effect on the viability of cells harboring EGFR mutants. These findings unveil a fundamental event in cell growth and suggest a promising strategy against cancers with EGFR mutations.

Phase separation, particularly liquid-liquid phase separation (LLPS), has emerged as a powerful tool in biological research, offering unique advantages for visualizing and analyzing biomolecular interactions. This review highlights recent advances in leveraging LLPS to develop experimental techniques for studying protein-protein interactions (PPIs), protein-RNA interactions, and enzyme activity. The integration of LLPS with advanced techniques has expanded its applications, offering new possibilities for unraveling the complexities of cellular function and disease mechanisms. Looking forward, the development of more versatile, sensitive, and targeted LLPS-based methods is poised to transform molecular biology, providing deeper insights into cellular dynamics and facilitating therapeutic advancements.

The PI3K/AKT/mTOR pathway is considered a key therapeutic target in triple-negative breast cancer (TNBC). In this issue of Developmental Cell, Remy et al. challenge this idea by demonstrating that mTORC1 inhibition activates TFEB, promoting MT1-MMP exocytosis, ECM degradation, and increased cell invasion, especially when combined with chemotherapy.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently hyperactivated in triple-negative breast cancers (TNBCs) associated with poor prognosis and is a therapeutic target in breast cancer management. Here, we describe the effects of repression of mTOR-containing complex 1 (mTORC1) through knockdown of several key mTORC1 components or with mTOR inhibitors used in cancer therapy. mTORC1 repression results in an ∼10-fold increase in extracellular matrix proteolytic degradation. Repression in several TNBC models, including in patient-derived xenografts (PDXs), induces nuclear translocation of transcription factor EB (TFEB), which drives a transcriptional program that controls endolysosome function and exocytosis. This response triggers a surge in endolysosomal recycling and the surface exposure of membrane type 1 matrix metalloproteinase (MT1-MMP) associated with invadopodia hyperfunctionality. Furthermore, repression of mTORC1 results in a basal-like breast cancer cell phenotype and disruption of ductal carcinoma in situ (DCIS)-like organization in a tumor xenograft model. Altogether, our data call for revaluation of mTOR inhibitors in breast cancer therapy.

Skeletal muscle is the most abundant tissue in the human body and is responsible for movement, metabolism, energy production and longevity. Muscle atrophy is a frequent complication of several diseases and occurs when protein degradation exceeds protein synthesis. Genetics, ageing, nerve injury, weightlessness, cancer, chronic diseases, the accumulation of metabolic byproducts and other stimuli can lead to muscle atrophy. Muscular dystrophy is a neuromuscular disorder, part of which is caused by the deficiency of dystrophin protein and is mostly related to genetics. Muscle atrophy and muscular dystrophy are accompanied by dynamic changes in transcriptomic, translational and epigenetic regulation. Multiple signalling pathways, such as the transforming growth factor-β (TGF-β) signalling pathway, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway, inflammatory signalling pathways, neuromechanical signalling pathways, endoplasmic reticulum stress and glucocorticoids signalling pathways, regulate muscle atrophy. A large number of long noncoding RNAs (lncRNAs) have been found to be abnormally expressed in atrophic muscles and dystrophic muscles and regulate the balance of muscle protein synthesis and degradation or dystrophin protein expression. These lncRNAs may serve as potential targets for treating muscle atrophy and muscular dystrophy. In this review, we summarized the known lncRNAs related to muscular dystrophy and muscle atrophy induced by denervation, ageing, weightlessness, cachexia and abnormal myogenesis, along with their molecular mechanisms. Finally, we explored the potential of using these lncRNAs as therapeutic targets for muscle atrophy and muscular dystrophy, including the methods of discovery and clinical application prospects for functional lncRNAs.

mTORC1/2 play central roles as signaling hubs of cell growth and metabolism and are therapeutic targets for several diseases. However, the human genetic evidence linking mutations of mTORC1/2 to obesity remains elusive. Using whole-exome sequencing of 1944 cases with severe obesity and 2161 healthy lean controls, we identify a rare RICTOR p.I116V variant enriched in 9 unrelated cases. In Rictor null mouse embryonic fibroblasts, overexpression of the RICTOR p.I116V mutant increases phosphorylation of AKT, a canonical mTORC2 substrate, compared with wild-type RICTOR, indicating a gain-of-function change. Consistent with the human obesity phenotype, the knock-in mice carrying homogenous Rictor p.I116V variants gain more body weight under a high-fat diet. Additionally, the stromal vascular fraction cells derived from inguinal white adipose tissue of knock-in mice display an enhanced capacity for adipocyte differentiation via AKT activity. These findings demonstrate that the rare gain-of-function RICTOR p.I116V mutation activates AKT signaling, promotes adipogenesis, and contributes to obesity in humans.

Lipids are an important energy source and are utilized as substrates for various physiological processes in insects. Comparative gene identification 58 (CGI-58), also known as α/β hydrolase domain-containing 5 (ABHD5), is a highly conserved and multifunctional gene involved in regulating lipid metabolism and cellular energy balance in many organisms. However, the biological functions of ABHD5 in insects are poorly understood. In the current study, we describe the identification and characterization of the ABHD5 gene in the lepidopteran model insect, Bombyx mori. The tissue expression profile investigated using quantitative reverse transcription polymerase chain reaction (RT-qPCR) reveals that BmABHD5 is widely expressed in all tissues, with particularly high levels found in the midgut and testis. A binary transgenic CRISPR/Cas9 system was employed to conduct a functional analysis of BmABHD5, with the mutation of BmABHD5 leading to the dysregulation of lipid metabolism and excessive lipid accumulation in the larval midgut. Histological and physiological analysis further reveals a significant accumulation of lipid droplets in the midgut of mutant larvae. RNA-seq and RT-qPCR analysis showed that genes related to metabolic pathways were significantly affected by the absence of BmABHD5. Altogether, our data prove that BmABHD5 plays an important role in regulating tissue-specific lipid metabolism in the silkworm midgut.

Major depressive disorder, also known as MDD, affects more than 264 million people globally, making it a prevalent and critical health challenge. Traditional treatments show limited efficacy in many patients. Therefore, exploring new treatment methods is particularly crucial. Mitophagy, as a regulatory process, can help understand and treat MDD. This paper focuses on the molecular mechanisms of mitophagy, starting from proteins and related pathways, and its role in MDD. The study also explores the associations between mitophagy and neuroinflammation, oxidative stress, neurotransmitter synthesis, and neuroplasticity in MDD and discusses the progress of clinical research on the role of mitophagy in MDD. In addition, the article describes the current pharmaceutical and non-pharmaceutical interventions that can regulate mitophagy in MDD and unravels the potential and challenges of these therapeutic strategies in clinical settings. This article offers a deeper insight into the pathogenesis of MDD and offers a scientific basis for the development of new treatment strategies.

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.

Rescue therapies of relapsed/refractory (r/r) Hodgkin's lymphoma (HL) in the third to sixth-line provide major, yet unresolved problems. The MEPED regimen includes nuclear receptor agonists such as pioglitazone and dexamethasone, which counterbalance HL homeostasis, HL stress response inhibitors, everolimus and COX-2 inhibitor, and a stress response inducer, low-dose metronomic treosulfan. CR (six of seven patients) and long-term cCR in patients receiving no consolidating allogeneic stem cell transplantation highlight MEPED as a potent salvage therapy in advanced refractory HL. MEPED edits everolimus activities in such a way that mTORC1 becomes a non-oncogene addiction bottleneck, hence determining long-term therapy outcome. The implications of the therapeutic paradigm shift toward editing of HL tissue, and particularly mTOR addiction, could prove to be profound for clinical practice, both in terms of outcome and treatment tolerability. The long-term results of MEPED treatment indicate the urgent evaluation of the schedule in a multicenter trial for r/r HL.