Dietary restriction reveals sex-specific expression of the mTOR pathway genes in Japanese quails.

Limited resources affect an organism's physiology through the conserved metabolic pathway, the mechanistic target of rapamycin (mTOR). Males and females often react differently to nutritional limitation, but whether it leads to differential mTOR pathway expression remains unknown. Recently, we found that dietary restriction (DR) induced significant changes in the expression of mTOR pathway genes in female Japanese quails (Coturnix japonica). We simultaneously exposed 32 male and female Japanese quails to either 20%, 30%, 40% restriction or ad libitum feeding for 14 days and determined the expression of six key genes of the mTOR pathway in the liver to investigate sex differences in the expression patterns. We found that DR significantly reduced body mass, albeit the effect was milder in males compared to females. We observed sex-specific liver gene expression. DR downregulated mTOR expression more in females than in males. Under moderate DR, ATG9A and RPS6K1 expressions were increased more in males than in females. Like females, body mass in males was correlated positively with mTOR and IGF1, but negatively with ATG9A and RS6K1 expressions. Our findings highlight that sexes may cope with nutritional deficits differently and emphasise the importance of considering sexual differences in studies of dietary restriction.

Endosymbiont Tremblaya phenacola influences the reproduction of cotton mealybugs by regulating the mechanistic target of rapamycin pathway.

The intricate evolutionary dynamics of endosymbiotic relationships result in unique characteristics among the genomes of symbionts, which profoundly influence host insect phenotypes. Here, we investigated an endosymbiotic system in Phenacoccus solenopsis, a notorious pest of the subfamily Phenacoccinae. The endosymbiont, "Candidatus Tremblaya phenacola" (T. phenacola PSOL), persisted throughout the complete life cycle of female hosts and was more active during oviposition, whereas there was a significant decline in abundance after pupation in males. Genome sequencing yielded an endosymbiont genome of 221.1 kb in size, comprising seven contigs and originating from a chimeric arrangement between betaproteobacteria and gammaproteobacteria. A comprehensive analysis of amino acid metabolic pathways demonstrated complementarity between the host and endosymbiont metabolism. Elimination of T. phenacola PSOL through antibiotic treatment significantly decreased P. solenopsis fecundity. Weighted gene coexpression network analysis demonstrated a correlation between genes associated with essential amino acid synthesis and those associated with host meiosis and oocyte maturation. Moreover, altering endosymbiont abundance activated the host mechanistic target of rapamycin pathway, suggesting that changes in the amino acid abundance affected the host reproductive capabilities via this signal pathway. Taken together, these findings demonstrate a mechanism by which the endosymbiont T. phenacola PSOL contributed to high fecundity in P. solenopsis and provide new insights into nutritional compensation and coevolution of the endosymbiotic system.

PI3K/AKT/mTORC1 signalling pathway regulates MMP9 gene activation via transcription factor NF-κB in mammary epithelial cells of dairy cows.

Matrix metalloproteinase 9 (MMP9) plays a pivotal role in mammary ductal morphogenesis, angiogenesis and glandular tissue architecture remodeling. However, the molecular mechanism of MMP9 expression in mammary epithelial cells of dairy cows remains unclear. This study aimed to explore the underlying mechanism of MMP9 expression. In this study, to determine whether the PI3K/AKT/mTORC1/NF-κB signalling pathway participates in the regulation of MMP9 expression, we treated mammary epithelial cells with specific pharmacological inhibitors of PI3K (LY294002), mTORC1 (Rapamycin) or NF-κB (Celastrol), respectively. Western blotting results indicated that LY294002, Rapamycin and Celastrol markedly decreased MMP9 expression and P65 nuclear translocation. Furthermore, we found that NF-κB (P65) overexpression resulted in elevated expression of MMP9 protein and activation of MMP9 promoter. In addition, we observed that Celastrol markedly decreases P65-overexpression-induced MMP9 promoter activity. Moreover, the results of the promoter assay indicated that the core regulation sequence for MMP9 promoter activation may be located at -420 ∼ -80 bp downstream from the transcription start site. These observations indicated that the PI3K/AKT/mTORC1 signalling pathway is involved in MMP9 expression by regulating MMP9 promoter activity via NF-κB in the mammary epithelial cells of dairy cows.

Impact of the TOR pathway on plant growth via cell wall remodeling.

Plant growth is intimately linked to the availability of carbon and energy status. The Target of rapamycin (TOR) pathway is a highly relevant metabolic sensor and integrator of plant-assimilated C into development and growth. The cell wall accounts for around a third of the cell biomass, and the investment of C into this structure should be finely tuned for optimal growth. The plant C status plays a significant role in controlling the rate of cell wall synthesis. TOR signaling regulates cell growth and expansion, which are fundamental processes for plant development. The availability of nutrients and energy, sensed and integrated by TOR, influences cell division and elongation, ultimately impacting the synthesis and deposition of cell wall components. The plant cell wall is crucial in environmental adaptation and stress responses. TOR senses and internalizes various environmental cues, such as nutrient availability and stresses. These environmental factors influence TOR activity, which modulates cell wall remodeling to cope with changing conditions. Plant hormones, including auxins, gibberellins, and brassinosteroids, also regulate TOR signaling and cell wall-related processes. The connection between nutrients and cell wall pathways modulated by TOR are discussed.

Suvorexant improves mitochondrial dynamics with the regulation of orexinergic and mTOR activation in rats exhibiting PTSD-like symptoms.

BACKGROUND: Increasing evidence suggests that mitochondrial dysfunction plays a significant role in PTSD. However, the exact mechanism is still unclear. Mitochondrial dynamics could be one of the mechanisms, as it is crucial for mitochondrial homeostasis and is widely affected in traumatic situations. Mitochondrial dynamics regulate mitochondrial homeostasis via orexinergic receptors, and it is shown that antagonism of orexinergic receptors attenuates PTSD-like symptoms. Therefore, the present study aimed to determine how orexin antagonists affect mitochondrial dynamics in rats exhibiting PTSD-like symptoms. METHODS: Using rats, a stress-re-stress (SRS) model with PTSD-like symptoms was established. On day 2 (D-2), the animals were exposed to variable stressors including 2 h of restraint followed by brief mild foot shock and exposure to 4%halothane. Foot shock was performed as a re-stress from D-8 to D-32 at six-day intervals. RESULTS: SRS exposure caused PTSD-like phenotype, hypothalamic-pituitary-adrenal axis dysfunction, activation of mammalian target of rapamycin (mTOR), and mitochondrial-fission-process-1 (MTFP-1). SRS-subjected rats exhibited enhanced expression of fission-regulating proteins, including dynamin-related protein-1 and mitochondrial-fission-protein-1 and reduced expression of fusion-regulating proteins, including optic-atrophy-1 and mitofusin-2, in the amygdala. TEM analysis revealed that SRS exposure further damaged the mitochondria. Treatment with suvorexant with rapamycin significantly mitigated PTSD-like symptoms and improved mitochondrial dynamics in SRS-exposed rats. However, their combination showed a more pronounced effect. Further, suvorexant in combination with rapamycin significantly mitigated mTOR and MTFP-1 activation. Sertraline attenuated PTSD-like symptoms without affecting SRS-induced activation of mTOR and disparity in mitochondrial dynamics. Suvorexant pharmacological effects on mitochondrial biogenesis also involve the mTOR pathway. LIMITATION: The role of orexinergic pathway in SRS-induced mitochondrial mitophagy was not explored. CONCLUSIONS: Targeting both the orexinergic and mTOR pathways might exert a beneficial synergistic effect for treating PTSD.

The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells.

BACKGROUND: Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive. RESULTS: Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein-protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles. CONCLUSIONS: These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically).

Elevated Level of PINK1/Parkin-Mediated Mitophagy Pathway Involved to the Inhibited Activity of Mitochondrial Superoxide Dismutase in Rat Brains and Primary Hippocampal Neurons Exposed to High Level of Fluoride.

To reveal the molecular mechanism of brain damage induced by chronic fluorosis, expression of PTEN-induced kinase 1 (PINK1)/parkin RBR E3 ubiquitin-protein ligase (Parkin)-mediated mitophagy pathway and activity of mitochondrial superoxide dismutase (SOD) were investigated in rat brains and primary cultured neurons exposed to high level of fluoride. Sprague-Dawley (SD) rats were treated with fluoride (0, 5, 50, and 100 ppm) for 3 and 6 months. The primary neurons were exposed to 0.4 mM (7.6 ppm) fluoride and thereafter treated with 100 nM rapamycin (a stimulator of mitophagy) or 50 µM 3-methyladenine (3-MA, an inhibitor of mitophagy) for 24 h. The expressions of PINK1/Parkin at the protein level and the activity of SOD in mitochondria of rat brains and cultured neurons were determined by Western blotting and biochemical method, respectively. The results showed that the rats exposed to fluoride exhibited different degrees of dental fluorosis. In comparison to controls, the expressions of PINK1 and Parkin were significantly higher in the rat brains and primary neurons exposed to high fluoride. In addition, a declined activity of mitochondrial SOD was determined. Interestingly, rapamycin treatment enhanced but 3-MA inhibited the changes of PINK1/Parkin pathway and SOD activity, and the correlations between the inhibited SOD activity and the elevated PINK1/Parkin proteins were observed. The results suggest that the inhibition of mitochondrial SOD activity induced by fluorosis may stimulate the expressions of mitophagy (PINK1/ Parkin) pathway to maintain the mitochondrial homeostasis.

Role of mammalian target of rapamycin in the formation and progression of retinopathy of prematurity-like vascular abnormalities in neonatal rats.

Retinopathy of prematurity (ROP), a retinal disease that can occur in premature infants, can lead to severe visual impairment. In this study, we examined the preventive and therapeutic effects of mammalian target of rapamycin complex 1 (mTORC1) inhibition on abnormal retinal blood vessels in a rat model of ROP. To induce ROP-like vascular abnormalities, rats were subcutaneously treated with KRN633, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, on postnatal day 7 (P7) and P8. KRN633-treated (ROP) rats were treated subcutaneously with the mTORC1 inhibitor rapamycin according to preventive and therapeutic protocols, i.e., from P11 to P13 (P11-P13) and from P14 to P20 (P14-P20), respectively. To compare with the effects of VEGF inhibition, KRN633 was administered according to similar protocols. Changes in retinal vasculature, phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, and the proliferative status of vascular cells were evaluated at P14 and P21 using immunohistochemistry. Rapamycin treatment from P11 to P13 prevented increases in arteriolar tortuosity, capillary density, and the number of proliferating vascular cells, and eliminated pS6 immunoreactivity in ROP rats. KRN633 treatment at P11 and P12 (P11/P12) also prevented the appearance of ROP-like retinal blood vessels. Rapamycin treatment from P14 to P20 failed to attenuate arteriolar tortuosity but prevented increases in capillary density and proliferating vascular cell number at the vascular front, but not at the central zone. KRN633 treatment from P14 to P20 significantly reduced abnormalities in the retinal vasculature; however, the effects were inferior to those of KRN633 treatment on P11/P12. These results suggest that activation of the mTORC1 pathway in proliferating endothelial cells contributes to the appearance and progression of ROP-like retinal blood vessels. Therefore, inhibition of mTORC1 may be a promising approach for selectively targeting abnormal retinal blood vessels in ROP.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 µM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Rapamycin extenuates experimental colitis by modulating the gut microbiota.

BACKGROUND AND AIM: Autophagy and gut microbiota correlates closely with the inflammatory bowel disease. Herein, we aimed to study the roles of rapamycin on the gut microbiota in inflammatory bowel disease. METHODS: Acute colitis was induced with dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzenesulfonic acid solution in mice. Mice were administered with rapamycin or hydroxychloroquine. Weight loss, disease activity index scores, histopathological score, serum inflammatory cytokines, intestinal permeability, and colonic autophagy-related proteins were detected. Cecal content was also preserved in liquid nitrogen and subsequently analyzed following the 16S DNA sequencing. The antibiotic cocktail-induced microbiome depletion was performed to further investigate the relationship between autophagy activation and gut microbiota. RESULTS: Compared with the control group, the colonic autophagy-related proteins of P62, mTOR, and p-mTOR increased significantly, while the levels of LC3B and ATG16L1 decreased (all P < 0.05) in the model group. After rapamycin intervention, the colonic pathology of mice improved, while the disease activity index score decreased substantially; the colon length increased, and the expression of IL-6 and TNF-α decreased. Following hydroxychloroquine treatment, some indicators suggested aggravation of colitis. Principal coordinates analysis showed that the DSS group was located on a separate branch from the rapamycin group but was closer to the hydroxychloroquine group. Compared with the DSS group, the rapamycin group was associated with higher abundances of f_Lactobacillaceae (P = 0.0151), f_Deferribacteraceae (P = 0.0290), g_Lactobacillus (P = 0.0151), g_Mucispirillum (P = 0.0137), s_Lactobacillus_reuteri (P = 0.0028), and s_Clostridium_sp_Culture_Jar-13 (P = 0.0082) and a lower abundance of s_Bacteroides_sartorii (P = 0.0180). Linear discriminant analysis effect size showed that rapamycin increased the abundances of Lactobacillus-reuteri, Prevotellaceae, Paraprevotella, Christensenella and Streptococcus and decreased those of Peptostreptococcaceae and Romboutsia Bacteroides-sartorii. Besides, the improvement effect of autophagy activation on colitis disappears following gut microbiome depletion. CONCLUSION: The therapeutic effects of rapamycin on extenuating experimental colitis may be related to the gut microbiota.

Autophagy protects against vocal fold injury-induced fibrosis.

Vocal fold scar formation due to vocal fold injury (VFI) is a common cause of surgery or trauma-induced voice disorders. Severe scar formation can lead to reduced voice quality or even be life-threatening. Here, we investigated the role of autophagy in VFI, focusing on fibrosis as a consequence of autophagy in inducing VFI. A VFI model was constructed in rats by dissecting the lamina propria tissue from the thyroarytenoid muscle. Real-time PCR and Western blot were used to analyze expressions of autophagy markers, including Beclin1 and Atg7, in VFI. Tgfb1 and Col1a1 were assessed to determine the correlation of fibrosis with VFI progression and autophagy levels. Rat vocal fold fibroblasts were also treated with TGF-ß1 or rapamycin, which activates and suppresses autophagy respectively, to explore how autophagy regulates fibrosis in VFI. Initially, we observed that autophagy was downregulated in vocal fold mucosa after VFI in rats. This was particularly evident by the time-dependent downregulation of Beclin1 and Atg7 following VFI. Concurrently, levels of Tgfb1 and Col1a1 also surged, hinting at elevated fibrosis levels. Furthermore, our experiments with TGF-ß1 stimulation revealed that it inhibited autophagy in rat vocal fold fibroblasts. Interestingly, when we introduced rapamycin, this effect was reversed. Our data suggest that autophagy is a suppressor of VFI by alleviating fibrosis, making targeting autophagy a potential therapeutic route in VFI.NEW & NOTEWORTHY The study has demonstrated that autophagy is a suppressor of VFI by alleviating fibrosis, making autophagy a potential therapeutic target in VFI.

mTOR inhibition abrogates human mammary stem cells and early breast cancer progression markers.

BACKGROUND: Mammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers. METHODS: We analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5-7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry. RESULTS: Sirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression. CONCLUSION: Overall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression. Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015.

[Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy].

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

The role of glycerol-water mixtures in the stability of FKBP12-rapalog-FRB complexes.

The thermodynamic and biophysical implications of the introduction of a co-solvent during protein-ligand binding remain elusive. Using ternary complexes of 12-kDa FK506 binding protein (FKBP12), FKBP-rapamycin binding (FRB) domain of the mammalian/mechanistic target of rapamycin (mTOR) kinase, and rapamycin analogs (rapalogs) in glycerol-water mixtures, the influence of solvent composition on ligand binding dynamics was explored. The pharmaceutical potential of rapalogs and the utility of glycerol as a co-solvent in drug delivery applications were critical in deciding the system to be studied. Consolidation of existing studies on rapamycin modification was first performed to strategically design a new rapalog called T1. The results from 100-ns dual-boost Gaussian accelerated molecular dynamics simulations showed that protein stability was induced in the presence of glycerol. Reweighting of the trajectories revealed that the glycerol-rich solvent system lowers the energy barrier in the conformational space of the protein while also preserving native contacts between the ligand and the residues in the binding site. Calculated binding free energies using MM/GBSA also showed that electrostatic energy and polar contribution of solvation energy are heavily influenced by the changes in solvation. Glycerol molecules are preferentially excluded through electrostatic interactions from the solvation shell which induce complex stability as seen in existing experiments. Hence, using glycerol as a co-solvent in rapamycin delivery has a significant role in maintaining stability. In addition, compound T1 is a potential mTORC1-selective inhibitor with strong affinity for the FKBP12-FRB complex. This study aims to provide insights on the design of new rapalogs, and the applicability of glycerol as co-solvent for FKBP12-rapalog-FRB complexes.

The histone H2B Arg95 residue efficiently recruits the transcription factor Spt16 to mediate Ste5 expression of the pheromone response pathway.

In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds.

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

RapaCaspase-9-based suicide gene applied to the safety of IL-1RAP CAR-T cells.

Even if adoptive cell transfer (ACT) has already shown great clinical efficiency in different types of disease, such as cancer, some adverse events consistently occur, and suicide genes are an interesting system to manage these events. Our team developed a new medical drug candidate, a chimeric antigen receptor (CAR) targeting interleukin-1 receptor accessory protein (IL-1RAP), which needs to be evaluated in clinical trials with a clinically applicable suicide gene system. To prevent side effects and ensure the safety of our candidate, we devised two constructs carrying an inducible suicide gene, RapaCasp9-G or RapaCasp9-A, containing a single-nucleotide polymorphism (rs1052576) affecting the efficiency of endogenous caspase 9. These suicide genes are activated by rapamycin and based on the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization. RapaCasp9-G- and RapaCasp9-A-expressing gene-modified T cells (GMTCs) were produced from healthy donors (HDs) and acute myeloid leukemia (AML) donors. The RapaCasp9-G suicide gene demonstrated better efficiency, and we showed its in vitro functionality in different clinically relevant culture conditions. Moreover, as rapamycin is not pharmacologically inert, we also demonstrated its safe use as part of our therapy.

Transcription by the Three RNA Polymerases under the Control of the TOR Signaling Pathway in Saccharomyces cerevisiae.

Ribosomes are the basis for protein production, whose biogenesis is essential for cells to drive growth and proliferation. Ribosome biogenesis is highly regulated in accordance with cellular energy status and stress signals. In eukaryotic cells, response to stress signals and the production of newly-synthesized ribosomes require elements to be transcribed by the three RNA polymerases (RNA pols). Thus, cells need the tight coordination of RNA pols to adjust adequate components production for ribosome biogenesis which depends on environmental cues. This complex coordination probably occurs through a signaling pathway that links nutrient availability with transcription. Several pieces of evidence strongly support that the Target of Rapamycin (TOR) pathway, conserved among eukaryotes, influences the transcription of RNA pols through different mechanisms to ensure proper ribosome components production. This review summarizes the connection between TOR and regulatory elements for the transcription of each RNA pol in the budding yeast Saccharomyces cerevisiae. It also focuses on how TOR regulates transcription depending on external cues. Finally, it discusses the simultaneous coordination of the three RNA pols through common factors regulated by TOR and summarizes the most important similarities and differences between S. cerevisiae and mammals.

Ox-LDL aggravates contrast-induced injury of renal tubular epithelial cells.

Hypercholesterolemia can aggravate contrast-induced acute kidney injury, and the exacerbation of renal tubular epithelial cell (RTEC) injury is a major cause. However, the exact mechanisms remain obscure. Mitophagy, a type of autophagy, selectively eliminates damaged mitochondria and reduces mitochondrial oxidative stress, which is strongly implicated in cell homeostasis and acute kidney injury. Oxidized low-density lipoprotein (Ox-LDL) is accumulated in hypercholesterolemia and has a cytotoxic effect. This study aimed to determine whether and how ox-LDL exacerbates contrast-induced injury in RTECs and to further explore whether PINK1/Parkin-dependent mitophagy is involved in this process. Iohexol and ox-LDL were used alone or in combination to treat HK-2 cells. Rapamycin pretreatment was utilized to enhance mitophagy. Cell viability, apoptosis, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) were detected by cell counting kit-8, TUNEL staining, JC-1 kit and MitoSOX fluorescence, respectively. The expression of mitophagy-related proteins (including PINK1, Parkin, and so on) and cleaved caspase-3 was confirmed by western blot. Colocalization of MitoTracker-labeled mitochondria and LysoTracker-labeled lysosomes was observed by fluorescence microscopy to evaluate mitophagy. The results of our study showed that ox-LDL aggravated MMP decline, mtROS release and apoptosis in iohexol-treated HK-2 cells, accompanied by a further increased autophagy level. Enhancement of PINK1/Parkin-dependent mitophagy by rapamycin alleviated apoptosis and mitochondrial injury in HK-2 cells in response to iohexol under ox-LDL condition. Therefore, our findings indicate that ox-LDL aggravates contrast-induced injury of RTECs by increasing mitochondrial damage and mitochondrial oxidative stress, which may be associated with the relative insufficiency of PINK1/Parkin-dependent mitophagy.

TMEM16A partners with mTOR to influence pathways of cell survival, proliferation, and migration in cholangiocarcinoma.

Expression of transmembrane protein 16 A (TMEM16A), a calcium activated chloride channel, is elevated in some human cancers and impacts tumor cell proliferation, metastasis, and patient outcome. Evidence presented here uncovers a molecular synergy between TMEM16A and mechanistic/mammalian target of rapamycin (mTOR), a serine-threonine kinase that is known to promote cell survival and proliferation in cholangiocarcinoma (CCA), a lethal cancer of the secretory cells of bile ducts. Analysis of gene and protein expression in human CCA tissue and CCA cell line detected elevated TMEM16A expression and Cl- channel activity. The Cl- channel activity of TMEM16A impacted the actin cytoskeleton and the ability of cells to survive, proliferate, and migrate as revealed by pharmacological inhibition studies. The basal activity of mTOR, too, was elevated in the CCA cell line compared with the normal cholangiocytes. Molecular inhibition studies provided further evidence that TMEM16A and mTOR were each able to influence the regulation of the other's activity or expression respectively. Consistent with this reciprocal regulation, combined TMEM16A and mTOR inhibition produced a greater loss of CCA cell survival and migration than their individual inhibition alone. Together these data reveal that the aberrant TMEM16A expression and cooperation with mTOR contribute to a certain advantage in CCA.NEW & NOTEWORTHY This study points to the dysregulation of transmembrane protein 16 A (TMEM16A) expression and activity in cholangiocarcinoma (CCA), the inhibition of which has functional consequences. Dysregulated TMEM16A exerts an influence on the regulation of mechanistic/mammalian target of rapamycin (mTOR) activity. Moreover, the reciprocal regulation of TMEM16A by mTOR demonstrates a novel connection between these two protein families. These findings support a model in which TMEM16A intersects the mTOR pathway to regulate cell cytoskeleton, survival, proliferation, and migration in CCA.