PP2A-dependent TFEB activation is blocked by PIKfyve-induced mTORC1 activity

Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of TFEB at multiple sites including Ser-211. Here we show that inhibition of PIKfyve, which produces phosphatidylinositol 3,5-bisphosphate on endosomes and lysosomes, causes a loss of Ser-211 phosphorylation and concomitant nuclear localization of TFEB. We found that while mTORC1 activity toward S6K1, as well as other major mTORC1 substrates, is not impaired, PIKfyve inhibition specifically impedes the interaction of TFEB with mTORC1. This suggests that mTORC1 activity on TFEB is selectively inhibited due to loss of mTORC1 access to TFEB. In addition, we found that TFEB activation during inhibition of PIKfyve relies on the ability of protein phosphatase 2A (PP2A) but not calcineurin/PPP3, to dephosphorylate TFEB Ser-211. Thus, when PIKfyve is inhibited, PP2A is dominant over mTORC1 for control of TFEB phosphorylation at Ser-S211. Together these findings suggest that mTORC1 and PP2A have opposing roles on TFEB via phosphorylation and dephosphorylation of Ser-211, respectively, and further, that PIKfyve inhibits TFEB activity by facilitating mTORC1-dependent phosphorylation of TFEB.

Mechanistic/mammalian target of rapamycin and side effects of antipsychotics: insights into mechanisms and implications for therapy

Antipsychotic pharmacotherapy has been widely recommended as the standard of care for the treatment of acute schizophrenia and psychotic symptoms of other psychiatric disorders. However, there are growing concerns regarding antipsychotic-induced side effects, including weight gain, metabolic syndrome (MetS), and extrapyramidal motor disorders, which not only decrease patient compliance, but also predispose to diabetes and cardiovascular diseases. To date, most studies and reviews on the mechanisms of antipsychotic-induced metabolic side effects have focused on central nervous system mediation of appetite and food intake. However, disturbance in glucose and lipid metabolism, and hepatic steatosis induced by antipsychotic drugs might precede weight gain and MetS. Recent studies have demonstrated that the mechanistic/mammalian target of rapamycin (mTOR) pathway plays a critical regulatory role in the pathophysiology of antipsychotic drug-induced disorders of hepatic glucose and lipid metabolism. Furthermore, antipsychotic drugs promote striatal mTOR pathway activation that contributes to extrapyramidal motor side effects. Although recent findings have advanced the understanding of the role of the mTOR pathway in antipsychotic-induced side effects, few reviews have been conducted on this emerging topic. In this review, we synthesize key findings by focusing on the roles of the hepatic and striatal mTOR pathways in the pathogenesis of metabolic and extrapyramidal side effects, respectively. We further discuss the potential therapeutic benefits of normalizing excessive mTOR pathway activation with mTOR specific inhibitors. A deeper understanding of pathogenesis may inform future intervention strategies using the pharmacological or genetic inhibitors of mTOR to prevent and manage antipsychotic-induced side effects.

Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis.

Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant (raptor1b), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b, can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes.

mTOR-mediated phosphorylation of VAMP8 and SCFD1 regulates autophagosome maturation

The mammalian target of rapamycin (mTORC1) has been shown to regulate autophagy at different steps. However, how mTORC1 regulates the N-ethylmaleimide-sensitive protein receptor (SNARE) complex remains elusive. Here we show that mTORC1 inhibits formation of the SNARE complex (STX17-SNAP29-VAMP8) by phosphorylating VAMP8, thereby blocking autophagosome-lysosome fusion. A VAMP8 phosphorylation mimic mutant is unable to promote autophagosome-lysosome fusion in vitro. Furthermore, we identify SCFD1, a Sec1/Munc18-like protein, that localizes to the autolysosome and is required for SNARE complex formation and autophagosome-lysosome fusion. VAMP8 promotes SCFD1 recruitment to autolysosomes when dephosphorylated. Consistently, phosphorylated VAMP8 or SCFD1 depletion inhibits autophagosome-lysosome fusion, and expression of phosphomimic VAMP8 leads to increased lipid droplet accumulation when expressed in mouse liver. Thus, our study supports that mTORC1-mediated phosphorylation of VAMP8 blocks SCFD1 recruitment, thereby inhibiting STX17-SNAP29-VAMP8 complex formation and autophagosome-lysosome fusion.