Target Of Rapamycin Regulation Research Articles

TOR coordinates cytokinin and gibberellin signals mediating development and defense.

Plants constantly perceive and process environmental signals and balance between the energetic demands of growth and defense. Growth arrest upon pathogen attack was previously suggested to result from a redirection of the plants' metabolic resources towards the activation of plant defense. The energy sensor Target of Rapamycin (TOR) kinase is a conserved master coordinator of growth and development in all eukaryotes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms by which TOR may potentially regulate the relationship between these two modalities. The plant hormones cytokinin (CK) and gibberellin (GA) execute various aspects of plant development and defense. The ratio between CK and GA was reported to determine the outcome of developmental programmes. Here, investigating the interplay between TOR-mediated development and TOR-mediated defense in tomato, we found that TOR silencing resulted in rescue of several different aberrant developmental phenotypes, demonstrating that TOR is required for the execution of developmental cues. In parallel, TOR inhibition enhanced immunity in genotypes with a low CK/GA ratio but not in genotypes with a high CK/GA ratio. TOR-inhibition mediated disease resistance was found to depend on developmental status, and was abolished in strongly morphogenetic leaves, while being strongest in mature, differentiated leaves. CK repressed TOR activity, suggesting that CK-mediated immunity may rely on TOR downregulation. At the same time, TOR activity was promoted by GA, and TOR silencing reduced GA sensitivity, indicating that GA signalling requires normal TOR activity. Our results demonstrate that TOR likely acts in concert with CK and GA signalling, executing signalling cues in both defense and development. Thus, differential regulation of TOR or TOR-mediated processes could regulate the required outcome of development-defense prioritisation.

The Case for the Target of Rapamycin Pathway as a Candidate Circadian Oscillator.

The molecular mechanisms that drive circadian (24 h) rhythmicity have been investigated for many decades, but we still do not have a complete picture of eukaryotic circadian systems. Although the transcription/translation feedback loop (TTFL) model has been the primary focus of research, there are many examples of circadian rhythms that persist when TTFLs are not functioning, and we lack any good candidates for the non-TTFL oscillators driving these rhythms. In this hypothesis-driven review, the author brings together several lines of evidence pointing towards the Target of Rapamycin (TOR) signalling pathway as a good candidate for a non-TTFL oscillator. TOR is a ubiquitous regulator of metabolism in eukaryotes and recent focus in circadian research on connections between metabolism and rhythms makes TOR an attractive candidate oscillator. In this paper, the evidence for a role for TOR in regulating rhythmicity is reviewed, and the advantages of TOR as a potential oscillator are discussed. Evidence for extensive feedback regulation of TOR provides potential mechanisms for a TOR-driven oscillator. Comparison with ultradian yeast metabolic cycles provides an example of a potential TOR-driven self-sustained oscillation. Unanswered questions and problems to be addressed by future research are discussed.

The Arabidopsis Target of Rapamycin kinase regulates ammonium assimilation and glutamine metabolism.

In eukaryotes, a target of rapamycin (TOR) is a well-conserved kinase that controls cell metabolism and growth in response to nutrients and environmental factors. Nitrogen (N) is an essential element for plants, and TOR functions as a crucial N and amino acid sensor in animals and yeast. However, knowledge of the connections between TOR and the overall N metabolism and assimilation in plants is still limited. In this study, we investigated the regulation of TOR in Arabidopsis (Arabidopsis thaliana) by the N source as well as the impact of TOR deficiency on N metabolism. Inhibition of TOR globally decreased ammonium uptake while triggering a massive accumulation of amino acids, such as Gln, but also of polyamines. Consistently, TOR complex mutants were hypersensitive to Gln. We also showed that the glutamine synthetase inhibitor glufosinate abolishes Gln accumulation resulting from TOR inhibition and improves the growth of TOR complex mutants. These results suggest that a high level of Gln contributes to the reduction in plant growth resulting from TOR inhibition. Glutamine synthetase activity was reduced by TOR inhibition while the enzyme amount increased. In conclusion, our findings show that the TOR pathway is intimately connected to N metabolism and that a decrease in TOR activity results in glutamine synthetase-dependent Gln and amino acid accumulation.

Ectopic expression of Jatropha curcas JcTAW1 improves the vegetative growth, yield, and drought resistance of tobacco

BackgroundJatropha curcas is a promising alternative bio-energy resource. However, underrun limited its broad application in the industry. Luckily, TAW1 is a high-productivity promoting gene that increases the lateral branches by prolonging the identification of inflorescence meristems to generate more spikes and flowers.ResultsIn the current study, we introduced the Jatropha JcTAW1 gene into tobacco to depict its functional profile. Ectopically expressed JcTAW1 increased the lateral branches and ultimate yield of the transgenic tobacco plants. Moreover, the JcTAW1 lines had significantly higher plant height, longer roots, and better drought resistance than those of wild-type (W.T.). We performed RNA sequencing and weighted gene co-expression network analysis to determine which biological processes were affected by JcTAW1. The results showed that biological processes such as carbon metabolism, cell wall biosynthesis, and ionization transport were extensively promoted by the ectopic expression of JcTAW1. Seven hub genes were identified. Therein, two up-regulated genes affect glucose metabolism and cell wall biosynthesis, five down-regulated genes are involved in DNA repair and negative regulation of TOR (target-of-rapamycin) signaling which was identified as a central regulator to promote cell proliferation and growth.ConclusionsOur study verified a new promising candidate for Jatropha productive breeding and discovered several new features of JcTAW1. Except for boosting flowering, JcTAW1 was found to promote stem and root growth. Additionally, transcriptome analysis indicated that JcTAW1 might promote glucose metabolism while suppressing the DNA repair system.

Analyzing the impact of autotrophic and heterotrophic metabolism on the nutrient regulation of TOR.

The target of rapamycin (TOR) protein kinase is a master regulator of cell growth in all eukaryotes, from unicellular yeast and algae to multicellular animals and plants. Target of rapamycin balances the synthesis and degradation of proteins, lipids, carbohydrates and nucleic acids in response to nutrients, growth factors and cellular energy to promote cell growth. Among nutrients, amino acids (AAs) and glucose are central regulators of TOR activity in evolutionary distant eukaryotes such as mammals, plants and algae. However, these organisms obtain the nutrients through totally different metabolic processes. Although photosynthetic eukaryotes can use atmospheric CO2 as the sole carbon (C) source for all reactions in the cell, heterotrophic organisms get nutrients from other sources of organic C including glucose. Here, we discuss the impact of autotrophic and heterotrophic metabolism on the nutrient regulation of TOR, focusing on the role of AAs and C sources upstream of this signaling pathway.

Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes

The target of rapamycin (TOR) kinase is a Ser/Thr kinase and master regulator, with roles in the regulation of nutritional sensing, protein translation, and autophagy, among others. In Chlamydomonas reinhardtii, a unicellular green algae and model organism for biofuel production and optimization, TOR has been linked to increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target is responsible for the elusive “lipid switch,” the protein or proteins responsible for increasing TAG formation under nutrient limitation and environmental stressors. However, when this lipid switch is activated, overall culture growth is arrested, truncating the alga’s ability to continuously produce TAGs at a level needed for widespread biofuel use.In order to engineer an effective strain for algal biofuels, the targets and signaling pathways of lipid regulatory networks must be determined. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems. Previous research has used TOR inhibitors to follow phosphorylation pathways and identify potential downstream targets of TOR but little work has uncovered the role of oxidative signaling in regulation. Since TOR inhibition results in an increase in reactive oxygen species analogous to that of nitrogen deprivation, the opportunity for redox control mechanisms is significant.This study inhibited TOR using AZD8055 and monitored reversible thiol oxidation while simultaneously tracking physiological changes. By pairing these data together, reversible thiol oxidation was related to nearly every major metabolic pathway in the cell, including TAG accumulation, the tricarboxylic acid cycle, and protein translation. Additionally, reversible thiol oxidation corresponded to changes in photosynthetic activity measured through chlorophyll a fluorescence, showing a novel role for TOR regulation of photosynthesis. The delineation of redox‐controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.Support or Funding InformationThis research was supported by a National Science Foundation CAREER award (MCB‐1552522) awarded to L.M.H and a National Science Foundation award (Cooperative Agreement OIA‐1458,952) to D.R.J.K. A.L.S. was funded by the NASA West Virginia Space Grant Consortium (NNX15AK74A and NNX15AI01H).

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of Rapamycin (TOR) Kinase Activation.

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O2 consumption rate (RN) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour RN measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf RN Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling RN Many amino acids, as well as Glc analogs, were found to potently inhibit the RN stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- and Ala-stimulated RN were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes.

The target of rapamycin (TOR) kinase is a master metabolic regulator with roles in nutritional sensing, protein translation, and autophagy. In Chlamydomonas reinhardtii, a unicellular green alga, TOR has been linked to the regulation of increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target(s) is responsible for the elusive “lipid switch” in control of increasing TAG accumulation under nutrient limitation. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems, and little work has been done to show the role of oxidative signaling in TOR regulation. In this study, the TOR inhibitor AZD8055 was used to relate reversible thiol oxidation to the physiological changes seen under TOR inhibition, including increased TAG content. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 401 proteins were determined to have significant changes in oxidation following TOR inhibition. These oxidative changes mirrored characterized physiological modifications, supporting the role of reversible thiol oxidation in TOR regulation of TAG production, protein translation, carbohydrate catabolism, and photosynthesis through the use of reversible thiol oxidation. The delineation of redox-controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.

The magic 'hammer' of TOR: the multiple faces of a single pathway in the metabolic regulation of plant growth and development.

The target of rapamycin (TOR) pathway has emerged as a central hub synchronizing plant growth according to the nutrient/energy status and environmental inputs. Molecular mechanisms through which TOR promotes plant growth involve the positive regulation of transcription of cell proliferation-associated genes, mRNA translation initiation and ribosome biogenesis, to cite a few examples. Phytohormones, light, sugars, and sulfur have been found to broadly regulate TOR activity. TOR operates as a metabolic homeostat to fine-tune anabolic processes and efficiently enable plant growth under different circumstances. However, little is known about the multiple effectors that act up- and downstream of TOR. Here, we mainly discuss recent findings related to the TOR pathway in the context of plant metabolism and highlight areas of interest that need to be addressed to keep unravelling the intricate networks governing the regulation of TOR and its function in controlling biosynthetic growth.

Target of rapamycin, a master regulator of multiple signalling pathways and a potential candidate gene for crop improvement

The target of rapamycin (TOR) protein regulates growth and development in photosynthetic and non-photosynthetic eukaryotes. Although the TOR regulatory networks are involved in nutrient and energy signalling, and transcriptional and translational control of multiple signalling pathways, the molecular mechanism of TOR regulation of plant abiotic stress responses is still unclear. The TOR-mediated transcriptional regulation of genes encoding ribosomal proteins (RP) is a necessity under stress conditions for balanced growth and productivity in plants. The activation of SnRKs (sucrose non-fermenting-related kinases) and the inactivation of TOR signalling in abiotic stresses is in line with the accumulation of ABA and transcriptional activation of stress responsive genes. Autophagy is induced under abiotic stress conditions, which results in degradation of proteins and the release of amino acids, which might possibly induce phosphorylation of TOR and, hence, its activation. TOR signalling also has a role in regulating ABA biosynthesis for transcriptional regulation of stress-related genes. The switch between activation and inactivation of TOR by its phosphorylation and de-phosphorylation maintains balanced growth in response to stresses. In the present review, we discuss the important signalling pathways that are regulated by TOR and try to assess the relationship between TOR signalling and tolerance to abiotic stresses in plants. The review also discusses possible cross-talk between TOR and RP genes in response to abiotic stresses.

Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii.

Rapamycin is an inhibitor of the evolutionary conserved Target of Rapamycin (TOR) kinase which promotes and coordinates translation with cell growth and division. In heterotrophic organisms, TOR regulation is based on intra- and extracellular stimuli such as amino acids level and insulin perception. However, how plant TOR pathways have evolved to integrate plastid endosymbiosis is a remaining question. Despite the close association of the TOR signaling with the coordination between protein turn-over and growth, proteome and phosphoproteome acclimation to a rapamycin treatment have not yet been thoroughly investigated in Chlamydomonas reinhardtii. In this study, we have used in vivo label-free phospho-proteomic analysis to profile both protein and phosphorylation changes at 0, 24, and 48 h in Chlamydomonas cells treated with rapamycin. Using multivariate statistics we highlight the impact of TOR inhibition on both the proteome and the phosphoproteome. Two-way ANOVA distinguished differential levels of proteins and phosphoproteins in response either to culture duration and rapamycin treatment or combined effects. Finally, protein–protein interaction networks and functional enrichment analysis underlined the relation between plastid and mitochondrial metabolism. Prominent changes of proteins involved in sulfur, cysteine, and methionine as well as nucleotide metabolism on the one hand, and changes in the TCA cycle on the other highlight the interplay of chloroplast and mitochondria metabolism. Furthermore, TOR inhibition revealed changes in the endomembrane trafficking system. Phosphoproteomics data, on the other hand, highlighted specific differentially regulated phosphorylation sites for calcium-regulated protein kinases as well as ATG7, S6K, and PP2C. To conclude we provide a first combined Chlamydomonas proteomics and phosphoproteomics dataset in response to TOR inhibition, which will support further investigations.

The Arabidopsis THADA homologue modulates TOR activity and cold acclimation.

Low temperature is one of the most important environmental factors that affect global survival of humans and animals and equally importantly the distribution of plants and crop productivity. Survival of metazoan cells under cold stress requires regulation of the sensor-kinase Target Of Rapamycin (TOR). TOR controls growth of eukaryotic cells by adjusting anabolic and catabolic metabolism. Previous studies identified the Thyroid Adenoma Associated (THADA) gene as the major effect locus by positive selection in the evolution of modern human adapted to cold. Here we investigate the role of THADA in TOR signaling and cold acclimation of plants. We applied BLAST searches and homology modeling to identify the AtTHADA (AT3G55160) in Arabidopsis thaliana as the highly probable orthologue protein. Reverse genetics approaches were combined with immunological detection of TOR activity and metabolite profiling to address the role of the TOR and THADA for growth regulation and cold acclimation. Depletion of the AtTHADA gene caused complete or partial loss of full-length mRNA, respectively, and significant retardation of growth under non-stressed conditions. Furthermore, depletion of AtTHADA caused hypersensitivity towards low-temperatures. Atthada displayed a lowered energy charge. This went along with decreased TOR activity, which offers a molecular explanation for the slow growth phenotype of Atthada. Finally, we used TOR RNAi lines to identify the de-regulation of TOR activity as one determinant for sensitivity towards low-temperatures. Taken together our results provide evidence for a conserved function of THADA in cold acclimation of eukaryotes and suggest that cold acclimation in plants requires regulation of TOR.

Sulfur Partitioning between Glutathione and Protein Synthesis Determines Plant Growth.

Photoautotrophic organisms must efficiently allocate their resources between stress-response pathways and growth-promoting pathways to be successful in a constantly changing environment. In this study, we addressed the coordination of sulfur flux between the biosynthesis of the reactive oxygen species scavenger glutathione (GSH) and protein translation as one example of a central resource allocation switch. We crossed the Arabidopsis (Arabidopsis thaliana) GSH synthesis-depleted cadmium-sensitive cad2-1 mutant, which lacks glutamate cysteine (Cys) ligase, into the sulfite reductase sir1-1 mutant, which suffers from a significantly decreased flux of sulfur into Cys and, consequently, is retarded in growth. Surprisingly, depletion of GSH synthesis promoted the growth of the sir1-1 cad2-1 double mutant (s1c2) when compared with sir1-1 Determination of GSH levels and in vivo live-cell imaging of the reduction-oxidation-sensitive green fluorescent protein sensor demonstrated significant oxidation of the plastidic GSH redox potential in cad2-1 and s1c2 This oxidized GSH redox potential aligned with significant activation of plastid-localized sulfate reduction and a significantly higher flux of sulfur into proteins. The specific activation of the serine/threonine sensor kinase Target of Rapamycin (TOR) in cad2-1 and s1c2 was the trigger for reallocation of Cys from GSH biosynthesis into protein translation. Activation of TOR in s1c2 enhanced ribosome abundance and partially rescued the decreased meristematic activity observed in sir1-1 mutants. Therefore, we found that the coordination of sulfur flux between GSH biosynthesis and protein translation determines growth via the regulation of TOR.

The impaired immune function and structural integrity by dietary iron deficiency or excess in gill of fish after infection with Flavobacterium columnare: Regulation of NF-κB, TOR, JNK, p38MAPK, Nrf2 and MLCK signalling

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKβ, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively.

Glucose-ABL1-TOR Signaling Modulates Cell Cycle Tuning to Control Terminal Appressorial Cell Differentiation.

The conserved target of rapamycin (TOR) pathway integrates growth and development with available nutrients, but how cellular glucose controls TOR function and signaling is poorly understood. Here, we provide functional evidence from the devastating rice blast fungus Magnaporthe oryzae that glucose can mediate TOR activity via the product of a novel carbon-responsive gene, ABL1, in order to tune cell cycle progression during infection-related development. Under nutrient-free conditions, wild type (WT) M. oryzae strains form terminal plant-infecting cells (appressoria) at the tips of germ tubes emerging from three-celled spores (conidia). WT appressorial development is accompanied by one round of mitosis followed by autophagic cell death of the conidium. In contrast, Δabl1 mutant strains undergo multiple rounds of accelerated mitosis in elongated germ tubes, produce few appressoria, and are abolished for autophagy. Treating WT spores with glucose or 2-deoxyglucose phenocopied Δabl1. Inactivating TOR in Δabl1 mutants or glucose-treated WT strains restored appressorium formation by promoting mitotic arrest at G1/G0 via an appressorium- and autophagy-inducing cell cycle delay at G2/M. Collectively, this work uncovers a novel glucose-ABL1-TOR signaling axis and shows it engages two metabolic checkpoints in order to modulate cell cycle tuning and mediate terminal appressorial cell differentiation. We thus provide new molecular insights into TOR regulation and cell development in response to glucose.

Mechanistic target of rapamycin regulation of the trophoblast secretome

Mechanistic target of rapamycin regulation of the trophoblast secretome

Colletotrichum orbiculare WHI2, a Yeast Stress-Response Regulator Homolog, Controls the Biotrophic Stage of Hemibiotrophic Infection Through TOR Signaling.

The hemibiotrophic fungus Colletotrichum orbiculare first establishes a biotrophic infection stage in cucumber (Cucumber sativus) epidermal cells and subsequently transitions to a necrotrophic stage. Here, we found that C. orbiculare established hemibiotrophic infection via C. orbiculare WHI2, a yeast stress regulator homolog, and TOR (target of rapamycin) signaling. Plant defense responses such as callose deposition, H2O2, and antimicrobial proteins were strongly induced by the C. orbiculare whi2Δ mutant, resulting in defective pathogenesis. Expression analysis of biotrophy-specific genes evaluated by the promoter VENUS fusion gene indicated weaker VENUS signal intensity in the whi2Δ mutant, thereby suggesting that C. orbiculare WHI2 plays a key role in regulating biotrophic infection of C. orbiculare. The involvement of CoWHI2 in biotrophic infection was further explored with a DNA microarray. In the Cowhi2Δ mutant, TOR-dependent ribosomal protein-related genes were strikingly upregulated compared with the wild type. Moreover, callose deposition in the host plant after inoculation with the Cowhi2Δ mutant treated with rapamycin, which inhibits TOR activity, was reduced, and the mutant remained biotrophic in contrast to the untreated mutant. Thus, regulation of TOR by Whi2 is apparently crucial to the biotrophic stage of hemibiotrophic infection in C. orbiculare.

The mucolipidosis IV Ca2+ channel TRPML1 (MCOLN1) is regulated by the TOR kinase.

Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs). During autophagy, aberrant regulation of the lysosomal Ca(2+) efflux channel TRPML1 [transient receptor potential mucolipin 1 (MCOLN1)], also known as MCOLN1, is solely responsible for the human LSD mucolipidosis type IV (MLIV); however, the exact mechanisms involved in the development of the pathology of this LSD are unknown. In the present study, we provide evidence that the target of rapamycin (TOR), a nutrient-sensitive protein kinase that negatively regulates autophagy, directly targets and inactivates the TRPML1 channel and thereby functional autophagy, through phosphorylation. Further, mutating these phosphorylation sites to unphosphorylatable residues proved to block TOR regulation of the TRPML1 channel. These findings suggest a mechanism for how TOR activity may regulate the TRPML1 channel.

TOR and Proteasomal Regulation of Ribosomal Protein Gene Expression

Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID) dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Ribosomal protein (RP) genes are important class of TFIID-dependent genes, and are crucial for ribosome biogenesis, protein synthesis, and hence cellular growth and proliferation. Since RP genes are TFIID-dependent and the role of the 19S proteasome subcomplex in their transcription remain unknown, we investigated whether 19S proteasome subcomplex regulates the transcription of RP genes. We find that the 19S proteasome subcomplex is recruited to the promoters of RP genes and enhances the targeting of co-activator NuA4 HAT (histone acetyl transferase) to activator Rap1. Such enhanced targeting of NuA4 HAT is essential to bring down TFIID complex in a HAT-dependent manner. Intriguingly, the HAT activity of NuA4 complex is regulated by environmental factors such as heat, nutrition and osmotic pressure via TOR (Target of Rapamycin) signaling pathway. We also find that TOR facilitates the targeting of 19S proteasome subcomplex to the promoters of RP genes to promote NuA4 HAT association, histone H4 acetylation and the formation of pre-initiation complex at their promoters. Together, our results delineate new regulatory pathway of RP gene expression via TOR, 19S proteasome subcomplex and NuA4 HAT.

Forkhead, a new cross regulator of metabolism and innate immunity downstream of TOR in Drosophila

Antimicrobial peptides (AMPs) are conserved cationic peptides which act both as defense molecules of the host immune system and as regulators of the commensal microbiome. Expression of AMPs is induced in response to infection by the Toll and Imd pathway. Under non-infected conditions, the transcription factor dFOXO directly regulates a set of AMP expression at low levels when nutrients are limited.Here we have analyzed whether target of rapamycin (TOR), another major regulator of growth and metabolism, also modulates AMP responses in Drosophila. We found that downregulation of TOR by feeding the drug rapamycin or by overexpressing the negative TOR regulators TSC1/TSC2, resulted in a specific induction of the AMPs Diptericin (Dpt) and Metchnikowin (Mtk). In contrast, overexpression of Rheb, which positively regulates TOR led to a repression of the two AMPs. Genetic and pharmacological experiments indicate that Dpt and Mtk activation is controlled by the transcription factor Forkhead (FKH), the founding member of the FoxO family. Shuttling of FKH from the cytoplasm to the nucleus is induced in the fat body and in the posterior midgut in response to TOR downregulation. The FKH-dependent induction of Dpt and Mtk can be triggered in dFOXO null mutants and in immune-compromised Toll and IMD pathway mutants indicating that FKH acts in parallel to these regulators.Together, we have discovered that FKH is the second conserved member of the FoxO family cross-regulating metabolism and innate immunity. dFOXO and FKH, which are activated upon downregulation of insulin or TOR activities, respectively, act in parallel to induce different sets of AMPs, thereby modulating the immune status of metabolic tissues such as the fat body or the gut in response to the oscillating energy status of the organism.