Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

AKT/PKB Signaling: Navigating the Network.

The Ser and Thr kinase AKT, also known as protein kinase B (PKB), was discovered 25 years ago and has been the focus of tens of thousands of studies in diverse fields of biology and medicine. There have been many advances in our knowledge of the upstream regulatory inputs into AKT, key multifunctional downstream signaling nodes (GSK3, FoxO, mTORC1), which greatly expand the functional repertoire of AKT, and the complex circuitry of this dynamically branching and looping signaling network that is ubiquitous to nearly every cell in our body. Mouse and human genetic studies have also revealed physiological roles for the AKT network in nearly every organ system. Our comprehension of AKT regulation and functions is particularly important given the consequences of AKT dysfunction in diverse pathological settings, including developmental and overgrowth syndromes, cancer, cardiovascular disease, insulin resistance and type 2 diabetes, inflammatory and autoimmune disorders, and neurological disorders. There has also been much progress in developing AKT-selective small molecule inhibitors. Improved understanding of the molecular wiring of the AKT signaling network continues to make an impact that cuts across most disciplines of the biomedical sciences.

Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling.

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8+ T cells.

CASTORing New Light on Amino Acid Sensing.

The activation state of mTORC1, a master regulator of cell growth, is particularly sensitive to changes in the intracellular levels of the amino acid arginine, but the sensing mechanisms are poorly understood. In this issue of Cell, Chantranupong et al. identify CASTOR1 as a direct arginine sensor that acts through the GATOR2 complex to regulate mTORC1.

Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome.

mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. We demonstrate that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals.

Insulin signaling: inositol phosphates get into the Akt.

An acute but transient response to insulin is essential for glucose homeostasis in mammals. Chakraborty et al. (2010) uncover a new feedback mechanism regulating insulin signaling. They show that the inositol pyrophosphate IP7, which is produced in response to insulin, inhibits the Akt kinase, a primary effector of insulin signaling.

Chewing the fat on tumor cell metabolism.

Tumor cells undergo a metabolic shift toward specific bioenergetic (glycolysis) and anabolic (protein and lipid synthesis) processes that promote rapid growth. Nomura et al. (2010) now demonstrate that an increase in monoacylglycerol lipase (MAGL) drives tumorigenesis through the lipolytic release and remodeling of free fatty acids.

Corrigendum: Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans.

This corrects the article DOI: 10.1038/nature20789.

Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans.

Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.

ZBTB7A acts as a tumor suppressor through the transcriptional repression of glycolysis.

Elevated glycolysis is a common metabolic trait of cancer, but what drives such metabolic reprogramming remains incompletely clear. We report here a novel transcriptional repressor-mediated negative regulation of glycolysis. ZBTB7A, a member of the POK (POZ/BTB and Krüppel) transcription repressor family, directly binds to the promoter and represses the transcription of critical glycolytic genes, including GLUT3, PFKP, and PKM. Analysis of The Cancer Genome Atlas (TCGA) data sets reveals that the ZBTB7A locus is frequently deleted in many human tumors. Significantly, reduced ZBTB7A expression correlates with up-regulation of the glycolytic genes and poor survival in colon cancer patients. Remarkably, while ZBTB7A-deficient tumors progress exceedingly fast, they exhibit an unusually heightened sensitivity to glycolysis inhibition. Our study uncovers a novel tumor suppressor role of ZBTB7A in directly suppressing glycolysis.

Coordinated regulation of protein synthesis and degradation by mTORC1.

Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.

TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1.

The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.

Improved detection of synthetic lethal interactions in Drosophila cells using variable dose analysis (VDA).

Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells, but this approach generally suffers from low consistency between screens. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called Variable Dose Analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC), and generated a SS/L network for TSC. Using this network, we identified four Food and Drug Administration-approved drugs that selectively affect viability of TSC-deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.

Activation of a metabolic gene regulatory network downstream of mTOR complex 1.

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.

Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis.

Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes, including growth, proliferation, and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.

Tuberous sclerosis complex 2 loss increases lysophosphatidylcholine synthesis in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease affecting women. LAM is caused by mutations in the tuberous sclerosis complex (TSC) genes. The TSC protein complex inhibits the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), which is a master regulator of cellular metabolism. Using mass spectrometry-based lipid profiling, we analyzed plasma from patients with LAM and discovered elevated levels of four lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, and C20:4) compared with those in healthy control women. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro preclinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid and neutral lipid species. Treatment with rapamycin or torin1 or down-regulation of sterol regulatory element-binding protein (SREBP), a lipogenic transcription factor, did not suppress LPC in TSC2-deficient cells. Inhibition of distinct isoforms of phospholipase A2 decreased the proliferation of TSC2-deficient cells. Collectively, these results demonstrate that TSC2-deficient cells have enhanced choline phospholipid metabolism and reveal a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies.