Biology subjects

Scott, D. A.

Publications and source records attributed to Scott, D. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Reversible, Orally Bioavailable Small-Molecule Inhibitors Occupy the Substrate Tunnel of the Thioesterase Domain of Fatty Acid Synthase

Fatty acid synthase (FASN) is the sole enzyme capable of lipogenesis in cells and is upregulated in many cancers. Herein we report the discovery and development of SBP-5917 (78S), a novel, reversible, small-molecule probe which targets the thioesterase (TE) domain of FASN. SBP-5917 and other compounds in the series possess IC50 < 1 M and < 5 M for inhibiting recombinantly purified FASN-TE and cellular FASN respectively. Co-crystal structures of our inhibitors bound to the FASN-TE domain show that they are positioned in the substrate tunnel of the enzyme. Biochemical and biophysical analyses quantify the strength of compound binding and demonstrate their selectivity for FASN-TE. Preliminary pharmacokinetic studies indicate promising in vivo potency for many of the compounds in the series. SBP-5917 is the first structurally validated, reversible inhibitor of FASN-TE with demonstrated cellular target engagement and can serve as a chemical probe to investigate FASN as a therapeutic target.

cell biology↗

bioRxiv2026

Tryptophan degradation by intestinal Bacteroides induces anti-tumor immunity and limits melanoma growth

Defining mechanisms used by gut microbiota to control anti-tumor immunity may offer novel therapeutic modalities. Here, we demonstrate that Bacteroides rodentium and closely related Bacteroides uniformis species induce anti-tumor immunity and limit melanoma development when colonized in either germ-free (GF) mice, mice with a complex microbiome, or WT mice. Enhanced CD8+ T cell infiltration seen in tumors of mice harboring B. rodentium coincided with increased expression of immune-stimulating pathways and activation of bone marrow-derived dendritic cells that were co-cultured with the B. rodentium secretome. Metabolomic analyses of cecal samples from GF mice colonized with Altered Shedlar Flora (ASF) plus B. rodentium revealed lower tryptophan levels than in ASF-colonized controls, and WT mice fed a tryptophan-deficient diet exhibited inhibition of melanoma development. In silico genomic reconstruction of metabolic pathways revealed that both B. rodentium and B. uniformis harbor tryptophanase A (TnaA) and aromatic amino transferase (ArAT) genes, both of which function in tryptophan degradation. Administration of a B. uniformis harboring TnaA mutant failed to inhibit melanoma growth in gnotobiotic mice. Notably, administration of indoles, but not kynurenines, also effectively inhibited melanoma development, increasing immune cell infiltration into the tumors. Correspondingly, levels of bacterially encoded tryptophan-degrading enzymes were higher in cohorts of melanoma patients responding to immune checkpoint blockade. These findings highlight a novel mechanism of anti-tumor immunity and tumor growth inhibition dependent on the tryptophan degradation products, indoles, produced by intestinal Bacteroides species.

cancer biology↗

bioRxiv2025

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

biochemistry↗

bioRxiv2025

PI5P4Kα Regulates Iron Balance to Promote Metabolic Fitness in Pancreatic Cancer

Phosphoinositide kinases generate distinct phosphoinositides that regulate processes that maintain cellular fitness. Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) have garnered interest for their role in cancer metabolism and cellular trafficking; however, their function in pancreatic ductal adenocarcinoma (PDAC) remains unexplored. Given the unique metabolic demands of PDAC cells, which heavily rely on altered trafficking pathways to support their growth, investigating PI5P4Ks in this context may reveal critical insights. We identify PI5P4K as a regulator of PDAC cell fitness through its key role in maintaining iron homeostasis. PI5P4K depletion causes metabolic disruptions and reduced intracellular iron import, leading to the induction of apoptosis in PDAC cells that is reversed by iron supplementation. Notably, we find that PI5P4K knockdown suppresses tumor growth in a xenograft mouse model of PDAC. These results not only illuminate the mechanistic underpinnings of PI5P4K function in PDAC but also position it as a promising therapeutic target for this disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/647654v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@17f52e5org.highwire.dtl.DTLVardef@1b78c1org.highwire.dtl.DTLVardef@194e09aorg.highwire.dtl.DTLVardef@125b3c9_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic representation of the novel function and implication of PI5P4K in PDAC. PI5P4K is upregulated in PDAC to support the ferro-addiction and heightened metabolic requirements of the tumor. When PI5P4K is inhibited, the PDAC cells undergo apoptotic cell death due to iron depletion. PI5P4K-depleted PDAC cells upregulate autophagy as an adaptive response but is not sufficient to defend against apoptotic cell death.

cancer biology↗

bioRxiv2024

Macropinocytosis controls metabolic stress-driven CAF subtype identity in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) tumors are deficient in glutamine, an amino acid that tumor cells and CAFs use to sustain their fitness. In PDAC, both cell types stimulate macropinocytosis as an adaptive response to glutamine depletion. CAFs play a critical role in sculpting the tumor microenvironment, yet how adaptations to metabolic stress impact the stromal architecture remains elusive. In this study, we find that macropinocytosis functions to control CAF subtype identity when glutamine is limiting. Our data demonstrate that metabolic stress leads to an intrinsic inflammatory CAF (iCAF) program driven by MEK/ERK signaling. Utilizing in vivo models, we find that blocking macropinocytosis alters CAF subtypes and reorganizes the tumor stroma. Importantly, these changes in stromal architecture can be exploited to sensitize PDAC to immunotherapy and chemotherapy. Our findings demonstrate that metabolic stress plays a role in shaping the tumor microenvironment, and that this attribute can be harnessed for therapeutic impact. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/625709v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@40b38eorg.highwire.dtl.DTLVardef@1cc6a3borg.highwire.dtl.DTLVardef@15283e8org.highwire.dtl.DTLVardef@5214d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Genetically encoded fluorescent sensors for visualizing polyamine levels, uptake, and distribution

Polyamines are abundant and physiologically essential biomolecules that play a role in numerous processes, but are disrupted in diseases such as cancer, and cardiovascular and neurological disorders. Despite their importance, measuring free polyamine concentrations and monitoring their metabolism and uptake in cells in real-time remains impossible due to the lack of appropriate biosensors. Here we engineered, characterized, and validated the first genetically encoded biosensors for polyamines, named iPASnFRs. We demonstrate the utility of iPASnFR for detecting polyamine import into mammalian cells, to the cytoplasm, mitochondria, and the nucleus. We demonstrate that these sensors are useful to probe the activity of polyamine transporters and to uncover biochemical pathways underlying the distribution of polyamines amongst organelles. The sensors powered a high-throughput small molecule compound library screen, revealing multiple compounds in different chemical classes that strongly modulate cellular polyamine levels. These sensors will be powerful tools to investigate the complex interplay between polyamine uptake and metabolic pathways, address open questions about their role in health and disease, and enable screening for therapeutic polyamine modulators.

cell biology↗

bioRxiv2024

Confinement and Catalysis Within De Novo Designed Peptide Barrels

De novo protein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses -helical barrel (HB) peptide assemblies, which comprise 5 or more -helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, HBs are analogous to molecular flasks made in supramolecular, polymer, and materials chemistry. Using Forster resonance energy transfer as a readout, we demonstrate that specific HBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an HB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the HB.

synthetic biology↗

bioRxiv2024

ZBTB11 Depletion Targets Metabolic Vulnerabilities in K-Ras Inhibitor Resistant PDAC

Over 95% of pancreatic ductal adenocarcinomas (PDAC) harbor oncogenic mutations in K-Ras. Upon treatment with K-Ras inhibitors, PDAC cancer cells undergo metabolic reprogramming towards an oxidative phosphorylation-dependent, drug-resistant state. However, direct inhibition of complex I is poorly tolerated in patients due to on-target induction of peripheral neuropathy. In this work, we develop molecular glue degraders against ZBTB11, a C2H2 zinc finger transcription factor that regulates the nuclear transcription of components of the mitoribosome and electron transport chain. Our ZBTB11 degraders leverage the differences in demand for biogenesis of mitochondrial components between human neurons and rapidly-dividing pancreatic cancer cells, to selectively target the K-Ras inhibitor resistant state in PDAC. Combination treatment of both K-Ras inhibitor-resistant cell lines and multidrug resistant patient-derived organoids resulted in superior anti-cancer activity compared to single agent treatment, while sparing hiPSC-derived neurons. Proteomic and stable isotope tracing studies revealed mitoribosome depletion and impairment of the TCA cycle as key events that mediate this response. Together, this work validates ZBTB11 as a vulnerability in K-Ras inhibitor-resistant PDAC and provides a suite of molecular glue degrader tool compounds to investigate its function.

cancer biology↗

bioRxiv2024

Metabolic Deficiencies Underlie Plasmacytoid Dendritic Cell Exhaustion After Viral Infection

Type I Interferons (IFN-I) are central to host protection against viral infections1. While any cell can produce IFN-I, Plasmacytoid Dendritic Cells (pDCs) make greater quantities and more varieties of these cytokines than any other cell type2. However, following an initial burst of IFN- I, pDCs lose their exceptional IFN-I production capacity and become "exhausted", a phenotype that associates with enhanced susceptibility to secondary infections3-5. Despite this apparent cost for the host, pDC exhaustion is conserved across multiple species and viral infections, but the underlying mechanisms and the potential evolutionary advantages are not well understood. Here we characterize pDC exhaustion and demonstrate that it is associated with a reduced capacity of pDCs to engage both oxidative and glycolytic metabolism. Mechanistically, we identify lactate dehydrogenase B (LDHB) as a novel positive regulator of pDC IFN-I production in mice and humans, show that LDHB deficiency is associated with suppressed IFN-I production, pDC metabolic capacity, and viral control following a viral infection, and demonstrate that preservation of LDHB expression is sufficient to partially restore exhausted pDC function in vitro and in vivo. Furthermore, restoring LDHB in vivo in exhausted pDCs increased IFNAR dependent infection- associated pathology. Therefore, our work identifies a novel and conserved mechanism for balancing immunity and pathology during viral infections, while also providing insight into the highly preserved but previously unexplained phenomenon of pDC exhaustion.

immunology↗

bioRxiv2023

Rationally seeded computational protein design

Computational protein design is advancing rapidly. Here we describe efficient routes to two families of -helical-barrel proteins with central channels that bind small molecules. The designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides. Adjacent helices are connected using computational loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels computationally. Throughout these pipelines, residues that define open states of the barrels are maintained. This minimises sequence sampling and accelerates routes to successful designs. For each of 6 targets, just 2 - 6 synthetic genes are made for expression in E. coli. On average, 80% express to give soluble monomeric proteins that are characterized fully, including high-resolution structures for most targets that match the seed structures and design models with high accuracy.

synthetic biology↗

bioRxiv2022

Serine-129 phosphorylation of α-synuclein is a trigger for physiologic protein-protein interactions and synaptic function

Phosphorylation of -synuclein at the Serine-129 site (-syn Ser129P) is an established pathologic hallmark of synucleinopathies, and also a therapeutic target. In physiologic states, only a small fraction of total -syn is phosphorylated at this site, and consequently, almost all studies to date have focused on putative pathologic roles of this post-translational modification. We noticed that unlike native (total) -syn that is widely expressed throughout the brain, the overall pattern of -syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser-129 site blocked the ability of -syn to attenuate activity-dependent synaptic vesicle (SV) recycling - widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments -syn Ser-129P, and this phosphorylation is required for -syn binding to VAMP2 and synapsin - two functional binding-partners that are necessary for -syn function. AlphaFold2-driven modeling suggests a scenario where Ser129P induces conformational changes in the C-terminus that stabilizes this region and facilitates protein-protein interactions. Our experiments indicate that the pathology-associated Ser129P is an unexpected physiologic trigger of -syn function, which has broad implications for pathophysiology and drug-development.

neuroscience↗

bioRxiv2022

Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Despite this established dependency, the targeting of specific enzymes involved in glutamine metabolism is yet to yield any clinical benefit. Here, we have examined the therapeutic potential of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism. We found that DON treatment significantly blocks PDAC tumor growth and attenuates metastasis. Interestingly, we link the effectiveness of DON in PDAC to asparagine (Asn) metabolism. By inhibiting asparagine synthetase (ASNS), DON significantly reduces intracellular Asn production and Asn supplementation rescues the anti-proliferative effects of DON. We discern that PDAC cells upregulate expression of ASNS as a metabolic adaptation and that modulating ASNS levels can impact DON efficacy. Strikingly, in patient-derived organoids, DON responsiveness is inversely correlated with ASNS expression, a feature that is not observed for other metabolic enzymes targeted by DON. We find that treatment with L-asparaginase (ASNase), an enzyme that catabolizes free Asn, synergizes with DON to impact the viability of PDAC cells. Finally, we identify that a combination therapy of DON and ASNase has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of co-targeting adaptive responses to control PDAC progression.

cancer biology↗

bioRxiv2022

Biphasic response of CD8 T cell to asparagine restriction maximizes its metabolic fitness and antitumoral functionality

Robust and effective T cell immune surveillance and cancer immunotherapy require properly allocating metabolic resources to sustain energetically costly processes, including growth and cytokine production. Amino acids are major cellular constituents that serve as protein building blocks, energy sources, and signaling molecules. Although T cells can synthesize all nonessential amino acids, including asparagine (Asn), activated CD8 T cells still consume considerable quantities of exogenous Asn. Unexpectedly, Asn restriction on CD8 T cells induced a biphasic response, consisting of sequential actions with opposing effects at two conceptually separated phases after activation. Asn restriction suppressed activation and cell cycle entry in the early phase by depleting the intracellular Asn pool while rapidly engaging an ATF4/NRF2-dependent stress response, conferring robust proliferation and effector function of CD8 T cells in the late phase. Mechanistically, ATF4 and NRF2 activation rendered CD8 T cells to utilize de novo biosynthesis of Asn, consuming less glucose and glutamine but producing more intracellular nucleotides for proliferation. Moreover, NRF2 activation promoted the expression of inflammatory and effector genes to enhance effector functions in CD8 T cells. Accordingly, Asn restriction or overexpression of ATF4 or NRF2 potentiated T cell-mediated antitumoral response in the metabolically restricted tumor microenvironment. Our studies revealed Asn as a critical metabolic node in directing the stress signaling to shape T cell metabolic fitness and effector functions. Asn restriction is a promising and clinically relevant strategy to enhance cancer immunotherapy.

immunology↗

bioRxiv2022

gamma aminobutyrate (GAB) functions as a bioenergetic and signaling gatekeeper to control T cell inflammation

{gamma}-Aminobutyrate (GAB) is the biochemical form of {gamma}-aminobutyric acid (GABA) at physiological pH and functions as an essential neurotransmitter in the vertebrates central nervous system (CNS). Growing evidence suggests that GAB may also mediate intercellular communications to shape various physiological processes, including immune response. Beyond acting as a paracrine signaling molecule, how GAB metabolism is controlled to exert many distinct functions remains elusive. By an integrated analysis of the extracellular metabolome, stable isotope traced metabolic pathway analysis, and metabolic transcriptome, we revealed that GAB is one of the most abundant metabolites produced through glutamine and arginine catabolism in CD4+ T help 17 (TH17) and induced T regulatory (iTreg) cells. GAB functions as a bioenergetic and signaling gatekeeper by reciprocally controlling pro-inflammatory TH17 cell and anti-inflammatory iTreg cell differentiation through distinct mechanisms. The expression of 4-aminobutyrate aminotransferase (ABAT) funnels GAB, as an anaplerotic substrate, into the TCA cycle to maximize carbon allocation in promoting TH17 cell differentiation. By contrast, the absence of ABAT activities in iTreg cells enables GAB exporting to the extracellular environment and acting as an autocrine signaling metabolite to promote iTreg cell differentiation. Accordingly, genetic or pharmacological ablation of ABAT activity in T cells confers protection against experimental autoimmune encephalomyelitis (EAE) pathogenic progression. Conversely, genetic ablation of GABA(A) receptor in T cells deteriorates EAE pathogenic progression. Collectively, our results suggest that the cell-autonomous control exerted by GAB on CD4+ T cell is bimodal and consists of the sequential action of two discrete processes, ABAT-dependent mitochondrial anaplerosis and the receptor-dependent autocrine signaling response, both of which are required for a properly controlled T cell-mediated inflammation.

immunology↗

bioRxiv2021

Metabolic heritage mapping: heterogenous pools of cytoplasmic nucleotide sugars are selectively utilized by various glycosyltransferases

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have general applicability.

biochemistry↗

bioRxiv2021

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation

Robust and effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain energetically costly processes like growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By employing complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fuels biosynthetic processes while controlling the ratio of -ketoglutarate/succinate to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells causes proliferation and survival defects when the TCA cycle is truncated, blocking carbon flux to support nucleosides biosynthesis. Accordingly, replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. Conversely, SDH deficiency induces a pro-inflammatory gene signature in T cells and promotes T helper 1 (TH1) and T helper 17 (TH17) lineage differentiation. Mechanistically, the hypoxia-inducible factor 1 (HIF-1) is not required for succinate-induced inflammation in T cells. A reduced -ketoglutarate/succinate ratio in SDH deficient T cells promotes inflammation through changing the pattern of the transcriptional and chromatin-accessibility signatures and consequentially increasing the expression of the transcription factor, B lymphocyte-induced maturation protein-1 (Blimp-1). Collectively, our studies revealed a critical role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

immunology↗

bioRxiv2021

Melanoma Addiction to GCDH is Mediated by NRF2 Tumor Suppressor Function

Tumor dependency on specific metabolic signals has guided numerous therapeutic approaches. Here we identify melanoma addiction to the mitochondrial protein Glutaryl-CoA dehydrogenase (GCDH), a component in lysine metabolism which controls protein glutarylation. GCDH knockdown promoted apoptotic Unfolded Protein Response signaling and cell death in melanoma cells, an activity blocked by knockdown of the upstream lysine catabolism enzyme DHTKD1. Correspondingly, reduced GCDH expression correlated with improved survival of melanoma patients. A key mediator of GCDH-dependent melanoma cell death programs is the transcription factor NRF2, which induces ATF3, CHOP, and CHAC1 transcription linking lysine catabolism with the UPR signaling. NRF2 glutarylation upon GCDH KD increased its stability and DNA binding activity, which coincided with increased transcriptional activity, promoting apoptotic UPR signaling and tumor suppression. In vivo, genetic GCDH inhibition effectively inhibited melanoma tumor growth. Overall, these findings demonstrate an addiction of melanoma cells to GCDH, which by controlling NRF2 glutarylation limits apoptotic UPR signaling. Inhibiting the GCDH pathway could represent a novel therapeutic modality to treat melanoma.

cancer biology↗

bioRxiv2021

OGT controls mammalian cell viability by regulating the proteasome/mTOR/ mitochondrial axis

O-GlcNAc transferase (OGT) catalyzes the modification of serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We employed a genome-wide CRISPR-Cas9 viability screen in mouse embryonic stem cells (mESCs) with inducible Ogt gene deletion and showed that the block in cell viability induced by OGT deficiency stems from mitochondrial dysfunction secondary to mTOR hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phosphoproteomic screen. Our study highlights a novel series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.

cell biology↗