Amino Acid Starvation Response Research Articles

The EPRS-ATF4-COLI pathway axis is a potential target for anaplastic thyroid carcinoma therapy

BackgroundAnaplastic thyroid carcinoma (ATC) is recognized as the most aggressive and malignant form of thyroid cancer, underscoring the critical need for effective therapeutic strategies to curb its progression and improve patient prognosis. Halofuginone (HF), a derivative of febrifugine, has displayed antitumor properties across various cancer types. However, there is a paucity of published research focused on the potential of HF to enhance the clinical efficacy of treating ATC. ObjectiveIn this study, we thoroughly investigated the antitumor effects and mechanisms of HF in ATC, aiming to discover lead compounds for treating ATC and reveal novel therapeutic targets for ATC tumors. MethodsA series of assays, including CCK8, colony formation, tumor xenograft models, and ATC tumor organoid experiments, were conducted to evaluate the anticancer properties of HF both in vitro and in vivo. Techniques such as drug affinity responsive target stability (DARTS), western blot, immunofluorescence, and immunohistochemistry were employed to pinpoint HF target proteins within ATC. Furthermore, we harnessed the GEPIA and GEO databases and performed immunohistochemistry to validate the therapeutic potential of the glutamyl-prolyl-tRNA-synthetase (EPRS)- activating transcription factor 4 (ATF4)- type I collagen (COLI) pathway axis in the context of ATC. The study also incorporated RNA sequencing analysis, confocal imaging, and flow cytometry to delve into the molecular mechanisms of HF in ATC. ResultsHF exhibited a substantial inhibitory impact on cell proliferation in vitro and on tumor growth in vivo. The DARTS results highlighted HF's influence on EPRS within ATC cells, triggering an amino acid starvation response (AASR) by suppressing EPRS expression, consequently leading to a reduction in COLI expression in ATC cells. The introduction of proline mitigated the effect of HF on ATF4 and COLI expression, indicating that the EPRS-ATF4-COLI pathway axis was a focal target of HF in ATC. Analysis of the expression levels of the EPRS, ATF4, and COLI proteins in thyroid tumors, along with an examination of the relationship between COLI expression and thyroid tumor stage, revealed that HF significantly inhibited the growth of ATC tumor organoids, demonstrating the therapeutic potential of targeting the EPRS-ATF4-COLI pathway axis in ATC. RNA sequencing analysis revealed significant differences in the pathways associated with metastasis and apoptosis between control and HF-treated cells. Transwell assays and flow cytometry experiments provided evidence of the capacity of HF to impede cell migration and induce apoptosis in ATC cells. Furthermore, HF hindered cell metastasis by suppressing the epithelial-mesenchymal transition (EMT) pathway, acting through the inhibition of FAK-AKT-NF-κB/Wnt-β-catenin signaling and restraining angiogenesis via the VEGF pathway. HF also promoted apoptosis through the mitochondrial apoptotic pathway. ConclusionThis study provided inaugural evidence suggesting that HF could emerge as a promising therapeutic agent for the treatment of ATC. The EPRS-ATF4-COLI pathway axis stood out as a prospective biomarker and therapeutic target for ATC.

Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy

The manipulation of Tcell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ Tcells exvivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ Tcells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated Tcells synergized invivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ Tcells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance Tcell metabolism and anti-tumor activity.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

Clinical pathological and genetic mutation characteristics of conjunctival lymphoepithelial carcinoma

Objective: To analyze the clinical pathological and genetic mutation characteristics of conjunctival lymphoepithelial carcinoma. Methods: A retrospective case series study was conducted. Data from three patients diagnosed with conjunctival lymphoepithelial carcinoma and treated with tumor resection surgery at Tianjin Eye Hospital from January 2006 to December 2022 were collected. Four paraffin specimens (including one patient undergoing two surgeries) were subjected to immunohistochemical staining for epithelial antigen and lymphocytic antigen. Epstein-Barr virus (EBV)-encoded RNA (EBER) was detected using in situ hybridization, and whole-exome sequencing was performed on three specimens from two patients using next-generation sequencing methods. Results: All three patients were males aged over 65, with a disease duration ranging from 3 to 44 months. The tumors were unilateral, located on the bulbar or limbal conjunctiva, appearing red, with a maximum diameter of 4-20 mm. Imaging examinations revealed anterior location of the tumors with no involvement of the orbital bone, extraocular muscles, optic nerve, or paranasal sinuses. No local lymph node metastasis was observed in any patient. Pathological findings included undifferentiated carcinoma nests with significant reactive lymphocytic and plasma cell infiltration. Tumor cells were positive for pan-cytokeratin (CK-pan), epithelial membrane antigen (EMA), tumor protein 40 (p40), and tumor protein 63 (p63), with a cell proliferation index (Ki67) exceeding 80%. Cluster of differentiation 20 (CD20), CD3, and CD8 were positive for lymphocytes. In situ hybridization showed partial tumor cell expression of EBER in two specimens of one patient. Whole-exome sequencing revealed 58, 50, and 36 mutated genes in the three specimens, with enriched signaling pathways including melanoma signaling pathway, Notch1 signaling pathway, and RHOQ GTP cycle; enriched biochemical processes included amino acid starvation response, programmed cell death, regulation of lipid synthesis, sodium ion transport, and chromosome segregation. The common mutated gene in all three specimens was SZT2, and SZT2 was involved in the amino acid starvation response. One patient underwent a second complete resection surgery 40 months after partial excision, while the other two underwent complete resection surgery without recurrence. Two patients did not undergo radiation or chemotherapy, and one was lost to follow-up. Conclusions: Conjunctival lymphoepithelial carcinoma is associated with prominent lymphocytic and plasma cell infiltration, some cases are associated with EBV infection, and SZT2 mutations are present in conjunctival lymphoepithelial carcinoma.

Abstract 11029: Bioenergetic Independent Stress Signaling in the Heart Due to Mitochondrial Protein Import Stress

Mitochondrial diseases are some of the most common genetically inherited disorders, yet the prognosis for affected patients has not drastically improved in recent years. Most therapies are supportive as the underlying biochemical derangements in these diseases are incompletely understood. Mitochondrial functions rely on the import of nuclear-encoded proteins, and it has recently been appreciated that defective import of mitochondrial proteins can result in the cytosolic accumulation of these preproteins. The accumulation of these preproteins results in cellular toxicity independently of any changes to the mitochondrial bioenergetic function, raising the possibility that mitochondrial protein import stress may be a yet uncharacterized pathway that links mitochondrial dysfunction with pathology. Here, we model this cytosolic mitochondrial precursor overaccumulation stress (mPOS) in mice through overexpression of the inner mitochondrial membrane protein Adenine Nucleotide Translocase 1 (ANT1). The goal of this study is to determine whether ANT1 can induce mPOS and tissue remodeling prior to the onset of cardiac bioenergetic defects. Using isolated cardiac mitochondria from these mice, we detected a moderate decrease in the levels of Complex I, II ,and IV. By measuring oxygen consumption in these mitochondria from 2-month-old animals, we found only a mild reduction in complex II based state 3 and 4 respirations. No change in complex I-based state 3 and 4 respirations was observed. Using RNAseq and western blot analysis, we find the robust activation of the integrated stress response (ISR). Transcriptomic data further demonstrates a strong upregulation of transcripts involved with 1-carbon metabolism, the amino acid starvation response, and proteolysis. Using echocardiographic evaluation of heart function on anesthetized mice at 6 months old, we find no significant alteration in systolic function. Together these data support the idea that mPOS may be a potent trigger of the ISR in a bioenergetics-independent manner. Whether activation of the ISR can alter cardiac function later in life is being evaluated.

Abstract 10708: Dietary Methionine Restriction Protects Against Abdominal Aortic Aneurysm via Amino Acid Starvation Response

Introduction: Dietary intervention emerges as a promising non-medicinal avenue for the treatment of a wide range of diseases. Limiting the intake of single amino acid has been shown to produce health benefits similar to that of calorie restriction yet without significant risks of malnutrition. One of the established regimen, methionine restriction (MR), was recently shown to confer pleiotropic benefits in cardiometabolic disease models, largely attributed to the activation of the GCN2-mediated amino acid starvation response pathway. However, the therapeutic and mechanistic implications of dietary amino acid restriction remain unknown in abdominal aortic aneurysm (AAA), a lethal vascular disease that is without a cure other than the traumatic surgical repairs. Herein, we sought to probe the outcome of MR as well as the role of GCN2 in AAA. Methods and Results: Following a 2-week pre-treatment with MR (20% methionine level) or iso-caloric control diet, male Sprague Dawley rats were subjected to intraluminal elastase perfusion. At day 7, animals in the MR group showed significantly less aneurysmal dilation in comparison to control. To investigate the contribution from the GCN2-mediated amino acid starvation response, we subjected animals to the same dietary regimen of MR or control, followed by GCN2 silencing in the elastase-challenged aortic segments through localized infusion with GCN2-targeting or scrambled shRNA lentivirus. Indeed, tuning down aortic GCN2 effectively compromised the benefits exerted by MR against AAA. Finally, we determined whether GCN2 constitutes an intrinsic protector against aortic degeneration using GCN2 knockout mice ( Gcn2 -/- ). Following periaortic elastase challenge and β-aminopropionitrile treatment, Gcn2 -/- mice displayed exacerbated expansion of aneurysmal lesions in comparison to Gcn2 +/+ control mice at day 28 post procedure. Conclusions: Our study provides the first experimental evidence supporting a potential role of dietary amino acid restriction (e.g., MR) in the prevention of AAA development. Additionally, GCN2-mediated amino acid starvation response possibly underlies the anti-AAA benefit exerted by MR and serves as an intrinsic protector against AAA pathogenesis.

Halofuginone for cancer treatment: A systematic review of efficacy and molecular mechanisms

• We provide a comprehensive review of the most recent anticancer studies reported for Halofuginone (HF). • HF exhibits significant anticancer activity and synergistic effects in chemotherapy/radiotherapy/virus for the treatment of various cancers in vitro and in vivo. • HF could induce cell cycle arrest, apoptosis and autophagy, inhibit cell proliferation, invasion, migration and angiogenesis, and disturb the tumor microenvironment in tumor cells. • The anticancer effect of HF might be associated with the induction of the amino acid starvation response by HF in response to the inhibition of glutamyl-prolyl-tRNA synthetase. Cancer is still considered a “hopeless case”, which seriously endangers human health. Therefore, it is of great significance to research and develop novel anticancer drugs. Active natural products play an important role in anticancer drug discovery by providing lead compounds. Halofuginone (HF), as a derivative of febrifugine, an alkaloid originally isolated from the plant Dichroa febrifuga , is used for the prevention and treatment of coccidiosis and malaria in poultry. In recent years, HF has shown excellent anticancer activity and great clinical potential for treating various cancers. Despite enormous progress, a thorough elaboration of the proposed mechanism and mode of action is absent. Herein, we provide a comprehensive review of the most recent anticancer studies reported for HF and a rationale for further exploring the potential application of HF in overcoming cancer in the future.

Bioinformatics analysis of the prognostic and immunotherapeutic significance of NPRL2 in stomach adenocarcinoma.

BackgroundStomach adenocarcinoma (STAD) is a major type of gastric cancer with high morbidity and mortality. NPRL2, a candidate cancer suppressor gene, has been shown to have anti-cancer effects in various types of cancers. Therefore, comprehensive analyses of NPRL2 in STAD may provide a potential prognostic marker and clinical target for the management of gastric cancer.MethodsGenomic expression and methylation were analysed based on data from the Human Protein Atlas, Gene Expression Omnibus and Oncomine database. Survival analyses were conducted with the Kaplan-Meier method, using data from The Cancer Genome Atlas database. Immune correlation analyses and prediction of response to immunotherapy were performed using the online Immune Cell Abundance Identifier. Co-expression analyses, functional clustering analyses and construction of a prognostic risk model were conducted in R, with the clinical covariates balanced by the inverse probability treatment weighting method.ResultsNPRL2 was abnormally downregulated in STAD (P<0.05). Survival analysis highlighted a positive association between the expression of NPRL2 and clinical outcomes for patients (P<0.05). Based on co-expression analyses, we found that NPRL2 may be involved in epithelial-mesenchymal transition, gastric cancer stem cells, and responsiveness to chemotherapeutic agents in STAD (P<0.05). Furthermore, functional clustering analysis revealed that NPRL2 was involved in the mTOR signalling pathway, autophagy, and the amino acid starvation response (adjust P<0.05). In addition, NPRL2 was negatively associated with tumour-infiltrating immune cells while positively associated with immunotherapeutic biomarkers in STAD (P<0.05). Meanwhile, patients with high NPRL2 expression were predicted to have a better response to immunotherapy (P<0.05). Finally, a prognostic model constructed based on NPRL2-related genes could predict the prognosis of STAD patients (AUC =0.641), and the risk score was an independent prognostic factor for STAD patients (HR =4.855, 95% CI: 2.683–8.785, P<0.001).ConclusionsThe present study provided a comprehensive analysis of the role and potential mechanisms of NPRL2 in STAD, suggesting that NPRL2 is a potential biomarker for the survival and prediction of immunotherapy response in STAD.

Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces-Aspergillus interaction.

Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid (ORS) and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact is a prerequisite for the metabolic reaction that involves a fungal amino acid starvation response and activating chromatin modifications at the biosynthetic gene cluster (BGC). Here we show that not physical contact, but a guanidine containing macrolide, named polaramycin B, triggers the response. The substance is produced constitutively by the bacterium and above a certain concentration, provokes the production of ORS. In addition, several other secondary metabolites were induced by polaramycin B. Our genome-wide transcriptome analysis showed that polaramycin B treatment causes downregulation of fungal genes necessary for membrane stability, general metabolism and growth. A compensatory genetic response can be observed in the fungus that included upregulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a novel chemical communication, in which the antifungal bacterial metabolite polaramycin B leads to the production of antibacterial defence chemicals and to the upregulation of genes necessary to compensate for the cellular damage caused by polaramycin B.

S107: SOD2 PROMOTES ACUTE LEUKEMIA ADAPTATION TO AMINO ACID STARVATION THROUGH THE N-DEGRON PATHWAY

Background: The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood. Aims: We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2, a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate. Methods: To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays. Results: Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines (p<0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity (p<0.001). The sensitization was rescued by either overexpressing SOD2 cDNA (p<0.0001), or by adding the functional SOD2 mimetic MnTBAP (p<0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine (p=ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2-inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability (p<0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions. Summary/Conclusion: The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias.

Abstract 5684: Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress

Abstract Cancer cells survive under extreme environments that alter protein homeostasis, thereby provoking cellular stress responses, such as the unfolded protein response (UPR) and the integrated stress response (ISR). These pathways increase the expression of molecular chaperones to prevent the accumulation of toxic proteins in the cell. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor. We first showed that established breast cancer cell lines bin into classes based on their sensitivity to the drug. We next discovered that resistant cells have higher autophagy levels compared to the more sensitive lines. Moreover, gene and protein expression studies and imaging methods showed that autophagy was further induced by Hsp70 inhibition in resistant breast cancer cells. Consistent with the fact that Hsp70 normally targets aberrant proteins for degradation, our data indicated that resistance to compromised Hsp70 activity arose from elevated levels of autophagy. We next showed that Hsp70 inhibition specifically triggers the ISR pathway. Unexpectedly, protection against the Hsp70 inhibitor was dependent on the ISR sensor, GCN2, which is most commonly associated with the amino acid starvation response. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating the PERK pathway. These data reveal an unexpected route through which cancer cells can adapt to chaperone targeted-proteotoxicity and position GCN2 and autophagy as critical compensatory mechanisms for cancer cells when proteostasis is compromised. Citation Format: Sara Sannino, Jeffrey L. Brodsky. Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5684.

Inducible Systemic Gcn1 Deletion in Mice Leads to Transient Body Weight Loss upon Tamoxifen Treatment Associated with Decrease of Fat and Liver Glycogen Storage.

GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial β-oxidation that accompanied increases of peroxisomal β-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.

The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

Origin of translational control by eIF2α phosphorylation: insights from genome-wide translational profiling studies in fission yeast.

During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question.

Anxiolytic Drug FGIN-1-27 Ameliorates Autoimmunity by Metabolic Reprogramming of Pathogenic Th17 Cells

Th17 cells are critical drivers of autoimmune diseases and immunopathology. There is an unmet need to develop therapies targeting pathogenic Th17 cells for the treatment of autoimmune disorders. Here, we report that anxiolytic FGIN-1-27 inhibits differentiation and pathogenicity of Th17 cells in vitro and in vivo using the experimental autoimmune encephalomyelitis (EAE) model of Th17 cell-driven pathology. Remarkably, we found that the effects of FGIN-1-27 were independent of translocator protein (TSPO), the reported target for this small molecule, and instead were driven by a metabolic switch in Th17 cells that led to the induction of the amino acid starvation response and altered cellular fatty acid composition. Our findings suggest that the small molecule FGIN-1-27 can be re-purposed to relieve autoimmunity by metabolic reprogramming of pathogenic Th17 cells.

The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels.

During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

Anesthetic pretreatment confers thermotolerance on Saccharomyces cerevisiae yeast

Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52 °C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 μM), and carbon tetrabromide (32 μM). Thermoprotection conferred by pretreatment with elevated temperatures was “left shifted” by anesthetic co-treatment from (in °C) 35.3 ± 0.1 to 32.2 ± 0.1 with trifluoroethanol (177.8 mM), to 31.2 ± 0.1 with trichloroethanol (17.8 mM), and to 29.1 ± 0.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.

Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production.

Specific reduction in the intake of proteins or amino acids (AAs) offers enormous health benefits, including increased life span, protection against age-associated disorders, and improved metabolic fitness and immunity. Cells respond to conditions of AA starvation by activating the amino acid starvation response (AAR). Here, we showed that mimicking AAR with halofuginone (HF) enhanced the magnitude and affinity of neutralizing, antigen-specific antibody responses in mice immunized with dengue virus envelope domain III protein (DENVrEDIII), a potent vaccine candidate against DENV. HF enhanced the formation of germinal centers (GCs) and increased the production of the cytokine IL-10 in the secondary lymphoid organs of vaccinated mice. Furthermore, HF promoted the transcription of genes associated with memory B cell formation and maintenance and maturation of GCs in the draining lymph nodes of vaccinated mice. The increased abundance of IL-10 in HF-preconditioned mice correlated with enhanced GC responses and may promote the establishment of long-lived plasma cells that secrete antigen-specific, high-affinity antibodies. Thus, these data suggest that mimetics of AA starvation could provide an alternative strategy to augment the efficacy of vaccines against dengue and other infectious diseases.

Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

BackgroundIn the event of amino acid starvation, the cell activates two main protective pathways: Amino Acid starvation Response (AAR), to inhibit global translation, and autophagy, to recover the essential substrates from degradation of redundant self-components. Whether and how AAR and autophagy (ATG) are cross-regulated and at which point the two regulatory pathways intersect remain unknown. Here, we provide experimental evidence that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) specifically located at the lysosome level links the AAR with the autophagy pathway.MethodsAs an inducer of the AAR, we used halofuginone (HF), an alkaloid that binds to the prolyl-tRNA synthetase thus mimicking the unavailability of proline (PRO). Induction of AAR was determined assessing the phosphorylation of the eukaryotic translation initiation factor (eIF) 2α. Autophagy was monitored by assessing the processing and accumulation of microtubule-associated protein 1 light chain 3 isoform B (LC3B) and sequestosome-1 (p62/SQSTM1) levels. The activity of mTORC1 was monitored through assessment of the phosphorylation of mTOR, (rp)S6 and 4E-BP1. Global protein synthesis was determined by puromycin incorporation assay. mTORC1 presence on the membrane of the lysosomes was monitored by cell fractionation and mTOR expression was determined by immunoblotting.ResultsIn three different types of human cancer cells (thyroid cancer WRO cells, ovarian cancer OAW-42 cells, and breast cancer MCF-7 cells), HF induced both the AAR and the autophagy pathways time-dependently. In WRO cells, which showed the strongest induction of autophagy and of AAR, global protein synthesis was little if any affected. Consistently, 4E-BP1 and (rp)S6 were phosphorylated. Concomitantly, mTOR expression and activation declined along with its detachment from the lysosomes and its degradation by the proteasome, and with the nuclear translocation of transcription factor EB (TFEB), a transcription factor of many ATG genes. The extra supplementation of proline rescued all these effects.ConclusionsWe demonstrate that the AAR and autophagy are mechanistically linked at the level of mTORC1, and that the lysosome is the central hub of the cross-talk between these two metabolic stress responses.

Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1β (IL-1β) and provides protection from intestinal inflammation in mice. HF inhibits IL-1β through general control nonderepressible 2 kinase (GCN2)–dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1β mRNA from translationally active polysomes to inactive ribocluster complexes—such as stress granules (SGs)—via recruitment of RNA-binding proteins (RBPs) T cell–restricted intracellular antigen-1(TIA-1)/TIA-1–related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1β production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1β is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.