The use of antiretroviral (ART) treatment during pregnancy has dramatically reduced rates of perinatally-acquired human immunodeficiency virus 1 (HIV-1) infection to <1% in the United States. Despite this success, we have limited knowledge of how ART drugs that cross the placental barrier affect fetal development, particularly in the central nervous system (CNS). During gestation, large populations of oligodendroglia are produced that are responsible for critical postnatal CNS myelination enabling appropriate neurological function. Previous studies have shown that antiretrovirals impair oligodendrocyte (OL) differentiation leading us to hypothesize that OL maturation might be inhibited by exposure to a frontline ART drug cocktail (Triumeq®) prescribed during pregnancy containing dolutegravir (DTG), abacavir (ABC), and lamivudine (3TC). In this study, we demonstrated that exposing primary rat oligodendrocyte precursor cells (OPCs) and OLs to the Triumeq drug combination decreased OL maturation and myelin protein production in a concentration-dependent manner, and that DTG was solely responsible. Regardless of the timing of exposure during OL development, a high concentration of DTG inhibited OL maturation. Bulk RNA sequencing revealed transcriptional changes after DTG exposure related to a variety of cellular mechanisms, including cellular responses to stress pathways, amino acid starvation, and mitochondrial dysfunction. Although we found that DTG robustly activated the integrated stress response (ISR), attempted rescue experiments showed that DTG primarily inhibits OL maturation independently of the ISR. Collectively, our novel data on DTG underscore the necessity of investigating how ART drugs that are administered during pregnancy and cross the placental barrier can affect fetal CNS development.

The Juvenile form of Batten disease is a neurodegenerative disease with symptoms starting in the first decade and ending in death in the third decade of life.The gene defective in this form of Batten disease, CLN3, is conserved in eukaryotes, suggesting that the gene product serves a basic function in the cell, though the function is unknown. We have investigated the expression and regulation of the yeast homolog BTN1.Reanalysis of publicly available gene expression data suggests that transcription of BTN1 increases in response to oxidative stress, treatment with rapamycin or arsenate, amino acid starvation, and sporulation conditions. Similar to GCN4, there are upstream open reading frames (uORF) in front of BTN1, suggesting translational regulation. We developed reporter strains in which the HIS3 open reading frame replaced that of the BTN1 gene, with and without the uORFs.These reporters show that one or more of the uORFs decrease the expression of the HIS3 reporter.When expressed in the reporter strain using a high copy vector, GCN3, tRNA , and tRNA , increase expression, suggesting the involvement of the TORC1 pathway.BIT61 abuts BTN1 but is encoded on the opposite strand; 3' RACE analysis indicates that the mRNA of BIT61 overlaps with that of BTN1.BIT61 is involved in the TORC2 pathway, which interacts with the TORC1 pathway, suggesting a possible cis-acting mechanism of co-regulation.Lastly, we demonstrate that a yeast strain with a null mutation in BTN1 is sensitive to selective amino acid starvation, further supporting the association of BTN1 with TORC1.

Mechanistic insights into L-asparaginase resistance in cancers.

Peripheral amylin modulation rebalances brain glycolysis and Tau-Ser214 phosphorylation via cAMP-PKA signaling

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous, and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we set out to create a quantitative dataset of autophagy across multiple stages in single, living cells, measured under normal growth conditions and during nutrient starvation of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions.

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous, and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we set out to create a quantitative dataset of autophagy across multiple stages in single, living cells, measured under normal growth conditions and during nutrient starvation of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions.

Several mammarenaviruses (MaAv), chiefly Lassa virus (LASV) in Western Africa and Junin virus (JUNV) in the Argentinean Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen of clinical significance, especially in congenital infections, and LCMV poses a serious risk for immunocompromised individuals. There are no FDA-approved MaAv vaccines or antivirals, and current anti-MaAv therapy is limited to an off-label use of ribavirin, whose efficacy remains controversial. This highlights an urgent unmet need for developing antivirals against human pathogenic MaAv. Halofuginone (HF), a derivative of the natural alkaloid febrifugine, has been shown to exhibit antiviral activity against several RNA viruses. Here, we present evidence that HF exhibits potent dose-dependent antiviral activity against LCMV, and against the hemorrhagic fever causing MaAv LASV and JUNV. HF binds to the bifunctional enzyme glutamyl-prolyl-tRNA synthetase 1 (EPRS1) and specifically inhibits its prolyl-tRNA synthetase (PRS) activity, resulting in translation inhibition via the amino acid starvation (AAS) response with preferential impact on proline-rich proteins. HF anti-LCMV activity was prevented by the addition of exogenous proline supporting that inhibition of PRS activity plays a critical role in the anti-MaAv activity of HF. We found that HF did not affect LCMV cell entry, modestly (twofold) reduced the activity of the virus ribonucleoprotein (vRNP), but strongly inhibited (>90%) Z budding activity, a process involving the Z proline-rich late domain motifs.

The general control nonderepressible 2 (GCN2) is a conserved stress-responsive protein that plays a critical role in restoring cellular homeostasis in the integrated stress response (ISR). In response to amino acid starvation or ribosome stalling and collisions, GCN2 phosphorylates the translation initiation factor eIF2α, conferring translational control to alleviate stress. GCN2 is a multidomain protein, containing a tandem kinase domain (KD) and a catalytically inactive pseudokinase domain (ψKD). Stress-induced activation of the kinase domain requires allosteric regulation and dimerization mediated by its regulatory domains. While the pseudokinase domain is essential for GCN2 function in yeast, its mechanistic role remains unclear and underexplored in other organisms. Here, we present the first crystal structure of the human GCN2 ψKD, revealing its distinct structural features. The structure visualizes an insertion N-terminal to helix αC unique to the GCN2 ψKD that interacts with the pseudoactivation loop, stabilizing an inactive conformation. Further structural analysis shows that the ψKD forms a dimer in the crystal lattice via a network of hydrophobic and electrostatic interactions spanning both the N- and C-lobes. Mutations that disrupt the dimer interface reduced downstream ATF4 expression that is important for stress adaptation, underscoring the functional significance of the GCN2 ψKD dimer in regulating GCN2 activity. Complementary AI-guided structure predictions indicate that the dimeric GCN2 ψKD architecture is conserved across evolution. These results support the role of ψKD dimerization as a regulatory feature in GCN2-mediated ISR signaling.

Several mammarenaviruses (MaAv), chiefly Lassa virus (LASV) in Western Africa and Junin virus (JUNV) in the Argentinean Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen of clinical significance especially in congenital infections and LCMV poses a serious risk for immunocompromised individuals. There are no FDA-approved MaAv vaccines or antivirals and current anti-MaAv therapy is limited to an off-label use of ribavirin whose efficacy remains controversial. This highlights an urgent unmet need for developing antivirals against human pathogenic MaAv. Halofuginone (HF), a derivative of the natural alkaloid febrifugine, has been shown to exhibit antiviral activity against several RNA viruses. Here, we present evidence that HF exhibits a potent dose-dependent antiviral activity against LCMV, and the hemorrhagic fever causing MaAv LASV and JUNV. HF binds to the bifunctional enzyme glutamyl-prolyl-tRNA synthetase 1 (EPRS1) and specifically inhibits its prolyl-tRNA synthetase (PRS) activity, resulting in translation inhibition via the amino acid starvation (AAS) response with preferential impact on proline-rich proteins. HF anti-LCMV activity was prevented by the addition of exogenous proline supporting that inhibition of PRS activity plays a critical role on the anti-MaAv activity of HF. We found that HF did not affect LCMV cell entry, modestly (twofold) reduced the activity of the virus ribonucleoprotein (vRNP) but strongly inhibited (>90%) Z budding activity, a process involving the Z proline-rich late domain motifs.

BackgroundAcute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with poor overall survival (OS). Resistance to chemotherapeutic drugs such as idarubicin (IDA) remains a major cause of treatment failure. This study investigated the anti-leukemic activity of halofuginone (HF) a synthetic derivative of the natural compound from hydrangea Dichroa febrifuge and its potential to overcome IDA resistance in AML cells.MethodsApoptosis, proliferation, cell cycle, and colony formation were assessed in AML cells treated with HF. RNA sequencing (RNA-seq) was performed to identify the potential molecular targets of HF. The anti-leukemic efficacy of HF was further assessed in NOD/SCID-IL2Rγ (NSG) mice xenografted with human relapsed/refractory (R/R) AML samples.ResultsHF treatment significantly inhibited cell proliferation, reduced colony formation, and induced apoptosis in AML cells. By RNA-seq analysis, S100A8 and S100A9 (S100A8/A9) were identified as potential targets of HF, and HF treatment markedly suppressed their expression. Overexpression of S100A8/A9 abrogated the anti-leukemic effects of HF, indicating that S100A8/A9 are critical mediators of HF activity. Mechanistically, HF activated the amino acid starvation response (AAR), leading to phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α), subsequent downregulation of S100A8/A9, and elevation of cytoplasmic Ca2⁺ levels. Knockdown of eIF2α prevented HF-induced downregulation of S100A8/A9, confirming that HF regulates S100A8/A9 expression via the eIF2α pathway. Furthermore, HF treatment inhibited global protein synthesis, enhanced the cytotoxicity of chemotherapeutic drugs, and reversed IDA resistance by suppressing S100A8/A9 expression. Finally, HF inhibits leukemic infiltration and extended OS in MLL-AF9-transduced AML mice and enhanced IDA-induced anti-leukemic effects in R/R AML-xenografted NSG mice model.ConclusionsThese findings reveal that HF exerts anti-leukemic effects by modulating the p-eIF2α–S100A8/A9–Ca2⁺ signaling axis in AML cells. HF represents a promising therapeutic candidate for AML, particularly for patients with IDA-resistant disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13020-025-01278-9.

Abstract Background Ulcerative colitis (UC) is a chronic, relapsing inflammatory disease of the colon characterized by impaired intestinal epithelial barrier function, a key feature of its complex pathogenesis. SLC6A19 is an important intestinal amino acid transporter that contributes to amino acid homeostasis and barrier integrity. This study investigates the role and mechanisms of SLC6A19 in regulating the intestinal epithelial barrier in UC, with potential implications for novel therapies. Methods This study integrates clinical samples, SLC6A19 knockout mice, in vitro cell models, and transcriptomics to elucidate the role and underlying mechanisms of SLC6A19 in the amino acid metabolic axis that maintains the homeostasis of the intestinal epithelial barrier in inflammatory bowel disease (IBD). First, we will characterize SLC6A19 expression in the intestinal mucosa of IBD patients and examine its association with barrier injury severity and clinical phenotype. In SLC6A19 knockout mouse models, we will assess how SLC6A19 influences the progression of intestinal inflammation and the integrity of the epithelial barrier. Finally, at the cellular level, we will examine how SLC6A19 maintains epithelial barrier integrity and regulates tight junction proteins. Results Compared with healthy controls, SLC6A19 expression in intestinal tissues from patients with IBD is significantly reduced; furthermore, SLC6A19 mRNA levels are significantly negatively correlated with Mayo scores in UC patients. SLC6A19-/- mice show increased susceptibility to DSS-induced colitis than WT mice. Amino acid-targeted metabolomics revealed significant reductions in valine, ornithine, and other amino acids in SLC6A19-/- mice. Transcriptomic sequencing of gut tissue indicated upregulation of genes such as KDM5D, and enrichment of signaling pathways including branched-chain amino acid degradation, the urea cycle, and the TCA cycle. In vitro lipopolysaccharide(LPS) exposure reduces SLC6A19 expression in a dose-dependent manner. Moreover, SLC6A19 knockdown increased barrier permeability in vitro, reduceed ZO-1 and E-cadherin expression, increased α-KG/succinate ratio, phosphorylation of eIF2α, increased expression of the amino acid starvation response effector ATF4, and increased transcription of the downstream target gene KDM5D. Conclusion SLC6A19 plays a crucial role in maintaining the homeostasis of the intestinal epithelial barrier in IBD. This finding provides a new potential therapeutic target for IBD, namely improving the intestinal epithelial barrier function by modulating the expression or function of SLC6A19, and may represent a promising new strategy for future IBD therapy.

Borrelia burgdorferi, the causative agent of Lyme disease, is well known for its unique physiology and enzootic cycle. Building on previous work showing peptide transport is essential for viability, we endeavored to clearly define the impact of peptide starvation on the spirochete and directly compare peptide starvation to targeted free amino acid starvation. Herein, we confirm the ability of a putative glutamate transporter, BB0401, to transport glutamate and aspartate as well as demonstrate its requirement for viability. Using conditional mutants for both peptide transport and BB0401, we characterize these systems throughout the enzootic cycle, confirming their essential role during murine infection and revealing that they are dispensable during prolonged colonization of the tick midgut. We broadly define the metabolic perturbations resulting from these starvation models and show, even under the most severe amino acid stress, B. burgdorferi is unable to modulate its physiological response via the canonical (p)ppGpp-driven stringent response.

The soil-borne fungus Verticillium dahliae is a devastating pathogen responsible for substantial losses in cotton production. This study elucidated the key functions of VdARO2 and VdCPC1 in fungal pathogenicity. VdARO2 encodes a Chalmoic acid synthase involved in the biosynthesis of aromatic amino acids, while VdCPC1 is a central regulator of amino acid starvation response and reveals a key regulatory relationship between VdARO2 and VdCPC1 to jointly control fungal virulence. We demonstrate that both genes are essential for growth, conidiation, and microsclerotia formation in V. dahliae. The VdΔaro2 mutant exhibited severe developmental defects and a complete loss of microsclerotia production, accompanied by widespread transcriptional dysregulation. Disruption of VdARO2 significantly upregulated VdCPC1, triggering a compensatory starvation response that nonetheless failed to restore pathogenicity. Silencing VdCPC1 similarly impaired fungal development and attenuated virulence. Our findings reveal a crucial regulatory axis in which VdARO2 and VdCPC1 coordinate metabolic homeostasis and stress adaptation to facilitate host colonization, thereby identifying promising targets for the control of Verticillium wilt.

PRRC2B is an intrinsically disordered RNA-binding protein that is part of the cell’s translation machinery. Here, we show that PRRC2B has two alternatively spliced mRNA transcripts producing major long and minor short isoforms. Mass spectrometry-based interaction studies indicated that both isoforms associate with the 40S ribosomal subunit and translation initiation factors. Importantly, the long isoform also interacted with additional RNA-binding proteins through its unique Arg/Gly-rich region. Among these is LARP1, a regulator of 5′ terminal oligopyrimidine (TOP) mRNAs under conditions of mTOR inhibition. We discovered that, like LARP1, PRRC2B-long isoform binds to 5′ TOP mRNAs. Moreover, it is necessary for the post-transcriptional preservation of their mRNA levels, particularly those encoding ribosomal proteins, during amino acid starvation. In its absence, the rapid de novo translation of ribosomal proteins that takes place upon nutrient recovery is impeded. Overall, our study elucidates a newly discovered function for PRRC2B as an RNA-binding protein that regulates ribosomal biogenesis upon metabolic shift, in addition to its established function in initiating translation of specific mRNA targets.

Autophagy is a conserved self-digestion pathway essential for maintaining cellular homeostasis. While the transcriptional and epigenetic activation of autophagy under nutrient-deprived condition is well studied, the repression mechanisms of autophagy under basal conditions remain poorly understood. Here, we identify plant homeodomain finger protein 23 (PHF23) as an epigenetic repressor of autophagy through a CRISPR interference screen. Importantly, PHF23 inhibits autophagy gene expression via two distinct mechanisms: by recruiting the nucleosome remodeling and deacetylase (NuRD) complex to autophagy gene promoters, and by reducing chromatin accessibility at enhancers through downregulation of AP-1 and C/EBPβ transcription factors. This dual repression requires an intact plant homeodomain (PHD) and is relieved following PHF23 degradation under amino acid starvation or mTOR inhibition. Notably, genetic or pharmacological inhibition of PHF23 induces autophagy and promotes the autophagic clearance of pathological protein aggregates, including Tau and α1-antitrypsin Z (ATZ) variant, highlighting PHF23 as a potential therapeutic target in proteotoxic diseases.

Mot1 in budding yeast regulates transcription by dissociating general initiation factor TBP (TATA-binding protein) from DNA. Previous studies suggested that Mot1 preferentially removes TBP from stress-responsive promoters containing consensus TATA elements that utilize coactivator SAGA while enhancing TBP binding at "house-keeping" genes with TATA-like elements that employ TFIID for TBP recruitment. In stress conditions of amino acid starvation, by contrast, we found that Mot1 promotes TBP binding at genes activated by transcription factor Gcn4, enriched for TATA/SAGA-dependent promoters, and at SAGA-dependent genes expressed constitutively at high-levels, while suppressing TBP binding at SAGA-dependent genes only expressed at lower levels. Importantly, Mot1's influence on genes induced by starvation or oxidative stress switches from increased to decreased TBP binding when transcribed at low levels in nonstressed cells. Mot1's role at TFIID-dependent promoters also scales with transcription level, enhancing TBP binding only for the highly expressed subset. Notably, reduced TBP binding on Mot1 depletion impairs transcription of highly expressed TFIID genes but not highly expressed SAGA/stress-activated genes, suggesting that SAGA produces a surfeit of incomplete preinitiation complexes dependent on Mot1 for assembly.

The opportunistic human fungal pathogen Candida albicans possesses a remarkable metabolic plasticity, which is essential for both fungal commensalism and virulence and influences its physiology and behavior in multiple ways. The investigation of such processes particularly benefits from the emergence of multi-omics and in silico approaches. In this study, we combined a multi-omics approach with genome-scale metabolic modeling to investigate the fungal metabolic adaptation to amino acid utilization and starvation. Most strikingly, we found an altered activity of the shikimate pathway upon amino acid starvation, accompanied by a simultaneous induction of two metabolic gene clusters required for the metabolism of hydroxybenzenes. Further analyses revealed so far unknown potential functional and regulatory links between both metabolic pathways, which provide starting points for future research leading to a better understanding of the fungal adaptation to dynamic host conditions.

Research advances on amino acid starvation interventions for hepatocellular carcinoma.

BackgroundCells regulate protein synthesis in response to fluctuating nutrient availability through mechanisms that affect both translation initiation and elongation. Branched-chain amino acids, leucine, isoleucine, and valine, are essential nutrients. However, how their depletion affects translation remains largely unclear. Here, we investigate the immediate effects of single, double, and triple branched-chain amino acid deprivation on translational dynamics in NIH3T3 cells using RNA-seq and ribosome profiling.ResultsAll starvation conditions increased ribosome dwell times, with pronounced stalling at all valine codons during valine and triple starvation, whereas leucine and isoleucine starvation produced milder, codon-specific effects. Notably, stalling under isoleucine deprivation largely decreased under triple starvation. Positional enrichment of valine codons near the 5′ end and downstream isoleucine codons potentially contributes to these patterns, suggesting a possible elongation bottleneck that influences translational responses under branched-chain amino acid starvation. The presence of multiple valine stalling sites was associated with decreased protein levels. Finally, codon-specific dwell time changes correlated strongly with patterns of tRNA isoacceptor charging.ConclusionsTogether, these findings suggest that differential ribosome stalling under branched-chain amino acid starvation reflects a balance between amino acid supply, tRNA charging dynamics, codon position, and stress-response signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03800-6.

The integrated stress response (ISR) is a cellular signaling pathway that reduces protein synthesis in the face of cellular stress, including viral infection. Two eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and general control nonderepressible 2 (GCN2), are commonly activated during viral infections. Mouse adenovirus type 1 (MAV-1) infection leads to a steep reduction of PKR levels by proteasomal degradation. We assayed whether GCN2, a sensor of amino acid starvation and UV damage, plays a role in the ISR to MAV-1 infection. There was more phosphorylated GCN2 in MAV-1-infected cells, and its activation was dependent on virus replication since UV-inactivated virus was not able to increase the phosphorylation of GCN2. Infected Eif2ak4tm1.2Dron mice (designated here Gcn2-/- mice) had lower survival than wild-type (WT) mice, but results indicated that this was not due to increased viral replication. Both Gcn2-/- and WT mice developed multifocal brain parenchymal microhemorrhages during infection. While Gcn2-/- animals had more lesions, their higher mortality is likely not due to the microhemorrhages alone. Cytokine RNA and protein assays of WT and Gcn2-/- mice only showed a difference for IL- β levels, which were higher in Gcn2-/- mice. Our results also indicate that of the two eIF2α kinases, PKR and GCN2, GCN2 is the primary inducer of phosphorylated-eIF2α during MAV-1 infection. GCN2 is thus antiviral and contributes to the host response to MAV-1 infection.IMPORTANCECells often respond to viral infection by activation of the host protein kinase R (PKR), part of the integrated stress response (ISR). We show that a second host protein kinase, general control nonderepressible 2 (GCN2), is activated by phosphorylation in response to mouse adenovirus type 1 (MAV-1) infection. Our results indicate GCN2 is antiviral: without it, the mortality in MAV-1-infected mouse is higher. Furthermore, the data show that GCN2, rather than PKR, is the main inducer of eIf2α phosphorylation (and thus the ISR) upon MAV-1 infection. This is consistent with PKR exerting antiviral effects in MAV-1 infections through a pathway independent of eIf2α phosphorylation.