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The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.

NRF2 is emerging as a major regulator of cellular metabolism. However, most studies have been performed in cancer cells, where co-occurring mutations and tumor selective pressures complicate the influence of NRF2 on metabolism. Here we use genetically engineered, non-transformed primary murine cells to isolate the most immediate effects of NRF2 on cellular metabolism. We find that NRF2 promotes the accumulation of intracellular cysteine and engages the cysteine homeostatic control mechanism mediated by cysteine dioxygenase 1 (CDO1), which catalyzes the irreversible metabolism of cysteine to cysteine sulfinic acid (CSA). Notably, CDO1 is preferentially silenced by promoter methylation in human non-small cell lung cancers (NSCLC) harboring mutations in KEAP1, the negative regulator of NRF2. CDO1 silencing promotes proliferation of NSCLC by limiting the futile metabolism of cysteine to the wasteful and toxic byproducts CSA and sulfite (SO32-), and depletion of cellular NADPH. Thus, CDO1 is a metabolic liability for NSCLC cells with high intracellular cysteine, particularly NRF2/KEAP1 mutant cells.

[This corrects the article DOI: 10.1371/journal.pone.0219436.].

RAS is the founding member of a superfamily of GTPases and regulates signaling pathways involved in cellular growth control. While recent studies have shown that the activation state of RAS can be controlled by lysine ubiquitylation and acetylation, the existence of lysine methylation of the RAS superfamily GTPases remains unexplored. In contrast to acetylation, methylation does not alter the side chain charge and it has been challenging to deduce its impact on protein structure by conventional amino acid substitutions. Herein, we investigate lysine methylation on RAS and RAS-related GTPases. We developed GoMADScan (Go language-based Modification Associated Database Scanner), a new user-friendly application that scans and extracts posttranslationally modified peptides from databases. The GoMADScan search on PhosphoSitePlus databases identified methylation of conserved lysine residues in the core GTPase domain of RAS superfamily GTPases, including residues corresponding to RAS Lys-5, Lys-16, and Lys-117. To follow up on these observations, we immunoprecipitated endogenous RAS from HEK293T cells, conducted mass spectrometric analysis and found that RAS residues, Lys-5 and Lys-147, undergo dimethylation and monomethylation, respectively. Since mutations of Lys-5 have been found in cancers and RASopathies, we set up molecular dynamics (MD) simulations to assess the putative impact of Lys-5 dimethylation on RAS structure. Results from our MD analyses predict that dimethylation of Lys-5 does not significantly alter RAS conformation, suggesting that Lys-5 methylation may alter existing protein interactions or create a docking site to foster new interactions. Taken together, our findings uncover the existence of lysine methylation as a novel posttranslational modification associated with RAS and the RAS superfamily GTPases, and putative impact of Lys-5 dimethylation on RAS structure.

Vibrio cholerae is a diarrheal pathogen that induces accumulation of lipid droplets in enterocytes, leading to lethal infection of the model host Drosophila melanogaster. Through untargeted lipidomics, we provide evidence that this process is the product of a host phospholipid degradation cascade that induces lipid droplet coalescence in enterocytes. This infection-induced cascade is inhibited by mutation of the V. cholerae glycine cleavage system due to intestinal accumulation of methionine sulfoxide (MetO), and both dietary supplementation with MetO and enterocyte knock-down of host methionine sulfoxide reductase A (MsrA) yield increased resistance to infection. MsrA converts both free and protein-associated MetO to methionine. These findings support a model in which dietary MetO competitively inhibits repair of host proteins by MsrA. Bacterial virulence strategies depend on functional host proteins. We propose a novel virulence paradigm in which an intestinal pathogen ensures the repair of host proteins essential for pathogenesis through consumption of dietary MetO.

Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway.

Horse urine is easily collected and contains molecules readily measurable using mass spectrometry that can be used as biomarkers representative of health, disease or drug tampering. This study aimed at analyzing microliter levels of horse urine to purify, identify and quantify proteins, polar metabolites and non-polar lipids. Urine from a healthy 12 year old quarter horse mare on a diet of grass hay and vitamin/mineral supplements with limited pasture access was collected for serial-omics characterization. The urine was treated with methyl tert-butyl ether (MTBE) and methanol to partition into three distinct layers for protein, non-polar lipid and polar metabolite content from a single liquid-liquid extraction and was repeated two times. Each layer was analyzed by high performance liquid chromatography-high resolution tandem mass spectrometry (LC-MS/MS) to obtain protein sequence and relative protein levels as well as identify and quantify small polar metabolites and lipids. The results show 46 urine proteins, many related to normal kidney function, structural and circulatory proteins as well as 474 small polar metabolites but only 10 lipid molecules. Metabolites were mostly related to urea cycle and ammonia recycling as well as amino acid related pathways, plant diet specific molecules, etc. The few lipids represented triglycerides and phospholipids. These data show a complete mass spectrometry based-omics characterization of equine urine from a single 333 μL mid-stream urine aliquot. These omics data help serve as a baseline for healthy mare urine composition and the analyses can be used to monitor disease progression, health status, monitor drug use, etc.

Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-β) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-β isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.

Abstract Increased de novo lipogenesis (DNL) in white adipose tissue is associated with insulin sensitivity. Under both Normal-Chow-Diet and High-Fat-Diet, mice expressing a kinase inactive Cyclin-dependent kinase 6 (Cdk6) allele (K43M) display an increase in DNL in visceral white adipose tissues (VAT) as compared to wild type mice (WT), accompanied by markedly increased lipogenic transcriptional factor Carbohydrate-responsive element-binding proteins (CHREBP) and lipogenic enzymes in VAT but not in the liver. Treatment of WT mice under HFD with a CDK6 inhibitor recapitulates the phenotypes observed in K43M mice. Mechanistically, CDK6 phosphorylates AMP-activated protein kinase, leading to phosphorylation and inactivation of acetyl-CoA carboxylase, a key enzyme in DNL. CDK6 also phosphorylates CHREBP thus preventing its entry into the nucleus. Ablation of runt related transcription factor 1 in K43M mature adipocytes reverses most of the phenotypes observed in K43M mice. These results demonstrate a role of CDK6 in DNL and a strategy to alleviate metabolic syndromes.

Abstract Degradation of unliganded androgen receptor (AR) in prostate cancer cells can be prevented by proteasome inhibition, but this is associated with only modest increases in polyubiquitylated AR. An inhibitor (VLX1570) of the deubiquitylases associated with the proteasome did not increase ubiquitylation of unliganded AR, indicating that AR is not targeted by these deubiquitylases. We then identified a series of AR ubiquitylation sites, including a not previously identified site at K911, as well as methylation sites and previously identified phosphorylation sites. Mutagenesis of K911 increases AR stability, chromatin binding, and transcriptional activity. We further found that K313, a previously reported ubiquitylation site, could also be methylated and acetylated. Mutagenesis of K313, in combination with K318, increases AR transcriptional activity, indicating that distinct posttranslational modifications at K313 differentially regulate AR activity. Together these studies expand the spectrum of AR posttranslational modifications, and indicate that the K911 site may regulate AR turnover on chromatin.

Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

The relatively new field of onco-metabolomics attempts to identify relationships between various cancer phenotypes and global metabolite content. Previous metabolomics studies utilized either nuclear magnetic resonance spectroscopy or gas chromatography/mass spectrometry, and analyzed metabolites present in urine and serum. However, direct metabolomic assessment of tumor tissues is important for determining altered metabolism in cancers. In this respect, the ability to obtain reliable data from archival specimens is desirable and has not been reported to date. In this feasibility study, we demonstrate the analysis of polar metabolites extracted directly from ten formalin-fixed, paraffin-embedded (FFPE) specimens, including five soft tissue sarcomas and five paired normal samples. Using targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) via selected reaction monitoring (SRM), we detect an average of 106 metabolites across the samples with excellent reproducibility and correlation between different sections of the same specimen. Unsupervised hierarchical clustering and principal components analysis reliably recovers a priori known tumor and normal tissue phenotypes, and supervised analysis identifies candidate metabolic markers supported by the literature. In addition, we find that diverse biochemical processes are well-represented in the list of detected metabolites. Our study supports the notion that reliable and broadly informative metabolomic data may be acquired from FFPE soft tissue sarcoma specimens, a finding that is likely to be extended to other malignancies.

Recent reports have suggested a possible involvement of Single-minded homolog 2 (SIM2) in human solid cancers, including prostate cancer. However, the exact role of SIM2 in cancer in general, and in prostate cancer in particular, remains largely unknown. This study was designed to elucidate the role of SIM2 in prostate cancer using a shRNA-based approach in the PC3 prostate cancer cell line.Lentiviral shRNAs were used to inhibit SIM2 gene and protein levels in PC3 cells. Quantitative RT-PCR and branched DNA were performed to evaluate transcript expression. SIM2 protein expression level was measured by western blot. Profiling of gene expression spanning the whole genome, as well as polar metabolomics of several major metabolic pathways was performed to identify major pathway dysregulations.SIM2 gene and protein products were significantly downregulated by lenti-shRNA in PC3 cell line. This low expression of SIM2 affected gene expression profile, revealing significant changes in major signaling pathways, networks and functions. In addition, major metabolic pathways were affected.Taken together, our results suggest an involvement of SIM2 in key traits of prostate tumor cell biology and might underlie a contribution of this transcription factor to prostate cancer onset and progression.

The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.

Abstract We present a comprehensive multi-omic analysis of the EPISTOP prospective clinical trial of early intervention with vigabatrin for pre-symptomatic epilepsy treatment in Tuberous Sclerosis Complex (TSC), in which 93 infants with TSC were followed from birth to age 2 years, seeking biomarkers of epilepsy development. Vigabatrin had profound effects on many metabolites, increasing serum deoxycytidine monophosphate (dCMP) levels 52-fold. Most serum proteins and metabolites, and blood RNA species showed significant change with age. Thirty-nine proteins, metabolites, and genes showed significant differences between age-matched control and TSC infants. Six also showed a progressive difference in expression between control, TSC without epilepsy, and TSC with epilepsy groups. A multivariate approach using enrollment samples identified multiple 3-variable predictors of epilepsy, with the best having a positive predictive value of 0.987. This rich dataset will enable further discovery and analysis of developmental effects, and associations with seizure development in TSC.

Preoperative risk biomarkers for delirium may aid in identifying high-risk patients and developing intervention therapies, which would minimize the health and economic burden of postoperative delirium. Previous studies have typically used single omics approaches to identify such biomarkers. Preoperative cerebrospinal fluid (CSF) from the Healthier Postoperative Recovery study of adults ≥ 63 years old undergoing elective major orthopedic surgery was used in a matched pair delirium case–no delirium control design. We performed metabolomics and lipidomics, which were combined with our previously reported proteomics results on the same samples. Differential expression, clustering, classification, and systems biology analyses were applied to individual and combined omics datasets. Probabilistic graph models were used to identify an integrated multi-omics interaction network, which included clusters of heterogeneous omics interactions among lipids, metabolites, and proteins. The combined multi-omics signature of 25 molecules attained an AUC of 0.96 [95% CI: 0.85–1.00], showing improvement over individual omics-based classification. We conclude that multi-omics integration of preoperative CSF identifies potential risk markers for delirium and generates new insights into the complex pathways associated with delirium. With future validation, this hypotheses-generating study may serve to build robust biomarkers for delirium and improve our understanding of its pathophysiology.

Abstract Activation of the cGAS/STING innate immunity pathway is essential and effective for anti-tumor immunotherapy. However, it remains largely elusive how tumor-intrinsic cGAS signaling is suppressed to facilitate tumorigenesis by escaping immune surveillance. Here, we report that the protein arginine methyltransferase, PRMT1, methylates cGAS at the conserved Arg133 residue, which prevents cGAS dimerization and suppresses the cGAS/STING signaling in cancer cells. Notably, genetic or pharmaceutical ablation of PRMT1 leads to activation of cGAS/STING-dependent DNA sensing signaling, and robustly elevates the transcription of type I and II interferon response genes. As such, PRMT1 inhibition elevates tumor-infiltrating lymphocytes in a cGAS-dependent manner, and promotes tumoral PD-L1 expression. Thus, combination therapy of PRMT1 inhibitor with anti-PD-1 antibody augments the anti-tumor therapeutic efficacy in vivo. Our study therefore defines the PRMT1/cGAS/PD-L1 regulatory axis as a critical factor in determining immune surveillance efficacy, which serves as a promising therapeutic target for boosting tumor immunity.

Excessive renal fibrosis is a common pathology in progressive chronic kidney diseases. Inflammatory injury and aberrant repair processes contribute to the development of kidney fibrosis. Myeloid cells, particularly monocytes/macrophages, play a crucial role in kidney fibrosis by releasing their proinflammatory cytokines and extracellular matrix components such as collagen and fibronectin into the microenvironment of the injured kidney. Numerous signaling pathways have been identified in relation to these activities. However, the involvement of metabolic pathways in myeloid cell functions during the development of renal fibrosis remains understudied. In our study, we initially reanalyzed single-cell RNA sequencing data of renal myeloid cells from Dr. Denby’s group and observed an increased gene expression in glycolytic pathway in myeloid cells that are critical for renal inflammation and fibrosis. To investigate the role of myeloid glycolysis in renal fibrosis, we utilized a model of unilateral ureteral obstruction in mice deficient of Pfkfb3, an activator of glycolysis, in myeloid cells (Pfkfb3ΔMϕ) and their wild type littermates (Pfkfb3WT). We observed a significant reduction in fibrosis in the obstructive kidneys of Pfkfb3ΔMϕ mice compared to Pfkfb3WT mice. This was accompanied by a substantial decrease in macrophage infiltration, as well as a decrease of M1 and M2 macrophages and a suppression of macrophage to obtain myofibroblast phenotype in the obstructive kidneys of Pfkfb3ΔMϕ mice. Mechanistic studies indicate that glycolytic metabolites stabilize HIF1α, leading to alterations in macrophage phenotype that contribute to renal fibrosis. In conclusion, our study implicates that targeting myeloid glycolysis represents a novel approach to inhibit renal fibrosis.

The PI3K/AKT/mTOR pathway is commonly dysregulated in cancer. Rapalogs exhibit modest clinical benefit, likely owing to their lack of effects on 4EBP1. We hypothesized that bi-steric mTORC1-selective inhibitors would have greater potential for clinical benefit than rapalogs in tumors with mTORC1 dysfunction. We assessed this hypothesis in tumor models with high mTORC1 activity both in vitro and in vivo. Bi-steric inhibitors had strong growth inhibition, eliminated phosphorylated 4EBP1, and induced more apoptosis than rapamycin or MLN0128. Multiomics analysis showed extensive effects of the bi-steric inhibitors in comparison with rapamycin. De novo purine synthesis was selectively inhibited by bi-sterics through reduction in JUN and its downstream target PRPS1 and appeared to be the cause of apoptosis. Hence, bi-steric mTORC1-selective inhibitors are a therapeutic strategy to treat tumors driven by mTORC1 hyperactivation.

Inactivation of the RB1 tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies against RB1-deficient cancers remain elusive. Here, we showed that RB1 loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression of ACSL4 and enriching ACSL4-dependent arachidonic acid–containing phospholipids, which are key components of ferroptosis execution. ACSL4 appeared to be a direct E2F target gene and was critical to RB1 loss–induced sensitization to ferroptosis. Importantly, using cell line–derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blocked RB1-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis and also suggest a promising approach for the treatment of RB1-deficient malignancies.

Renal fibrosis, a hallmark of chronic kidney diseases, is driven by the activation of renal fibroblasts. Recent studies have highlighted the role of glycolysis in this process. Nevertheless, one critical glycolytic activator, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), remains unexplored in renal fibrosis. Upon reanalyzing the single-cell sequencing data from Dr. Humphreys’ lab, we noticed an upregulation of glycolysis, gluconeogenesis, and the TGFβ signaling pathway in myofibroblasts from fibrotic kidneys after unilateral ureter obstruction (UUO) or kidney ischemia/reperfusion. Furthermore, our experiments showed significant induction of PFKFB3 in mouse kidneys following UUO or kidney ischemia/reperfusion. To delve deeper into the role of PFKFB3, we generated mice with Pfkfb3 deficiency, specifically in myofibroblasts (Pfkfb3f/f/PostnMCM). Following UUO or kidney ischemia/reperfusion, a substantial decrease in fibrosis in the injured kidneys of Pfkfb3f/f/PostnMCM mice was identified compared to their wild-type littermates. Additionally, in cultured renal fibroblast NRK-49F cells, PFKFB3 was elevated upon exposure to TGFβ1, accompanied by an increase in α-SMA and fibronectin. Notably, this upregulation was significantly diminished with PFKFB3 knockdown, correlated with glycolysis suppression. Mechanistically, the glycolytic metabolite lactate promoted the fibrotic activation of NRK-49F cells. In conclusion, our study demonstrates the critical role of PFKFB3 in driving fibroblast activation and subsequent renal fibrosis.

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor governs a variety of biological processes, including metabolism, by acting on distinct molecular targets in different subcellular compartments. In the cytosol, inactive PTEN can be recruited to the plasma membrane where it dimerizes and functions as a lipid phosphatase to regulate metabolic processes mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin complex 1 (mTORC1) pathway. However, the metabolic regulation of PTEN in the nucleus remains undefined. Here, using a gain-of-function approach to targeting PTEN to the plasma membrane and nucleus, we show that nuclear PTEN contributes to pyrimidine metabolism, in particular de novo thymidylate (dTMP) biosynthesis. PTEN appears to regulate dTMP biosynthesis through interaction with methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), a key enzyme that generates 5,10-methylenetetrahydrofolate, a cofactor required for thymidylate synthase (TYMS) to catalyze deoxyuridylate (dUMP) into dTMP. Our findings reveal a nuclear function for PTEN in controlling dTMP biosynthesis and may also have implications for targeting nuclear-excluded PTEN prostate cancer cells with antifolate drugs.