GTP Levels Research Articles

Unraveling the Mystery of Energy-Sensing Enzymes and Signaling Pathways in Tumorigenesis and Their Potential as Therapeutic Targets for Cancer.

Cancer research has advanced tremendously with the identification of causative genes, proteins, and signaling pathways. Numerous antitumor drugs have been designed and screened for cancer therapeutics; however, designing target-specific drugs for malignant cells with minimal side effects is challenging. Recently, energy-sensing- and homeostasis-associated molecules and signaling pathways playing a role in proliferation, apoptosis, autophagy, and angiogenesis have received increasing attention. Energy-metabolism-based studies have shown the contribution of energetics to cancer development, where tumor cells show increased glycolytic activity and decreased oxidative phosphorylation (the Warburg effect) in order to obtain the required additional energy for rapid division. The role of energy homeostasis in the survival of normal as well as malignant cells is critical; therefore, fuel intake and expenditure must be balanced within acceptable limits. Thus, energy-sensing enzymes detecting the disruption of glycolysis, AMP, ATP, or GTP levels are promising anticancer therapeutic targets. Here, we review the common energy mediators and energy sensors and their metabolic properties, mechanisms, and associated signaling pathways involved in carcinogenesis, and explore the possibility of identifying drugs for inhibiting the energy metabolism of tumor cells. Furthermore, to corroborate our hypothesis, we performed meta-analysis based on transcriptomic profiling to search for energy-associated biomarkers and canonical pathways.

Abstract Mo109: Importance of Myocyte Serine De Novo Biosynthesis and its Downregulation in Heart Failure

Introduction: The failing heart exhibits metabolic inflexibility and dysregulated metabolism. Serine is a non-essential amino acid that contributes to purine synthesis, redox, and amino acid synthesis. It is transported into cells from the circulation or formed by de novo synthesis (SdS). We have found human failing myocardium has reduced serine (Circ 2023;147:1147) and lower expression of SdS genes (Circ 2021;143:120). Yet, little is known about the role of SdS or its depression in myocardium. Hypothesis: Reduced serine biosynthesis impairs myocyte energy and redox balance that could contribute to heart failure. Methods/Results: Immunoblots of human HF myocardium found phosphoglycerate dihydrogen-ase (PHGDH, rate limiting SdS enzyme) and other SdS enzymes were reduced in HF, and moreso in HF with preserved ejection fraction. Expression of plasmalemmal serine transporters SLC1A4, SLC1A5, SLC12A4 were consistently depressed in HFpEF. To assess the relative contribution of exogenous serine versus SdS, we inhibited PHGDH by siRNA or an inhibitor (NCT503). The former reduced serine by 71% despite exogenous sources, and both induced greater cytotoxicity than from solely depleting exogenous sources. Inhibiting PHGDH in serine/glycine-free media induced cytotoxicity after 1hr, whereas with exogenous serine, this occurred by 24 hours. 24 hr PHGDH knockdown reduced ATP and GTP levels, mitochondrial DNA. In response to endothelin-1 stimulation, basal and maximal mitochondrial O 2 consumption increased along with PHGDH gene expression. These changes in mitochondrial respiration were essentially prevented by gene silencing of PHDGH coupled to a decline in TCA metabolites. PHGDH inhibition also increased oxidative stress (increased GSSG/GSH) and reduced NAD levels. The cytotoxicity induced by 24 hr SdS inhibition by PHGDH knockdown could be offset by supplementing myocytes with ribose (purine precursor) and DTT (antioxidant). Conclusion: This study identifies SdS as a pivotal metabolic pathway in myocyte energy metabolism homeostasis, and its depression may play a role in HF, particularly HFpEF. These results support enhancing serine metabolic pathways as a therapeutic strategy for these diseases.

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of a childhood neurodegenerative disorder caused by AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that mouse models of AMPD2 deficiency exhibit predominant degeneration of the hippocampal dentate gyrus, despite a general reduction of brain GTP levels. Neurodegeneration-resistant regions accumulate micron-sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis, while these filaments are barely detectable in the hippocampal dentate gyrus. Furthermore, we show that IMPDH2 filament disassembly reduces GTP levels and impairs growth of neural progenitor cells derived from individuals with human AMPD2 deficiency. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation, opening the possibility of exploring the induction of IMPDH2 assembly as a therapy for neurodegeneration.

The bioenergetics of nucleocytoplasmic transport.

How nucleocytoplasmic transport (NCT) rates change due to cellular physiology-mediated fluctuations in GTP availability remains unclear. In this issue, Scott et al. (https://doi.org/10.1083/jcb.202308152) demonstrate that cell migration, spreading, and nucleocytoskeletal coupling impact GTP levels, thereby regulating NCT, RNA export, and protein synthesis.

Ruthenium red alleviates post-resuscitation myocardial dysfunction by upregulating mitophagy through inhibition of USP33 in a cardiac arrest rat model

Cardiac arrest (CA) remains a leading cause of death, with suboptimal survival rates despite efforts involving cardiopulmonary resuscitation and advanced life-support technology. Post-resuscitation myocardial dysfunction (PRMD) is an important determinant of patient outcomes. Myocardial ischemia/reperfusion injury underlies this dysfunction. Previous reports have shown that ruthenium red (RR) has a protective effect against cardiac ischemia-reperfusion injury; however, its precise mechanism of action in PRMD remains unclear. This study investigated the effects of RR on PRMD and analyzed its underlying mechanisms. Ventricular fibrillation was induced in rats, which were then subjected to cardiopulmonary resuscitation to establish an experimental CA model. At the onset of return of spontaneous circulation, RR (2.5 mg/kg) was administered intraperitoneally. Our study showed that RR improved myocardial function and reduced the production of oxidative stress markers such as malondialdehyde (MDA), glutathione peroxidase (GSSG), and reactive oxygen species (ROS) production. RR also helped maintain mitochondrial structure and increased ATP and GTP levels. Additionally, RR effectively attenuated myocardial apoptosis. Furthermore, we observed downregulation of proteins closely related to mitophagy, including ubiquitin-specific protease 33 (USP33) and P62, whereas LC3B (microtubule-associated protein light chain 3B) was upregulated. The upregulation of mitophagy may play a critical role in reducing myocardial injury. These results demonstrate that RR may attenuate PRMD by promoting mitophagy through the inhibition of USP33. These effects are likely mediated through diverse mechanisms, including antioxidant activity, apoptosis suppression, and preservation of mitochondrial integrity and energy metabolism. Consequently, RR has emerged as a promising therapeutic approach for addressing post-resuscitation myocardial dysfunction.

Abstract 5865: Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines

Abstract Background: The enzyme purine nucleoside phosphorylase (PNP) is involved in the purine salvage pathway and is present at high levels in lymphoid tissues. Children born with PNP deficiency are known to have significantly reduced T-cell counts but relatively normal B-cell immunity, suggesting PNP as one of the most promising therapeutic targets for T-cell malignancies. Forodesine (For, BCX-1777), a novel PNP inhibitor, was approved in Japan for the treatment of relapsed/refractory PTCL. To understand the cytotoxic effect of For and its mechanism of action, we established a For-resistant (For-R) leukemia cell line and explored the mechanism of resistance. Methods: We established two For-R T-lymphoblastic leukemia cell lines (CCRF-CEM and MOLT4) through repeated escalating exposure to For and 2’-deoxyguanosined(dGuo). Gene expression analysis was performed to identify the mechanisms of resistance. Results: For-R cells showed very high resistance compared to parental cells (IC50 value, CEM/CEM-R 4.2nM/>5x105nM, MOLT4/MOLT4-R 8nM/>5x105nM). When cross-resistance was examined in these For-R cells showed mild resistance to cytarabine. The expression of hENT1, a nucleoside transporter, was examined and found to be upregulated in both CEM-R and MOLT4-R cells, while the expression level of deoxycytidine kinase (dCK) was unchanged in both cells. Next, high performance liquid chromatography was used to assess the intracellular dGTP in each parental and its For-R cells. In the parental cells, intracellular dGTP levels increased promptly 4-6 hours after fosteredin administration, whereas in resistant cells, intracellular dGTP levels were higher than before fosteredin administration, and there was little change in dGTP levels after administration. Conclusion: The established For-R cell line acquired a high resistance to For and showed high intracellular dGTP levels. Imbalance in intracellular GTP levels may be involved in the induction of cell death by For. IC50 value of each cell lines Citation Format: Naoko Hosono, Kana Oiwa, Toshiki Tasaki, Rie Nishi, Luigi Scott, Owen A. O’Connor, Takahiro Yamauchi. Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5865.

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function.

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, β, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor-knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.

Promoting Nucleic Acid Synthesis in Saccharomyces cerevisiae through Enhanced Expression of Rrn7p, Rrn11p, IMPDH, and Pho84p.

Saccharomyces cerevisiae has emerged as a preferred source for industrial production of ribonucleic acids (RNAs) and their derivatives, which find wide applications in the food and pharmaceutical sectors. In this study, we employed a modified RNA polymerase I-mediated green fluorescent protein expression system, previously developed by our team, to screen and identify an industrial S. cerevisiae strain with an impressive 18.2% increase in the RNA content. Transcriptome analysis revealed heightened activity of genes and pathways associated with rRNA transcription, purine metabolism, and phosphate transport in the high nucleic acid content mutant strains. Our findings highlighted the crucial role of the transcription factor Sfp1p in enhancing the expression of two key components of the transcription initiation factor complex, Rrn7p and Rrn11p, thereby promoting rRNA synthesis. Moreover, elevated expression of 5'-inosine monophosphate dehydrogenases, regardless of the specific isoform (IMD2, 3, or 4), resulted in increased rRNA synthesis through heightened GTP levels. Additionally, exogenous phosphate application, coupled with overexpression of the phosphate transporter PHO84, led to a 61.4% boost in the RNA yield, reaching 2050.4 mg/L. This comprehensive study provides valuable insights into the mechanism of RNA synthesis and serves as a reference for augmenting RNA production in the food industry.

Hypoxia-induced TMTC3 expression in esophageal squamous cell carcinoma potentiates tumor angiogenesis through Rho GTPase/STAT3/VEGFA pathway

BackgroundHypoxia is one of most typical features in the tumor microenvironment of solid tumor and an inducer of endoplasmic reticulum (ER) stress, and HIF-1α functions as a key transcription factor regulator to promote tumor angiogenesis in the adaptive response to hypoxia. Increasing evidence has suggested that hypoxia plays an important regulatory role of ER homeostasis. We previously identified TMTC3 as an ER stress mediator under nutrient-deficiency condition in esophageal squamous cell carcinoma (ESCC), but the molecular mechanism in hypoxia is still unclear.MethodsRNA sequencing data of TMTC3 knockdown cells and TCGA database were analyzed to determine the association of TMTC3 and hypoxia. Moreover, ChIP assay and dual-luciferase reporter assay were performed to detect the interaction of HIF-1α and TMTC3 promoter. In vitro and in vivo assays were used to investigate the function of TMTC3 in tumor angiogenesis. The molecular mechanism was determined using co-immunoprecipitation assays, immunofluorescence assays and western blot. The TMTC3 inhibitor was identified by high-throughput screening of FDA-approved drugs. The combination of TMTC3 inhibitor and cisplatin was conducted to confirm the efficiency in vitro and in vivo.ResultsThe expression of TMTC3 was remarkably increased under hypoxia and regulated by HIF-1α. Knockdown of TMTC3 inhibited the capability of tumor angiogenesis and ROS production in ESCC. Mechanistically, TMTC3 promoted the production of GTP through interacting with IMPDH2 Bateman domain. The activity of Rho GTPase/STAT3, regulated by cellular GTP levels, decreased in TMTC3 knockdown cells, whereas reversed by IMPDH2 overexpression. Additionally, TMTC3 regulated the expression of VEGFA through Rho GTPase/STAT3 pathway. Allopurinol inhibited the expression of TMTC3 and further reduced the phosphorylation and activation of STAT3 signaling pathway in a dose-dependent manner in ESCC. Additionally, the combination of allopurinol and cisplatin significantly inhibited the cell viability in vitro and tumor growth in vivo, comparing with single drug treatment, respectively.ConclusionsCollectively, our study clarified the molecular mechanism of TMTC3 in regulating tumor angiogenesis and highlighted the potential therapeutic combination of TMTC3 inhibitor and cisplatin, which proposed a promising strategy for the treatment of ESCC.

Predictive Modeling of Riboflavin Production in Lactiplantibacillus plantarum MTCC 25432 Using Fuzzy Inference System.

Riboflavin (Vitamin B2) is an essential vitamin and a microbial metabolite produced by some lactic acid bacteria (LAB). This investigation aims to study the overproduction of riboflavin in selected Lactiplantibacillus plantarum strain by using the one factor at a time (OFAT) tool coupled with the Fuzzy Inference System (FIS) and its validation through fermentative production in semi-defined media. Out of three Lactiplantibacillus strains used in this study, the maximum riboflavin producing strain was selected based on its ability to grow and produce higher levels of riboflavin. In results, Lactiplantibacillus plantarum strain MTCC 25432 was able to produce 346 µg/L riboflavin in riboflavin deficient assay medium and was investigated further. By using the OFAT-fuzzy FIS system, casamino acid in the range of 5-20 g/L, GTP 0.01-0.04 g/L, sodium acetate 5-15 g/L, and glycine 5-15 g/L were used to predict their effect on riboflavin production. The conditions optimized with modeling showed a 24% increment in riboflavin production (429 µg/L) by Lactiplantibacillus plantarum MTCC 25432 vis-a-vis the unoptimized counterpart (346 µg/L). In conclusion, an FIS-based predictive model was effectively implemented to estimate the riboflavin within an acceptable limit of 3.4%. Riboflavin production enhancing effects observed with various levels of sodium acetate, casamino acid, and GTP could be useful to re-design matrices for riboflavin production.

Specific inhibitory effects of guanosine on breast cancer cell proliferation

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death. Drug therapy for breast cancer is currently selected based on the subtype classification; however, many anticancer drugs are highly cytotoxic. Since intracellular levels of GTP are elevated in many cancer cells that undergo a specific cell proliferation cycle, GTP has potential as a target for cancer therapy. The present study focused on nucleosides and nucleotides and examined intracellular GTP-dependent changes in cell proliferation rates in normal (MCF-12A) and cancer (MCF-7) breast cell lines. Decreased cell proliferation due to a reduction in intracellular GTP levels by mycophenolic acid (MPA), an inosine monophosphate dehydrogenase inhibitor, was observed in both cell lines. The inhibitory effects of MPA on cell proliferation were suppressed when it was applied in combination with Guanosine (Guo), a substrate for GTP salvage synthesis, while the single exposure to Guo suppressed the proliferation of MCF-7 cells only. Although the underlying mechanisms remain unclear, since the inhibitory effects of Guo on cell proliferation did not correlate with GTP or ATP intracellular levels or the GTP/ATP ratio, there may be another cause besides GTP metabolism. Guo inhibited the proliferation of MCF-7, a human breast cancer cell line, but not MCF-12A, a human normal breast cell line. Further studies are needed to investigate the potential of applying Guo as a target for the development of a novel cancer treatment system.

Resveratrol-induced SIRT1 activation inhibits glycolysis-fueled angiogenesis under rheumatoid arthritis conditions independent of HIF-1α.

This study investigated the impacts of SIRT1 activation on rheumatoid arthritis (RA)-related angiogenesis. HUVECs were cultured by different human serum. Intracellular metabolites were quantified by UPLC-MS. Next, HUVECs and rat vascular epithelial cells under different inflammatory conditions were treated by a SIRT1 agonist resveratrol (RSV). Cytokines and biochemical indicators were detected by corresponding kits. Protein and mRNA expression levels were assessed by immunoblotting and PCR methods, respectively. Angiogenesis capabilities were evaluated by migration, wound-healing and tube-formation experiments. To down-regulate certain signals, gene-specific siRNA were applied. Metabolomics study revealed the accelerated glycolysis in RA serum-treated HUVECs. It led to ATP accumulation, but did not affect GTP levels. RSV inhibited pro-angiogenesis cytokines production and glycolysis in both the cells, and impaired the angiogenesis potentials. These effects were mimicked by an energy metabolism interrupter bikini in lipopolysaccharide (LPS)-primed HUVECs, largely independent of HIF-1α. Both RSV and bikinin can inhibit the activation of the GTP-dependent pathway Rho/ROCK and reduce VEGF production. Abrogation of RhoA signaling reinforced HIF-1α silencing-brought changes in LPS-stimulated HUVECs, and overshadowed the anti-angiogenesis potentials of RSV. Glycolysis provides additional energy to sustain Rho/ROCK activation in RA subjects, which promotes VEGF-driven angiogenesis and can be inhibited by SIRT1 activation.

Abstract 3677: DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma

Abstract Glioblastoma (GBM) is uniformly fatal due to inherent radiation (RT) and chemotherapy resistance. We have found this therapeutic resistance is mediated by alterations in tumor cellular metabolic activity. Our group and others have found that metabolites can regulate DNA repair and RT resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here, we show that DNA damage acutely increases guanine-containing purine metabolites in multiple in vitro and intracranial GBM models. By interrogating metabolic fluxes in vitro using a variety of stable isotope tracers, we confirmed RT-induced elevation in guanylates was due to increased de novo purine synthesis (DNPS) rather than activation of purine salvage. By developing and using novel stable isotope tracing methods to directly measure DNPS in awake, unrestrained mice, we confirmed that orthotopic GBMs have higher DNPS rates than adjacent cortical tissue that further increase after treatment with RT. Neither salvage synthesis of purines nor pyrimidine synthesis were impacted by RT in any intracranial tissues. With these findings, we opened a clinical study to directly measure purine synthesis in patients, and we found that human GBMs have similarly high purine synthesis rates compared to normal brain tissue. Because DNA damage activated DNPS without affecting purine salvage or pyrimidine synthesis, we reasoned that active signaling may be involved. Indeed, therapy-induced DNPS increases are lost in vitro and in vivo upon pharmacological or genomic inhibition of the DNA-damage sensing kinase DNA-PK. Moreover, RT and DNA-PK have direct influence over the spatial organization of DNPS enzymes, including IMPDH, the rate-limiting step in guanylate synthesis. Because purines can promote DNA repair, these findings suggest that DNA-PK signaling helps promote DNA repair in part by causing the spatial reorganization of DNPS enzymes, thereby activating purine synthesis. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models, suggesting a potential therapeutic targeting opportunity in patients. In this study, we have developed novel methodology to directly measure purine synthesis in brain tumors in mice and humans. With these tools, we discovered that after DNA damage, DNA-PK mediates a novel pathway controlling the spatial reorganization of purine synthesis enzymes and subsequent DNPS increases. The resulting elevation of GTP levels promotes therapy resistance in tumors, and we are now directly measuring and inhibiting this molecular activity in patients with GBM in an effort to improve standard therapy. Citation Format: Andrew J. Scott, Alexandra M. O'Brien, Weihua Zhou, Vidhi Pareek, Zhou Sha, Sravya Palavalasa, Ayesha U. Kothari, Kari Wilder-Romans, Li Zhang, Anthony C. Andren, Sriram Chandrasekaran, Jason Heth, Yoshie Umemura, Nathan Qi, John Woulfe, Sriram Venneti, Meredith A. Morgan, Theodore S. Lawrence, Wajd N. Al-Holou, Costas A. Lyssiotis, Daniel R. Wahl. DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3677.

Anticancer Drug Doxorubicin Spontaneously Reacts with GTP and dGTP.

Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (Kd) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.

Deficiency of gluconeogenic enzyme PCK1 promotes metabolic-associated fatty liver disease through PI3K/AKT/PDGF axis activation in male mice

Metabolic associated fatty liver disease (MAFLD) encompasses a broad spectrum of hepatic disorders, including steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis. We demonstrated that phosphoenolpyruvate carboxykinase 1 (PCK1) plays a central role in MAFLD progression. Male mice with liver Pck1 deficiency fed a normal diet displayed hepatic lipid disorder and liver injury, whereas fibrosis and inflammation were aggravated in mice fed a high-fat diet with drinking water containing fructose and glucose (HFCD-HF/G). Forced expression of hepatic PCK1 by adeno-associated virus ameliorated MAFLD in male mice. PCK1 deficiency stimulated lipogenic gene expression and lipid synthesis. Moreover, loss of hepatic PCK1 activated the RhoA/PI3K/AKT pathway by increasing intracellular GTP levels, increasing secretion of platelet-derived growth factor-AA (PDGF-AA), and promoting hepatic stellate cell activation. Treatment with RhoA and AKT inhibitors or gene silencing of RhoA or AKT1 alleviated MAFLD progression in vivo. Hepatic PCK1 deficiency may be important in hepatic steatosis and fibrosis development through paracrine secretion of PDGF-AA in male mice, highlighting a potential therapeutic strategy for MAFLD.

Host Cell Amplification of Nutritional Stress Contributes To Persistence in Chlamydia trachomatis.

Persistence, a viable but non-replicating growth state, has been implicated in diseases caused by Chlamydia trachomatis. Starvation of distinct nutrients produces a superficially similar persistent state, implying convergence on a common intracellular environment. We employed host-pathogen dual RNA-sequencing under both iron- and tryptophan-starved conditions to systematically characterize the persistent chlamydial transcriptome and to define common contributions of the host cell transcriptional stress response in shaping the intracellular environment. The transcriptome of the infected host cells was highly specific to each nutritional stress, despite comparable effects on chlamydial growth and development in each condition. In contrast, the chlamydial transcriptomes between nutritional conditions were highly similar, suggesting some overlap in host cell responses to iron limitation and tryptophan starvation that contribute to a common persistent phenotype. We demonstrate that a commonality in the host cell responses is the suppression of GTP biosynthesis, a nucleotide for which Chlamydia are auxotrophic. Pharmacological inhibition of host IMP dehydrogenase (IMPDH1), which catalyzes the rate-limiting step in de novo guanine nucleotide synthesis, resulted in comparable GTP depletion to both iron and tryptophan starvation and induced chlamydial persistence. Moreover, IMPDH1 inhibition and iron starvation acted synergistically to control chlamydial growth. Thus, host cell reduction in GTP levels amplifies the nutritional stress to intracellular chlamydiae in infection-relevant models of persistence, illustrating the determinative role the infected host cell plays in bacterial stress responses. IMPORTANCE Bacteria respond to nutritional stress through universal and unique mechanisms. Genome reduction in the Chlamydiaceae, a consequence of coevolution with their obligate eukaryotic hosts, has reduced their repertoire of stress response mechanisms. Here, we demonstrate that the infected host cell may provide the context within which universal stress responses emerge for Chlamydia trachomatis. We report that during starvation of the essential nutrients iron or tryptophan, a common response of the infected epithelial cell is the suppression of GTP biosynthesis, which induces a persistent developmental state in the pathogen. Thus, chlamydial persistence results from the combined effects of primary stresses on the pathogen and the host, with the latter eliciting a secondary host cell response that intensifies the inhospitable intracellular environment.

Human Thg1 displays tRNA-inducible GTPase activity

tRNAHis guanylyltransferase (Thg1) catalyzes the 3′-5′ incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74–76), how HsThg1 prevents successive 3′-5′ incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.

Renal Transcriptome and Metabolome in Mice with Principal Cell-Specific Ablation of the Tsc1 Gene: Derangements in Pathways Associated with Cell Metabolism, Growth and Acid Secretion.

Tuberous sclerosis complex (TSC) is caused by mutations in the hamartin (TSC1) or tuberin (TSC2) genes. Using a mouse model of TSC renal cystogenesis that we have previously described, the current studies delineate the metabolic changes in the kidney and their relation to alterations in renal gene expression. To accomplish this, we compared the metabolome and transcriptome of kidneys from 28-day-old wildtype (Wt) and principal cell-specific Tsc1 KO (Tsc1 KO) mice using targeted 1H nuclear magnetic resonance targeted metabolomic and RNA-seq analyses. The significant changes in the kidney metabolome of Tsc1 KO mice included reductions in the level of several amino acids and significant decreases in creatine, NADH, inosine, UDP-galactose, GTP and myo-inositol levels. These derangements may affect energy production and storage, signal transduction and synthetic pathways. The pertinent derangement in the transcriptome of Tsc1 KO mice was associated with increased collecting duct acid secretion, active cell division and the up-regulation of signaling pathways (e.g., MAPK and AKT/PI3K) that suppress the TSC2 GTPase-activating function. The combined renal metabolome and transcriptome alterations observed in these studies correlate with the unregulated growth and predominance of genotypically normal A-intercalated cells in the epithelium of renal cysts in Tsc1 KO mice.

Compartmentalization and regulation of GTP in control of cellular phenotypes.

Genetic or pharmacological inhibition of enzymes involved in GTP biosynthesis has substantial biological effects, underlining the need to better understand the function of GTP levels in regulation of cellular processes and the significance of targeting GTP biosynthesis enzymes for therapeutic intervention. Our current understanding of spatiotemporal regulation of GTP metabolism and its role in physiological and pathological cellular processes is far from complete. Novel methodologies such as genetically encoded sensors of free GTP offered insights into intracellular distribution and function of GTP molecules. In the current Review, we provide analysis of recent discoveries in the field of GTP metabolism and evaluate the key enzymes as molecular targets.

Citrate Regulates the Saccharomyces cerevisiae Mitochondrial GDP/GTP Carrier (Ggc1p) by Triggering Unidirectional Transport of GTP.

The yeast mitochondrial transport of GTP and GDP is mediated by Ggc1p, a member of the mitochondrial carrier family. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria in exchange for GDP generated in the matrix. ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in mitochondria, are unable to grow on nonfermentable substrates and lose mtDNA. Because in yeast, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space, Ggc1p is the only supplier of mitochondrial GTP required for the maturation of proteins containing Fe-S clusters, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. In this work, it was demonstrated that citrate is a regulator of purified and reconstituted Ggc1p by trans-activating unidirectional transport of GTP across the proteoliposomal membrane. It was also shown that the binding site of Ggc1p for citrate is different from the binding site for the substrate GTP. It is proposed that the citrate-induced GTP uniport (CIGU) mediated by Ggc1p is involved in the homeostasis of the guanine nucleotide pool in the mitochondrial matrix.