bims-celmim 2025-04-06 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2025–04–06
twenty-two papers selected by
Marc Segarra Mondejar

Three's company: Membrane waltz among organelles.
Method for Detecting Mitochondrial ATP by Hybridization Chain Reaction.
Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis.
Conversion of glutamate into proline by the leucine analog BCH enhances biphasic insulin secretion in pancreatic β-cells.
A dual reporter system for intracellular and extracellular amino acid sensing in budding yeast.
Mitochondrial heat production: the elephant in the lab….
MACAW: a method for semi-automatic detection of errors in genome-scale metabolic models.
Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina.
Calcium signals shape metabolic control of H3K27ac and H3K18la to regulate EGA.
Hexokinase2-engineered T cells display increased anti-tumor function.
At the crossroads of calcium signaling, protein synthesis and neuropeptide release.
Activating Transcription Factor 3 regulates hepatic Apolipoprotein A4 upon metabolic stress.
PDZD8 promotes autophagy at ER-lysosome membrane contact sites to regulate activity-dependent synaptic growth.
RPE-specific MCT2 expression promotes cone survival in models of retinitis pigmentosa.
Resilience and vulnerabilities of tumor cells under purine shortage stress.
Uridine-sensitized screening identifies genes and metabolic regulators of nucleotide synthesis.
Protein succinylome analysis identifies citrate synthase as a central regulator of osteoclast metabolic activity.
Seizure and redox rescue in a model of glucose transport deficiency.
Moonlighting functions of glucose metabolic enzymes and metabolites in cancer.
De novo NAD+ synthesis is ineffective for NAD+ supply in axenically cultured Caenorhabditis elegans.
Rab10 regulates neuropeptide release by maintaining Ca2+ homeostasis and protein synthesis.
Gang of 3: How the Krebs cycle-linked metabolites itaconate, succinate, and fumarate regulate macrophages and inflammation.

Biochim Biophys Acta Bioenerg. 2025 Apr 01. pii: S0005-2728(25)00021-0. [Epub ahead of print] 149555

Three's company: Membrane waltz among organelles.

Valentin Guyard, Francesca Giordano.

  The study of membrane contact sites (MCS) has profoundly transformed our understanding of inter-organelle communication. These sites, where the membranes of two organelles are closely apposed, facilitate the transfer of small molecules such as lipids and ions. They are especially crucial for the maintenance of the structure and function of organelles like mitochondria and lipid droplets, which are largely excluded from vesicular trafficking. The significant advancements in imaging techniques, and molecular and cell biology research have shown that MCS are more complex than what originally thought and can involve more than two organelles. This has revealed the intricate nature and critical importance of these subcellular connections. Here, we provide an overview of newly described three-way inter-organelles associations, and the proteins involved in these MCS. We highlight the roles these contacts play in key cellular processes such as lipid droplet biogenesis and mitochondrial division. Additionally, we discuss the latest advances in super-resolution imaging that enable the study of these complex three-way interactions. Ongoing research, driven by technological innovations, promises to uncover further insights into their roles in fundamental cellular processes and their implications for health and disease.

Keywords:  Lipid droplet biogenesis; Membrane contact sites; Mitochondria; Mitochondria dynamics

DOI:  https://doi.org/10.1016/j.bbabio.2025.149555
Methods Mol Biol. 2025 ;2901 159-165

Method for Detecting Mitochondrial ATP by Hybridization Chain Reaction.

Lei Luo, Xiaohai Yang, Kemin Wang.

  Adenosine triphosphate (ATP) plays a central role in energy transduction and signaling in living cells. For mitochondrial ATP detection, the appropriate probes should include the abilities to enter target cells noninvasively, target mitochondria, and then respond to the ATP reliably. Here, we provide a detailed protocol for imaging mitochondrial ATP in living cells exploiting the hybridization chain reaction (HCR).

Keywords:  Adenosine triphosphate (ATP); Hybridization chain reaction; Imaging; Mitochondria

DOI:  https://doi.org/10.1007/978-1-0716-4394-5_12
bioRxiv. 2025 Mar 13. pii: 2025.03.10.642426. [Epub ahead of print]

Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis.

Patrick B Jonker, Mumina Sadullozoda, Guillaume Cognet, Juan J Apiz Saab, Kelly H Sokol, Violet X Wu, Deepa Kumari, Colin Sheehan, Mete E Ozgurses, Darby Agovino, Grace Croley, Smit A Patel, Althea Bock-Hughes, Kay F Macleod, Hardik Shah, Jonathan L Coloff, Evan C Lien, Alexander Muir.

Nutrient limitation is a characteristic feature of poorly perfused tumors. In contrast to well-perfused tissues, nutrient deficits in tumors perturb cellular metabolic activity, which imposes metabolic constraints on cancer cells. The metabolic constraints created by the tumor microenvironment can lead to vulnerabilities in cancers. Identifying the metabolic constraints of the tumor microenvironment and the vulnerabilities that arise in cancers can provide new insight into tumor biology and identify promising antineoplastic targets. To identify how the microenvironment constrains the metabolism of pancreatic tumors, we challenged pancreatic cancer cells with microenvironmental nutrient levels and analyzed changes in cell metabolism. We found that arginine limitation in pancreatic tumors perturbs saturated and monounsaturated fatty acid synthesis by suppressing the lipogenic transcription factor SREBP1. Synthesis of these fatty acids is critical for maintaining a balance of saturated, monounsaturated, and polyunsaturated fatty acids in cellular membranes. As a consequence of microenvironmental constraints on fatty acid synthesis, pancreatic cancer cells and tumors are unable to maintain lipid homeostasis when exposed to polyunsaturated fatty acids, leading to cell death by ferroptosis. In sum, arginine restriction in the tumor microenvironment constrains lipid metabolism in pancreatic cancers, which renders these tumors vulnerable to polyunsaturated-enriched fat sources.

DOI: https://doi.org/10.1101/2025.03.10.642426
J Biol Chem. 2025 Mar 26. pii: S0021-9258(25)00298-4. [Epub ahead of print] 108449

Conversion of glutamate into proline by the leucine analog BCH enhances biphasic insulin secretion in pancreatic β-cells.

Sevda Gheibi, Luis Rodrigo Cataldo, Hamidreza Ardalani, Lisa Nocquet, Peter Spégel, Susanne G Straub, Geoffrey W G Sharp, Malin Fex, Hindrik Mulder.

  Biphasic insulin secretion, which fails in Type 2 Diabetes, can be provoked by various nutrient stimuli, glucose being the superior physiological one. To identify pathways that may play a role in β-cell stimulus-secretion coupling, we compared β-cell and islet functional, secretory, and metabolic responses to glucose and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a leucine analog, that acts as an allosteric activator of glutamate dehydrogenase (GDH). We employed a range of techniques, including insulin secretion assays, mitochondrial activity measurements, ATP/ADP ratio assessments, and cytosolic Ca2+ level quantifications. Metabolomics was used to analyze cellular metabolite profiles in response to glucose and BCH. Additionally, we investigated the role of proline synthesis by silencing ALDH18A1, encoding proline 5-carboxylate (P5C) synthase, in both clonal β-cells and human islets. BCH and glucose similarly induced a biphasic insulin response in INS-1 832/13 cells, parallelled by increased mitochondrial activity and raised ATP/ADP ratios, plasma membrane depolarization, and elevated cytosolic Ca2+ levels. Metabolomics revealed that proline levels increased significantly only in BCH-stimulated β-cells. Silencing ALDH18A1 disrupted insulin secretion in response to both glucose and BCH, accompanied by reduced cytosolic Ca2+ levels, ATP/ADP ratios, and mitochondrial activity. Our findings demonstrated that BCH-induced activation of GDH leads to the conversion of glutamate into proline, which apparently enhances β-cell stimulus-secretion coupling. This work identifies a previously unrecognized role of proline metabolism in β-cell function and provides novel insights into the complex regulation of insulin secretion.

Keywords:  BCH; Insulin secretion; glutamate dehydrogenase; proline; β-cells

DOI:  https://doi.org/10.1016/j.jbc.2025.108449
Mol Biol Cell. 2025 Apr 02. mbcE24040162

A dual reporter system for intracellular and extracellular amino acid sensing in budding yeast.

Jurgita Paukštytė, Emma Cervera Tena, Juha Saarikangas.

Amino acid homeostasis is essential for cellular functions such as growth, metabolism, and signaling. In budding yeast Saccharomyces cerevisiae, the General Amino Acid Control (GAAC) and Target of Rapamycin Complex 1 (TORC1) pathways are utilized for intracellular amino acid sensing, while the Ssy1-Ptr3-Ssy5 (SPS) pathway is used for extracellular sensing. These pathways maintain homeostasis by responding to variations in amino acid levels to regulate amino acid biosynthesis and uptake. However, their interactions under various conditions and behavior at single-cell resolution remain insufficiently understood. We developed fluorescent transcriptional reporters to monitor amino acid biosynthesis and uptake pathways in single cells, revealing pathway engagement in response to different amino acid levels and types. Inhibition experiments demonstrated that the SPS pathway influences TORC1 and GAAC activities differently. Additionally, pathway engagement varied between liquid culture and colony environments. In colonies, some cells specialized in either amino acid synthesis or uptake. Disruption of the SPS pathway hindered this specialization and increased cell death rates in aging colonies, indicating a role for metabolic differentiation in maintaining colony viability. Collectively, this study introduces a new tool for exploring cellular amino acid homeostasis and highlights the importance of cellular differentiation in amino acid control for colony survival.

DOI: https://doi.org/10.1091/mbc.E24-04-0162
Trends Biochem Sci. 2025 Mar 31. pii: S0968-0004(25)00051-9. [Epub ahead of print]

Mitochondrial heat production: the elephant in the lab….

Pierre Rustin, Howard T Jacobs, Mügen Terzioglu, Paule Bénit.

  It has long been established that heat represents a major part of the energy released during the oxidation of mitochondrial substrates. However, with a few exceptions, the release of heat is rarely mentioned other than as being produced at the expense of ATP, without having any specific function. Here, after briefly surveying the literature on mitochondrial heat production, we argue for its cellular and organismal importance, sharing our opinions as to what could account for this unbalanced portrayal of mitochondrial energy transactions.

Keywords:  ATP; H(+)-ATPase; heat diffusion; mitochondria; nanoscale; respiratory chain

DOI:  https://doi.org/10.1016/j.tibs.2025.03.002
Genome Biol. 2025 Mar 28. 26(1): 79

MACAW: a method for semi-automatic detection of errors in genome-scale metabolic models.

Devlin C Moyer, Justin Reimertz, Daniel Segrè, Juan I Fuxman Bass.

  Genome-scale metabolic models (GSMMs) are used to predict metabolic fluxes, with applications ranging from identifying novel drug targets to engineering microbial metabolism. Erroneous or missing reactions, scattered throughout densely interconnected networks, are a limiting factor in these applications. We present Metabolic Accuracy Check and Analysis Workflow (MACAW), a suite of algorithms that helps to identify and visualize errors at the level of connected pathways, rather than individual reactions. We show how MACAW highlights inaccuracies of varying severity in manually curated and automatically generated GSMMs for humans, yeast, and bacteria and helps to identify systematic issues to be addressed in future model construction efforts.

Keywords:  Flux balance analysis; Genome-scale metabolic models; Human metabolism; Metabolic networks; Metabolic pathway analysis; Microbial metabolism

DOI:  https://doi.org/10.1186/s13059-025-03533-6
J Vis Exp. 2025 Mar 14.

Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina.

Olivia L Bossardet, Joseph M Holden, Lauren K Wareham.

The retina is a highly metabolic tissue with multiple cell types requiring glucose and its derivatives to produce energy in the form of ATP. Retinal cells, including endothelial cells, neurons, photoreceptors, and glial cells, express glucose transporters (GLUTs; e.g., GLUT1-4) to enable the uptake of glucose for energy production. GLUT1 is the most abundantly expressed glucose transporter in the retina. This protocol enables researchers to measure the uptake of glucose in the neural murine retina in ex vivo conditions using the fluorescent glucose analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG). After retinal dissection, total retinal 6-NBDG levels can be easily determined via fluorescence endpoint measurement using a plate reader. For consistency, we recommend normalizing results to total protein levels. Although 6-NBDG is highly specific for GLUT1, uptake of this analog is detected in the presence of GLUT1 inhibitor BAY-876. As such, this assay provides a relatively quick and inexpensive method to measure glucose uptake ex vivo in whole mouse neural retina, which is partially mediated by GLUT1.

DOI: https://doi.org/10.3791/67921
bioRxiv. 2025 Mar 16. pii: 2025.03.14.643362. [Epub ahead of print]

Calcium signals shape metabolic control of H3K27ac and H3K18la to regulate EGA.

Virginia Savy, Paula Stein, Don Delker, Martín A Estermann, Brian N Papas, Zongli Xu, Lenka Radonova, Carmen J Williams.

  The use of assisted reproductive technologies (ART) has enabled the birth of over 9 million babies; but it is associated with increased risks of negative metabolic outcomes in offspring. Yet, the underlying mechanism remains unknown. Calcium (Ca2+) signals, which initiate embryo development at fertilization, are frequently disrupted in human ART. In mice, abnormal Ca2+ signals at fertilization impair embryo development and adult offspring metabolism. Changes in intracellular Ca2+ drive mitochondrial activity and production of metabolites used by the epigenetic machinery. For example, acetyl-CoA (derived mainly from pyruvate) and lactyl-CoA (derived from lactate) are used for writing H3K27ac and H3K18la marks that orchestrate initiation of development. Using both a genetic mouse model and treatment with ionomycin to raise intracellular Ca2+ of wild-type fertilized eggs, we found that excess Ca2+ at fertilization changes metabolic substrate availability, causing epigenetic changes that impact embryo development and offspring health. Specifically, increased Ca2+ exposure at fertilization led to increased H3K27ac levels and decreased H3K18la levels at the 1-cell (1C) stage, that persisted until the 2-cell (2C) stage. Ultralow input CUT&Tag revealed significant differences in H3K27ac and H3K18la genomic profiles between control and ionomycin groups. In addition, increased Ca2+ exposure resulted in a marked reduction in global transcription at the 1C stage that persisted through the 2C stage due to diminished activity of RNA polymerase I. Excess Ca2+ following fertilization increased pyruvate dehydrogenase activity (enzyme that converts pyruvate to acetyl-CoA) and decreased total lactate levels. Provision of exogenous lactyl-CoA before ionomycin treatment restored H3K18la levels at the 1C and 2C stages and rescued global transcription to control levels. Our findings demonstrate conclusively that Ca2+ dynamics drive metabolic regulation of epigenetic reprogramming at fertilization and alter EGA.

Keywords:  DOHaD; Metabolofertility; artificial oocyte activation (AOA); lactylation; zygotic genome activation (ZGA)

DOI:  https://doi.org/10.1101/2025.03.14.643362
Front Immunol. 2025 ;16 1477929

Hexokinase2-engineered T cells display increased anti-tumor function.

Raphaëlle Toledano Zur, Shiran Didi Zurinam, Maria Radman, Elia Funaro Balouka, Tatiana Borodianskiy-Shteinberg, Dieter Saur, Cyrille J Cohen.

   Background: T cells face significant metabolic challenges in the tumor microenvironment (TME), where cancer cells monopolize critical nutrients like glucose and amino acids. This metabolic competition supports tumor growth while impairing T-cell anti-tumor responses, partly by reducing glycolytic function. Hexokinase 2 (HK2), a key enzyme in glycolysis, plays a pivotal role in maintaining T-cell functionality.
Methods: To enhance T-cell function, primary human T cells were genetically engineered to overexpress HK2 alongside a tumor-specific receptor. These engineered T cells were tested in vitro and in vivo to evaluate their metabolic and therapeutic efficacy.
Results: HK2-engineered T cells exhibited increased glycolytic capacity, leading to enhanced cytokine secretion, activation marker expression, and metabolic activity compared to controls. In vivo studies using a human tumor xenograft model demonstrated the superior therapeutic efficacy of HK2-engineered T cells, including delayed tumor growth and improved survival.
Conclusion: HK2 overexpression improves T-cell metabolic fitness and functionality in hostile TMEs, offering a promising foundation for the development of next-generation immunotherapies targeting T-cell metabolism.

Keywords:  T-cells; TCR; cellular immunotherapy; hexokinase 2; immunometabolism

DOI:  https://doi.org/10.3389/fimmu.2025.1477929
Elife. 2025 Apr 02. pii: e106553. [Epub ahead of print]14

At the crossroads of calcium signaling, protein synthesis and neuropeptide release.

Jakob Rupert, Dragomir Milovanovic.

  By influencing calcium homeostasis, local protein synthesis and the endoplasmic reticulum, a small protein called Rab10 emerges as a crucial cytoplasmic regulator of neuropeptide secretion.

Keywords:  Rab10; endoplasmic reticulum; exocytosis; mouse; neuropeptide; neuroscience; protein synthesis; synaptic transmission

DOI:  https://doi.org/10.7554/eLife.106553
J Biol Chem. 2025 Mar 28. pii: S0021-9258(25)00317-5. [Epub ahead of print] 108468

Activating Transcription Factor 3 regulates hepatic Apolipoprotein A4 upon metabolic stress.

Jasmine Encarnacion, Danielle M Smith, Joseph Choi, Joseph Scafidi, Michael J Wolfgang.

  The liver plays essential roles in maintaining systemic glucolipid homeostasis under ever changing metabolic stressors. Metabolic dysregulation can lead to both adaptive and maladaptive changes that impact systemic physiology. Here we examined disparate genetic and environmental metabolic stressors and identified Apolipoprotein A4 (ApoA4) as a circulating protein upregulated in liver-specific knockouts for Carnitine Palmitoyltransferase 2 and Pyruvate Carboxylase. We found this upregulation to be exacerbated by fasting and high fat or ketogenic diets. Unique among these models was a concomitant increase in Activating Transcription Factor 3 (Atf3). Liver-specific overexpression of Atf3 resulted in increased ApoA4 expression in a sex-dependent manner. To understand the requirement of Atf3 to metabolic stress, we generated liver-specific Atf3, Cpt2 double knockout mice. These experiments demonstrated the requirement for Atf3 in the induction of ApoA4 mRNA, ApoA4 protein, and serum triglycerides that were also sex dependent. These experiments reveal the roles of hepatic Atf3 and ApoA4 in response to metabolic stress in vivo.

Keywords:  Apolipoprotein A4; Atf3; Fatty acid oxidation; Liver metabolism; gene knockout; metabolic regulation; sex-dependent; transcription factor

DOI:  https://doi.org/10.1016/j.jbc.2025.108468
Cell Rep. 2025 Mar 28. pii: S2211-1247(25)00254-2. [Epub ahead of print]44(4): 115483

PDZD8 promotes autophagy at ER-lysosome membrane contact sites to regulate activity-dependent synaptic growth.

Rajan S Thakur, Kate M O'Connor-Giles.

  Building synaptic connections requires coordinating a host of cellular activities from cell signaling to protein turnover, placing a high demand on intracellular communication. Membrane contact sites (MCSs) formed between organelles have emerged as key signaling hubs for coordinating diverse cellular activities, yet their roles in the developing nervous system remain obscure. We investigate the in vivo function of the endoplasmic reticulum (ER) MCS tethering and lipid-transfer protein PDZD8, which was recently linked to intellectual disability, in the nervous system. We find that PDZD8 is required for activity-dependent synaptic bouton formation in multiple paradigms. PDZD8 is sufficient to drive excess synaptic bouton formation through an autophagy-dependent mechanism and required for synapse development when autophagy is limited. PDZD8 accelerates autophagic flux by promoting lysosome maturation at ER-late endosome/lysosome MCSs. We propose that PDZD8 functions in the nervous system to increase autophagy during periods of high demand, including activity-dependent synaptic growth.

Keywords:  CP: Cell biology; CP: Neuroscience; Drosophila; autophagy; lipid transfer protein; lysosomes; membrane contact sites; neurodevelopment; synapse

DOI:  https://doi.org/10.1016/j.celrep.2025.115483
Proc Natl Acad Sci U S A. 2025 Apr 08. 122(14): e2421978122

RPE-specific MCT2 expression promotes cone survival in models of retinitis pigmentosa.

Laurel C Chandler, Apolonia Gardner, Constance L Cepko.

  Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration worldwide. It is characterized by the sequential death of rod and cone photoreceptors, the cells responsible for night and daylight vision, respectively. Although the expression of most RP genes occurs only in rods, there is a secondary degeneration of cones. One possible mechanism of cone death is metabolic dysregulation. Photoreceptors are highly metabolically active, consuming large quantities of glucose and producing substantial amounts of lactate. The retinal pigment epithelium (RPE) mediates the transport of glucose from the blood to photoreceptors and, in turn, removes lactate, which can influence the rate of consumption of glucose by the RPE. One model for metabolic dysregulation in RP suggests that following the death of rods, lactate levels are substantially diminished causing the RPE to withhold glucose, resulting in nutrient deprivation for cones. Here, we present adeno-associated viral vector-mediated delivery of monocarboxylate transporter 2 (MCT2, Slc16a7) into the eye, with expression limited to RPE cells, with the aim of promoting lactate uptake from the blood and encouraging the passage of glucose to cones. We demonstrate prolonged survival and function of cones in rat and mouse RP models, revealing a possible gene-agnostic therapy for preserving vision in RP. We also present the use of fluorescence lifetime imaging-based biosensors for lactate and glucose within the eye. Using this technology, we show changes to lactate and glucose levels within MCT2-expressing RPE, suggesting that cone survival is impacted by changes in RPE metabolism.

Keywords:  gene therapy; metabolism; retinal pigment epithelium; retinitis pigmentosa

DOI:  https://doi.org/10.1073/pnas.2421978122
bioRxiv. 2025 Mar 20. pii: 2025.03.19.644180. [Epub ahead of print]

Resilience and vulnerabilities of tumor cells under purine shortage stress.

Jianpeng Yu, Chen Jin, Cheng Su, David Moon, Michael Sun, Hong Zhang, Xue Jiang, Fan Zhang, Nomi Tserentsoodol, Michelle L Bowie, Christopher J Pirozzi, Daniel J George, Robert Wild, Xia Gao, David M Ashley, Yiping He, Jiaoti Huang.

Purine metabolism is a promising therapeutic target in cancer; however how cancer cells respond to purine shortage,particularly their adaptation and vulnerabilities, remains unclear. Using the recently developed purine shortage-inducing prodrug DRP-104 and genetic approaches, we investigated these responses in prostate, lung and glioma cancer models. We demonstrate that when de novo purine biosynthesis is compromised, cancer cells employ microtubules to assemble purinosomes, multi-protein complexes of de novo purine biosynthesis enzymes that enhance purine biosynthesis efficiency. While this process enables tumor cells to adapt to purine shortage stress, it also renders them more susceptible to the microtubule-stabilizing chemotherapeutic drug Docetaxel. Furthermore, we show that although cancer cells primarily rely on de novo purine biosynthesis, they also exploit Methylthioadenosine Phosphorylase (MTAP)-mediated purine salvage as a crucial alternative source of purine supply, especially under purine shortage stress. In support of this finding, combining DRP-104 with an MTAP inhibitor significantly enhances tumor suppression in prostate cancer (PCa) models in vivo. Finally, despite the resilience of the purine supply machinery, purine shortage-stressed tumor cells exhibit increased DNA damage and activation of the cGAS-STING pathway, which may contribute to impaired immunoevasion and provide a molecular basis of the previously observed DRP-104-induced anti-tumor immunity. Together, these findings reveal purinosome assembly and purine salvage as key mechanisms of cancer cell adaptation and resilience to purine shortage while identifying microtubules, MTAP, and immunoevasion deficits as therapeutic vulnerabilities.

DOI: https://doi.org/10.1101/2025.03.19.644180
bioRxiv. 2025 Mar 13. pii: 2025.03.11.642569. [Epub ahead of print]

Uridine-sensitized screening identifies genes and metabolic regulators of nucleotide synthesis.

Abigail Strefeler, Zakery N Baker, Sylvain Chollet, Rachel M Guerra, Julijana Ivanisevic, Hector Gallart-Ayala, David J Pagliarini, Alexis A Jourdain.

Nucleotides are essential for nucleic acid synthesis, signaling, and metabolism, and can be synthesized de novo or through salvage. Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited target for cancer therapy. However, resistance frequently emerges, highlighting the need for a deeper understanding of nucleotide regulation. Here, we harness uridine salvage and CRISPR-Cas9 screening to reveal regulators of de novo pyrimidine synthesis. We identify several factors and report that pyrimidine synthesis can continue in the absence of coenzyme Q (CoQ), the canonical electron acceptor in de novo synthesis. We further investigate NUDT5 and report its conserved interaction with PPAT, the rate-limiting enzyme in purine synthesis. We show that in the absence of NUDT5, hyperactive purine synthesis siphons the phosphoribosyl pyrophosphate (PRPP) pool at the expense of pyrimidine synthesis, promoting resistance to chemotherapy. Intriguingly, the interaction between NUDT5 and PPAT appears to be disrupted by PRPP, highlighting intricate allosteric regulation. Our findings reveal a fundamental mechanism for maintaining nucleotide balance and position NUDT5 as a potential biomarker for predicting resistance to chemotherapy.

DOI: https://doi.org/10.1101/2025.03.11.642569
FEBS J. 2025 Apr 02.

Protein succinylome analysis identifies citrate synthase as a central regulator of osteoclast metabolic activity.

Dayoung Yu, Yue Gao, Marcin Luzarowski, Elisabeth Seebach, Thomas Heitkamp, Michael Börsch, Thomas Ruppert, Katharina F Kubatzky.

  Tumour necrosis factor ligand superfamily member 11 (TNFSF11; RANKL) and macrophage colony-stimulating factor 1 receptor (M-CSF) differentiate macrophages into osteoclasts. This process is characterised by changes in metabolic activity that support energy-consuming processes. Treatment with RANKL triggers a phenotype of accelerated metabolism with enhanced glycolysis and an initial disruption of the tricarboxylic acid cycle (TCA) through increased expression of the enzyme aconitate decarboxylase (ACOD1), which results in an upregulation of intracellular succinate levels. Succinate then causes post-translational succinylation of lysine residues. ACOD1 as an inducer of protein succinylation and the desuccinylase NAD-dependent protein deacylase sirtuin-5, mitochondrial (SIRT5) are regulated differentially, and the initially high expression of ACOD1 decreases towards the end of differentiation, whereas SIRT5 levels increase. To mimic the effect of protein succinylation, diethyl succinate or a SIRT5 inhibitor was added during differentiation, which reduced the formation of large osteoclasts, showing its relevance for osteoclastogenesis. To identify succinylated proteins, we used an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. Most lysine succinylated proteins were mitochondrial metabolic enzymes. Citrate synthase (CS), the enzyme catalysing the first reaction of the TCA cycle, showed a notable difference in succinylation levels before and after RANKL stimulation, with succinylation detected exclusively in stimulated cells. Immunoprecipitation assays confirmed CS succinylation. Using whole cell extracts, we observed that RANKL treatment decreased CS activity in a concentration-dependent manner. This suggests that CS could be critical in the context of energy production during osteoclastogenesis and that protein succinylation modulates the differentiation program of osteoclasts.

Keywords:  PTM scan; RANKL; citrate synthase; metabolism; mitochondria; osteoclast; post‐translational modification; succinylation

DOI:  https://doi.org/10.1111/febs.70090
PLoS Comput Biol. 2025 Apr 04. 21(4): e1012959

Seizure and redox rescue in a model of glucose transport deficiency.

Jay S Coggan, Polina Shichkova, Henry Markram, Daniel Keller.

Disruptions of energy supply to the brain are associated with many neurodegenerative pathologies and are difficult to study due to numerous interlinked metabolic pathways. We explored the effects of diminished energy supply on brain metabolism using a computational model of the neuro-glia-vasculature ensemble, in the form of a neuron, an astrocyte and local blood supply. As a case study, we investigated the glucose transporter type-1 deficiency syndrome (GLUT1-DS), a childhood affliction characterized by impaired glucose utilization and associated with phenotypes including seizures. Compared to neurons, astrocytes exhibited markedly higher metabolite concentration variabilities for all but a few redox species. This effect could signal a role for astrocytes in absorbing the shock of blood nutrient fluctuations. Redox balances were disrupted in GLUT1-DS with lower levels of reducing equivalent carriers NADH and ATP. The best non-glucose nutrient or pharmacotherapies for re-establishing redox normalcy involved lactate, the keto-diet (β-hydroxybutyrate), NAD and Q10 supplementation, suggesting a possible glucose sparing mechanism. GLUT1-DS seizures resulted from after-discharge neuronal firing caused by post-stimulus ATP reductions and impaired Na+/K+-ATPase, which can be rescued by restoring either normal glucose or by relatively small increases in neuronal ATP.

DOI: https://doi.org/10.1371/journal.pcbi.1012959
Nat Rev Cancer. 2025 Apr 02.

Moonlighting functions of glucose metabolic enzymes and metabolites in cancer.

Dong Guo, Ying Meng, Gaoxiang Zhao, Qingang Wu, Zhimin Lu.

Glucose metabolic enzymes and their metabolites not only provide energy and building blocks for synthesizing macromolecules but also possess non-canonical or moonlighting functions in response to extracellular and intracellular signalling. These moonlighting functions modulate various cellular activities, including gene expression, cell cycle progression, DNA repair, autophagy, senescence and apoptosis, cell proliferation, remodelling of the tumour microenvironment and immune responses. These functions integrate glucose metabolism with other essential cellular activities, driving cancer progression. Targeting these moonlighting functions could open new therapeutic avenues and lead to cancer-specific treatments.

DOI: https://doi.org/10.1038/s41568-025-00800-3
Commun Biol. 2025 Apr 02. 8(1): 545

De novo NAD+ synthesis is ineffective for NAD+ supply in axenically cultured Caenorhabditis elegans.

Shihao Zhu, Runshuai Zhang, Luxia Yao, Zhirong Lin, Yanjie Li, Siyuan Li, Lianfeng Wu.

To secure an adequate nicotinamide adenine dinucleotide (NAD+) supply for survival, organisms typically rely on two complementary mechanisms: the de novo synthesis pathway and the salvage pathway. Notably, the classic quinolinic acid phosphoribosyltransferase (QPRTase) for de novo NAD+ synthesis is absent in Caenorhabditis elegans (C. elegans), despite the reported alternative mechanism involving uridine monophosphate phosphoribosyltransferase (UMPS). However, the effectiveness of this proposed mechanism for NAD+ production of C. elegans remains unclear. Here, using a chemically defined medium, we observed that removing NAD+ salvage precursors from the medium results in a significant decrease in NAD+ levels, causing severe developmental delay and fecundity loss in C. elegans. Strikingly, these defects cannot be restored by any metabolites from the de novo synthesis pathway, including the direct QPRTase substrate quinolinic acid (QA). Furthermore, the deficiency of umps-1 does not cause any significant changes in the NAD+ levels of C. elegans. Moreover, the growth defects of the umps-1 mutant could be rescued by uridine, but not the salvage NAD+ supply. Additionally, we discovered that commercially available QA products contain substantial amounts of nicotinic acid, potentially producing misleading information. Collectively, our results demonstrate that C. elegans lacks the necessary mechanisms for de novo synthesis of NAD+.

DOI: https://doi.org/10.1038/s42003-025-07984-2
Elife. 2025 Apr 02. pii: RP94930. [Epub ahead of print]13

Rab10 regulates neuropeptide release by maintaining Ca2+ homeostasis and protein synthesis.

Jian Dong, Mian Chen, Jan R T van Weering, Ka Wan Li, August B Smit, Ruud F Toonen, Matthijs Verhage.

  Dense core vesicles (DCVs) transport and release various neuropeptides and neurotrophins that control diverse brain functions, but the DCV secretory pathway remains poorly understood. Here, we tested a prediction emerging from invertebrate studies about the crucial role of the intracellular trafficking GTPase Rab10, by assessing DCV exocytosis at single-cell resolution upon acute Rab10 depletion in mature mouse hippocampal neurons, to circumvent potential confounding effects of Rab10's established role in neurite outgrowth. We observed a significant inhibition of DCV exocytosis in Rab10-depleted neurons, whereas synaptic vesicle exocytosis was unaffected. However, rather than a direct involvement in DCV trafficking, this effect was attributed to two ER-dependent processes, ER-regulated intracellular Ca2+ dynamics, and protein synthesis. Gene Ontology analysis of differentially expressed proteins upon Rab10 depletion identified substantial alterations in synaptic and ER/ribosomal proteins, including the Ca2+ pump SERCA2. In addition, ER morphology and dynamics were altered, ER Ca2+ levels were depleted, and Ca2+ homeostasis was impaired in Rab10-depleted neurons. However, Ca2+ entry using a Ca2+ ionophore still triggered less DCV exocytosis. Instead, leucine supplementation, which enhances protein synthesis, largely rescued DCV exocytosis deficiency. We conclude that Rab10 is required for neuropeptide release by maintaining Ca2+ dynamics and regulating protein synthesis. Furthermore, DCV exocytosis appeared more dependent on (acute) protein synthesis than synaptic vesicle exocytosis.

Keywords:  ER; Rab10; exocytosis; mouse; neuropeptide; neuroscience; protein synthesis; synaptic transmission

DOI:  https://doi.org/10.7554/eLife.94930
Cell Metab. 2025 Mar 24. pii: S1550-4131(25)00107-X. [Epub ahead of print]

Gang of 3: How the Krebs cycle-linked metabolites itaconate, succinate, and fumarate regulate macrophages and inflammation.

Eva M Pålsson-McDermott, Luke A J O'Neill.

  The reprogramming of metabolic pathways and processes in immune cells has emerged as an important aspect of the immune response. Metabolic intermediates accumulate as a result of metabolic adaptations and mediate functions outside of metabolism in the regulation of immunity and inflammation. In macrophages, there has been a major focus on 3 metabolites linked to the Krebs cycle, itaconate, succinate, and fumarate, which have been shown to regulate multiple processes. Here, we discuss recent progress on these 3 metabolites with regard to their effect on macrophages in host defense and inflammatory diseases. We also consider the therapeutic opportunities presented from the mimicry of these metabolites or by targeting the enzymes that make or metabolize them in order to leverage the body's own anti-inflammatory response.

Keywords:  ETC; Krebs cycle; immunometabolism; immunometabolites; inflammation; therapeutic targets

DOI:  https://doi.org/10.1016/j.cmet.2025.03.004