bims-celmim Biomed News
on Cellular and mitochondrial metabolism
Issue of 2025–07–20
twenty-two papers selected by
Marc Segarra Mondejar
  1. Dynamic assembly of malate dehydrogenase-citrate synthase multienzyme complex in the mitochondria.
  2. Deletion of Skeletal Muscle Mitochondrial Glutamic-Oxaloacetic Transaminase (GOT2) Enhances Oxaloacetate Inhibition of Succinate Dehydrogenase and Alters Substrate Selectivity.
  3. mtKO: A dedicated guide RNA library for mitochondria research.
  4. Organic cation transporter 3 on neuronal mitochondria mediates MPP+-induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner.
  5. Cancer cells differentially modulate mitochondrial respiration to alter redox state and enable biomass synthesis in nutrient-limited environments.
  6. Nucleoside diphosphate kinase strongly promotes GDP and ADP metabolism in the cell and affects endogenous proton leak in mitochondria - the kinase is hampered by oxidative phosphorylation inhibitors.
  7. Ensemble quantitation of absolute metabolite concentrations in T cells reveals conserved features of immunometabolism.
  8. Measuring mitochondrial oxygenation and respiration during systemic inflammation in humans in vivo.
  9. Mitochondrial origins of the pressure to sleep.
  10. Human glycogenins maintain glucose homeostasis by regulating glycogen metabolism.
  11. Glucose-induced STUB1-GOT2 axis promotes aspartate synthesis and mitochondrial dysfunction in bladder cancer.
  12. 13 C tracing in synaptosomes reveals that SGLT2 inhibition with dapagliflozin prevents metabolic deficits in the 5X-FAD model of Alzheimer's Disease.
  13. "Glutaminolysis Fuels Reactive Astrocytes, Exacerbating Amyloid Pathology in Alzheimer's Disease".
  14. Lipoyl deglutarylation by ABHD11 regulates mitochondrial and T cell metabolism.
  15. ATP synthase inhibition, an overlooked confounding factor in the mitochondrial stress test.
  16. CAR-T cells containing CD28 versus 4-1BB co-stimulatory domains show distinct metabolic profiles in patients.
  17. De novo designed protein guiding targeted protein degradation.
  18. Nutrient-Responsive Formation of Mitochondrial-Derived Structures in Caenorhabditis elegans.
  19. Bi-directional metabolic reprogramming between cancer cells and T cells reshapes the anti-tumor immune response.
  20. Two-Photon FLIM Imaging of Mitochondrial Microenvironment in Apoptosis, Ferroptosis, and Fatty Liver Disease Models Using a Carbazole-Pyridinium Probe.
  21. Old mitochondria regulate niche renewal via α-ketoglutarate metabolism in stem cells.
  22. Specialized high-capacity mitochondria fuel cell invasion.
  1. bioRxiv. 2025 Jun 20. pii: 2025.06.16.659985. [Epub ahead of print]
    Dynamic assembly of malate dehydrogenase-citrate synthase multienzyme complex in the mitochondria.
    Joy Omini, Inga Krassovskaya, Taiwo Dele-Osibanjo, Connor Pedersen, Toshihiro Obata.
      The tricarboxylic acid (TCA) cycle enzymes, malate dehydrogenase (MDH1) and citrate synthase (CIT1), form a multienzyme complex called 'metabolon' that channels intermediate, oxaloacetate, between the reaction centers of the enzymes. Since the MDH1-CIT1 metabolon enhances the pathway reactions in vitro, it is postulated to regulate the TCA cycle flux through dynamic assembly in response to cellular metabolic demands. Here, we demonstrated that yeast mitochondrial MDH1 and CIT1 dissociated when aerobic respiration was suppressed by the Crabtree effect and associated when the pathway flux was enhanced by acetate. Pharmacological TCA cycle inhibitions dissociated the complex, while electron transport chain inhibition enhanced the interaction. The multienzyme complex assembly was related to the mitochondrial matrix acidification and oxidation, as well as cellular levels of malate, fumarate, and citrate. These factors significantly affected the MDH1-CIT1 complex affinity in vitro. Especially the buffer pH significantly changed the MDH1-CIT1 affinity within the pH range between 6.0 and 7.0, which is observed in the mitochondrial matrix under physiological conditions. These results show a dynamic association and dissociation of a metabolon in the mitochondria and its relationship with pathway flux, supporting the metabolon's role in metabolic regulation. Multiple factors, including pH and metabolite availabilities, possibly regulate MDH1-CIT1 interaction.
    DOI:  https://doi.org/10.1101/2025.06.16.659985
  2. FASEB J. 2025 Jul 31. 39(14): e70825
    Deletion of Skeletal Muscle Mitochondrial Glutamic-Oxaloacetic Transaminase (GOT2) Enhances Oxaloacetate Inhibition of Succinate Dehydrogenase and Alters Substrate Selectivity.
    Brian D Fink, Ritu Som, Liping Yu, William I Sivitz.
      Oxaloacetate (OAA) is converted to aspartate by mitochondrial glutamic-oxaloacetic transaminase 2 (GOT2) along with the conversion of glutamate to alpha-ketoglutarate (α-KG). Glutamate can also be directly converted to α-KG by glutamate dehydrogenase. In past work, we found that in skeletal muscle mitochondria energized by succinate alone, oxaloacetate accumulates and inhibits succinate dehydrogenase (complex II) in a manner dependent on inner membrane potential (ΔΨ). Here, we tested the hypothesis that deleting GOT2 would increase OAA concentrations, decrease complex II-energized respiration, and alter the selectivity of succinate versus glutamate for energy. Incubating wild-type mitochondria with succinate and glutamate revealed that increments in ADP increased OAA and caused a preferential use of glutamate for energy. Deletion of GOT2 compared to wild-type decreased complex II energized respiration, increased OAA, and decreased consumption of glutamate relative to succinate. OAA accumulation was also associated with decreased conversion of succinate to fumarate and malate. These findings are consistent with GOT2 control of metabolite flow through succinate dehydrogenase via regulation of OAA and consequent inhibition of succinate dehydrogenase. In contrast to respiration energized at complex II, when mitochondria were energized at complex I by pyruvate + malate, respiration did not differ between GOT2KO and WT mitochondria, and oxaloacetate was not detectable. In summary, GOT2 and OAA mediate complex II respiration and mitochondrial energy substrate selectivity.
    Keywords:  glutamic‐oxaloacetic transaminase‐2; mitochondria; mitochondrial complex II; mitochondrial inner membrane potential; oxaloacetate; respiration; skeletal muscle; succinate dehydrogenase
    DOI:  https://doi.org/10.1096/fj.202501071R
  3. Proc Natl Acad Sci U S A. 2025 Jul 22. 122(29): e2502285122
    mtKO: A dedicated guide RNA library for mitochondria research.
    Karambir Kaur, Javeria Zaheer, Fengchao Lang, Diego Luis Ribeiro, Meili Zhang, Hua Song, Wei Zhang, Raj Chari, Hussam Alkaissi, Chunzhang Yang.
      Mitochondria are multifunctional organelles central to both physiological and pathological processes. In malignant cancer cells, mitochondrial reprogramming establishes the metabolic foundation to meet cellular demands, which is particularly important in tumor cells with existing metabolic perturbations. To identify key mitochondrial pathways supporting cancer development, we developed mitochondria Knockout (mtKO), a robust and unbiased CRISPR screening platform to pinpoint critical mitochondria-associated pathways. The mtKO screen revealed that the mitochondrial antioxidant enzyme SOD2 is essential for cells harboring IDH1 mutations. Mechanistically, SOD2 activity determines the disease manifestation of IDH1-mutated cancers, through maintaining redox homeostasis and mitochondrial fitness. This study introduces a powerful functional genomic tool to identify mitochondrial-centered pathways and reveals the selective mitochondrial vulnerability in Krebs cycle-deficient cancers for future therapeutic intervention.
    Keywords:  CRISPR screen; IDH1; SOD2; metabolism; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2502285122
  4. BMC Biol. 2025 Jul 15. 23(1): 214
    Organic cation transporter 3 on neuronal mitochondria mediates MPP+-induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner.
    Ao Guan, Sida Han, Suzhen Liang, Weiwei Shen, Min Guo, Mei Cui.
       BACKGROUND: Mitochondria play crucial roles in cellular metabolism, and metabolite compartmentalization significantly impacts mitochondrial function and disease pathophysiology. MPP+ accumulation in mitochondria, a key factor in MPTP-induced neurodegeneration, leads to mitochondrial dysfunction, such as respiratory chain inhibition, ultimately leading to neuronal death. However, the mechanisms underlying mitochondrial MPP+ accumulation remain poorly understood. Organic cation transporter 3 (OCT3), a passive transporter mediating MPP+ transport, has been observed on the mitochondrial membrane, but it remains unclear whether mitochondrial OCT3 is involved in MPP+ accumulation in mitochondria.
    RESULTS: OCT3 was detected in the mitochondria fraction of SH-SY5Y cells, located on both the inner membrane and outer membrane. Following MPP+ incubation, there was a significant increase in mitochondrial uptake of MPP+, which was mitigated by OCT3 inhibition. Knockdown of the translocase of inner mitochondrial membrane 22 (TIMM22), an important component of the mitochondrial protein import apparatus, successfully reduced OCT3 levels on mitochondria without impairing mitochondrial morphology or mitochondrial membrane potential. TIMM22 knockdown reduced mitochondrial MPP+ uptake, which in turn rescued MPP+-induced mitochondrial fragmentation, complex I inhibition, and mitochondrial membrane potential reduction. Furthermore, TIMM22 knockdown suppressed caspase-9 and caspase-3 activation and reversed the alterations of BAX and BCL-xL induced by mitochondrial MPP+ accumulation.
    CONCLUSIONS: Here we found that OCT3 on neuronal mitochondria serves as an effective MPP+ transporter, crucial for mitochondrial MPP+ uptake and MPP+-induced neurotoxicity. Furthermore, TIMM22 downregulation can selectively reduce mitochondrial OCT3 and reverse MPP+-induced mitochondrial dysfunction and neurotoxicity, highlighting TIMM22 and OCT3 as potential therapeutic targets for MPP+-associated neurodegeneration and diseases.
    Keywords:  MPP+ ; Mitochondria; Neurodegeneration; Organic cation transporter 3; TIMM22
    DOI:  https://doi.org/10.1186/s12915-025-02318-4
  5. bioRxiv. 2025 May 10. pii: 2025.05.09.653205. [Epub ahead of print]
    Cancer cells differentially modulate mitochondrial respiration to alter redox state and enable biomass synthesis in nutrient-limited environments.
    Sarah M Chang, Muhammad Bin Munim, Sonia E Trojan, Anna Shevzov-Zebrun, Keene L Abbott, Matthew G Vander Heiden.
      The cell NAD+/NADH ratio can constrain biomass synthesis and influence proliferation in nutrient-limited environments. However, which cell processes regulate the NAD+/NADH ratio is not known. Here, we find that some cancer cells elevate the NAD+/NADH ratio in response to serine deprivation by increasing mitochondrial respiration. Cancer cells that elevate mitochondrial respiration have higher serine production and proliferation in serine limiting conditions than cells with no mitochondrial respiration response, independent of serine synthesis enzyme expression. Increases in mitochondrial respiration and the NAD+/NADH ratio promote serine synthesis regardless of whether serine is environmentally limiting. Lipid deprivation can also increase the NAD+/NADH ratio via mitochondrial respiration in some cells, including cells that do not increase respiration following serine deprivation. Thus, in cancer cells where lipid depletion raises the NAD+/NADH ratio, proliferation in serine depleted environments improves when lipids are also depleted. Taken together, these data suggest that changes in mitochondrial respiration in response to nutrient deprivation can influence the NAD+/NADH ratio in a cell-specific manner to impact oxidative biomass synthesis and proliferation. Given the complexity of tumor microenvironments, this work provides a metabolic framework for understanding how levels of more than one environmental nutrient affects cancer cell proliferation.
    DOI:  https://doi.org/10.1101/2025.05.09.653205
  6. J Enzyme Inhib Med Chem. 2025 Dec;40(1): 2520611
    Nucleoside diphosphate kinase strongly promotes GDP and ADP metabolism in the cell and affects endogenous proton leak in mitochondria - the kinase is hampered by oxidative phosphorylation inhibitors.
    Andrzej M Woyda-Ploszczyca.
      Rapid GDP metabolism in mitochondria isolated from wild-type yeast is postulated. The hallmark of exogenous GDP is convergence with the effect of exogenous ADP, typically inducing oxidative phosphorylation (OXPHOS). The GDP-provoked changes in the presence of ATP, i.e. increased respiratory rate accompanied by decreased inner mitochondrial membrane electrical potential, were curtailed by OXPHOS inhibitors, such as carboxyatractyloside, which apparently merged the GDP effect with OXPHOS. However, all performed tests indicated that the response of mitochondria to GDP is indirect and involves two steps. First, GDP is transphosphorylated via nucleoside diphosphate kinase (NDPK), ATP + GDP → ADP + GTP, which is followed by ADP-induced OXPHOS. Importantly, in mitochondria isolated from mutant yeast with a deleted NDPK gene, the stimulatory effect of GDP was eliminated. Therefore, a prerequisite for GDP metabolic action is the cooperation of NDPK with the OXPHOS apparatus. This biological model can help elucidate the molecular basis of some diseases treatment, such as cancer.
    Keywords:  ADP/ATP carrier; mitochondria; nucleoside-diphosphate kinase (NDPK); nucleotide metabolism; proton (H+) leak
    DOI:  https://doi.org/10.1080/14756366.2025.2520611
  7. bioRxiv. 2025 Jun 12. pii: 2025.06.09.658709. [Epub ahead of print]
    Ensemble quantitation of absolute metabolite concentrations in T cells reveals conserved features of immunometabolism.
    Samantha O'Keeffe, Harin Sim, Elle McCue, Junyoung O Park.
      Cells regulate metabolite levels to efficiently utilize metabolic networks and avoid toxic buildup. In turn, metabolites dictate metabolic activity by acting as substrates, products, and effectors. Despite their foundational role in cell physiology, kinetics, and thermodynamics, absolute metabolite concentrations are seldom known. Here we develop an ensemble method for absolute metabolite quantitation and quantify 84 metabolites in T cells. Liquid chromatography-mass spectrometry of metabolites co-extracted from T cells and 13 C-labeled reference cells reveals absolute concentrations en masse . Across subtypes and individuals, T cell metabolomes resemble one another. T cells possess high adenylate energy charge and favorable redox ratios for energy and biomass production without compromising the forward driving force in glycolysis. Across metabolism, metabolite concentrations exceed their associated Michaelis constants and inhibitor constants two thirds and half of the time, respectively. The conserved features of T cell metabolomes underlie a design principle: metabolite levels prime cells for adaptive immune response.
    DOI:  https://doi.org/10.1101/2025.06.09.658709
  8. Sci Rep. 2025 Jul 16. 15(1): 25815
    Measuring mitochondrial oxygenation and respiration during systemic inflammation in humans in vivo.
    Mark A Wefers Bettink, Jelle Zwaag, Bernard Schockaert, Peter Pickkers, Matthijs Kox, Egbert G Mik.
      Disturbances in mitochondrial function are implicated in several chronic and acute diseases. Systemic inflammation has been described to affect mitochondrial respiration. Yet, in vivo measurement of mitochondrial respiration is notoriously difficult. We measured mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) using the bedside COMET (Cellular Oxygen METabolism) system during systemic inflammation elicited by intravenous administration of 2 ng/kg lipopolysaccharide (LPS) in 42 healthy male volunteers. Four male subjects who did not receive LPS served as uninflamed controls. MitoPO2 and mitoVO2 were measured immediately prior to LPS administration, and at 1.45 h, 4 h, and 7 h, as well as at the corresponding timepoints in the control group. Compared to the control group, MitoPO2 significantly decreased over time in the LPS group (p = 0.002), with the nadir observed at 1.45 h post-LPS administration (45.8 ± 1.8 vs. 75.2 ± 2.6 mmHg); while mitoVO2 did not change. Concluding, the COMET monitor detects changes in mitochondrial parameters in a relatively mild model of systemic inflammation. This study paves the way for bedside monitoring of alterations in mitochondrial oxygenation and respiration, which may represent a vital next step in early diagnosis of mitochondrial dysfunction and stratification of patients in the intensive care unit.Trial registration: ClinicalTrials.gov NCT03240497. (12/04/2016) toetsingonline.nl NL56686.091.16 (11/04/2016) and NL65767.078.18 (01/05/2018).
    Keywords:  5-aminolevulinic acid; Endotoxemia; Healthy volunteers; Lipopolysaccharides; Mitochondria
    DOI:  https://doi.org/10.1038/s41598-025-10715-6
  9. Nature. 2025 Jul 16.
    Mitochondrial origins of the pressure to sleep.
    Raffaele Sarnataro, Cecilia D Velasco, Nicholas Monaco, Anissa Kempf, Gero Miesenböck.
      To gain a comprehensive, unbiased perspective on molecular changes in the brain that may underlie the need for sleep, we have characterized the transcriptomes of single cells isolated from rested and sleep-deprived flies. Here we report that transcripts upregulated after sleep deprivation, in sleep-control neurons projecting to the dorsal fan-shaped body1,2 (dFBNs) but not ubiquitously in the brain, encode almost exclusively proteins with roles in mitochondrial respiration and ATP synthesis. These gene expression changes are accompanied by mitochondrial fragmentation, enhanced mitophagy and an increase in the number of contacts between mitochondria and the endoplasmic reticulum, creating conduits3,4 for the replenishment of peroxidized lipids5. The morphological changes are reversible after recovery sleep and blunted by the installation of an electron overflow6,7 in the respiratory chain. Inducing or preventing mitochondrial fission or fusion8-13 in dFBNs alters sleep and the electrical properties of sleep-control cells in opposite directions: hyperfused mitochondria increase, whereas fragmented mitochondria decrease, neuronal excitability and sleep. ATP concentrations in dFBNs rise after enforced waking because of diminished ATP consumption during the arousal-mediated inhibition of these neurons14, which augments their mitochondrial electron leak7. Consistent with this view, uncoupling electron flux from ATP synthesis15 relieves the pressure to sleep, while exacerbating mismatches between electron supply and ATP demand (by powering ATP synthesis with a light-driven proton pump16) precipitates sleep. Sleep, like ageing17,18, may be an inescapable consequence of aerobic metabolism.
    DOI:  https://doi.org/10.1038/s41586-025-09261-y
  10. Nat Commun. 2025 Jul 16. 16(1): 6556
    Human glycogenins maintain glucose homeostasis by regulating glycogen metabolism.
    Tzu-Han Weng, Yu-Chung Pien, Ching-Jou Chen, Po-Pang Chen, Yu-Ting Tseng, Ying-Chen Chen, Wen-Po Hsiao, Ying-Ting Lee, Yi-An Chen, Yao-Chi Chen, Carmay Lim, Tzu-Han Hsu, Sung-Jan Lin, Hsin-Yung Yen, Kuo-Chiang Hsia, Su-Yi Tsai.
      Proper regulation of glycogen metabolism is fundamental to cellular energy homeostasis, and its disruption is associated with various metabolic disorders, including glycogen storage diseases (GSDs) and potentially diabetes. Despite glycogen's role as an essential energy reservoir, the mechanisms governing its synthesis and structural diversity across tissues remain unclear. Here, we uncover the distinct physiological roles of the human glycogenins GYG1 and GYG2 in glycogen synthesis. Through cellular models, structural biology, and biochemical analyses, we demonstrate that, unlike GYG1, GYG2 exhibits minimal autoglycosylation activity and acts as a suppressor of glycogen formation. Together, these two glycogenins coordinate glycogen synthase activity and influence glycogen assembly in a cell-type-dependent manner. Importantly, these glycogenins modulate glucose metabolic pathways, thereby ensuring cellular glucose homeostasis. These findings address longstanding questions in glycogen metabolism and establish both GYG1 and GYG2 as critical regulators of glycogen synthesis and breakdown in human, providing insights with potential therapeutic implications for treating GSDs and metabolic diseases.
    DOI:  https://doi.org/10.1038/s41467-025-61862-3
  11. Cell Death Dis. 2025 Jul 12. 16(1): 516
    Glucose-induced STUB1-GOT2 axis promotes aspartate synthesis and mitochondrial dysfunction in bladder cancer.
    Yunqiang Xiong, Qianxi Dong, Hongji Hu, Zhongqi Li, Xiangpeng Zhan, Fuchun Zheng, Hao Wan, Jiahao Liu, Shuyu Wu, Wang Pan, Ruize Yuan, Jing Xiong, Ju Guo, Songhui Xu, Bin Fu.
      Aberrant glucose metabolism, a characteristic of malignant tumors, contributes to the development and progression of bladder cancer (BCa). However, the underlying mechanism by which aberrant glucose metabolism promotes BCa progression is still incompletely understood. Here, we demonstrate that low levels of STUB1 are associated with worse progression and poor prognosis of BCa patients. STUB1 overexpression attenuates BCa cell proliferation, migration and amino acid metabolism, especial aspartate metabolism. Mechanistically, we identify that STUB1 induces K6- and K48-linked polyubiquitination of GOT2 at K73 lysine residue to decrease its stability, which attenuates mitochondrial aspartate (Asp) synthesis and regulates mitochondrial dysfunction. GOT2 was significantly up-regulated in BCa tissues and negatively associated with STUB1 expression. Furthermore, we reveal that high glucose stress promotes Asp synthesis and tumor growth through STUB1-GOT2 axis. Collectively, our findings identify that STUB1-GOT2 axis is an important regulator for maintaining Asp synthesis and mitochondrial function in BCa cell growth, which highlights that targeting STUB1-GOT2 axis could be a valuable strategy to ameliorate BCa progression by inhibiting amino acid metabolic function.
    DOI:  https://doi.org/10.1038/s41419-025-07840-5
  12. bioRxiv. 2025 May 03. pii: 2025.04.30.651373. [Epub ahead of print]
    13 C tracing in synaptosomes reveals that SGLT2 inhibition with dapagliflozin prevents metabolic deficits in the 5X-FAD model of Alzheimer's Disease.
    Marisa Mekkittikul, Cansheng Zhu, Bezawit T Danna, Xiaohong Zuo, Amy Rios, Krista Yang, Miranda Villanueva, Devanshi Agarwal, Daniel Castro, Xuelin Gu, Alexander R Strofs, Orian S Shirihai, Linsey Stiles, Sally A Frautschy, Jan Oscarsson, Russell L Esterline, Gregory M Cole, Ajit S Divakaruni.
      Metabolic dysfunction is linked to several forms of age-related neurodegeneration including Alzheimer's Disease (AD), and targeting brain energy metabolism is an increasingly attractive mode of therapeutic intervention. However, commonly used in vitro methods to identify specific metabolic pathways of interest in preclinical models of neurodegenerative disease have considerable limitations. They are prone to subselection of sample material, unable to identify cell type-specific effects, or cannot identify metabolic defects upstream of mitochondria. Here we address these challenges by validating a method for stable isotope tracing with isolated synaptic nerve terminals, or 'synaptosomes'. We further applied this approach to study glucose metabolism in synaptosomes isolated from the 5X-FAD mouse model of AD treated with the antidiabetic sodium-glucose linked transporter-2 (SGLT-2) inhibitor Dapagliflozin. Treatment with Dapagliflozin preserved steady-state levels of synaptosomal metabolites and enrichment from labeled glucose into citrate that was reduced in the 5X-FAD model. These changes correlated with trends towards improved spatial working memory but not amyloid burden. The results highlight the utility of stable isotope tracing in synaptosomes to identify precise sites of metabolic dysfunction and mechanisms of action for metabolic drug candidates in preclinical models of neurodegeneration.
    DOI:  https://doi.org/10.1101/2025.04.30.651373
  13. bioRxiv. 2025 Jun 15. pii: 2025.06.12.658977. [Epub ahead of print]
    "Glutaminolysis Fuels Reactive Astrocytes, Exacerbating Amyloid Pathology in Alzheimer's Disease".
    Shamulailatpam Shreedarshanee Devi, Sofia Luella Heese, Isabelle Leray, Gilbert Gallardo.
      Amyloid-β (Aβ) plaques with progressively increasing reactive astrocytes characterize Alzheimer's disease (AD). Reactive astrocytes are regulated by cellular and molecular mechanisms that are known to progress Aβ pathology. However, the metabolic adaptation and metabolites required to fuel these molecular changes in reactive astrocytes remain unknown. Using human AD samples, in vivo amyloid mouse models, and in vitro approaches, we demonstrate that reactive astrocytes utilize glutamine to fuel anaplerosis and meet their metabolic demands, thereby progressing amyloidosis. We show that reactive astrocytes increase Na + -coupled neutral amino acid transporters for glutamine uptake that are interdependent on Na + /K + ATPase. Furthermore, increasing brain-glutamine levels with a high-glutamine diet exacerbated reactive astrocytes, increasing Aβ burden in an amyloid mouse model. We demonstrate that glutamine undergoes glutaminolysis via glutaminase-2/glutamate dehydrogenase-1 enzymes to be incorporated into TCA metabolites for anaplerosis. Pharmacologically or genetically blocking glutaminolysis reduces reactive astrocytes and decreases Aβ pathology in an amyloid mouse. Our findings reveal the first glutamine-dependent metabolic adaptation of reactive astrocytes affecting Aβ pathology, which may be harnessed for AD therapeutic strategies.
    DOI:  https://doi.org/10.1101/2025.06.12.658977
  14. Nat Chem Biol. 2025 Jul 15.
    Lipoyl deglutarylation by ABHD11 regulates mitochondrial and T cell metabolism.
    Guinevere L Grice, Eleanor Minogue, Hudson W Coates, Mekdes Debela, Richard J Stopforth, Niek Wit, Zongyu Li, Joseph P Crowley, Arthur Kaser, Nicole Kaneider-Kaser, P Robin Antrobus, Marcia C Haigis, Randall S Johnson, James A Nathan.
      Glutarate is an intermediate of amino acid catabolism and an important metabolite for reprogramming T cell immunity. Glutarate exerts its effects either by directly inhibiting metabolite-dependent enzymes or through conjugation to substrates. Intriguingly, glutarylation can occur on protein and nonprotein substrates, but our understanding of these distinct glutaryl modifications is in its infancy. Here we uncover ABHD11 as a noncanonical deglutarylating enzyme critical for maintaining the tricarboxylic acid (TCA) cycle. Mechanistically, we find ABHD11 removes glutaryl adducts from lipoate-an essential fatty acid modification required for the TCA cycle. Loss of ABHD11 results in the accumulation of glutaryl-lipoyl adducts that drive an adaptive program, involving 2-oxoglutarate accumulation, that rewires mitochondrial metabolism. Functionally, this role of ABHD11 influences the metabolic programming of human CD8+ T cells. Therefore, our findings reveal lipoyl glutarylation as a reversible modification that regulates the TCA cycle.
    DOI:  https://doi.org/10.1038/s41589-025-01965-6
  15. PLoS One. 2025 ;20(7): e0328256
    ATP synthase inhibition, an overlooked confounding factor in the mitochondrial stress test.
    Jesse Corbin, Eric A Lehoux, Isabelle Catelas.
      The mitochondrial stress test, a widely used procedure to study energy metabolism using extracellular flux analysis, involves the inhibition of ATP synthase (a.k.a. complex V [CV]). This inhibition was recently shown to cause a glycolysis-dependent underestimation of two key mitochondrial respiration parameters, maximal respiration (MR) and spare respiratory capacity (SRC), in tumor cells. However, it is unknown if test substances (toxins, drugs, signaling molecules, etc.), especially those affecting glycolysis, can impact the underestimation of MR and SRC caused by CV inhibition and thereby produce potentially erroneous results. The objective of the present study was to determine if the inhibition of CV in the mitochondrial stress test can act as a confounding factor when measuring MR and SRC in intact non-tumor cells exposed to exemplificatory test substances that affect energy metabolism: Ni2+ and lipopolysaccharides (LPS). Murine bone marrow-derived macrophages were exposed to Ni2+ (0-72 ppm) or LPS (0 or 1 µg/mL), and oxygen consumption rates were measured by extracellular flux analysis using the mitochondrial stress test, with and without CV inhibition. Results showed that CV inhibition masked the decrease in MR induced by Ni2+ or LPS. It also caused the lack of a statistically significant effect of Ni2+ on SRC to present as an increase of SRC, and the LPS-induced decrease of SRC to be masked. Results further showed that these erroneous results arose because exposure to Ni2+ or LPS reduced the underestimation of MR and SRC caused by CV inhibition. This phenomenon was associated with increased glycolytic flux. Finally, results confirmed that underestimation of MR and SRC induced by CV inhibition can occur in non-tumor cells. In conclusion, the present study demonstrates that CV inhibition can act as a confounding factor leading to erroneous conclusions when the mitochondrial stress test is used with intact cells exposed to test substances.
    DOI:  https://doi.org/10.1371/journal.pone.0328256
  16. Cell Rep. 2025 Jul 11. pii: S2211-1247(25)00744-2. [Epub ahead of print]44(7): 115973
    CAR-T cells containing CD28 versus 4-1BB co-stimulatory domains show distinct metabolic profiles in patients.
    Molly S Cook, Ellen King, Katie R Flaherty, Khadiza Siddika, Sophie Papa, Reuben Benjamin, Anna Schurich.
      Chimeric antigen receptor (CAR)-T cell therapy has led to unprecedented success in treating relapsed/refractory diffuse large B cell lymphoma (DLBCL). The most common CAR-T cell products currently in the clinic for DLBCL differ in their co-stimulation moiety, containing either CD28 or 4-1BB, which initiate distinct signaling pathways. Previous work has highlighted the importance of T cell metabolism in fueling anti-cancer function. We have studied the metabolic characteristics induced by CD28 versus 4-1BB co-stimulation in patient CAR-T cells ex vivo. Our data show that in patients, CD28 and 4-1BB drive significantly divergent metabolic profiles. CD28 signaling endows T cells with preferentially glycolytic metabolism supporting an effector phenotype and increased expansion capacity, while 4-1BB co-stimulation preserves mitochondrial fitness and results in memory-like differentiation. Despite the differences in metabolic programming, T cells in patients responding successfully to therapy were metabolically similar, irrespective of co-stimulator. In contrast, in non-responders, CD28- and 4-1BB-co-stimulated CAR-T cells were metabolically distinct from each other.
    Keywords:  4-1BB; CAR-T cells; CD28; CP: Cancer; CP: Metabolism; DLBCL; co-stimulation; glycolysis; lymphoma; metabolism; mitochondria; translational
    DOI:  https://doi.org/10.1016/j.celrep.2025.115973
  17. Nat Commun. 2025 Jul 17. 16(1): 6598
    De novo designed protein guiding targeted protein degradation.
    Zhendong Li, Gan Qiao, Xianghe Wang, Ming Wang, Jinyu Cheng, Guipeng Hu, Xiaomin Li, Jing Wu, Jia Liu, Cong Gao, Liming Liu.
      Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.
    DOI:  https://doi.org/10.1038/s41467-025-62050-z
  18. bioRxiv. 2025 Jun 26. pii: 2025.06.24.661357. [Epub ahead of print]
    Nutrient-Responsive Formation of Mitochondrial-Derived Structures in Caenorhabditis elegans.
    Miriam Valera-Alberni, Anne Lanjuin, Silvia Romero-Sanz, Kristopher Burkewitz, Adam L Hughes, William B Mair.
      Mitochondrial morphology is dynamically regulated through remodeling processes essential for maintaining mitochondrial function and ensuring cellular and metabolic homeostasis. While classical models of mitochondrial dynamics center on cycles of fragmentation and elongation, emerging evidence highlights additional membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and mitochondrial-derived compartments (MDCs). These mitochondrial-derived structures, however, have been predominantly characterized in cultured cells and unicellular organisms, leaving their relevance in multicellular systems largely unexplored. Here, we identify a previously uncharacterized class of mitochondrial-derived structures in Caenorhabditis elegans muscle cells that are induced in response to intermittent fasting. We show that these structures appear specifically during the refeeding phase- coinciding with mitochondrial elongation -and are absent during fasting. Consistent with MDCs, the structures, approximately 1 µm in size, are enriched in outer mitochondrial membrane markers such as TOMM-20 aa1-49 and TOMM-70, but notably lack components of the inner mitochondrial membrane. Their formation requires the microtubule-associated MIRO-1/2 proteins, and their size is modulated by the mitochondrial dynamics machinery. Together, our findings reveal a nutritionally regulated mitochondrial remodeling event in C. elegans muscle that may play a role in mitochondrial quality control and adaptation to metabolic cues.
    DOI:  https://doi.org/10.1101/2025.06.24.661357
  19. PLoS Biol. 2025 Jul 14. 23(7): e3003284
    Bi-directional metabolic reprogramming between cancer cells and T cells reshapes the anti-tumor immune response.
    Yajing Qiu, Yihan Xu, Xinyuan Ding, Congcong Zhao, Hongcheng Cheng, Guideng Li.
      Cancer cells and T cells engage in dynamic crosstalk within the tumor microenvironment (TME), shaping tumor progression and anti-tumor immunity. While cancer cells reprogram metabolism to support growth and immune evasion, T cells must adapt their metabolic states to maintain effector functions. Tumor-driven metabolic perturbations, such as nutrient depletion and accumulation of immunosuppressive metabolites, profoundly impair T cell function and fate. Conversely, metabolically reprogrammed T cells can modulate the TME and influence tumor growth. This reciprocal metabolic crosstalk represents both metabolic competition and intercellular communication, offering promising therapeutic targets.
    DOI:  https://doi.org/10.1371/journal.pbio.3003284
  20. Anal Chem. 2025 Jul 14.
    Two-Photon FLIM Imaging of Mitochondrial Microenvironment in Apoptosis, Ferroptosis, and Fatty Liver Disease Models Using a Carbazole-Pyridinium Probe.
    Tong Zhu, Shang Wan, Hong Wang, Zhihui Feng, Xin Yang, Linge Wang, Bin Song, Xiaohe Tian, Wenwu Ling.
      Mitochondria are dynamic organelles whose microenvironmental state is tightly linked to cell death pathways and metabolic disease progression. However, directly visualizing mitochondrial microenvironment dynamics (e.g., viscosity changes) in living systems remains challenging. Here, we report an innovative two-photon fluorescent probe with a donor-π-acceptor architecture - featuring a hexyl-carbazole donor and a pyridinium acceptor - that exhibits bright near-infrared two-photon fluorescence. The probe's design enables robust mitochondrial targeting and high-performance two-photon excitation in the NIR region. By employing two-photon fluorescence lifetime imaging microscopy (TP-FLIM), we achieve quantitative, real-time and high-resolution mapping of mitochondrial functional status in live cells and tissues. Using this TP-FLIM approach, the probe sensitively tracks dynamic mitochondrial alterations under stress. In cultured cells undergoing apoptosis or ferroptosis, it reports distinct microenvironmental changes associated with mitochondrial stress and remodeling - for instance, revealing increased mitochondrial viscosity during apoptotic condensation and compaction of the organelle during ferroptotic cell death. In a nonalcoholic fatty liver disease (NAFLD) mouse model, longitudinal imaging with the probe visualizes progressive mitochondrial dysfunction and remodeling across different disease stages, reflecting the mounting stress on hepatic mitochondria as NAFLD advances. Overall, this D-π-A based two-photon FLIM probe provides a powerful biosensing tool for functional imaging of mitochondria, highlighting dynamic mitochondrial remodeling and microenvironment changes in cell death and disease contexts with high spatiotemporal resolution.
    DOI:  https://doi.org/10.1021/acs.analchem.5c01996
  21. Nat Metab. 2025 Jul 14.
    Old mitochondria regulate niche renewal via α-ketoglutarate metabolism in stem cells.
    Simon Andersson, Hien Bui, Arto Viitanen, Daniel Borshagovski, Ella Salminen, Sami Kilpinen, Angelika Gebhart, Emilia Kuuluvainen, Swetha Gopalakrishnan, Nina Peltokangas, Martyn James, Kaia Achim, Eija Jokitalo, Petri Auvinen, Ville Hietakangas, Pekka Katajisto.
      Cellular metabolism is a key regulator of cell fate1, raising the possibility that the recently discovered metabolic heterogeneity between newly synthesized and chronologically old organelles may affect stem cell fate in tissues2,3. In the small intestine, intestinal stem cells (ISCs)4 produce metabolically distinct progeny5, including their Paneth cell (PC) niche6. Here we show that asymmetric cell division of mouse ISCs generates a subset enriched for old mitochondria (ISCmito-O), which are metabolically distinct, and form organoids independently of niche because of their ability to recreate the PC niche. ISCmito-O mitochondria produce more α-ketoglutarate, driving ten-eleven translocation-mediated epigenetic changes that promote PC formation. In vivo α-ketoglutarate supplementation enhanced PC turnover and niche renewal, aiding recovery from chemotherapy-induced damage in aged mice. Our results reveal a subpopulation of ISCs whose old mitochondria metabolically regulate cell fate, and provide proof of principle for metabolically promoted replacement of specific aged cell types in vivo.
    DOI:  https://doi.org/10.1038/s42255-025-01325-7
  22. bioRxiv. 2025 May 03. pii: 2025.05.02.651978. [Epub ahead of print]
    Specialized high-capacity mitochondria fuel cell invasion.
    Isabel W Kenny-Ganzert, Lena P Basta, Lianzijun Wang, Qiuyi Chi, Caitlin Su, Katherine S Morton, Joel N Meyer, David R Sherwood.
      Cell invasion through basement membrane (BM) is energetically intensive, and how an invading cell produces high ATP levels to power invasion is understudied. By generating 20 endogenously tagged mitochondrial proteins, we identified a specialized mitochondrial subpopulation within the C. elegans anchor cell (AC) that localizes to the BM breaching site and generates elevated ATP to fuel invasion. These ETC-enriched high-capacity mitochondria are compositionally unique, harboring increased protein import machinery and dense cristae enriched with ETC components. High-capacity mitochondria emerge at the time of AC specification and depend on the AC pro-invasive transcriptional program. Finally, we show that netrin signaling through a Src kinase directs microtubule polarization, which facilitates metaxin adaptor complex dependent ETC-enriched mitochondrial trafficking to the AC invasive front. Our studies reveal that an invasive cell produces high ATP by generating and localizing high-capacity mitochondria. This might be common strategy used by other cells to meet energy demanding processes.
    DOI:  https://doi.org/10.1101/2025.05.02.651978
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