bims-celmim 2025-06-15 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2025–06–15
thirty papers selected by
Marc Segarra Mondejar

Metabolic Pathway Tracing for NAD+ Synthesis and Consumption.
Mitochondrial fusion controls the development of specialized mitochondrial structure and metabolism in rod photoreceptor cells.
PFKFB2 is Pivotal for Metabolic Flexibility and Differential Glucose Utilization.
Impact of PARL-mediated mitochondrial protease activity on calcium regulation.
GCLM lactylation mediated by ACAT2 promotes ferroptosis resistance in KRASG12D-mutant cancer.
Metabolic profile of human non-small cell lung cancer cells through combined 13C and 2H NMR.
Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca 2+ levels without impacting glucagon secretion.
A mitochondria-targeted long-wavelength fluorescent probe for imaging sulfur dioxide in ferroptosis.
Leveraging autophagy and pyrimidine metabolism to target pancreatic cancer.
Lifting the veil on tumor metabolism: A GDH1-focused perspective.
An adaptive organelle triad houses lipid droplets for dynamic regulation.
Mitochondrial PTRH2 controls the deubiquitinase TRABID to regulate mt-ND5 stability and metabolism.
Promiscuous enzyme SQOR in cellular metabolism and ferroptosis regulation.
Aspartate in the Brain: A Review.
Cell death and cancer: Metabolic interconnections.
Metabolite Analog-Induced Proteome Changes.
Mitochondrial molecule has unexpected role in tissue healing.
Redirecting Intermediary Metabolism to Counteract Cyanide Poisoning.
An alternative mechanism by which If1 prevents ATP hydrolysis by the ATP synthase subcomplex in S. cerevisiae.
Impact of mtG3PDH inhibitors on proliferation and metabolism of androgen receptor-negative prostate cancer cells: Role of extracellular pyruvate.
Feedback regulation between histone lactylation and ALKBH3-mediated glycolysis regulates age-related macular degeneration pathology.
The intersection between metabolism and translation through a subcellular lens.
Complete Enzyme Clustering Enhances Coenzyme Q Biosynthesis via Substrate Channeling.
In-vivo dendrite injury drives local mitochondrial contraction and dendrite branching.
Cold exposure stimulates cross-tissue metabolic rewiring to fuel glucose-dependent thermogenesis in brown adipose tissue.
Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments.
Unraveling mitochondrial influence on mammalian pluripotency via enforced mitophagy.
Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD + metabolites in CD38 knockout mice exhibiting improved metabolism.
Cell-Type-Specific Autophagy in Human Leukocytes.
A Nucleic Acid Prodrug That Activates Mitochondrial Respiration, Promotes Stress Resilience, and Prolongs Lifespan.

Methods Mol Biol. 2025 ;2925 203-222

Metabolic Pathway Tracing for NAD+ Synthesis and Consumption.

Tumpa Dutta, Stephen J Gardell.

  NAD+ is an abundant cellular metabolite which plays vital roles in central metabolism while serving as a cofactor for oxidoreductases and cosubstrate for sirtuins and poly(ADP-ribose)polymerases (PARPs). Decreased tissue NAD+ levels have been linked to aging-associated metabolic decline and a host of chronic diseases. Cellular steady-state NAD+ levels are governed by contemporaneous synthetic and consumptive processes. Hence, lower NAD+ levels in aged tissues can arise from decreased synthesis or increased consumption. A static snapshot of the tissue levels of NAD+ is inadequate for assessing the highly dynamic pathway network which mediates NAD+ synthesis and consumption. Metabolic pathway tracing with stable isotope-labeled NAD+ precursors (e.g., nicotinamide (NAM), nicotinic acid (NA), tryptophan) and high-resolution mass spectrometry (HRMS) can unveil the individual contributions of synthesis and consumption to the steady-state NAD+ concentration. The metabolic fate of the NAD+ precursor can also be traced to metabolic products of NAD+ including NADH, NADP, and NADPH as well as intermediates in the various NAD+ biosynthetic pathways. Metabolic tracing of NAD+ synthesis and degradation as well as conversion of NAD+ to its downstream products is a highly versatile technique. It can be used to interrogate isolated cells, tissues slices, or specimens collected from preclinical or clinical in vivo studies (e.g., blood, urine, tissues). Bold claims about the pivotal role of NAD+ in human health and disease are typically fraught with uncertainty due to an incomplete understanding of NAD+ metabolism. Insight gleaned from metabolic pathway tracing can shed important new light on NAD+ metabolism and help to critically evaluate the intriguing link between cellular NAD+ levels and healthy aging.

Keywords:  Mass isotopomer distribution profiling; Mass spectrometry; NAD+ consumption; NAD+ flux; NAD+ metabolism; NAD+ synthesis; Stable isotope tracing

DOI:  https://doi.org/10.1007/978-1-0716-4534-5_14
bioRxiv. 2025 May 26. pii: 2025.05.21.655403. [Epub ahead of print]

Mitochondrial fusion controls the development of specialized mitochondrial structure and metabolism in rod photoreceptor cells.

Michael Landowski, Ryo Hagimori, Purnima Gogoi, Vijesh J Bhute, Sakae Ikeda, Tetsuya Takimoto, Akihiro Ikeda.

Mitochondria are dynamic organelles that undergo continuous morphological changes, yet exhibit unique, cell-type-specific structures. In rod photoreceptor cells of the retina, these structures include elongated mitochondria in the inner segments and a distinct, large, circular mitochondrion in each presynaptic terminal. The mechanisms underlying the establishment and maintenance of these specialized mitochondrial morphologies, along with their functional significance, are not well understood. Here, we investigate the roles of mitochondrial fusion proteins mitofusin 1 (MFN1) and mitofusin 2 (MFN2) in shaping these structures and maintaining photoreceptor cell health. Rod photoreceptor cell-specific ablation of MFN1 and MFN2 resulted in mitochondrial fragmentation by one month of age, suggesting that mitochondrial fusion is essential for the development of photoreceptor cell-specific mitochondrial structures. Notably, the layer structures of the retina examined by light microscopy appeared unaffected at this age. Following this time period, significant photoreceptor cell degeneration occurred by three months of age. Furthermore, we showed that impaired mitochondrial fusion perturbed the balance of proteins involved in glycolysis, oxidative phosphorylation (OXPHOS), and β-oxidation, highlighting the critical role of mitochondrial fusion in ensuring the proper levels of proteins necessary for optimal energy metabolism. Additionally, we identified upregulation of cellular stress pathways such as endoplasmic reticulum (ER) stress and unfolded protein response (UPR), which arise in response to energy deprivation, and cytoprotective biosynthetic pathways mediated by CCAAT/enhancer-binding protein gamma (C/EBPγ) and mammalian target of rapamycin complex 1 (mTORC1) signaling. In summary, our findings indicate that mitochondrial fusion through MFN1 and MFN2 is vital for the development of unique mitochondrial structures and proper energy production, underscoring the fundamental importance of mitochondrial dynamics in photoreceptor cell function and survival.
Significance Statements: Rod photoreceptor cells exhibit unique mitochondrial morphologies and high energy requirements. In this report, we examined how these unique mitochondrial structures are established and their biological significance. We identified that mitochondrial fusion is essential for the development of characteristic mitochondrial morphologies in rod photoreceptor cells. Furthermore, we demonstrated that impaired mitochondrial fusion disrupts the equilibrium of proteins associated with OXPHOS, glycolysis, and β-oxidation, ultimately leading to an imbalance in cellular energy homeostasis. Our findings also revealed activation of cellular stress pathways, including ER stress and the UPR, which are likely triggered by energy depletion. Additionally, we identified activation of cytoprotective biosynthetic pathways that are engaged to preserve cellular homeostasis and function.

DOI: https://doi.org/10.1101/2025.05.21.655403
bioRxiv. 2025 May 27. pii: 2025.05.26.656235. [Epub ahead of print]

PFKFB2 is Pivotal for Metabolic Flexibility and Differential Glucose Utilization.

Kylene M Harold, Satoshi Matsuzaki, Atul Pranay, Jie Zhu, Anna Faakye, Kenneth M Humphries.

Background: The heart's constant energy demands make metabolic flexibility critical to its function as nutrient availability varies. The enzyme phosphofructokinase-2/fructose 2,6-bisphosphatase (PFKFB2) contributes to this flexibility by acting as a positive or negative regulator of cardiac glycolysis. We have previously shown that PFKFB2 is degraded in the diabetic heart and that a cardiac-specific PFKFB2 knockout (cKO) impacts ancillary glucose pathways and mitochondrial substrate preference. Therefore, defining PFKFB2's role in mitochondrial metabolic flexibility is paramount to understanding both metabolic homeostasis and metabolic syndromes. Further, it is unknown how PFKFB2 loss impacts the heart's response to acute stress. Here we examined how cardiac mitochondrial flexibility and the post-translational modification O-GlcNAcylation are affected in cKO mice in response to fasting or pharmacologic stimulation.
Methods: cKO and litter-matched controls (CON) were sacrificed in the fed or fasted (12 hours) states, with or without a 20 minute stimulant stress of caffeine and epinephrine.Mitochondrial respiration, metabolomics, and changes to systemic glucose homeostasis were evaluated.
Results: cKO mice had moderate impairment in mitochondrial metabolic flexibility, affecting downstream glucose oxidation, respiration, and CPT1 activity. O-GlcNAcylation, a product of ancillary glucose metabolism, was upregulated in cKO hearts in the fed state, but this was ameliorated in the fasted state. Furthermore, metabolic remodeling in response to PFKFB2 loss was sufficient to impact circulating glucose in fasted and stressed states.
Conclusions: PFKFB2 is essential for fed-to-fasted changes in cardiac metabolism and plays an important regulatory role in protein O-GlcNAcylation. Its loss also affects systemic glucose homeostasis under stressed conditions.
Graphic Abstract:
Research Perspective: This study raises and answers three key questions: how PFKFB2 contributes to cardiac mitochondrial metabolic flexibility, how post-prandial status regulates O-GlcNAcylation in a PFKFB2-dependent manner, and how altered cardiac glucose use impacts systemic glucose homeostasis under stress.These findings highlight a novel role for nutrient state in regulating cardiac metabolism, and especially O-GlcNAcylation, with PFKFB2 loss.Future studies should investigate whether reducing O-GlcNAcylation through fasting is sufficient to ameliorate pathological changes observed in the absence of PFKFB2.

DOI: https://doi.org/10.1101/2025.05.26.656235
Biochim Biophys Acta Mol Cell Res. 2025 Jun 06. pii: S0167-4889(25)00103-X. [Epub ahead of print] 119998

Impact of PARL-mediated mitochondrial protease activity on calcium regulation.

Donato D'Angelo, Aya Al Saidi, Giorgia Ghirardo, Denis Vecellio Reane, Nicola Gasparini, Rosario Rizzuto, Anna Raffaello.

  The presenilin-associated rhomboid-like protein (PARL) is a mitochondrial inner membrane serine protease that is a key regulator of several cellular processes, including apoptosis, metabolism, inflammation and stress responses. While recent studies suggest that PARL may play a role in mitochondrial calcium homeostasis, the underlying mechanisms remain poorly understood. In this study, we investigated the effects of PARL modulation on mitochondrial and cytosolic calcium dynamics, as well as mitochondrial membrane potential. Our results show that altering PARL protein levels, through both overexpression and silencing, significantly affects mitochondrial calcium uptake, without influencing cytosolic calcium transients or mitochondrial membrane potential. Despite the observed changes in mitochondrial calcium dynamics, PARL does not interact with the mitochondrial calcium uniporter complex (mtCU) regulators MICU1 and MICU2, which are critical for regulating mitochondrial calcium influx. However, we observed alterations in the protein levels of MICU1 and MICU2, either in their monomeric or dimeric forms, suggesting that PARL may influence these mtCU components indirectly. Interestingly, the pore-forming subunit MCU, and the structural subunit EMRE, essential for the assembly of the mtCU, were unaffected by PARL modulation. These findings suggest that the role of PARL in modulating mitochondrial calcium homeostasis may involve indirect mechanisms, potentially involving other regulatory pathways. Overall, our study provides novel insights into the functional role of PARL in mitochondrial calcium regulation, offering potential avenues for further investigation into its broader cellular functions.

Keywords:  Calcium signaling; Mitochondria; Mitochondrial calcium uniporter; Mitochondrial intermembrane proteolysis; PARL; Rhomboid protease

DOI:  https://doi.org/10.1016/j.bbamcr.2025.119998
Cell Rep. 2025 Jun 09. pii: S2211-1247(25)00545-5. [Epub ahead of print]44(6): 115774

GCLM lactylation mediated by ACAT2 promotes ferroptosis resistance in KRASG12D-mutant cancer.

Yubin Chen, Qian Yan, Shiye Ruan, Jinwei Cui, Zhenchong Li, Zhongyan Zhang, Jiayu Yang, Jike Fang, SiYang Liu, Shanzhou Huang, Baohua Hou, Chuanzhao Zhang.

  KRAS mutations drive tumorigenesis, but their role in ferroptosis regulation remains unclear. Here, we construct wild-type KRAS (KRASWT) and KRASG12D-mutant cancer cells and demonstrate that G12D-mutant cells exhibit increased viability and reduced ferroptosis upon RSL3 or erastin treatment. These cells show diminished lipid peroxidation and mitochondrial damage, indicating ferroptosis resistance. KRASG12D activates MEK/ERK signaling to phosphorylate LDHA, enhancing glycolysis and lactate production. Exogenous lactate supplementation similarly protects WT cells from ferroptosis. Mechanistically, G12D-mutation-derived lactate induces glutamate-cysteine ligase (GCL) modifier (GCLM) lactylation, a process catalyzed by acetyl-coenzyme A (CoA) acetyltransferase 2 (ACAT2). Inhibition of GCLM lactylation either through the mutation of the lactylation site or by knockdown of ACAT2 diminished the enzymatic activity of GCL and suppressed glutathione synthesis. Importantly, ACAT2 depletion overcomes ferroptosis resistance in KRASG12D-mutant tumors in vivo. Our findings reveal a KRASG12D-driven metabolic adaptation linking GCLM lactylation to ferroptosis resistance, proposing ACAT2 inhibition as a therapeutic strategy for KRAS-mutant cancers.

Keywords:  CP: Cancer; CP: Metabolism; GCLM; KRAS mutation; ferroptosis; glutamate-cysteine ligase modifier; pancreatic cancer; protein lactylation

DOI:  https://doi.org/10.1016/j.celrep.2025.115774
Biochim Biophys Acta Mol Basis Dis. 2025 Jun 05. pii: S0925-4439(25)00297-2. [Epub ahead of print] 167949

Metabolic profile of human non-small cell lung cancer cells through combined 13C and 2H NMR.

Ludgero C Tavares, Ricardo Amorim, José Teixeira, Paulo J Oliveira, Rui A Carvalho.

  The metabolic remodeling occurring in carcinogenesis cells is firmly established. However, to understand the connection between the cellular metabolic profile and carcinogene sis, an accurate measurement of metabolic fluxes is required. In order to quantify the fluxes in these metabolic pathways, stable isotopes tracers and nuclear magnetic resonance (NMR) techniques were employed. For that purpose, two human non-small lung cancer cell lines (A549 and H1299) were used. For the quantification of carbon intermediary metabolism cells were grown in 13C labelled glucose while for de novo lipogenesis (DNL) assessment 2H2O was supplemented to the culture media. To better understand and characterize cellular bioenergetics, mitochondrial membrane potential, oxygen consumption, and energy charge were also assessed. Finally, to establish a bridge between metabolic fluxes and cancer proliferation, substrate dependency studies were performed. Several metabolic inhibitors were also tested, targeting glycolysis, TCA cycle, pentose phosphate pathway (PPP) and transaminases. Our results showed the occurrence of metabolic heterogeneity between the two non-small lung cancer cell lines: H1299 exhibited a relatively active TCA cycle, while A549 showed a more glycolytic phenotype. The overall mitochondrial bioenergetic parameters were in agreement with the metabolic profiles. The mitochondrial network was polarized and active in all cell lines, although the H1299 cell line exhibited higher basal oxygen consumption and spare respiratory capacity. Nonetheless, DNL rate did not differ in H1299 and A549 lung cancer cell lines. Additionally, α-ketoglutarate availability was proven a key determinant for H1299 non-small cell lung cancer cells survival and proliferation. In conclusion, this work revealed that cells derived from a lymph node metastasis (H1299) have a more active TCA cycle and altered oxidative stress levels when compared to cells derived from a primary tumor (A549). In the process, we successfully implemented a new 2H enrichment method for DNL assessment for the first time in in vitro cancer research.

Keywords:  A549; Glycolysis; H1299; Krebs cycle; Lipogenesis; NADPH; Stable isotope tracers

DOI:  https://doi.org/10.1016/j.bbadis.2025.167949
bioRxiv. 2025 May 28. pii: 2025.05.26.656009. [Epub ahead of print]

Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca 2+ levels without impacting glucagon secretion.

Erli Jin, Hannah R Foster, Evgeniy Potapenko, Shih Ming Huang, Xinhang Dong, Jing W Hughes, Matthew J Merrins.

Objective: Pancreatic islet α-cells are increasingly recognized as amino acid sensors for the organism, however the metabolic pathways that α-cells use to sense amino acids have not been identified. Building on our prior work in β-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in α-cell amino acid sensing.
Methods: To investigate amino acid regulation of α-cells at the cellular level, we measured intracellular Ca 2+ (GCaMP6s imaging), membrane potential (JEDI-2P imaging and patch-clamp), K ATP channel activity, and glucagon secretion. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with α-cell specific deletion of pyruvate kinase M1/M2 (PKM1/2-αKO) or mitochondrial PEP carboxykinase (PCK2-αKO).
Results: The mitochondrial fuels glutamine/leucine antagonized alanine/arginine-stimulated Ca 2+ influx and glucagon secretion under hypoglycemic conditions. Both pyruvate kinase and PCK2 were required for glutamine/leucine to close K ATP channels and limit amino acid-stimulated membrane depolarization. The Ca 2+ response to amino acids was blocked by pyruvate kinase activation with TEPP-46, and enhanced by α-cell deletion of pyruvate kinase or PCK2 - all without changing glucagon secretion. Finally, using diazoxide/KCl to probe the pathways downstream of membrane depolarization, we identified an essential role of the PEP cycle in homeostatically restoring intracellular Ca 2+ levels.
Conclusions: The α-cell mitochondrial PEP cycle senses glutamine/leucine and inhibits K ATP channels similarly to β-cells, while restricting amino acid stimulated membrane depolarization and Ca 2+ influx. However, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or intracellular Ca 2+ .
Highlights: Our studies identify a role for the α-cell PEP cycle in sensing amino acids under hypoglycemic conditions. Pyruvate kinase and PCK2 are required for glutamine/leucine to close α-cell K ATP channels and limit membrane depolarization and Ca 2+ influx. Glutamine/leucine oppose alanine/arginine-stimulated Ca 2+ influx and glucagon secretion. All of the amino acids tested regulate glucagon secretion, but none do so by modulating membrane depolarization or intracellular Ca 2+ levels.

DOI: https://doi.org/10.1101/2025.05.26.656009
Anal Methods. 2025 Jun 09.

A mitochondria-targeted long-wavelength fluorescent probe for imaging sulfur dioxide in ferroptosis.

Yulong Wu, Tianyu Zhang, Guangyi Jiang, Qingxia Duan, Chen Zuo, Wei Shu.

Ferroptosis, an iron-dependent, lipid peroxidation-mediated type of programmed cell death, is crucial in the pathogenesis of numerous diseases. Mitochondria, central to cellular energy production, are significantly involved in ferroptosis. Sulfur dioxide (SO2) is generated within both the mitochondria and cytoplasm, and its abnormal levels are linked to mitochondrial dysfunction and various diseases. To detect mitochondrial HSO3- in ferroptosis, we developed CMP, a long-wavelength fluorescent probe with high specificity and a rapid response. CMP utilizes a benzopyrylium cation for HSO3- recognition and mitochondrial targeting. It reacts with HSO3-via Michael addition, quenching fluorescence at 622 nm, and achieves ultrasensitive detection. CMP enables real-time HSO3- monitoring in HeLa cells and zebrafish, and successfully detected increased mitochondrial HSO3- levels during Erastin-induced ferroptosis and CCCP-induced apoptosis. CMP offers a valuable tool for studying ferroptosis-related diseases.

DOI: https://doi.org/10.1039/d5ay00578g
bioRxiv. 2025 Jun 05. pii: 2025.05.29.656904. [Epub ahead of print]

Leveraging autophagy and pyrimidine metabolism to target pancreatic cancer.

Suzanne Dufresne, Ramya S Kuna, Kristiana Wong, Anvita Komarla, Angelica Rock, Joel Rosada-Encarnación, Celina Shen, Payel Mondal, Louis R Parham, Fabiana Izidro Layng, Kristina L Peck, Alexandra Fowler, Andrew M Lowy, Dannielle Engle, Herve Tiriac, Reuben Shaw, Nicholas Cosford, Christian Metallo, Christina Towers.

Autophagy inhibitors are promising compounds to treat pancreatic ductal adenocarcinoma (PDA) but their efficacy in patients is unclear, highlighting a need to understand mechanisms of resistance. We used a novel approach to uncover metabolic adaptations that bypass autophagy inhibition. Utilizing PDA cells with acquired resistance to different autophagy inhibitors, we found that severe autophagy depletion induces metabolic rewiring to sustain TCA intermediates and nucleotides for biosynthesis. Long-term autophagy inhibition results in altered pyruvate metabolism likely regulated by lower pyrimidine pools. Cells adapting to loss of autophagy preferentially salvage pyrimidines to replenish these pools instead of synthesizing them de novo. Exploiting this metabolic vulnerability, we found that acquired resistance to autophagy inhibition promotes increased salvage and therefore sensitivity to pyrimidine analogues, including gemcitabine and trifluridine/tipiracil leading to combinatory effects with autophagy inhibitors and pyrimidine analogs. These studies provide mechanistic insight defining how autophagy inhibition can be leveraged to treat pancreatic cancer.

DOI: https://doi.org/10.1101/2025.05.29.656904
iScience. 2025 Jun 20. 28(6): 112551

Lifting the veil on tumor metabolism: A GDH1-focused perspective.

Sisi Zhou, Huaer Wu, Yun Chen, Jiawei Lv, Shufang Chen, Hua Yu, Tiezhu Shi, Xiongjun Wang, Lingyun Xiao.

  Tumors depend on glutamine for energy production, biosynthesis, and redox homeostasis. Glutamate dehydrogenase 1 (GDH1) primarily catalyzes the oxidative deamination of glutamate to α-ketoglutarate (α-KG) and ammonia, utilizing NAD+ or NADP+ as cofactors. α-KG is a tricarboxylic acid (TCA) cycle intermediate at the nexus of multiple metabolic pathways, fueling the TCA cycle for energy production or providing intermediates essential for lipid, amino acid, and nucleotide synthesis. Its derivatives, succinate and fumarate, function as oncometabolites that promote tumor progression through diverse mechanisms. Additionally, α-KG is an essential cofactor for α-KG-dependent dioxygenases (2-OGDDs), regulating epigenetic modifications that drive tumorigenesis. GDH1 may also catalyze the reductive amination of α-KG to glutamate under glutamine deprivation or hypoxic conditions. The roles of GDH1 in tumors are context-dependent, influencing progression through metabolic and epigenetic mechanisms. This review discusses GDH1's multifaceted functions and advances in targeting it for cancer therapy.

Keywords:  Cancer

DOI:  https://doi.org/10.1016/j.isci.2025.112551
Cell Rep. 2025 Jun 11. pii: S2211-1247(25)00584-4. [Epub ahead of print]44(6): 115813

An adaptive organelle triad houses lipid droplets for dynamic regulation.

Hong Qiu, Can Miao, Cunqi Ye.

  Cell organelles compartmentalize metabolic reactions and require inter-organelle communications to coordinate metabolic activities in fluctuating nutrient environments. While membrane contacts enable this communication by facilitating metabolite exchange, the functional organization of organelles through these contacts remains underexplored. Here, we show that excess lactate induces severe metabolic stress under nutrient deprivation in the budding yeast Saccharomyces cerevisiae, necessitating a rapid life cycle of lipid droplets (LDs) for cellular adaptation. This process uncovers a previously uncharacterized subcellular architecture-an organelle triad-comprising the vacuole, LDs, and the nuclear endoplasmic reticulum (ER). The vacuole undergoes expansion and deformation, enveloping the entire nucleus that is encircled by an orbit of LDs. Formation of this organelle triad depends on the timely and abundant expression of membrane-tethering proteins that mediate vacuole-LD contact sites and nuclear ER-vacuole junctions. This dynamic and reversible subcellular organization ensures efficient LD metabolism to support cell survival under nutrient stress.

Keywords:  CP: Cell biology; LDO proteins; lipid droplet; membrane contact; nutrient stress; nvj1; subcellular architecture; the nucleus–vacuole junction; vac8; vacuole deformation

DOI:  https://doi.org/10.1016/j.celrep.2025.115813
PNAS Nexus. 2025 Jun;4(6): pgaf178

Mitochondrial PTRH2 controls the deubiquitinase TRABID to regulate mt-ND5 stability and metabolism.

Carlotta Giorgi, Femke J Aan, Natalija Glibetic, Daniela Ramaccini, Lorenzo Modesti, Veronica A M Vitto, Vanessa Montoya-Uribe, Austin Corpuz, Sonia Missiroli, Inês Simões, Yaiza Potes, Mariusz R Wieckowski, Joe W Ramos, Paolo Pinton, Michelle L Matter.

  The integrin effector, PTRH2, associates with mitochondria in adherent cells where its function has not been elucidated (Jan Y, et al. 2004. A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell. 116:751-762; Griffiths GS, et al. 2011. Bit-1 mediates integrin-dependent cell survival through activation of the NF{kappa}B pathway. J Biol Chem. 286:14713-14723). PTRH2 loss-of-function mutations cause multisystem disease in children through an unknown mechanism. We sought to determine the role of mitochondrial PTRH2. We used immunoprecipitation/mass spectrometric proteomics to identify PTRH2 interacting partners: TRABID (a deubiquitinase [DUB]) and respiratory complex I NADH: ubiquinone oxidoreductase core subunit 5 (mt-ND5). We show for the first time that mitochondrial PTRH2 regulates TRABID's ability to deubiquitylate mt-ND5. In cells lacking PTRH2 expression, mt-ND5 stability is significantly increased due to aberrant TRABID-mediated deubiquitylation of mt-ND5. This increase in mt-ND5 stability promotes complex I activity and ATP production, which under stress conditions leads to mitochondrial Ca2+ overload. Reexpression of mitochondrial PTRH2 blocks TRABID-mediated mt-ND5 deubiquitylation, resulting in mt-ND5 polyubiquitylation and proteasomal degradation. Inhibiting complex I or TRABID activity rescued PTRH2 loss-of-function mutant patient cells from mitochondrial Ca2+ overload under stress. Immunostaining analysis of ptrh2+/+ and ptrh2-/- mouse skeletal muscle revealed a negative relationship between PTRH2 and mt-ND5, confirming a regulatory role for PTRH2 in controlling mt-ND5 stability. Taken together, mitochondrial PTRH2 is a regulator of metabolic homeostasis that, when lost, promotes mitochondrial Ca2+ overload when cells are exposed to stress signals. Targeting mt-ND5 stability through PTRH2-mediated regulation of TRABID's DUB function provides a novel mechanistic approach to inhibit mitochondrial Ca2+ overload in diseases that occur due to dysregulated mitochondria.

Keywords:  PTRH2; TRABID; metabolism; mitochondrial Ca2+ overload; mt-ND5

DOI:  https://doi.org/10.1093/pnasnexus/pgaf178
BMB Rep. 2025 Jun 11. pii: 6453. [Epub ahead of print]

Promiscuous enzyme SQOR in cellular metabolism and ferroptosis regulation.

Jumi Lee, Inhwan Yoo, Ihyeon Ahn, Namgyu Lee.

Ferroptosis, an iron-dependent form of programmed cell death, is primarily driven by the accumulation of lipid peroxides through radical generation, notably via the Fenton reaction. Emerging evidence highlights the intricate link between ferroptosis and cellular metabolism, with metabolic enzymes playing pivotal roles in its regulation. Sulfide quinone oxidoreductase (SQOR), traditionally recognized for its role in hydrogen sulfide (H2S) detoxification and electron transport chain (ETC) activation, has recently been identified as a promiscuous enzyme with a novel function in ferroptosis regulation. This review explores SQOR's canonical function in H2S metabolism and its emerging role in ferroptosis resistance through the production of ubiquinol and hydropersulfides, radical-trapping antioxidants. Additionally, we provide insights into potential future research directions, emphasizing SQOR's therapeutic relevance in ferroptosis-associated diseases.
Neurochem Res. 2025 Jun 12. 50(3): 199

Aspartate in the Brain: A Review.

Caroline D Rae, Benjamin D Rowlands, Vladimir J Balcar.

  L-Aspartate (aspartic acid; C4H7NO4; 2-aminobutanedoic acid) is a non-essential α-amino acid found ubiquitously throughout the body, including in the brain. Aspartate is one of the protein-forming amino acids and the formation of tRNA-aspartate complex is catalysed by aspartyl tRNA synthetase. Free aspartate, which is the main subject of this review, plays key roles in metabolism, as an amino donor and acceptor. It contributes to the synthesis of protein, arginine and nitric oxide, asparagine, N-acetylaspartate and N-methyl-D-aspartate. Its major metabolic role in the brain is recycling reducing equivalents (protons) between the cytoplasm and mitochondrial matrix as part of the malate-aspartate shuttle. L-Aspartate's actions on synaptic receptors, as well as its possible presence in nerve terminals and synaptic vesicles, are, in principle, consistent with a role as an excitatory neurotransmitter. The evidence is far from conclusive and at times controversial. The role of D-aspartate in brain function is even less certain but, it appears that, rather than being a minor neurotransmitter, D-aspartate is more likely to be involved in fine regulation of endocrine and homeostatic processes. Much research remains to be done in this area. The diversity of its functions and chemistry make aspartate a complex molecule to investigate and measure in vivo. Perturbations of aspartate metabolism have been described in a range of neurological deficits, particularly those of white matter. Here, we examine what is known about the various roles of aspartate in brain, its metabolism, transport and compartmentation, its role as a neurotransmitter or a more general signalling molecule, and what is currently known about its role(s) in disease processes.

Keywords:   d-aspartate; Energy metabolism; Malate aspartate shuttle; Neurotransmitter

DOI:  https://doi.org/10.1007/s11064-025-04454-3
Cell Rep. 2025 Jun 07. pii: S2211-1247(25)00575-3. [Epub ahead of print]44(6): 115804

Cell death and cancer: Metabolic interconnections.

Destiny Dalseno, Nikolai Gajic, Lyndsey Flanagan, Stephen W G Tait.

  Recent findings in the cell death field have transformed our understanding of the interplay between metabolism and cell death in the context of cancer. In this review, we discuss the relationships between metabolism and the cell death pathways of apoptosis, necroptosis, pyroptosis, and ferroptosis, with a particular focus on recent advancements. We will also explore the regulation of metabolism by the BCL-2 family and the participation of oncometabolites in the regulation of cell death. Finally, we examine the emerging links between cell death signaling and cellular persistence. As we highlight in this review, the intersection of metabolic and cell death pathways has implications for cancer cell survival, treatment resistance, and the tumor microenvironment.

Keywords:  BCL-2; CP: Cancer; CP: Metabolism; apoptosis; cancer; cell death; ferroptosis; metabolism; necroptosis

DOI:  https://doi.org/10.1016/j.celrep.2025.115804
Methods Mol Biol. 2025 ;2925 133-144

Metabolite Analog-Induced Proteome Changes.

Molly Kennedy, Nikhil Gandikota, Victoria Pereira, Sanjoy K Bhattacharya.

  Metabolites are small molecules crucial for metabolic processes, while their analogs are structurally similar compounds used to study and manipulate these pathways in medicine and science. Metabolite analogs serve as powerful tools in drug development, research, and therapeutics by mimicking or inhibiting natural metabolites to alter biochemical pathways, impacting both catabolism and anabolism. These analogs are pivotal in research and treatment, influencing pathways like catabolism and anabolism. Common metabolites like glucose, amino acids, and nucleotides play vital roles in pathways such as PTEN and mTOR, which are essential for regulating cell growth, proliferation, and survival. By influencing these pathways, metabolite analogs can help elucidate metabolic mechanisms and offer potential treatments for diseases like cancer and metabolic disorders. This protocol chapter will detail the preparation and handling of metabolites, specifically focusing on a PTEN inhibitor. We will outline the steps for dissolving and storing these compounds, followed by a comprehensive protein extraction protocol. Finally, the use of Proteome Discoverer software will be discussed to analyze the resultant proteome changes.

Keywords:  Metabolite; PTEN; Protein; Protein extraction; Proteome discover; mTOR pathway

DOI:  https://doi.org/10.1007/978-1-0716-4534-5_9
Nature. 2025 Jun 11.

Mitochondrial molecule has unexpected role in tissue healing.

Ram P Chakrabarty, Navdeep S Chandel.



Keywords:  Biochemistry; Cell biology; Metabolism; Stem cells

DOI:  https://doi.org/10.1038/d41586-025-01583-1
FASEB J. 2025 Jun 30. 39(12): e70709

Redirecting Intermediary Metabolism to Counteract Cyanide Poisoning.

Vik S Bebarta, Anjali K Nath.

  Cyanide is one of the oldest known poisons in human history. In the 1980s, seminal work began to elucidate the broad cellular mechanisms of cyanide toxicity beyond its canonical inhibition of cytochrome c oxidase. In the 1990s, endogenous metabolites were shown to sequester cyanide, and these became promising avenues for the development of a cyanide antidote. However, an FDA-approved metabolite-based cyanide antidote did not come to fruition. More recently, in the past 10 years, advances in mass spectrometry-based metabolomics profiling, subcellular drug targeting, and genome editing have brought fresh perspectives to the concept of a metabolism-based cyanide antidote. Here, we review the mechanisms of cyanide toxicity with a focus on intermediary metabolism. We discuss the current state of our knowledge and gaps in our understanding of the metabolic mechanisms that contribute to cyanide poisoning, in addition to highlighting recent findings that break new ground in the field. We present the theory of redirecting intermediary metabolism to counteract cyanide poisoning: while cyanide shifts metabolism from oxidative phosphorylation to glycolysis, the metabolome encompasses hundreds of pathways; thus, potential therapeutic opportunities may reside in activating metabolism into other pathways. Potential approaches to targeting metabolism as a therapeutic intervention for cyanide poisoning will also be discussed. These targets represent an opportunity for a significant paradigm shift from current FDA-approved treatments, which chelate the chemical toxicant but do not reverse the broad spectrum of cellular and metabolic damage caused by cyanide, to a treatment that may improve the long-term effects of cyanide poisoning.

Keywords:  drug development; emergency medicine; glyoxylate; histotoxic hypoxia; mass casualty incidents; mass spectrometry; metabolism; mitochondrial poisons; nicotinamide adenine dinucleotide; succinate

DOI:  https://doi.org/10.1096/fj.202400230RR
EMBO Rep. 2025 Jun 09.

An alternative mechanism by which If1 prevents ATP hydrolysis by the ATP synthase subcomplex in S. cerevisiae.

Orane Lerouley, Isabelle Larrieu, Tom Louis Ducrocq, Benoît Pinson, Marie-France Giraud, Arnaud Mourier.

  The mitochondrial F1F0-ATP synthase is crucial for maintaining the ATP/ADP balance which is critical for cell metabolism, ion homeostasis and cell proliferation. This enzyme, conserved across evolution, is found in the mitochondria or chloroplasts of eukaryotic cells and the plasma membrane of bacteria. In vitro studies have shown that the mitochondrial F1F0-ATP synthase is reversible, capable of hydrolyzing instead of synthesizing ATP. In vivo, its reversibility is inhibited by the endogenous peptide If1 (Inhibitory Factor 1), which specifically prevents ATP hydrolysis in a pH-dependent manner. Despite its presumed importance, the loss of If1 in various model organisms does not cause severe phenotypes, suggesting its role may be confined to specific stress or metabolic conditions yet to be discovered. Our analyses indicate that inhibitory peptides are crucial in mitigating mitochondrial depolarizing stress under glyco-oxidative metabolic conditions. Additionally, we found that the absence of If1 destabilizes the nuclear-encoded free F1 subcomplex. This mechanism highlights the role of If1 in preventing harmful ATP wastage, offering new insights into its function under physiological and pathological conditions.

Keywords:  ATP Synthase; Bioenergetics; F1 Subcomplex; IF1; Mitochondria

DOI:  https://doi.org/10.1038/s44319-025-00430-8
PLoS One. 2025 ;20(6): e0325509

Impact of mtG3PDH inhibitors on proliferation and metabolism of androgen receptor-negative prostate cancer cells: Role of extracellular pyruvate.

Floriana Jessica Di Paola, Luiza Hd Cardoso, Efterpi Nikitopoulou, Bianca Kulik, Sandra Rühl, Alexander Eva, Natascha Sommer, Thomas Linn, Erich Gnaiger, Klaus Failing, Kathrin Büttner, Christian Frezza, Sybille Mazurek.

Mitochondrial glycerol 3-P dehydrogenase (mtG3PDH) plays a significant role in cellular bioenergetics by serving as a rate-limiting element in the glycerophosphate shuttle, which connects cytosolic glycolysis to mitochondrial oxidative metabolism. mtG3PDH was identified as an important site of electron leakage leading to ROS production to the mitochondrial matrix and intermembrane space. Our research focused on the role of two published mtG3PDH inhibitors (RH02211 and iGP-1) on the proliferation and metabolism of PC-3 and DU145 prostate cancer cells characterized by different mtG3PDH activities. Since pyruvate as a substrate of lactate dehydrogenase (LDH) may represent an escape mechanism for the recycling of cytosolic NAD+ via the glycerophosphate shuttle, we investigated the effect of pyruvate on the mode of action of the mtG3PDH inhibitors. Extracellular pyruvate weakened the growth-inhibitory effects of RH02211 and iGP-1 in PC-3 cells but not in DU145 cells, which correlated with higher H-type LDH and lower mitochondrial glutamate-oxaloacetate transaminase in DU145 cells. In the pyruvate-low medium, the strength of inhibition was more pronounced in PC-3 cells, characterized by higher mtG3PDH activities compared to DU145 cells. Pyruvate conversion rates (production in pyruvate-low and consumption in pyruvate-high PC-3 cells) were not impaired by RH02211 and iGP-1, suggesting that the conversion of extracellular pyruvate to lactate was not the primary factor responsible for the weakening effect of extracellular pyruvate on the RH02211-induced inhibition of PC-3 proliferation. In pyruvate-high PC-3 cells, the intracellular glycerol-3-P and dihydroxyacetone-P concentrations were consistent with an inhibition of mtG3PDH. In contrast, in pyruvate-low cells, the concentrations of these metabolites suggested an activation of mtG3PDH in parallel with an impairment of cytosolic G3PDH by RH02211. Of all metabolic characterizations recorded in this study (fluxes, intracellular intermediates, O2 consumption and H2O2 production), the decrease in glutaminolysis correlated best with the RH02211-induced inhibition of proliferation in pyruvate-low and pyruvate-high PC-3 cells.

DOI: https://doi.org/10.1371/journal.pone.0325509
Proc Natl Acad Sci U S A. 2025 Jun 17. 122(24): e2416046122

Feedback regulation between histone lactylation and ALKBH3-mediated glycolysis regulates age-related macular degeneration pathology.

Ying Wang, Yi-Chen Zhang, Zi-Qin Ding, Shi-Yao Xu, Ye-Ran Zhang, Hong-Jing Zhu, Chang Huang, Jia-Nan Wang, Meidong Zhu, Jiang-Dong Ji, Biao Yan, Qing-Huai Liu, Xue Chen.

  Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. It is characterized by degeneration of the retinal pigment epithelium (RPE), which can develop into choroidal neovascularization (CNV) to cause severe and rapid vision loss. Preventing this progression might help save vision, but the exact mechanisms remain unclear. In this study, using clinical AMD samples and the gene knockout mice, we reported that the m1A eraser ALKBH3 reshaped retinal metabolism to promote this progression. In RPE, the dm1ACRISPR system demonstrated that ALKBH3 demethylated the rate-limiting glycolytic enzyme HK2 to activate glycolysis, resulting in excess lactate production. This lactate promoted histone lactylation at H3K18, which in turn bound to ALKBH3 to amplify its transcription, establishing a positive feedback loop. The ALKBH3 inhibitor HUHS015 disrupted this loop, effectively mitigating RPE degeneration. Furthermore, ALKBH3 directly targeted the proangiogenic factor VEGFA to modulate the metabolic cross-talk between RPE and choroidal capillaries, thus promoting CNV. HUHS015 inhibited CNV synergistically with the anti-VEGF drug Aflibercept. Overall, our study provides critical insights into the molecular mechanisms and metabolic events that facilitates the progression from RPE degeneration to CNV in AMD, laying the groundwork for new treatments of age-related retinal disorders.

Keywords:  ALKBH3; N1-methyladenosine (m1A); age-related macular degeneration (AMD); glycolysis; retinal pigment epithelium (RPE)

DOI:  https://doi.org/10.1073/pnas.2416046122
Trends Cell Biol. 2025 Jun 09. pii: S0962-8924(25)00113-8. [Epub ahead of print]

The intersection between metabolism and translation through a subcellular lens.

Massimo M Santoro.

  The crosstalk between metabolism and mRNA translation (protein synthesis) is crucial for modulating cellular physiology. Signals from metabolic pathways or various metabolic states can influence multiple aspects of RNA biology and translation machinery. In turn, cells can reprogram their metabolism by controlling mRNA translation. Current studies have revealed that localized mRNA translation is specifically regulated by distinct metabolic states, suggesting the existence of specialized subcellular machinery that coordinates this interplay. This review aims to explore recent discoveries and provide an overview of the specialized methodologies developed in recent years on novel modes of translation-metabolism cross-regulation by subcellular localized cues. Spatial compartmentalization, especially in the context of metabolism and mRNA translation, offers a unique advantage, providing a novel mechanism for cellular regulation and function.

Keywords:  RNA condensate; glycolysis; mRNA translation; mTOR signaling; metabolism; organelles; stress granules

DOI:  https://doi.org/10.1016/j.tcb.2025.05.003
bioRxiv. 2025 May 28. pii: 2025.05.24.655883. [Epub ahead of print]

Complete Enzyme Clustering Enhances Coenzyme Q Biosynthesis via Substrate Channeling.

Dianzhuo Wang, Andrea Gottinger, Jio Jeong, Callum R Nicoll, Junlang Liu, Tereza Kadavá, Domiziana Cecchini, Marco Malatesta, Albert J R Heck, Andrea Mattevi, Eugene I Shakhnovich.

Metabolons - transient assemblies of sequential metabolic enzymes - facilitate the reactions of multi-step metabolic pathways, yet, how they mechanistically bolster metabolic flux remains unknown. Here, we investigate the molecular determinants of metabolon formation in coenzyme Q (CoQ) biosynthesis using coarse-grained molecular dynamics simulations and biochemical experiments. We show that the COQ metabolon forms at the critical region of a phase transition, where both metabolon clustering and metabolic flux exhibit coordinated sigmoidal responses to changes in protein-protein interaction strength. These complete metabolons enable substrate channeling between sequential enzymes, leading to a crucial enhancement of CoQ production efficiency. Selectively disrupting protein-protein interactions and randomly shuffling the interaction network demonstrate that protein-proximity rather than fine structure of the metabolon clusters is imperative for substrate channeling. Grounded in both experiment and simulation, these findings provide a framework for understanding the organization and function of metabolons across diverse metabolic pathways.

DOI: https://doi.org/10.1101/2025.05.24.655883
bioRxiv. 2025 Jun 06. pii: 2025.06.05.658131. [Epub ahead of print]

In-vivo dendrite injury drives local mitochondrial contraction and dendrite branching.

Patrick T Hwu, Lauren A Kim, Marcelo A Wood, Katherine L Thompson-Peer.

Mitochondria regulate cellular homeostasis in development and disease, and mitochondrial morphology plays a role in local injury signaling and wound repair. How mitochondria respond during dendrite injury remains an open fundamental question. Here we show that mitochondria contract rapidly and locally after laser dendrotomy. In the proximal intact dendrite, the extent of mitochondrial contraction diminishes with increasing distance from the injury site. We report that mitochondrial contraction is dependent on injury severity and that immediate contraction after injury results in a spatiotemporal increase in dendrite branching. Additionally, we find that mitochondrial contraction is inhibited by KCNJ2 (potassium inwardly rectifying channel subfamily J member 2), providing evidence that mitochondrial contraction is regulated by electrical activity. Mechanistically, we find that injury-induced mitochondrial contraction requires Drp1 (Dynamin related protein 1). In conclusion, these in-vivo findings characterize a dendrite response for mitochondria in neurons and provide insight into the regenerative outcomes of dendrites after injury.
Graphical abstract: In-vivo dendrite injury drives local mitochondrial contraction and dendrite branching.

DOI: https://doi.org/10.1101/2025.06.05.658131
Sci Adv. 2025 Jun 13. 11(24): eadt7369

Cold exposure stimulates cross-tissue metabolic rewiring to fuel glucose-dependent thermogenesis in brown adipose tissue.

Harry B Cutler, Sigrid Jall-Rogg, Senthil Thillainadesan, Kristen C Cooke, Stewart W C Masson, James M Sligar, Jonathan G Crowston, Luke Carroll, Jacqueline Stöckli, David E James, Søren Madsen.

To gain insight into the root causes of metabolic dysfunction, it is essential to understand how tissues communicate and coordinate their metabolic functions. Here, we sought to address this in the context of cold exposure, a well-studied metabolic perturbation. We performed proteomics across six metabolic tissues and plasma, quantifying 11,394 proteins. Beginning our investigation in brown adipose tissue (BAT), we identified a mechanism to explain enhanced glucose utilization in cold-adapted BAT. This was characterized by select remodeling of upper glycolysis and pentose cycling to increase oxygen consumption, likely by increasing uncoupling protein 1 activity through the production of reactive oxygen species. Cold-induced remodeling of the plasma proteome appeared to underpin the ability of BAT to modify its fuel preference, stimulating lipolysis in white adipose tissue and glucose production in the liver. These findings emphasize the importance of considering metabolic adaptations in the context of the whole body and suggest overlap between the mechanisms of cold adaptation and obesity.

DOI: https://doi.org/10.1126/sciadv.adt7369
bioRxiv. 2025 May 30. pii: 2025.05.28.656717. [Epub ahead of print]

Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments.

Julian Najera, Hao Chen, Bianca Batista, Frank Ketchum, Aktar Ali, Pinar Zorlutuna, Scott Howard, Meenal Datta.

The breast peritumor microenvironment (pTME) is increasingly recognized as a mediator of breast cancer progression and treatment resistance. However, if and how growth-induced tumor compressive forces (i.e., solid stresses) influence the breast pTME remains unclear. Here we show using instant fluorescence lifetime imaging microscopy (FLIM)-a frequency-domain FLIM system capable of simultaneous image acquisition and instantaneous data processing-that breast tumor-mimicking in vitro compression promotes metabolic changes in stromal cells found in the breast pTME. Namely, compression shifts NIH3T3 fibroblasts and differentiated 3T3-L1 (d3T3-L1) adipocytes toward a more glycolytic state, while it promotes increased oxidative phosphorylation in 3T3-L1 undifferentiated adipocytes. The gold-standard Seahorse extracellular flux assay fails to capture these changes, underscoring the superior sensitivity of instant FLIM in detecting metabolic shifts. We validate these phenotypic findings at the transcriptomic level via RNA sequencing, confirming that compressed fibroblasts downregulate oxidative phosphorylation and upregulate glycolysis compared to uncompressed controls. We further demonstrate that compression induces mitochondrial dysregulation in undifferentiated adipocytes, driven in part by upregulated mitophagy and disrupted fusion dynamics. Finally, we confirm that these stromal cell types recapitulate these distinct metabolic states in human breast cancer patient samples, consistent with our in vitro findings. By elucidating mechano-metabolic interactions occurring at the tumor-host interface, these results will inform the development of innovative mechano-metabolic reprogramming treatment strategies to improve breast cancer patient survival.

DOI: https://doi.org/10.1101/2025.05.28.656717
Cell. 2025 Jun 05. pii: S0092-8674(25)00570-7. [Epub ahead of print]

Unraveling mitochondrial influence on mammalian pluripotency via enforced mitophagy.

Daniel A Schmitz, Seiya Oura, Leijie Li, Yi Ding, Rashmi Dahiya, Emily Ballard, Carlos Pinzon-Arteaga, Masahiro Sakurai, Daiji Okamura, Leqian Yu, Peter Ly, Jun Wu.

  Mitochondrial abundance and genome are crucial for cellular function, with disruptions often associated with disease. However, methods to modulate these parameters for direct functional dissection remain limited. Here, we eliminate mitochondria from pluripotent stem cells (PSCs) by enforced mitophagy and show that PSCs survived for several days in culture without mitochondria. We then leverage enforced mitophagy to generate interspecies PSC fusions that harbor either human or non-human hominid (NHH) mitochondrial DNA (mtDNA). Comparative analyses indicate that human and NHH mtDNA are largely interchangeable in supporting pluripotency in these PSC fusions. However, species divergence between nuclear and mtDNA leads to subtle species-specific transcriptional and metabolic variations. By developing a transgenic enforced mitophagy approach, we further show that reducing mitochondrial abundance leads to delayed development in pre-implantation mouse embryos. Our study opens avenues for investigating the roles of mitochondria in development, disease, and interspecies biology.

Keywords:  cell fusion; great apes; interspecies composite; interspecies hybrid; metabolism; mitochondria; mitophagy; mtDNA; pluripotent stem cells

DOI:  https://doi.org/10.1016/j.cell.2025.05.020
bioRxiv. 2025 May 26. pii: 2025.05.25.655991. [Epub ahead of print]

Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD + metabolites in CD38 knockout mice exhibiting improved metabolism.

Charles A Schurman, Joanna Bons, Prasanna Vadhana Ashok Kumaar, Jingji Fang, Andrea Roberts, Genesis Vega Hormazabal, Rebeccah R Riley, Nannan Tao, Eric Verdin, Birgit Schilling.

Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from wild-type and CD38 knockout mice (CD38 -/- ). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD + ) metabolism, significantly influences liver metabolic function and contributes to age-related NAD + decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and revealed increased NAD + and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD + hydrolysis, in CD38 -/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.

DOI: https://doi.org/10.1101/2025.05.25.655991
FASEB J. 2025 Jun 30. 39(12): e70708

Cell-Type-Specific Autophagy in Human Leukocytes.

Linh V P Dang, Alexis Martin, Julian M Carosi, Jemima Gore, Sanjna Singh, Timothy J Sargeant.

  Autophagy is a naturally conserved mechanism crucial for degrading and recycling damaged organelles and proteins to support cell survival. This process slows biological aging and age-related disease in preclinical models. However, there has been little translation of autophagy to the clinic, and we have identified a lack of measurement tools for physiological human autophagy as a barrier. To address this, we have previously developed a direct measurement tool for autophagy in pooled human peripheral blood mononuclear cells (PBMCs) in the context of whole blood. In order to better understand how autophagy behaves and changes in humans, we measured human autophagic flux using flow cytometry in 19 cell subpopulations in whole blood to retain physiological flux. Autophagic flux was different between different cell types, being different within different monocyte, B lymphocyte, natural killer cell, and T lymphocyte subtypes. Autophagic flux also varied with sex, being higher in monocytes in females compared with males. In keeping with previous observations in humans, autophagy also increased with aging at subpopulation levels. Importantly, we found that only monocytes-specifically, nonclassical monocytes-displayed robust increased autophagic flux following amino acid withdrawal, underscoring the importance of population selection for measurement of autophagic flux during nutrient restriction studies in humans. Collectively, these data show PBMC population-level analysis improves sensitivity of human autophagic flux measurement.

Keywords:  aging; autophagic flux; autophagy; blood; human; sex

DOI:  https://doi.org/10.1096/fj.202402377R
J Am Chem Soc. 2025 Jun 13.

A Nucleic Acid Prodrug That Activates Mitochondrial Respiration, Promotes Stress Resilience, and Prolongs Lifespan.

Takahisa Anada, Michiharu Kawahara, Taisei Shimada, Ryotaro Kuroda, Hidenori Okamura, Daiki Setoyama, Fumi Nagatsugi, Yuya Kunisaki, Eriko Kage-Nakadai, Shingo Kobayashi, Masaru Tanaka.

Mitochondrial dysfunction caused by aging leads to decreased energy metabolism, resulting in functional decline and increased frailty in multiple tissues. Strategies for protecting and activating mitochondria under stressful conditions are required to suppress aging and age-related diseases. However, it is challenging to develop drugs capable of boosting mitochondrial respiration and compensating for the reduced intracellular adenosine triphosphate (ATP) levels. In this study, we developed a prodrug that stimulates the metabolism of intracellular adenine nucleotides (AXP: adenosine monophosphate (AMP), adenosine diphosphate (ADP), and ATP). It enhances AMP-activated protein kinase activity, fatty acid oxidation, oxidative stress resistance, and mitochondrial respiration, thereby increasing the intracellular ATP levels. Furthermore, this prodrug markedly extended the lifespan of Caenorhabditis elegans. AXP-driven stimulation of cellular energy metabolism proposed herein represents a novel geroprotective strategy and paves the way for the development of bioenergetic-molecule therapeutics.

DOI: https://doi.org/10.1021/jacs.5c06772