bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2024–11–03
sixty-two papers selected by
Christian Frezza, Universität zu Köln
  1. TrkB signalling regulates dopamine circuits and motor function through metabolic pathways.
  2. Glucose-dependent glycosphingolipid biosynthesis fuels CD8+ T cell function and tumor control.
  3. Bempedoic acid suppresses diet-induced hepatic steatosis independently of ATP-citrate lyase.
  4. Noncanonical TCA cycle fosters canonical TCA cycle and mitochondrial integrity in acute myeloid leukemia.
  5. A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones.
  6. An SLC12A9-dependent ion transport mechanism maintains lysosomal osmolarity.
  7. Rhythmic IL-17 production by γδ T cells maintains adipose de novo lipogenesis.
  8. Quantitation of metabolic activity from isotope tracing data using automated methodology.
  9. Branched-chain α-ketoacids aerobically activate HIF1α signalling in vascular cells.
  10. Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.
  11. High mitochondrial DNA levels accelerate lung adenocarcinoma progression.
  12. The immunometabolic roots of aging.
  13. Analysis of targeting signals for mitochondrial intermembrane space import.
  14. Methods to monitor mitochondrial disulfide bonds.
  15. Site of breast cancer metastasis is independent of single nutrient levels.
  16. The post-translational modification O-GlcNAc is a sensor and regulator of metabolism.
  17. Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.
  18. Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria.
  19. Understanding metabolic plasticity at single cell resolution.
  20. A thermodynamic bottleneck in the TCA cycle contributes to acetate overflow in Staphylococcus aureus.
  21. Metabolic regulation of the glioblastoma stem cell epitranscriptome by malate dehydrogenase 2.
  22. TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis.
  23. Modeling compound lipid homeostasis using stable isotope tracing.
  24. Super Resolved Single-Cell Spatial Metabolomics from Multimodal Mass Spectrometry Imaging guided by Imaging Mass Cytometry.
  25. Ultra-precise 3D maps of cancer cells unlock secrets of how tumours grow.
  26. Structure of the turnover-ready state of an ancestral respiratory complex I.
  27. Polyclonality overcomes fitness barriers in Apc-driven tumorigenesis.
  28. The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.
  29. Systems biology approaches identify metabolic signatures of dietary lifespan and healthspan across species.
  30. Temporal recording of mammalian development and precancer.
  31. Monitoring mitochondrial protein import by live cell imaging.
  32. Copper drives remodeling of metabolic state and progression of clear cell renal cell carcinoma.
  33. Caspase-2 kills cells with extra centrosomes.
  34. Tumour evolution and microenvironment interactions in 2D and 3D space.
  35. Methylmalonic acid induces metabolic abnormalities and exhaustion in CD8+ T cells to suppress anti-tumor immunity.
  36. Metabolic rewiring in fat-depleted Drosophila reveals triglyceride:glycogen crosstalk and identifies cDIP as a new regulator of energy metabolism.
  37. Acid affairs in anti-tumour immunity.
  38. Analysis of mitochondrial translation using click chemistry.
  39. Amino acid is a major carbon source for hepatic lipogenesis.
  40. Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
  41. Accumulation of F-actin drives brain aging and limits healthspan in Drosophila.
  42. Multiparameter imaging reveals clinically relevant cancer cell-stroma interaction dynamics in head and neck cancer.
  43. Metatranscriptomics-guided discovery and characterization of a polyphenol-metabolizing gut microbial enzyme.
  44. Subcellular fractionation by differential centrifugation for mitochondrial studies.
  45. RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax.
  46. Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
  47. H4K20me3-Mediated Repression of Inflammatory Genes is a Characteristic and Targetable Vulnerability of Persister Cancer Cells.
  48. Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
  49. HIF1α-regulated glycolysis promotes activation-induced cell death and IFN-γ induction in hypoxic T cells.
  50. Progressive plasticity during colorectal cancer metastasis.
  51. Profiling the transcriptomic age of single-cells in humans.
  52. LCoRL regulates growth and metabolism.
  53. Chemoproteogenomic stratification of the missense variant cysteinome.
  54. Work Around the Clock: How Work Hours Induce Social Jetlag and Sleep Deficiency.
  55. The Drosophila ribonucleoprotein Clueless is required for ribosome biogenesis in vivo.
  56. IRGQ-mediated autophagy in MHC class I quality control promotes tumor immune evasion.
  57. Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.
  58. A temperature-sensitive metabolic valve and a transcriptional feedback loop drive rapid homeoviscous adaptation in Escherichia coli.
  59. Hexokinase 2 senses fructose in tumor-associated macrophages to promote colorectal cancer growth.
  60. TFAM as a sensor of UVC-induced mitochondrial DNA damage.
  61. The de novo purine synthesis enzyme Adssl1 promotes cardiomyocyte proliferation and cardiac regeneration.
  62. Assays to monitor mitochondrial translation.
  1. Nat Metab. 2024 Oct 28.
    TrkB signalling regulates dopamine circuits and motor function through metabolic pathways.
      
    DOI:  https://doi.org/10.1038/s42255-024-01154-0
  2. bioRxiv. 2024 Oct 14. pii: 2024.10.10.617261. [Epub ahead of print]
    Glucose-dependent glycosphingolipid biosynthesis fuels CD8+ T cell function and tumor control.
    Joseph Longo, Lisa M DeCamp, Brandon M Oswald, Robert Teis, Alfredo Reyes-Oliveras, Michael S Dahabieh, Abigail E Ellis, Michael P Vincent, Hannah Damico, Kristin L Gallik, Shelby E Compton, Colt D Capan, Kelsey S Williams, Corinne R Esquibel, Zachary B Madaj, Hyoungjoo Lee, Dominic G Roy, Connie M Krawczyk, Brian B Haab, Ryan D Sheldon, Russell G Jones.
      Glucose is essential for T cell proliferation and function, yet its specific metabolic roles in vivo remain poorly defined. Here, we identify glycosphingolipid (GSL) biosynthesis as a key pathway fueled by glucose that enables CD8+ T cell expansion and cytotoxic function in vivo. Using 13C-based stable isotope tracing, we demonstrate that CD8+ effector T cells use glucose to synthesize uridine diphosphate-glucose (UDP-Glc), a precursor for glycogen, glycan, and GSL biosynthesis. Inhibiting GSL production by targeting the enzymes UGP2 or UGCG impairs CD8+ T cell expansion and cytolytic activity without affecting glucose-dependent energy production. Mechanistically, we show that glucose-dependent GSL biosynthesis is required for plasma membrane lipid raft integrity and aggregation following TCR stimulation. Moreover, UGCG-deficient CD8+ T cells display reduced granzyme expression and tumor control in vivo. Together, our data establish GSL biosynthesis as a critical metabolic fate of glucose-independent of energy production-required for CD8+ T cell responses in vivo.
    Keywords:  CD8+ T cells; UGCG; cytotoxic function; glucose; glycosphingolipids; immunometabolism; lipid rafts; lipidomics; metabolomics; nucleotide sugar metabolism
    DOI:  https://doi.org/10.1101/2024.10.10.617261
  3. Cell Metab. 2024 Oct 26. pii: S1550-4131(24)00410-8. [Epub ahead of print]
    Bempedoic acid suppresses diet-induced hepatic steatosis independently of ATP-citrate lyase.
    Joyce Y Liu, Ramya S Kuna, Laura V Pinheiro, Phuong T T Nguyen, Jaclyn E Welles, Jack M Drummond, Nivitha Murali, Prateek Sharma, Julianna G Supplee, Mia Shiue, Steven Zhao, Aimee T Farria, Avi Kumar, Mauren L Ruchhoeft, Christina Demetriadou, Daniel S Kantner, Adam Chatoff, Emily Megill, Paul M Titchenell, Nathaniel W Snyder, Christian M Metallo, Kathryn E Wellen.
      ATP citrate lyase (ACLY) synthesizes acetyl-CoA for de novo lipogenesis (DNL), which is elevated in metabolic dysfunction-associated steatotic liver disease. Hepatic ACLY is inhibited by the LDL-cholesterol-lowering drug bempedoic acid (BPA), which also improves steatosis in mice. While BPA potently suppresses hepatic DNL and increases fat catabolism, it is unclear if ACLY is its primary molecular target in reducing liver triglyceride. We show that on a Western diet, loss of hepatic ACLY alone or together with the acetyl-CoA synthetase ACSS2 unexpectedly exacerbates steatosis, linked to reduced PPARα target gene expression and fatty acid oxidation. Importantly, BPA treatment ameliorates Western diet-mediated triacylglyceride accumulation in both WT and liver ACLY knockout mice, indicating that its primary effects on hepatic steatosis are ACLY independent. Together, these data indicate that hepatic ACLY plays an unexpected role in restraining diet-dependent lipid accumulation and that BPA exerts substantial effects on hepatic lipid metabolism independently of ACLY.
    Keywords:  ACLY; ACSS2; PPARα; bempedoic acid; lipid metabolism; metabolic dysfunction-associated steatotic liver disease
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.014
  4. Cancer Sci. 2024 Oct 31.
    Noncanonical TCA cycle fosters canonical TCA cycle and mitochondrial integrity in acute myeloid leukemia.
    Atsushi Watanabe, Chartsiam Tipgomut, Haruhito Totani, Kentaro Yoshimura, Tomohiko Iwano, Hamed Bashiri, Lee Hui Chua, Chong Yang, Toshio Suda.
      Cancer cells rely on mitochondrial oxidative phosphorylation (OXPHOS) and the noncanonical tricarboxylic acid (TCA) cycle. In this paper, we shed light on the vital role played by the noncanonical TCA cycle in a host-side concession to mitochondria, especially in highly energy-demanding malignant tumor cells. Inhibition of ATP-citrate lyase (ACLY), a key enzyme in the noncanonical TCA cycle, induced apoptosis by increasing reactive oxygen species levels and DNA damage while reducing mitochondrial membrane potential. The mitochondrial membrane citrate transporter inhibitor, CTPI2, synergistically enhanced these effects. ACLY inhibition reduced cytosolic citrate levels and CTPI2 lowered ACLY activity, suggesting that the noncanonical TCA cycle is sustained by a positive feedback mechanism. These inhibitions impaired ATP production, particularly through OXPHOS. Metabolomic analysis of mitochondrial and cytosolic fractions revealed reduced levels of glutathione pathway-related and TCA cycle-related metabolite, except fumarate, in mitochondria following noncanonical TCA cycle inhibition. Despite the efficient energy supply to the cell by mitochondria, this symbiosis poses challenges related to reactive oxygen species and mitochondrial maintenance. In conclusion, the noncanonical TCA cycle is indispensable for the canonical TCA cycle and mitochondrial integrity, contributing to mitochondrial domestication.
    Keywords:  ATP‐citrate lyase; antimetabolites; apoptosis; cancer metabolism; cell lines; hematopoietic organ; mitochondria; noncanonical TCA cycle; others; reactive oxygen species
    DOI:  https://doi.org/10.1111/cas.16347
  5. Cell Metab. 2024 Oct 25. pii: S1550-4131(24)00409-1. [Epub ahead of print]
    A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones.
    Adam J Rauckhorst, Ryan D Sheldon, Daniel J Pape, Adnan Ahmed, Kelly C Falls-Hubert, Ronald A Merrill, Reid F Brown, Kshitij Deshmukh, Thomas A Vallim, Stanislaw Deja, Shawn C Burgess, Eric B Taylor.
      Hepatic de novo lipogenesis (DNL) is a fundamental physiologic process that is often pathogenically elevated in metabolic disease. Treatment is limited by incomplete understanding of the metabolic pathways supplying cytosolic acetyl-CoA, the obligate precursor to DNL, including their interactions and proportional contributions. Here, we combined extensive 13C tracing with liver-specific knockout of key mitochondrial and cytosolic proteins mediating cytosolic acetyl-CoA production. We show that the mitochondrial pyruvate carrier (MPC) and ATP-citrate lyase (ACLY) gate the major hepatic lipogenic acetyl-CoA production pathway, operating in parallel with acetyl-CoA synthetase 2 (ACSS2). Given persistent DNL after mitochondrial citrate carrier (CiC) and ACSS2 double knockout, we tested the contribution of exogenous and leucine-derived acetoacetate to acetoacetyl-CoA synthetase (AACS)-dependent DNL. CiC knockout increased acetoacetate-supplied hepatic acetyl-CoA production and DNL, indicating that ketones function as mitochondrial-citrate reciprocal DNL precursors. By delineating a mitochondrial-cytosolic DNL substrate supply network, these findings may inform strategies to therapeutically modulate DNL.
    Keywords:  AACS; ACLY; ACSS2; ATP-citrate lyase; CiC; DNL; MPC; acetoacetyl-CoA synthetase; acetyl-CoA synthetase 2; de novo lipogenesis; liver; metabolomics; mitochondrial citrate carrier; mitochondrial pyruvate carrier; stable isotope tracers
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.013
  6. Dev Cell. 2024 Oct 21. pii: S1534-5807(24)00604-X. [Epub ahead of print]
    An SLC12A9-dependent ion transport mechanism maintains lysosomal osmolarity.
    Roni Levin-Konigsberg, Koushambi Mitra, Kaitlyn Spees, AkshatKumar Nigam, Katherine Liu, Camille Januel, Pravin Hivare, Sophia M Arana, Laura M Prolo, Anshul Kundaje, Manuel D Leonetti, Yamuna Krishnan, Michael C Bassik.
      Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity cause ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH4+). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal-resident protein that preserves organelle homeostasis by controlling ammonium and chloride levels. SLC12A9 knockout (KO) cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 KO cells, and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 function is central for the handling of lysosomal ammonium and chloride, an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.
    Keywords:  SLC12A9; ammonium; chloride; ion transport; lysosome metabolism; lysosome volume regulation
    DOI:  https://doi.org/10.1016/j.devcel.2024.10.003
  7. Nature. 2024 Oct 30.
    Rhythmic IL-17 production by γδ T cells maintains adipose de novo lipogenesis.
    Aaron Douglas, Brenneth Stevens, Miguel Rendas, Harry Kane, Evan Lynch, Britta Kunkemoeller, Karl Wessendorf-Rodriguez, Emily A Day, Caroline Sutton, Martin Brennan, Katie O'Brien, Ayano C Kohlgruber, Hannah Prendeville, Amanda E Garza, Luke A J O'Neill, Kingston H G Mills, Christian M Metallo, Henrique Veiga-Fernandes, Lydia Lynch.
      The circadian rhythm of the immune system helps to protect against pathogens1-3; however, the role of circadian rhythms in immune homeostasis is less well understood. Innate T cells are tissue-resident lymphocytes with key roles in tissue homeostasis4-7. Here we use single-cell RNA sequencing, a molecular-clock reporter and genetic manipulations to show that innate IL-17-producing T cells-including γδ T cells, invariant natural killer T cells and mucosal-associated invariant T cells-are enriched for molecular-clock genes compared with their IFNγ-producing counterparts. We reveal that IL-17-producing γδ (γδ17) T cells, in particular, rely on the molecular clock to maintain adipose tissue homeostasis, and exhibit a robust circadian rhythm for RORγt and IL-17A across adipose depots, which peaks at night. In mice, loss of the molecular clock in the CD45 compartment (Bmal1∆Vav1) affects the production of IL-17 by adipose γδ17 T cells, but not cytokine production by αβ or IFNγ-producing γδ (γδIFNγ) T cells. Circadian IL-17 is essential for de novo lipogenesis in adipose tissue, and mice with an adipocyte-specific deficiency in IL-17 receptor C (IL-17RC) have defects in de novo lipogenesis. Whole-body metabolic analysis in vivo shows that Il17a-/-Il17f-/- mice (which lack expression of IL-17A and IL-17F) have defects in their circadian rhythm for de novo lipogenesis, which results in disruptions to their whole-body metabolic rhythm and core-body-temperature rhythm. This study identifies a crucial role for IL-17 in whole-body metabolic homeostasis and shows that de novo lipogenesis is a major target of IL-17.
    DOI:  https://doi.org/10.1038/s41586-024-08131-3
  8. Nat Metab. 2024 Oct 25.
    Quantitation of metabolic activity from isotope tracing data using automated methodology.
    Shiyu Liu, Xiaojing Liu, Jason W Locasale.
      
    DOI:  https://doi.org/10.1038/s42255-024-01144-2
  9. Nat Metab. 2024 Oct 29.
    Branched-chain α-ketoacids aerobically activate HIF1α signalling in vascular cells.
    Wusheng Xiao, Nishith Shrimali, Niv Vigder, William M Oldham, Clary B Clish, Huamei He, Samantha J Wong, Bradley M Wertheim, Elena Arons, Marcia C Haigis, Jane A Leopold, Joseph Loscalzo.
      Hypoxia-inducible factor 1α (HIF1α) is a master regulator of biological processes in hypoxia. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in normal (primary) cells, remain elusive. Here we show that HIF1α signalling is activated in several human primary vascular cells in normoxia and in vascular smooth muscle cells of normal human lungs. Mechanistically, aerobic HIF1α activation is mediated by paracrine secretion of three branched-chain α-ketoacids (BCKAs), which suppress PHD2 activity via direct inhibition and via LDHA-mediated generation of L-2-hydroxyglutarate. BCKA-mediated HIF1α signalling activation stimulated glycolytic activity and governed a phenotypic switch of pulmonary artery smooth muscle cells, which correlated with BCKA metabolic dysregulation and pathophenotypic changes in pulmonary arterial hypertension patients and male rat models. We thus identify BCKAs as previously unrecognized signalling metabolites that aerobically activate HIF1α and that the BCKA-HIF1α pathway modulates vascular smooth muscle cell function, an effect that may be relevant to pulmonary vascular pathobiology.
    DOI:  https://doi.org/10.1038/s42255-024-01150-4
  10. Sci Adv. 2024 Nov;10(44): eadp7725
    Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.
    Vasiliki Mavridou, Martin S King, Andre Bazzone, Roger Springett, Edmund R S Kunji.
      The mitochondrial adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier imports ADP into the mitochondrion and exports ATP to the cell. Here, we demonstrate that 3.3 positive charges are translocated with the negatively charged substrate in each transport step. They can be assigned to three positively charged residues of the central substrate-binding site and two asparagine/arginine pairs. In this way, the membrane potential stimulates not only the ATP4- export step, as a net -0.7 charge is transported, but also the ADP3- import step, as a net +0.3 charge is transported with the electric field. These positive charge movements also inhibit the import of ATP and export of ADP in the presence of a membrane potential, allowing these nucleotides to be maintained at high concentrations in the cytosol and mitochondrial matrix to drive the hydrolysis and synthesis of ATP, respectively. Thus, this is the mechanism by which the membrane potential drives adenine nucleotide exchange with high directional fluxes to fuel the cellular processes.
    DOI:  https://doi.org/10.1126/sciadv.adp7725
  11. Sci Adv. 2024 Nov;10(44): eadp3481
    High mitochondrial DNA levels accelerate lung adenocarcinoma progression.
    Mara Mennuni, Stephen E Wilkie, Pauline Michon, David Alsina, Roberta Filograna, Markus Lindberg, David E Sanin, Florian Rosenberger, Alina Schaaf, Erik Larsson, Erika L Pearce, Nils-Göran Larsson.
      Lung adenocarcinoma is a common aggressive cancer and a leading cause of mortality worldwide. Here, we report an important in vivo role for mitochondrial DNA (mtDNA) copy number during lung adenocarcinoma progression in the mouse. We found that lung tumors induced by KRASG12D expression have increased mtDNA levels and enhanced mitochondrial respiration. To experimentally assess a possible causative role in tumor progression, we induced lung cancer in transgenic mice with a general increase in mtDNA copy number and found that they developed a larger tumor burden, whereas mtDNA depletion in tumor cells reduced tumor growth. Immune cell populations in the lung and cytokine levels in plasma were not affected by increased mtDNA levels. Analyses of large cancer databases indicate that mtDNA copy number is also important in human lung cancer. Our study thus reports experimental evidence for a tumor-intrinsic causative role for mtDNA in lung cancer progression, which could be exploited for development of future cancer therapies.
    DOI:  https://doi.org/10.1126/sciadv.adp3481
  12. Curr Opin Immunol. 2024 Oct 25. pii: S0952-7915(24)00088-8. [Epub ahead of print]91 102498
    The immunometabolic roots of aging.
    Pierpaolo Ginefra, Helen C Hope, Girieca Lorusso, Patrizia D'Amelio, Nicola Vannini.
      Aging is one of the greatest risk factors for several chronic diseases and is accompanied by a progressive decline of cellular and organ function. Recent studies have highlighted the changes in metabolism as one of the main drivers of organism dysfunctions during aging and how that strongly deteriorate immune cell performance and function. Indeed, a dysfunctional immune system has been shown to have a pleiotropic impact on the organism, accelerating the overall aging process of an individual. Intrinsic and extrinsic factors are responsible for such metabolic alterations. Understanding the contribution, regulation, and connection of these different factors is fundamental to comprehend the process of aging and develop approaches to mitigate age-related immune decline. Here, we describe metabolic perturbations occurring at cellular and systemic levels. Particularly, we emphasize the interplay between metabolism and immunosenescence and describe novel interventions to protect immune function and promote health span.
    DOI:  https://doi.org/10.1016/j.coi.2024.102498
  13. Methods Enzymol. 2024 ;pii: S0076-6879(24)00366-5. [Epub ahead of print]706 243-262
    Analysis of targeting signals for mitochondrial intermembrane space import.
    Kostas Tokatlidis, Amiyo Haider.
      The mitochondrial intermembrane space (IMS) is the smallest sub-mitochondrial compartment, containing only 5%-10% of mitochondrial proteins. Despite its size, it exhibits the most diverse array of protein import mechanisms. These are underpinned by several different types of targeting signals that are quite distinct from targeting signals for other mitochondrial sub-compartments. In this chapter we outlined our current understanding of some of the main IMS import pathways, the primary oxidative protein folding targeting signal, and explore the remarkable variety of alternative import methods. Unlike proteins destined for the matrix or inner membrane (IM), IMS proteins need only traverse the outer mitochondrial membrane. This process doesn't require energy from ATP hydrolysis in the matrix or the IM electrochemical potential. We also examine unconventional IMS import pathways that remain poorly understood, often guided by ill-defined or unknown targeting peptides. Many IMS proteins are implicated in human diseases, making it crucial to comprehend how they reach their functional location within the IMS. The chapter concludes by discussing current insights into how understanding IMS targeting pathways can contribute to improved understanding of a wide range of human disorders.
    Keywords:  Chaperones; Disulfide bonds; In vitro protein import; Intermembrane space; MIA pathway; Oxidative folding; Redox; Targeting
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.030
  14. Methods Enzymol. 2024 ;pii: S0076-6879(24)00379-3. [Epub ahead of print]706 125-158
    Methods to monitor mitochondrial disulfide bonds.
    Ben Hur Marins Mussulini, Michal Wasilewski, Agnieszka Chacinska.
      Mitochondria contain numerous proteins that utilize the chemistry of cysteine residues, which can be reversibly oxidized. These proteins are involved in mitochondrial biogenesis, protection against oxidative stress, metabolism, energy transduction to adenosine triphosphate, signaling and cell death among other functions. Many proteins located in the mitochondrial intermembrane space are imported by the mitochondrial import and assembly pathway the activity of which is based on the reversible oxidation of cysteine residues and oxidative trapping of substrates. Oxidative modifications of cysteine residues are particularly difficult to study because of their labile character. Here we present techniques that allow for monitoring the oxidative state of mitochondrial proteins as well as to investigate the mitochondrial import and assembly pathway. This chapter conveys basic concepts on sample preparation and techniques to monitor the redox state of cysteine residues in mitochondrial proteins as well as the strategies to study mitochondrial import and assembly pathway.
    Keywords:  Direct thiol trap; Import assay; Indirect thiol trap; MIA40; Mitochondria
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.039
  15. bioRxiv. 2024 Oct 25. pii: 2024.10.24.616714. [Epub ahead of print]
    Site of breast cancer metastasis is independent of single nutrient levels.
    Keene L Abbott, Sonu Subudhi, Raphael Ferreira, Yetiş Gültekin, Sophie C Steinbuch, Muhammad Bin Munim, Sophie E Honeder, Ashwin S Kumar, Diya L Ramesh, Michelle Wu, Jacob A Hansen, Sharanya Sivanand, Lisa M Riedmayr, Mark Duquette, Ahmed Ali, Nicole Henning, Anna Shevzov-Zebrun, Florian Gourgue, Anna M Barbeau, Millenia Waite, Tenzin Kunchok, Gino B Ferraro, Brian T Do, Virginia Spanoudaki, Francisco J Sánchez-Rivera, Xin Jin, George M Church, Rakesh K Jain, Matthew G Vander Heiden.
      Cancer metastasis is a major contributor to patient morbidity and mortality 1 , yet the factors that determine the organs where cancers can metastasize are incompletely understood. In this study, we quantify the absolute levels of over 100 nutrients available across multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different organs. We then asked how tumor growth in different tissues relates to nutrient availability and tumor biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. Additionally, we identify purine synthesis as a requirement for tumor growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumor de novo nucleotide synthesis activity. These data suggest that a complex interplay of multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.
    DOI:  https://doi.org/10.1101/2024.10.24.616714
  16. Open Biol. 2024 Oct;14(10): 240209
    The post-translational modification O-GlcNAc is a sensor and regulator of metabolism.
    Murielle M Morales, Matthew R Pratt.
      Cells must rapidly adapt to changes in nutrient conditions through responsive signalling cascades to maintain homeostasis. One of these adaptive pathways results in the post-translational modification of proteins by O-GlcNAc. O-GlcNAc modifies thousands of nuclear and cytoplasmic proteins in response to nutrient availability through the hexosamine biosynthetic pathway. O-GlcNAc is highly dynamic and can be added and removed from proteins multiple times throughout their life cycle, setting it up to be an ideal regulator of cellular processes in response to metabolic changes. Here, we describe the link between cellular metabolism and O-GlcNAc, and we explore O-GlcNAc's role in regulating cellular processes in response to nutrient levels. Specifically, we discuss the mechanisms of elevated O-GlcNAc levels in contributing to diabetes and cancer, as well as the role of decreased O-GlcNAc levels in neurodegeneration. These studies form a foundational understanding of aberrant O-GlcNAc in human disease and provide an opportunity to further improve disease identification and treatment.
    Keywords:  O-GlcNAc; metabolism; modifications; post-translational; regulators; sensor
    DOI:  https://doi.org/10.1098/rsob.240209
  17. Sci Adv. 2024 Nov;10(44): eadk8801
    Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.
    Spencer D Shelton, Sara House, Luiza Martins Nascentes Melo, Vijayashree Ramesh, Zhenkang Chen, Tao Wei, Xun Wang, Claire B Llamas, Siva Sai Krishna Venigalla, Cameron J Menezes, Gabriele Allies, Jonathan Krystkiewicz, Jonas Rösler, Sven W Meckelmann, Peihua Zhao, Florian Rambow, Dirk Schadendorf, Zhiyu Zhao, Jennifer G Gill, Ralph J DeBerardinis, Sean J Morrison, Alpaslan Tasdogan, Prashant Mishra.
      Mitochondrial DNA (mtDNA) mutations are frequent in cancer, yet their precise role in cancer progression remains debated. To functionally evaluate the impact of mtDNA variants on tumor growth and metastasis, we developed an enhanced cytoplasmic hybrid (cybrid) generation protocol and established isogenic human melanoma cybrid lines with wild-type mtDNA or pathogenic mtDNA mutations with partial or complete loss of mitochondrial oxidative function. Cybrids with homoplasmic levels of pathogenic mtDNA reliably established tumors despite dysfunctional oxidative phosphorylation. However, these mtDNA variants disrupted spontaneous metastasis from primary tumors and reduced the abundance of circulating tumor cells. Migration and invasion of tumor cells were reduced, indicating that entry into circulation is a bottleneck for metastasis amid mtDNA dysfunction. Pathogenic mtDNA did not inhibit organ colonization following intravenous injection. In heteroplasmic cybrid tumors, single-cell analyses revealed selection against pathogenic mtDNA during melanoma growth. Collectively, these findings experimentally demonstrate that functional mtDNA is favored during melanoma growth and supports metastatic entry into the blood.
    DOI:  https://doi.org/10.1126/sciadv.adk8801
  18. bioRxiv. 2024 Oct 17. pii: 2024.10.15.618543. [Epub ahead of print]
    Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria.
    Yi Fu, Max Land, Ruobing Cui, Tamar Kavlashvili, Minsoo Kim, Toby Lieber, Keun Woo Ryu, Emily DeBitetto, Ignas Masilionis, Rahul Saha, Meril Takizawa, Daphne Baker, Marco Tigano, Ed Reznik, Roshan Sharma, Ronan Chaligne, Craig B Thompson, Dana Pe'er, Agnel Sfeir.
      Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ∼75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.
    Highlights: Combining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cellsEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunctionTwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing.
    DOI:  https://doi.org/10.1101/2024.10.15.618543
  19. Essays Biochem. 2024 Oct 28. pii: EBC20240002. [Epub ahead of print]
    Understanding metabolic plasticity at single cell resolution.
    Christina C Abbate, Jason Hu, John G Albeck.
      It is increasingly clear that cellular metabolic function varies not just between cells of different tissues, but also within tissues and cell types. In this essay, we envision how differences in central carbon metabolism arise from multiple sources, including the cell cycle, circadian rhythms, intrinsic metabolic cycles, and others. We also discuss and compare methods that enable such variation to be detected, including single-cell metabolomics and RNA-sequencing. We pay particular attention to biosensors for AMPK and central carbon metabolites, which when used in combination with metabolic perturbations, provide clear evidence of cellular variance in metabolic function.
    Keywords:  adenosine triphosphate; fluorescence resonance energy transfer; glycolysis; metabolic regulation; oxidative phosphorylation; systems biology
    DOI:  https://doi.org/10.1042/EBC20240002
  20. bioRxiv. 2024 Oct 18. pii: 2024.10.16.618751. [Epub ahead of print]
    A thermodynamic bottleneck in the TCA cycle contributes to acetate overflow in Staphylococcus aureus.
    Nabia Shahreen, Jongsam Ahn, Adil Alsiyabi, Niaz Bahar Chowdhury, Dhananjay Shinde, Sujata S Chaudhari, Kenneth W Bayles, Vinai C Thomas, Rajib Saha.
      During aerobic growth, S. aureus relies on acetate overflow metabolism, a process where glucose is incompletely oxidized to acetate, for its bioenergetic needs. Acetate is not immediately captured as a carbon source and is excreted as waste by cells. The underlying factors governing acetate overflow in S. aureus have not been identified. Here, we show that acetate overflow is favored due to a thermodynamic bottleneck in the TCA cycle, specifically involving the oxidation of succinate to fumarate by succinate dehydrogenase. This bottleneck reduces flux through the TCA cycle, making it more efficient for S. aureus to generate ATP via acetate overflow metabolism. Additionally, the protein allocation cost of maintaining ATP flux through the restricted TCA cycle is greater than that of acetate overflow metabolism. Finally, we show that the TCA cycle bottleneck provides S. aureus the flexibility to redirect carbon towards maintaining redox balance through lactate overflow when oxygen becomes limiting, albeit at the expense of ATP production through acetate overflow. Overall, our findings suggest that overflow metabolism offers S. aureus distinct bioenergetic advantages over a thermodynamically constrained TCA cycle, potentially supporting its commensal-pathogen lifestyle.
    DOI:  https://doi.org/10.1101/2024.10.16.618751
  21. Cell Metab. 2024 Oct 19. pii: S1550-4131(24)00396-6. [Epub ahead of print]
    Metabolic regulation of the glioblastoma stem cell epitranscriptome by malate dehydrogenase 2.
    Deguan Lv, Deobrat Dixit, Andrea F Cruz, Leo J Y Kim, Likun Duan, Xin Xu, Qiulian Wu, Cuiqing Zhong, Chenfei Lu, Zachary C Gersey, Ryan C Gimple, Qi Xie, Kailin Yang, Xiaojing Liu, Xiaoguang Fang, Xujia Wu, Reilly L Kidwell, Xiuxing Wang, Shideng Bao, Housheng H He, Jason W Locasale, Sameer Agnihotri, Jeremy N Rich.
      Tumors reprogram their metabolism to generate complex neoplastic ecosystems. Here, we demonstrate that glioblastoma (GBM) stem cells (GSCs) display elevated activity of the malate-aspartate shuttle (MAS) and expression of malate dehydrogenase 2 (MDH2). Genetic and pharmacologic targeting of MDH2 attenuated GSC proliferation, self-renewal, and in vivo tumor growth, partially rescued by aspartate. Targeting MDH2 induced accumulation of alpha-ketoglutarate (αKG), a critical co-factor for dioxygenases, including the N6-methyladenosine (m6A) RNA demethylase AlkB homolog 5, RNA demethylase (ALKBH5). Forced expression of MDH2 increased m6A levels and inhibited ALKBH5 activity, both rescued by αKG supplementation. Reciprocally, targeting MDH2 reduced global m6A levels with platelet-derived growth factor receptor-β (PDGFRβ) as a regulated transcript. Pharmacological inhibition of MDH2 in GSCs augmented efficacy of dasatinib, an orally bioavailable multi-kinase inhibitor, including PDGFRβ. Collectively, stem-like tumor cells reprogram their metabolism to induce changes in their epitranscriptomes and reveal possible therapeutic paradigms.
    Keywords:  ALKBH5; MDH2; PDGFRβ; alpha-ketoglutarate; cancer stem cell; epitranscriptomics; glioblastoma; m6A; malate-aspartate shuttle; metabolism
    DOI:  https://doi.org/10.1016/j.cmet.2024.09.014
  22. bioRxiv. 2024 Oct 15. pii: 2024.10.15.618357. [Epub ahead of print]
    TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis.
    Zhenghan Wang, Yassmin Elbanna, Inês Godet, Lila Peters, George Lampe, Yanyan Chen, Joao Xavier, Morgan Huse, Joan Massagué.
      The heterogeneity of epithelial-to-mesenchymal transition (EMT) programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers. In these cells, TGF-β induces growth inhibition accompanied by a full EMT response that subsequently transitions into an atypical mesenchymal state of round morphology and lacking actin stress fibers. TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy.
    DOI:  https://doi.org/10.1101/2024.10.15.618357
  23. bioRxiv. 2024 Oct 17. pii: 2024.10.16.618599. [Epub ahead of print]
    Modeling compound lipid homeostasis using stable isotope tracing.
    Karl Wessendorf-Rodriguez, Maureen L Ruchhoeft, Ethan L Ashley, Hector M Galvez, Christopher W Murray, Grace McGregor, Shrikaar Kambhampati, Reuben J Shaw, Christian M Metallo.
      Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states such as cancer and neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing compound lipid homeostasis, we applied stable isotope tracing, high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Compound lipid metabolic flux analysis (CL-MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Here, we resolve liver kinase B1 (LKB1)-mediated regulation of sphingolipid recycling in NSCLC cells and precision-cut lung slice cultures. We also demonstrate that widely used tissue culture conditions drive cells to upregulate fatty acid synthase flux to supraphysiological levels. Finally, we identify previously uncharacterized isozyme specificity of ceramide synthase inhibitors, highlighting the molecular detail revealed by CL-MFA.
    DOI:  https://doi.org/10.1101/2024.10.16.618599
  24. bioRxiv. 2024 Oct 24. pii: 2024.10.21.619323. [Epub ahead of print]
    Super Resolved Single-Cell Spatial Metabolomics from Multimodal Mass Spectrometry Imaging guided by Imaging Mass Cytometry.
    Efe Ozturk, Abhijeet Venkataraman, Felix G Rivera Moctezuma, Ahmet F Coskun.
      Mass spectrometry imaging (MSI) is a powerful technique for spatially resolved analysis of metabolites and other biomolecules within biological tissues. However, the inherent low spatial resolution of MSI often limits its ability to provide detailed cellular-level information. To address this limitation, we propose a guided super-resolution (GSR) approach that leverages high-resolution Imaging Mass Cytometry (IMC) images to enhance the spatial resolution of low-resolution MSI data. By using these detailed IMC images as guides, we improve the resolution of MSI images, creating high-resolution metabolite maps. This enhancement facilitates more precise analysis of cellular structures and tissue architectures, providing deeper insights into super-resolved spatial metabolomics at the single-cell level.
    DOI:  https://doi.org/10.1101/2024.10.21.619323
  25. Nature. 2024 Oct 30.
    Ultra-precise 3D maps of cancer cells unlock secrets of how tumours grow.
    Miryam Naddaf.
      
    Keywords:  Cancer; Cell biology
    DOI:  https://doi.org/10.1038/d41586-024-03539-3
  26. Nat Commun. 2024 Oct 29. 15(1): 9340
    Structure of the turnover-ready state of an ancestral respiratory complex I.
    Bozhidar S Ivanov, Hannah R Bridges, Owen D Jarman, Judy Hirst.
      Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-Å resolution structure of complex I from the α-proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.
    DOI:  https://doi.org/10.1038/s41467-024-53679-3
  27. Nature. 2024 Oct;634(8036): 1196-1203
    Polyclonality overcomes fitness barriers in Apc-driven tumorigenesis.
    Iannish D Sadien, Sam Adler, Shenay Mehmed, Sasha Bailey, Ashley Sawle, Dominique-Laurent Couturier, Matthew Eldridge, David J Adams, Richard Kemp, Filipe C Lourenço, Douglas J Winton.
      Loss-of-function mutations in the tumour suppressor APC are an initial step in intestinal tumorigenesis1,2. APC-mutant intestinal stem cells outcompete their wild-type neighbours through the secretion of Wnt antagonists, which accelerates the fixation and subsequent rapid clonal expansion of mutants3-5. Reports of polyclonal intestinal tumours in human patients and mouse models appear at odds with this process6,7. Here we combine multicolour lineage tracing with chemical mutagenesis in mice to show that a large proportion of intestinal tumours have a multiancestral origin. Polyclonal tumours retain a structure comprising subclones with distinct Apc mutations and transcriptional states, driven predominantly by differences in KRAS and MYC signalling. These pathway-level changes are accompanied by profound differences in cancer stem cell phenotypes. Of note, these findings are confirmed by introducing an oncogenic Kras mutation that results in predominantly monoclonal tumour formation. Further, polyclonal tumours have accelerated growth dynamics, suggesting a link between polyclonality and tumour progression. Together, these findings demonstrate the role of interclonal interactions in promoting tumorigenesis through non-cell autonomous pathways that are dependent on the differential activation of oncogenic pathways between clones.
    DOI:  https://doi.org/10.1038/s41586-024-08053-0
  28. bioRxiv. 2024 Oct 17. pii: 2024.10.16.617214. [Epub ahead of print]
    The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.
    Mingchong Yang, Zengshuo Mo, Kelly Walsh, Wen Liu, Xiaoyan Guo.
      Mitophagy is crucial for maintaining mitochondrial health, but how its levels adjust to different stress conditions remains unclear. In this study, we investigated the role of the DELE1-HRI axis of integrated stress response (ISR) in regulating mitophagy, a key mitochondrial stress pathway. Our findings show that the ISR suppresses mitophagy under non-depolarizing mitochondrial stress by positively regulating mitochondrial protein import, independent of ATF4 activation. Mitochondrial protein import is regulated by the rate of protein synthesis under both depolarizing and non-depolarizing stress. Without ISR, increased protein synthesis overwhelms the mitochondrial import machinery, reducing its efficiency. Under depolarizing stress, mitochondrial import is heavily impaired even with active ISR, leading to significant PINK1 accumulation. In contrast, non-depolarizing stress allows more efficient protein import in the presence of ISR, resulting in lower mitophagy. Without ISR, mitochondrial protein import becomes severely compromised, causing PINK1 accumulation to reach the threshold necessary to trigger mitophagy. These findings reveal a novel link between ISR-regulated protein synthesis, mitochondrial import, and mitophagy, offering potential therapeutic targets for diseases associated with mitochondrial dysfunction.
    DOI:  https://doi.org/10.1101/2024.10.16.617214
  29. Nat Commun. 2024 Oct 29. 15(1): 9330
    Systems biology approaches identify metabolic signatures of dietary lifespan and healthspan across species.
    Tyler A U Hilsabeck, Vikram P Narayan, Kenneth A Wilson, Enrique M Carrera, Daniel Raftery, Daniel Promislow, Rachel B Brem, Judith Campisi, Pankaj Kapahi.
      Dietary restriction (DR) is a potent method to enhance lifespan and healthspan, but individual responses are influenced by genetic variations. Understanding how metabolism-related genetic differences impact longevity and healthspan are unclear. To investigate this, we used metabolites as markers to reveal how different genotypes respond to diet to influence longevity and healthspan traits. We analyzed data from Drosophila Genetic Reference Panel (DGRP) strains raised under AL and DR conditions, combining metabolomic, phenotypic, and genome-wide information. We employed two computational and complementary methods across species-random forest modeling within the DGRP as our primary analysis and Mendelian randomization in human cohorts as a secondary analysis. We pinpointed key traits with cross-species relevance as well as underlying heterogeneity and pleiotropy that influence lifespan and healthspan. Notably, orotate was linked to parental age at death in humans and blocked the DR lifespan extension in flies, while threonine supplementation extended lifespan, in a strain- and sex-specific manner. Thus, utilizing natural genetic variation data from flies and humans, we employed a systems biology approach to elucidate potential therapeutic pathways and metabolomic targets for diet-dependent changes in lifespan and healthspan.
    DOI:  https://doi.org/10.1038/s41467-024-52909-y
  30. Nature. 2024 Oct;634(8036): 1187-1195
    Temporal recording of mammalian development and precancer.
    Mirazul Islam, Yilin Yang, Alan J Simmons, Vishal M Shah, Krushna Pavan Musale, Yanwen Xu, Naila Tasneem, Zhengyi Chen, Linh T Trinh, Paola Molina, Marisol A Ramirez-Solano, Iannish D Sadien, Jinzhuang Dou, Andrea Rolong, Ken Chen, Mark A Magnuson, Jeffrey C Rathmell, Ian G Macara, Douglas J Winton, Qi Liu, Hamim Zafar, Reza Kalhor, George M Church, Martha J Shrubsole, Robert J Coffey, Ken S Lau.
      Temporal ordering of cellular events offers fundamental insights into biological phenomena. Although this is traditionally achieved through continuous direct observations1,2, an alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible marks that enables retrospective temporal ordering3-5. Using a multipurpose, single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, with incorporation of cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during mouse embryonic development, unconventional developmental relationships between cell types and new epithelial progenitor states by their unique genetic histories. Analysis of mouse adenomas, coupled to multiomic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyclonal initiation in 15-30% of colonic precancers, showing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses, to explore the origins and timing of development and tumorigenesis in mammalian systems.
    DOI:  https://doi.org/10.1038/s41586-024-07954-4
  31. Methods Enzymol. 2024 ;pii: S0076-6879(24)00363-X. [Epub ahead of print]706 437-447
    Monitoring mitochondrial protein import by live cell imaging.
    F X Reymond Sutandy.
      The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.
    Keywords:  MTS; mitochondria; protein import; stress response
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.027
  32. Cancer Discov. 2024 Oct 31.
    Copper drives remodeling of metabolic state and progression of clear cell renal cell carcinoma.
    Megan E Bischoff, Behrouz Shamsaei, Juechen Yang, Dina Secic, Bhargav Vemuri, Julie A Reisz, Angelo D'Alessandro, Caterina Bartolacci, Rafal Adamczak, Lucas Schmidt, Jiang Wang, Amelia Martines, Jahnavi Venkat, Vanina Toffessi Tcheuyap, Jacek Biesiada, Catherine A Behrmann, Katherine E Vest, James Brugarolas, Pier Paolo Scaglioni, David R Plas, Krushna C Patra, Shuchi Gulati, Julio A Landero Figueroa, Jarek Meller, John T Cunningham, Maria F Czyzyk-Krzeska.
      Copper (Cu) is a cofactor of cytochrome c oxidase (CuCOX), indispensable for aerobic mitochondrial respiration. This study reveals that advanced clear cell renal cell carcinomas (ccRCCs) accumulate Cu, allocating it to CuCOX. Using a range of orthogonal approaches, including metabolomics, lipidomics, isotope-labeled glucose and glutamine flux analysis, and transcriptomics across tumor samples, cell lines, xenografts, and PDX models, combined with genetic and pharmacological interventions, we explored Cu's role in ccRCC. Elevated Cu levels stimulate CuCOX biogenesis, providing bioenergetic and biosynthetic benefits that promote tumor growth. This effect is complemented by glucose-dependent glutathione production, which facilitates detoxification and mitigates Cu-H2O2 toxicity. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics reveal increased oxidative metabolism, altered glutathione and Cu metabolism, and diminished HIF activity during ccRCC progression. Thus, Cu drives an integrated oncogenic remodeling of bioenergetics, biosynthesis, and redox homeostasis, fueling ccRCC growth, which can be targeted for new therapeutic approaches.
    DOI:  https://doi.org/10.1158/2159-8290.CD-24-0187
  33. Sci Adv. 2024 Nov;10(44): eado6607
    Caspase-2 kills cells with extra centrosomes.
    Dario Rizzotto, Vincenza Vigorito, Patricia Rieder, Filip Gallob, Gian Mario Moretta, Claudia Soratroi, Joel S Riley, Florian Bellutti, Stefano Li Veli, Alessia Mattivi, Michael Lohmüller, Sebastian Herzog, Beat C Bornhauser, Etienne D Jacotot, Andreas Villunger, Luca L Fava.
      Centrosomes are membrane-less organelles that orchestrate a wide array of biological functions by acting as microtubule organizing centers. Here, we report that caspase-2-driven apoptosis is elicited in blood cells failing cytokinesis and that extra centrosomes are necessary to trigger this cell death. Activation of caspase-2 depends on the PIDDosome multi-protein complex, and priming of PIDD1 at extra centrosomes is necessary for pathway activation. Accordingly, loss of its centrosomal adapter, ANKRD26, allows for cell survival and unrestricted polyploidization in response to cytokinesis failure. Mechanistically, cell death is initiated upstream of mitochondria via caspase-2-mediated processing of the BCL2 family protein BID, driving BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Remarkably, BID-deficient cells enforce apoptosis by engaging p53-dependent proapoptotic transcriptional responses initiated by caspase-2. Consistently, BID and MDM2 act as shared caspase-2 substrates, with BID being kinetically favored. Our findings document that the centrosome limits its own unscheduled duplication by the induction of PIDDosome-driven mitochondrial apoptosis to avoid potentially pathogenic polyploidization events.
    DOI:  https://doi.org/10.1126/sciadv.ado6607
  34. Nature. 2024 Oct;634(8036): 1178-1186
    Tumour evolution and microenvironment interactions in 2D and 3D space.
    Chia-Kuei Mo, Jingxian Liu, Siqi Chen, Erik Storrs, Andre Luiz N Targino da Costa, Andrew Houston, Michael C Wendl, Reyka G Jayasinghe, Michael D Iglesia, Cong Ma, John M Herndon, Austin N Southard-Smith, Xinhao Liu, Jacqueline Mudd, Alla Karpova, Andrew Shinkle, S Peter Goedegebuure, Abdurrahman Taha Mousa Ali Abdelzaher, Peng Bo, Lauren Fulghum, Samantha Livingston, Metin Balaban, Angela Hill, Joseph E Ippolito, Vesteinn Thorsson, Jason M Held, Ian S Hagemann, Eric H Kim, Peter O Bayguinov, Albert H Kim, Mary M Mullen, Kooresh I Shoghi, Tao Ju, Melissa A Reimers, Cody Weimholt, Liang-I Kang, Sidharth V Puram, Deborah J Veis, Russell Pachynski, Katherine C Fuh, Milan G Chheda, William E Gillanders, Ryan C Fields, Benjamin J Raphael, Feng Chen, Li Ding.
      To study the spatial interactions among cancer and non-cancer cells1, we here examined a cohort of 131 tumour sections from 78 cases across 6 cancer types by Visium spatial transcriptomics (ST). This was combined with 48 matched single-nucleus RNA sequencing samples and 22 matched co-detection by indexing (CODEX) samples. To describe tumour structures and habitats, we defined 'tumour microregions' as spatially distinct cancer cell clusters separated by stromal components. They varied in size and density among cancer types, with the largest microregions observed in metastatic samples. We further grouped microregions with shared genetic alterations into 'spatial subclones'. Thirty five tumour sections exhibited subclonal structures. Spatial subclones with distinct copy number variations and mutations displayed differential oncogenic activities. We identified increased metabolic activity at the centre and increased antigen presentation along the leading edges of microregions. We also observed variable T cell infiltrations within microregions and macrophages predominantly residing at tumour boundaries. We reconstructed 3D tumour structures by co-registering 48 serial ST sections from 16 samples, which provided insights into the spatial organization and heterogeneity of tumours. Additionally, using an unsupervised deep-learning algorithm and integrating ST and CODEX data, we identified both immune hot and cold neighbourhoods and enhanced immune exhaustion markers surrounding the 3D subclones. These findings contribute to the understanding of spatial tumour evolution through interactions with the local microenvironment in 2D and 3D space, providing valuable insights into tumour biology.
    DOI:  https://doi.org/10.1038/s41586-024-08087-4
  35. Oncogene. 2024 Oct 29.
    Methylmalonic acid induces metabolic abnormalities and exhaustion in CD8+ T cells to suppress anti-tumor immunity.
    Joanne D Tejero, Rebecca S Hesterberg, Stanislav Drapela, Didem Ilter, Devesh Raizada, Felicia Lazure, Hossein Kashfi, Min Liu, Leonardo Silvane, Dorina Avram, Juan Fernández-García, John M Asara, Sarah-Maria Fendt, John L Cleveland, Ana P Gomes.
      Systemic levels of methylmalonic acid (MMA), a byproduct of propionate metabolism, increase with age and MMA promotes tumor progression via its direct effects in tumor cells. However, the role of MMA in modulating the tumor ecosystem remains to be investigated. The proliferation and function of CD8+ T cells, key anti-tumor immune cells, declines with age and in conditions of vitamin B12 deficiency, which are the two most well-established conditions that lead to increased systemic levels of MMA. Thus, we hypothesized that increased circulatory levels of MMA would lead to a suppression of CD8+ T cell immunity. Treatment of primary CD8+ T cells with MMA induced a dysfunctional phenotype characterized by robust immunosuppressive transcriptional reprogramming and marked increases in the expression of the exhaustion regulator, TOX. Accordingly, MMA treatment upregulated exhaustion markers in CD8+ T cells and decreased their effector functions, which drove the suppression of anti-tumor immunity in vitro and in vivo. Mechanistically, MMA-induced CD8+ T cell exhaustion was associated with a suppression of NADH-regenerating reactions in the TCA cycle and concomitant defects in mitochondrial function. Thus, MMA has immunomodulatory roles, thereby highlighting MMA as an important link between aging, immune dysfunction, and cancer.
    DOI:  https://doi.org/10.1038/s41388-024-03191-1
  36. Res Sq. 2024 Oct 18. pii: rs.3.rs-4505077. [Epub ahead of print]
    Metabolic rewiring in fat-depleted Drosophila reveals triglyceride:glycogen crosstalk and identifies cDIP as a new regulator of energy metabolism.
    W Mike Henne, Rupali Ugrankar-Banerjee, Son Tran, Jade Bowerman, Blessy Paul, Lauren Zacharias, Thomas Mathews, Ralph DeBerardinis.
      Tissues store excess nutrients as triglyceride or glycogen, but how these reserves are sensed and communicate remains poorly understood. Here we identify molecular players orchestrating this metabolic balance during fat depletion. We show fat body (FB)-specific depletion of fatty acyl-CoA synthase FASN1 in Drosophila causes near-complete fat loss and metabolic remodeling that dramatically elevates glycogen storage and carbohydrate metabolism. Proteomics and metabolomics identify key factors necessary for rewiring including glycolysis enzymes and target-of-brain-insulin (tobi). FASN1-deficient flies are viable but starvation sensitive, oxidatively stressed, and infertile. We also identify CG10824/cDIP as upregulated in FASN1-depleted Drosophila. cDIP is a leucine-rich-repeat protein with homology to secreted adipokines that fine-tune energy signaling, and is required for fly development in the absence of FASN1. Collectively, we show fat-depleted Drosophila rewire their metabolism to complete development, and identify cDIP as a putative new cytokine that signals fat insufficiency and may regulate energy homeostasis.
    DOI:  https://doi.org/10.21203/rs.3.rs-4505077/v1
  37. Cancer Cell Int. 2024 Oct 27. 24(1): 354
    Acid affairs in anti-tumour immunity.
    Federica Cappellesso, Massimiliano Mazzone, Federico Virga.
      Metabolic rewiring of cancer cells is one of the hallmarks of cancer. As a consequence, the metabolic landscape of the tumour microenvironment (TME) differs compared to correspondent healthy tissues. Indeed, due to the accumulation of acid metabolites, such as lactate, the pH of the TME is generally acidic with a pH drop that can be as low as 5.6. Disruptions in the acid-base balance and elevated lactate levels can drive malignant progression not only through cell-intrinsic mechanisms but also by impacting the immune response. Generally, acidity and lactate dampen the anti-tumour response of both innate and adaptive immune cells favouring tumour progression and reducing the response to immunotherapy. In this review, we summarize the current knowledge on the functional, metabolic and epigenetic effects of acidity and lactate on the cells of the immune system. In particular, we focus on the role of monocarboxylate transporters (MCTs) and other solute carrier transporters (SLCs) that, by mediating the exchange of lactate (among other metabolites) and bicarbonate, participate in pH regulation and lactate transport in the cancer context. Finally, we discuss advanced approaches to target pH or lactate in the TME to enhance the anti-tumour immune response.
    Keywords:  Acidity; Epigenetics; Immune response; Immunotherapy; Lactate; SLC; Tumour microenvironment; pH
    DOI:  https://doi.org/10.1186/s12935-024-03520-0
  38. Methods Enzymol. 2024 ;pii: S0076-6879(24)00384-7. [Epub ahead of print]706 533-547
    Analysis of mitochondrial translation using click chemistry.
    Roya Yousefi, Sven Dennerlein.
      Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components.
    Keywords:  Microscopy; Mitochondria; Mitochondrial translation
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.044
  39. Cell Metab. 2024 Oct 22. pii: S1550-4131(24)00397-8. [Epub ahead of print]
    Amino acid is a major carbon source for hepatic lipogenesis.
    Yilie Liao, Qishan Chen, Lei Liu, Haipeng Huang, Jingyun Sun, Xiaojie Bai, Chenchen Jin, Honghao Li, Fangfang Sun, Xia Xiao, Yahong Zhang, Jia Li, Weiping Han, Suneng Fu.
      Increased de novo lipogenesis is a hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD) in obesity, but the macronutrient carbon source for over half of hepatic fatty acid synthesis remains undetermined. Here, we discover that dietary protein, rather than carbohydrates or fat, is the primary nutritional risk factor for MASLD in humans. Consistently, ex vivo tracing studies identify amino acids as a major carbon supplier for the tricarboxylic acid (TCA) cycle and lipogenesis in isolated mouse hepatocytes. In vivo, dietary amino acids are twice as efficient as glucose in fueling hepatic fatty acid synthesis. The onset of obesity further drives amino acids into fatty acid synthesis through reductive carboxylation, while genetic and chemical interventions that divert amino acid carbon away from lipogenesis alleviate hepatic steatosis. Finally, low-protein diets (LPDs) not only prevent body weight gain in obese mice but also reduce hepatic lipid accumulation and liver damage. Together, this study uncovers the significant role of amino acids in hepatic lipogenesis and suggests a previously unappreciated nutritional intervention target for MASLD.
    Keywords:  DNL; MASH; MASLD; NAFLD; amino acids; dietary protein; glucose; glutamine; lipogenesis
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.001
  40. J Cell Sci. 2024 Oct 28. pii: jcs.263548. [Epub ahead of print]
    Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
    Marta Lopez-Nieto, Zhaozhi Sun, Emily Relton, Rahme Safakli, Brian D Freibaum, J Paul Taylor, Alessia Ruggieri, Ioannis Smyrnias, Nicolas Locker.
      To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2a phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions.
    Keywords:  GADD34; Integrated stress response; Mitochondrial stress response; Stress granules; UPRmt
    DOI:  https://doi.org/10.1242/jcs.263548
  41. Nat Commun. 2024 Oct 25. 15(1): 9238
    Accumulation of F-actin drives brain aging and limits healthspan in Drosophila.
    Edward T Schmid, Joseph M Schinaman, Naomi Liu-Abramowicz, Kylie S Williams, David W Walker.
      The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan.
    DOI:  https://doi.org/10.1038/s41467-024-53389-w
  42. Cell. 2024 Oct 22. pii: S0092-8674(24)01146-2. [Epub ahead of print]
    Multiparameter imaging reveals clinically relevant cancer cell-stroma interaction dynamics in head and neck cancer.
    Karolina Punovuori, Fabien Bertillot, Yekaterina A Miroshnikova, Mirjam I Binner, Satu-Marja Myllymäki, Gautier Follain, Kai Kruse, Johannes Routila, Teemu Huusko, Teijo Pellinen, Jaana Hagström, Noemi Kedei, Sami Ventelä, Antti Mäkitie, Johanna Ivaska, Sara A Wickström.
      Epithelial tumors are characterized by abundant inter- and intra-tumor heterogeneity, which complicates diagnostics and treatment. The contribution of cancer-stroma interactions to this heterogeneity is poorly understood. Here, we report a paradigm to quantify phenotypic diversity in head and neck squamous cell carcinoma (HNSCC) with single-cell resolution. By combining cell-state markers with morphological features, we identify phenotypic signatures that correlate with clinical features, including metastasis and recurrence. Integration of tumor and stromal signatures reveals that partial epithelial-mesenchymal transition (pEMT) renders disease outcome highly sensitive to stromal composition, generating a strong prognostic and predictive signature. Spatial transcriptomics and subsequent analyses of cancer spheroid dynamics identify the cancer-associated fibroblast-pEMT axis as a nexus for intercompartmental signaling that reprograms pEMT cells into an invasive phenotype. Taken together, we establish a paradigm to identify clinically relevant tumor phenotypes and discover a cell-state-dependent interplay between stromal and epithelial compartments that drives cancer aggression.
    Keywords:  cancer; cancer biomarker; cancer stem cell; epithelial-to-mesenchyme-transition
    DOI:  https://doi.org/10.1016/j.cell.2024.09.046
  43. Cell Host Microbe. 2024 Oct 23. pii: S1931-3128(24)00360-3. [Epub ahead of print]
    Metatranscriptomics-guided discovery and characterization of a polyphenol-metabolizing gut microbial enzyme.
    Minwoo Bae, Chi Le, Raaj S Mehta, Xueyang Dong, Lindsey M Pieper, Lorenzo Ramirez, Margaret Alexander, Sina Kiamehr, Peter J Turnbaugh, Curtis Huttenhower, Andrew T Chan, Emily P Balskus.
      Gut microbial catechol dehydroxylases are a largely uncharacterized family of metalloenzymes that potentially impact human health by metabolizing dietary polyphenols. Here, we use metatranscriptomics (MTX) to identify highly transcribed catechol-dehydroxylase-encoding genes in human gut microbiomes. We discover a prevalent, previously uncharacterized catechol dehydroxylase (Gp Hcdh) from Gordonibacter pamelaeae that dehydroxylates hydrocaffeic acid (HCA), an anti-inflammatory gut microbial metabolite derived from plant-based foods. Further analyses suggest that the activity of Gp Hcdh may reduce anti-inflammatory benefits of polyphenol-rich foods. Together, these results show the utility of combining MTX analysis and biochemical characterization for gut microbial enzyme discovery and reveal a potential link between host inflammation and a specific polyphenol-metabolizing gut microbial enzyme.
    Keywords:  catechol dehydroxylase; diet; gut microbe; inflammation; metatranscriptomics; polyphenol
    DOI:  https://doi.org/10.1016/j.chom.2024.10.002
  44. Methods Enzymol. 2024 ;pii: S0076-6879(24)00377-X. [Epub ahead of print]706 61-73
    Subcellular fractionation by differential centrifugation for mitochondrial studies.
    Conny Steiert, Jon V Busto, Laura Melchionda, Nils Wiedemann.
      In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction. These principles help to collect supernatant fractions with less cross-contaminations from the corresponding pellets. These approaches are scalable and adaptable for the fractionation of other cell types and are also useful for the characterization of other organelles.
    Keywords:  Cell organelles; Cytosol; Endoplasmic reticulum; Microsomes; Mitochondria; Nucleus; Post nuclear supernatant; Saccharomyces cerevisiae; Yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.037
  45. Nature. 2024 Oct 30.
    RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax.
    Junya Sango, Saul Carcamo, Maria Sirenko, Abhishek Maiti, Hager Mansour, Gulay Ulukaya, Lewis E Tomalin, Nataly Cruz-Rodriguez, Tiansu Wang, Malgorzata Olszewska, Emmanuel Olivier, Manon Jaud, Bettina Nadorp, Benjamin Kroger, Feng Hu, Lewis Silverman, Stephen S Chung, Elvin Wagenblast, Ronan Chaligne, Ann-Kathrin Eisfeld, Deniz Demircioglu, Dan A Landau, Piro Lito, Elli Papaemmanuil, Courtney D DiNardo, Dan Hasson, Marina Konopleva, Eirini P Papapetrou.
      Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukaemia, upon progression or relapsed/refractory disease1-6. Here, by using human leukaemogenesis models, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte-monocyte progenitors) harbouring previously acquired driver mutations, showing that advanced leukaemic clones can originate from a different cell type in the haematopoietic hierarchy than ancestral clones. Furthermore, we demonstrate that RAS-mutant leukaemia stem cells (LSCs) give rise to monocytic disease, as observed frequently in patients with poor responses to treatment with the BCL2 inhibitor venetoclax. We show that this is because RAS-mutant LSCs, in contrast to RAS-wild-type LSCs, have altered BCL2 family gene expression and are resistant to venetoclax, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver shapes the non-genetic cellular hierarchy of acute myeloid leukaemia by imposing a specific LSC target cell restriction and critically affects therapeutic outcomes in patients.
    DOI:  https://doi.org/10.1038/s41586-024-08137-x
  46. Methods Enzymol. 2024 ;pii: S0076-6879(24)00370-7. [Epub ahead of print]706 365-390
    Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
    Jordan J Crameri, Diana Stojanovski.
      Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.
    Keywords:  in vitro; mitochondria; protein import; translocase
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.034
  47. Cancer Res. 2024 Oct 30.
    H4K20me3-Mediated Repression of Inflammatory Genes is a Characteristic and Targetable Vulnerability of Persister Cancer Cells.
    Valentina Ramponi, Laia Richart, Marta Kovatcheva, Camille Stephan-Otto Attolini, Jordi Capellades, Alice E Lord, Oscar Yanes, Gabriella Ficz, Manuel Serrano.
      Anti-cancer therapies can induce cellular senescence, which is highly stable, or drug-tolerant persistence, which is efficiently reversed upon therapy termination. While approaches to target senescent cells have been extensively studied, further understanding of the processes regulating persistence is needed to develop treatment strategies to suppress persister cell survival. Here, we used mTOR/PI3K inhibition to develop and characterize a model of persistence-associated arrest in human cancer cells of various origins. Persister and senescent cancer cells shared an expanded lysosomal compartment and hypersensitivity to BCL-XL inhibition. However, persister cells lacked other features of senescence, such as loss of lamin B1, senescence-associated β-galactosidase activity, upregulation of MHC-I, and an inflammatory and secretory phenotype (SASP). Genome-wide CRISPR/Cas9 screening for genes required for the survival of persister cells revealed that they are hypersensitive to the inhibition of one-carbon (1C) metabolism, which was validated by the pharmacological inhibition of SHMT, a key enzyme that feeds methyl groups from serine into 1C metabolism. Connecting 1C metabolism with the epigenetic regulation of transcription, the repressive heterochromatic mark H4K20me3 was enriched at the promoters of SASP and interferon response genes in persister cells, while it was absent in proliferative or senescent cells. Moreover, persister cells overexpressed the H4K20 methyltransferases KMT5B/C, and their downregulation unleashed inflammatory programs and compromised the survival of persister cells. In summary, this study defined distinctive features of persister cancer cells, identified actionable vulnerabilities, and provided mechanistic insight into their low inflammatory activity.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-24-0529
  48. Methods Enzymol. 2024 ;pii: S0076-6879(24)00356-2. [Epub ahead of print]706 287-311
    Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
    Klaudia K Maruszczak, Agnieszka Chacinska.
      The vast majority of mitochondrial precursor proteins is synthesized in the cytosol and subsequently imported into the organelle with the help of targeting signals that are present within these proteins. Disruptions in mitochondrial import will result in the accumulation of the organellar precursors in the cytosol of the cell. If mislocalized proteins exceed their critical concentrations, they become prone to aggregation. Under certain circumstances, protein aggregation becomes an irreversible process, which eventually endangers cellular health. Impairment in mitochondrial biogenesis and its effect on cellular protein homeostasis were recently linked to neurodegeneration, therefore placing this process in the center of attention. In this chapter, we are presenting a set of techniques that allows to monitor and study mitochondrial precursor protein aggregates upon mitochondrial dysfunction in the cytosol of both yeast and human cells.
    Keywords:  Mitochondria; Mitochondrial dysfunction; Mitochondrial import; Protein aggregates
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.020
  49. Nat Commun. 2024 Oct 30. 15(1): 9394
    HIF1α-regulated glycolysis promotes activation-induced cell death and IFN-γ induction in hypoxic T cells.
    Hongxing Shen, Oluwagbemiga A Ojo, Haitao Ding, Logan J Mullen, Chuan Xing, M Iqbal Hossain, Abdelrahman Yassin, Vivian Y Shi, Zach Lewis, Ewa Podgorska, Shaida A Andrabi, Maciek R Antoniewicz, James A Bonner, Lewis Zhichang Shi.
      Hypoxia is a common feature in various pathophysiological contexts, including tumor microenvironment, and IFN-γ is instrumental for anti-tumor immunity. HIF1α has long been known as a primary regulator of cellular adaptive responses to hypoxia, but its role in IFN-γ induction in hypoxic T cells is unknown. Here, we show that the HIF1α-glycolysis axis controls IFN-γ induction in both human and mouse T cells, activated under hypoxia. Specific deletion of HIF1α in T cells (Hif1α-/-) and glycolytic inhibition suppresses IFN-γ induction. Conversely, HIF1α stabilization by hypoxia and VHL deletion in T cells (Vhl-/-) increases IFN-γ production. Hypoxic Hif1α-/- T cells are less able to kill tumor cells in vitro, and tumor-bearing Hif1α-/- mice are not responsive to immune checkpoint blockade (ICB) therapy in vivo. Mechanistically, loss of HIF1α greatly diminishes glycolytic activity in hypoxic T cells, resulting in depleted intracellular acetyl-CoA and attenuated activation-induced cell death (AICD). Restoration of intracellular acetyl-CoA by acetate supplementation re-engages AICD, rescuing IFN-γ production in hypoxic Hif1α-/- T cells and re-sensitizing Hif1α-/- tumor-bearing mice to ICB. In summary, we identify HIF1α-regulated glycolysis as a key metabolic control of IFN-γ production in hypoxic T cells and ICB response.
    DOI:  https://doi.org/10.1038/s41467-024-53593-8
  50. Nature. 2024 Oct 30.
    Progressive plasticity during colorectal cancer metastasis.
    A R Moorman, E K Benitez, F Cambuli, Q Jiang, A Mahmoud, M Lumish, S Hartner, S Balkaran, J Bermeo, S Asawa, C Firat, A Saxena, F Wu, A Luthra, C Burdziak, Y Xie, V Sgambati, K Luckett, Y Li, Z Yi, I Masilionis, K Soares, E Pappou, R Yaeger, P Kingham, W Jarnagin, P Paty, M R Weiser, L Mazutis, M D'Angelica, J Shia, J Garcia-Aguilar, T Nawy, T J Hollmann, R Chaligné, F Sanchez-Vega, R Sharma, D Pe'er, K Ganesh.
      As cancers progress, they become increasingly aggressive-metastatic tumours are less responsive to first-line therapies than primary tumours, they acquire resistance to successive therapies and eventually cause death1,2. Mutations are largely conserved between primary and metastatic tumours from the same patients, suggesting that non-genetic phenotypic plasticity has a major role in cancer progression and therapy resistance3-5. However, we lack an understanding of metastatic cell states and the mechanisms by which they transition. Here, in a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that, although primary tumours largely adopt LGR5+ intestinal stem-like states, metastases display progressive plasticity. Cancer cells lose intestinal cell identities and reprogram into a highly conserved fetal progenitor state before undergoing non-canonical differentiation into divergent squamous and neuroendocrine-like states, a process that is exacerbated in metastasis and by chemotherapy and is associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues compared with their intestinal lineage-restricted primary tumour counterparts. We identify PROX1 as a repressor of non-intestinal lineage in the fetal progenitor state, and show that downregulation of PROX1 licenses non-canonical reprogramming.
    DOI:  https://doi.org/10.1038/s41586-024-08150-0
  51. Commun Biol. 2024 Oct 26. 7(1): 1397
    Profiling the transcriptomic age of single-cells in humans.
    Enikő Zakar-Polyák, Attila Csordas, Róbert Pálovics, Csaba Kerepesi.
      Although aging clocks predicting the age of individual organisms have been extensively studied, the age of individual cells remained largely unexplored. Most recently single-cell omics clocks were developed for the mouse, however, extensive profiling the age of human cells is still lacking. To fill this gap, here we use available scRNA-seq data of 1,058,909 blood cells of 508 healthy, human donors (between 19 and 75 years), for developing single-cell transcriptomic clocks and predicting the age of human blood cells. By the application of the proposed cell-type-specific single-cell clocks, our main observations are that (i) transcriptomic age is associated with cellular senescence; (ii) the transcriptomic age of classical monocytes as well as naive B and T cells is decreased in moderate COVID-19 followed by an increase for some cell types in severe COVID-19; and (iii) the human embryo cells transcriptomically rejuvenated at the morulae and blastocyst stages. In summary, here we demonstrate that single-cell transcriptomic clocks are useful tools to investigate aging and rejuvenation at the single-cell level.
    DOI:  https://doi.org/10.1038/s42003-024-07094-5
  52. Endocrinology. 2024 Oct 28. pii: bqae146. [Epub ahead of print]
    LCoRL regulates growth and metabolism.
    Steven C Wyler, Surbhi Gahlot, Lara Bideyan, Cecilia Yip, Jasmine Dushime, Bandy Chen, Jenny J Lee, Arely Tinajero, Chelsea Limboy, Staci Bordash, Samuel R Heaselgrave, Tammy-Nhu Nguyen, Syann Lee, Angie Bookout, Loise Lantier, John L Fowlkes, Young-Jai You, Teppei Fujikawa, Joel K Elmquist.
      Genome Wide Association Studies (GWAS) in humans and livestock have identified genes associated with metabolic traits. However, the causality of many of these genes on metabolic homeostasis is largely unclear due to lack of detailed functional analyses. Here we report Ligand Dependent Corepressor-Like (LCoRL) as a metabolic regulator for body weight and glucose homeostasis. Although GWAS data show that LCoRL is strongly associated with body size, glucose homeostasis, and other metabolic traits in humans and livestock, functional investigations had not been performed. We generated Lcorl knockout mice (Lcorl-/-) and characterized the metabolic traits. We found that Lcorl-/- pups are born smaller than the wildtype littermates (WT) before reaching normal weight by 7-9 weeks of age. While aging, Lcorl-/- mice remain lean compared to WT mice, which is associated with a decrease in daily food intake. Glucose tolerance and insulin sensitivity are improved in Lcorl-/- mice. Mechanistically, this stunted growth is linked to a reduction of circulating levels of insulin like growth factor-1. The expression of the genes downstream of growth hormone signaling and the genes involved in glucose and lipid metabolism are altered in the liver of Lcorl-/- mice. Furthermore, Lcorl-/- mice are protected against a high-fat diet (HFD) challenge and show reduced exercise capacity in an exercise stress test. Collectively, our results are congruent with many of the metabolic parameters linked to the Lcorl locus as reported in GWAS in humans and livestock.
    Keywords:  Lcorl; adiposity; body weight; exercise; glucose homeostasis
    DOI:  https://doi.org/10.1210/endocr/bqae146
  53. Nat Commun. 2024 Oct 28. 15(1): 9284
    Chemoproteogenomic stratification of the missense variant cysteinome.
    Heta Desai, Katrina H Andrews, Kristina V Bergersen, Samuel Ofori, Fengchao Yu, Flowreen Shikwana, Mark A Arbing, Lisa M Boatner, Miranda Villanueva, Nicholas Ung, Elaine F Reed, Alexey I Nesvizhskii, Keriann M Backus.
      Cancer genomes are rife with genetic variants; one key outcome of this variation is widespread gain-of-cysteine mutations. These acquired cysteines can be both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain unidentified via chemoproteomics; identification is a critical step to enable functional analysis, including assessment of potential druggability and susceptibility to oxidation. Here, we pair cysteine chemoproteomics-a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues-with genomics to reveal the hidden landscape of cysteine genetic variation. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized two-stage false discovery rate (FDR) error controlled proteomic search, which is further enhanced with a user-friendly FragPipe interface. Chemoproteogenomics analysis reveals that cysteine acquisition is a ubiquitous feature of both healthy and cancer genomes that is further elevated in the context of decreased DNA repair. Reference cysteines proximal to missense variants are also found to be pervasive, supporting heretofore untapped opportunities for variant-specific chemical probe development campaigns. As chemoproteogenomics is further distinguished by sample-matched combinatorial variant databases and is compatible with redox proteomics and small molecule screening, we expect widespread utility in guiding proteoform-specific biology and therapeutic discovery.
    DOI:  https://doi.org/10.1038/s41467-024-53520-x
  54. Sleep Med Clin. 2024 Dec;pii: S1556-407X(24)00067-5. [Epub ahead of print]19(4): 569-579
    Work Around the Clock: How Work Hours Induce Social Jetlag and Sleep Deficiency.
    Joseph T Hebl, Josie Velasco, Andrew W McHill.
      A growing body of evidence has placed an increasing emphasis on how sleep affects health. Not only does insufficient sleep make one subjectively feel worse, but is associated with chronic diseases that are considered epidemics in industrialized nations. This is partly caused by the growing need for prolonged work and social schedules, exemplified by shift work, late-night weekends, and early morning work/school start times (social jetlag). Here, we consider fundamental relationships between the circadian clock and biologic processes and discuss how common practices, such as shift work and social jetlag, contribute to sleep disruption, circadian misalignment, and adverse health outcomes.
    Keywords:  Chronic disease; Chronotherapy; Circadian misalignment; Shift work; Sleep and circadian medicine; Sleep hygiene
    DOI:  https://doi.org/10.1016/j.jsmc.2024.07.006
  55. J Biol Chem. 2024 Oct 29. pii: S0021-9258(24)02448-7. [Epub ahead of print] 107946
    The Drosophila ribonucleoprotein Clueless is required for ribosome biogenesis in vivo.
    Aditya Sen, Ambar Rodriguez-Martinez, Sara K Young-Baird, Rachel T Cox.
      As hubs of metabolism, mitochondria contribute critical processes to coordinate and optimize energy and intermediate metabolites. Drosophila Clueless (Clu) and vertebrate CLUH are ribonucleoproteins critical for supporting mitochondrial function yet do so in multiple ways. Clu/CLUH bind mRNAs and CLUH regulates mRNA localization and translation of mRNAs encoding proteins destined for mitochondrial import. In addition, Clu associates with ribosomal proteins and translation factors, yet whether it is required for fundamental ribosome function in vivo is not clear. In this study, we examine the Clu interactome and probe Clu's requirement in ribosome biogenesis. We previously showed that Clu associates with ribosomal proteins. In this study, we extend these observations to show that clu null mutants display a significant decrease in overall protein synthesis. In addition, Clu associates with ribosomal proteins in an mRNA-independent manner, suggesting Clu's core ribosomal function may be separate from its role in localizing and translating specific mRNAs. We find that Clu is present in the nucleus and associates with the ribosomal RNA (rRNA) processing protein fibrillarin but, surprisingly, that processed rRNA products are normal in the absence of Clu. Furthermore, Clu loss does not affect ribosomal protein levels, but does result in a decrease in 40S and 60S ribosomal subunits abundance. Together, these results demonstrate that Clu is present in the nucleus and required for 40S and 60S biogenesis and global translation in vivo. These results highlight the multifaceted role of Clu in supporting cell function through regulation of mRNA encoding mitochondrial proteins and ribosome biogenesis.
    Keywords:  Clu; Drosophila; mitochondria; nucleus; protein synthesis; ribosomal RNA; ribosome; ribosome biogenesis
    DOI:  https://doi.org/10.1016/j.jbc.2024.107946
  56. Cell. 2024 Oct 21. pii: S0092-8674(24)01148-6. [Epub ahead of print]
    IRGQ-mediated autophagy in MHC class I quality control promotes tumor immune evasion.
    Lina Herhaus, Uxía Gestal-Mato, Vinay V Eapen, Igor Mačinković, Henry J Bailey, Cristian Prieto-Garcia, Mohit Misra, Anne-Claire Jacomin, Aparna Viswanathan Ammanath, Ivan Bagarić, Jolina Michaelis, Joshua Vollrath, Ramachandra M Bhaskara, Georg Bündgen, Adriana Covarrubias-Pinto, Koraljka Husnjak, Jonathan Zöller, Ajami Gikandi, Sara Ribičić, Tobias Bopp, Gerbrand J van der Heden van Noort, Julian D Langer, Andreas Weigert, J Wade Harper, Joseph D Mancias, Ivan Dikic.
      The autophagy-lysosome system directs the degradation of a wide variety of cargo and is also involved in tumor progression. Here, we show that the immunity-related GTPase family Q protein (IRGQ), an uncharacterized protein to date, acts in the quality control of major histocompatibility complex class I (MHC class I) molecules. IRGQ directs misfolded MHC class I toward lysosomal degradation through its binding mode to GABARAPL2 and LC3B. In the absence of IRGQ, free MHC class I heavy chains do not only accumulate in the cell but are also transported to the cell surface, thereby promoting an immune response. Mice and human patients suffering from hepatocellular carcinoma show improved survival rates with reduced IRGQ levels due to increased reactivity of CD8+ T cells toward IRGQ knockout tumor cells. Thus, we reveal IRGQ as a regulator of MHC class I quality control, mediating tumor immune evasion.
    Keywords:  GABARAPL2; IRGQ; LC3B; MHC class I; autophagy; hepatocellular carcinoma; immune evasion; quality control
    DOI:  https://doi.org/10.1016/j.cell.2024.09.048
  57. EMBO Mol Med. 2024 Oct 28.
    Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.
    Louise Nuyttens, Jolien Vandewalle, Claude Libert.
      Sepsis is a heterogeneous syndrome resulting from a dysregulated host response to infection. It is considered as a global major health priority. Sepsis is characterized by significant metabolic perturbations, leading to increased circulating metabolites such as lactate. In mammals, pyruvate is the primary substrate for lactate production. It plays a critical role in metabolism by linking glycolysis, where it is produced, with the mitochondrial oxidative phosphorylation pathway, where it is oxidized. Here, we provide an overview of all cytosolic and mitochondrial enzymes involved in pyruvate metabolism and how their activities are disrupted in sepsis. Based on the available data, we also discuss potential therapeutic strategies targeting these pyruvate-related enzymes leading to enhanced survival.
    Keywords:  Lactate; Metabolism; Mitochondria; Pyruvate; Sepsis
    DOI:  https://doi.org/10.1038/s44321-024-00155-6
  58. Nat Commun. 2024 Oct 30. 15(1): 9386
    A temperature-sensitive metabolic valve and a transcriptional feedback loop drive rapid homeoviscous adaptation in Escherichia coli.
    Loles Hoogerland, Stefan Pieter Hendrik van den Berg, Yixing Suo, Yuta W Moriuchi, Adja Zoumaro-Djayoon, Esther Geurken, Flora Yang, Frank Bruggeman, Michael D Burkart, Gregory Bokinsky.
      All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. Here, we quantify enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, to describe how this organism measures temperature and restores optimal membrane fluidity within a single generation after a temperature shock. A first element of this regulatory system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways via the branchpoint enzymes FabI and FabB. A second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This strategy is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a mathematical model, which we use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across temperatures. We hypothesize that core features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems.
    DOI:  https://doi.org/10.1038/s41467-024-53677-5
  59. Cell Metab. 2024 Oct 28. pii: S1550-4131(24)00398-X. [Epub ahead of print]
    Hexokinase 2 senses fructose in tumor-associated macrophages to promote colorectal cancer growth.
    Huiwen Yan, Zhi Wang, Da Teng, Xiaodong Chen, Zijing Zhu, Huan Chen, Wen Wang, Ziyuan Wei, Zhenzhen Wu, Qian Chai, Fei Zhang, Youwang Wang, Kaile Shu, Shaotang Li, Guizhi Shi, Mingzhao Zhu, Hai-Long Piao, Xian Shen, Pengcheng Bu.
      Fructose is associated with colorectal cancer tumorigenesis and metastasis through ketohexokinase-mediated metabolism in the colorectal epithelium, yet its role in the tumor immune microenvironment remains largely unknown. Here, we show that a modest amount of fructose, without affecting obesity and associated complications, promotes colorectal cancer tumorigenesis and growth by suppressing the polarization of M1-like macrophages. Fructose inhibits M1-like macrophage polarization independently of fructose-mediated metabolism. Instead, it serves as a signal molecule to promote the interaction between hexokinase 2 and inositol 1,4,5-trisphophate receptor type 3, the predominant Ca2+ channel on the endoplasmic reticulum. The interaction reduces Ca2+ levels in cytosol and mitochondria, thereby suppressing the activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1) as well as NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Consequently, this impedes M1-like macrophage polarization. Our study highlights the critical role of fructose as a signaling molecule that impairs the polarization of M1-like macrophages for tumor growth.
    Keywords:  M1-like macrophage polarization; calcium signaling; fructose; hexokinase 2; inositol 1,4,5-trisphophate receptor type 3
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.002
  60. bioRxiv. 2024 Oct 24. pii: 2024.10.24.620005. [Epub ahead of print]
    TFAM as a sensor of UVC-induced mitochondrial DNA damage.
    Dillon E King, Emily E Beard, Matthew J Satusky, Ian Ryde, Alex George, Caitlin Johnson, Emma L Dolan, Yuning Zhang, Wei Zhu, Hunter Wilkins, Evan Corden, Susan K Murphy, Dorothy Erie, Raluca Gordân, Joel N Meyer.
      Mitochondria lack nucleotide excision DNA repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids. As such, TFAM has emerged as a candidate for protecting DNA or sensing damage. To examine these possibilities, we used live-cell imaging, cell-based assays, atomic force microscopy, and high-throughput protein-DNA binding assays to characterize the binding properties of TFAM to UVC-irradiated DNA and cellular consequences of UVC irradiation. Our data indicate an increase in mtDNA degradation and turnover, without a loss in mitochondrial membrane potential that might trigger mitophagy. We identified a reduction in sequence specificity of TFAM associated with UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA, we do not observe any protective effect on DNA damage accumulation in cells or in vitro . Taken together, these studies indicate that UVC-induced DNA damage promotes compaction by TFAM, suggesting that TFAM may act as a damage sensor, sequestering damaged genomes to prevent mutagenesis by direct removal or suppression of replication.
    DOI:  https://doi.org/10.1101/2024.10.24.620005
  61. Sci Signal. 2024 Oct 29. 17(860): eadn3285
    The de novo purine synthesis enzyme Adssl1 promotes cardiomyocyte proliferation and cardiac regeneration.
    Zhigang Li, Xiaxi Dong, Lingfang Zhuang, Kangni Jia, Haomai Cheng, Hang Sun, Yuke Cui, Wenqi Ma, Keying Wei, Pupu Zhang, Hongyang Xie, Lei Yi, Zhiyong Chen, Lin Lu, Tao Li, Ruiyan Zhang, Xiaoxiang Yan.
      There is a short window during which the neonatal heart has the proliferative capacity to completely repair damage, an ability that is lost in adulthood. Inducing proliferation in adult cardiomyocytes by reactivating cell cycle reentry after myocardial infarction (MI) improves cardiac function. De novo purine synthesis is a critical source of nucleotides for cell proliferation. Here, using loss- and gain-of-function genetic approaches, we explored the role of the muscle-specific de novo purine synthesis enzyme Adssl1 in cardiac regeneration. Deletion of Adssl1 in mouse neonatal hearts reduced cardiomyocyte proliferation and attenuated heart regeneration after apical resection. Conversely, cardiomyocyte-specific Adssl1 overexpression extended the postnatal regenerative window and induced robust cell cycle reentry after MI, which decreased fibrotic scar size and improved cardiac function. RNA sequencing analysis suggested that Adssl1 overexpression induced strong dedifferentiation and cell cycle entry. Moreover, LC-MS/MS analysis showed that Adssl1 overexpression was associated with increased amounts of purine metabolites, including inosine, which is in clinical use. Administration of exogenous inosine promoted cardiac repair after MI in adult mice. At a molecular level, the increase in purine metabolite production mediated by Adssl1 enhanced the activity of the proliferation-promoting mTORC1 pathway. Our study identifies a role for Adssl1 in supporting cardiomyocyte proliferation and cardiac regeneration.
    DOI:  https://doi.org/10.1126/scisignal.adn3285
  62. Methods Enzymol. 2024 ;pii: S0076-6879(24)00385-9. [Epub ahead of print]706 519-532
    Assays to monitor mitochondrial translation.
    Sung-Jun Jung, Martin Ott.
      The complexes of the oxidative phosphorylation (OXPHOS) system found in the mitochondrial inner membrane comprises nuclear and mitochondrial-encoded proteins. The mitochondrial-encoded subunits of the OXPHOS complexes play vital catalytic roles for OXPHOS. These subunits are inserted co-translationally into the inner membrane, where they are matured and assembled with nuclear encoded subunits, requiring a set of OXPHOS assembly and quality control factors. Hence, monitoring the fate of newly synthesized mitochondrial-encoded polypeptides is a basic and essential approach for exploring OXPHOS biogenesis and the related protein quality control processes. Here, we describe a detailed protocol for labeling mitochondrial encoded proteins with 35S-methionine for pulse and pulse/chase experiments, both in vivo and in organello, using the yeast Saccharomyces cerevisiae as the model. These methods enable analyses of the early steps during the biogenesis and turnover of mitochondrial-encoded proteins.
    Keywords:  35S-methionine; Mitochondrial translation; isolated mitochondria; protein stability; protein synthesis; yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.045
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