bims-celmim Biomed News
on Cellular and mitochondrial metabolism
Issue of 2024–08–11
24 papers selected by
Marc Segarra Mondejar
  1. Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.
  2. Methods for Monitoring ER-Phagy.
  3. Engineering new-to-nature biochemical conversions by combining fermentative metabolism with respiratory modules.
  4. Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity.
  5. Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.
  6. SLC25A48 controls mitochondrial choline import and metabolism.
  7. Hierarchical tricarboxylic acid cycle regulation by hepatocyte arginase 2 links the urea cycle to oxidative metabolism.
  8. Nrf2 as a regulator of energy metabolism and mitochondrial function.
  9. Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation.
  10. Purinosomes spatially co-localize with mitochondrial transporters.
  11. Unlocking the potential: Targeting metabolic pathways in the tumor microenvironment for Cancer therapy.
  12. Organelle communication maintains mitochondrial and endosomal homeostasis during podocyte lipotoxicity.
  13. AMPK activation induces RALDH+ tolerogenic dendritic cells by rewiring glucose and lipid metabolism.
  14. Analysis of Mitophagy Reporters in Drosophila.
  15. Quantitative principles of microbial metabolism shared across scales.
  16. In Vivo Monitoring of Nucleophagy in Caenorhabditis elegans.
  17. Aspartate in tumor microenvironment and beyond: Metabolic interactions and therapeutic perspectives.
  18. Methods for Monitoring Mitophagy Using mt-Keima.
  19. Targeting fatty acid oxidation enhances response to HER2-targeted therapy.
  20. Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.
  21. Methods to Monitor Nucleophagy in Yeast.
  22. Monitoring Mitophagy Dynamics in Live Cells.
  23. Protocol for the production and reconstitution of VDAC1 for functional assays.
  24. Structural basis of adenine nucleotides regulation and neurodegenerative pathology in ClC-3 exchanger.
  1. Nat Commun. 2024 Aug 08. 15(1): 6777
    Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.
    Soeun Kang, Maciek R Antoniewicz, Nissim Hay.
      Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.
    DOI:  https://doi.org/10.1038/s41467-024-51117-y
  2. Methods Mol Biol. 2024 ;2845 109-126
    Methods for Monitoring ER-Phagy.
    Marisa Di Monaco, Marian Peteri, Jin Rui Liang.
      The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.
    Keywords:  Autophagy; ER-phagy; Endoplasmic reticulum; FACS; Fluorescent reporters; Immunofluorescence; Selective autophagy; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_9
  3. Nat Commun. 2024 Aug 07. 15(1): 6725
    Engineering new-to-nature biochemical conversions by combining fermentative metabolism with respiratory modules.
    Helena Schulz-Mirbach, Jan Lukas Krüsemann, Theofania Andreadaki, Jana Natalie Nerlich, Eleni Mavrothalassiti, Simon Boecker, Philipp Schneider, Moritz Weresow, Omar Abdelwahab, Nicole Paczia, Beau Dronsella, Tobias J Erb, Arren Bar-Even, Steffen Klamt, Steffen N Lindner.
      Anaerobic microbial fermentations provide high product yields and are a cornerstone of industrial bio-based processes. However, the need for redox balancing limits the array of fermentable substrate-product combinations. To overcome this limitation, here we design an aerobic fermentative metabolism that allows the introduction of selected respiratory modules. These can use oxygen to re-balance otherwise unbalanced fermentations, hence achieving controlled respiro-fermentative growth. Following this design, we engineer and characterize an obligate fermentative Escherichia coli strain that aerobically ferments glucose to stoichiometric amounts of lactate. We then re-integrate the quinone-dependent glycerol 3-phosphate dehydrogenase and demonstrate glycerol fermentation to lactate while selectively transferring the surplus of electrons to the respiratory chain. To showcase the potential of this fermentation mode, we direct fermentative flux from glycerol towards isobutanol production. In summary, our design permits using oxygen to selectively re-balance fermentations. This concept is an advance freeing highly efficient microbial fermentation from the limitations imposed by traditional redox balancing.
    DOI:  https://doi.org/10.1038/s41467-024-51029-x
  4. Cell Mol Life Sci. 2024 Aug 09. 81(1): 340
    Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity.
    Saiya Zhu, Yangyang Niu, Wenqian Zhou, Yuqing Liu, Jing Liu, Xi Liu, Limin Lu, Chen Yu.
      Copper is a trace element essential for numerous biological activities, whereas the mitochondria serve as both major sites of intracellular copper utilization and copper reservoir. Here, we investigated the impact of mitochondrial copper overload on the tricarboxylic acid cycle, renal senescence and fibrosis. We found that copper ion levels are significantly elevated in the mitochondria in fibrotic kidney tissues, which are accompanied by reduced pyruvate dehydrogenase (PDH) activity, mitochondrial dysfunction, cellular senescence and renal fibrosis. Conversely, lowering mitochondrial copper levels effectively restore PDH enzyme activity, improve mitochondrial function, mitigate cellular senescence and renal fibrosis. Mechanically, we found that mitochondrial copper could bind directly to lipoylated dihydrolipoamide acetyltransferase (DLAT), the E2 component of the PDH complex, thereby changing the interaction between the subunits of lipoylated DLAT, inducing lipoylated DLAT protein dimerization, and ultimately inhibiting PDH enzyme activity. Collectively, our study indicates that mitochondrial copper overload could inhibit PDH activity, subsequently leading to mitochondrial dysfunction, cellular senescence and renal fibrosis. Reducing mitochondrial copper overload might therefore serve as a strategy to rescue renal fibrosis.
    Keywords:  Copper; Mitochondria; Pyruvate dehydrogenase; Renal fibrosis; Tricarboxylic acid (TCA) cycle
    DOI:  https://doi.org/10.1007/s00018-024-05358-1
  5. Methods Mol Biol. 2024 ;2845 1-14
    Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.
    Yuki Nakayama, Koji Okamoto.
      Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.
    Keywords:  Atg32; Budding yeast; Fluorescence microscopy; Immunoblotting; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_1
  6. Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00281-X. [Epub ahead of print]
    SLC25A48 controls mitochondrial choline import and metabolism.
    Anthony R P Verkerke, Xu Shi, Mark Li, Yusuke Higuchi, Tadashi Yamamuro, Daisuke Katoh, Hiroshi Nishida, Christopher Auger, Ichitaro Abe, Robert E Gerszten, Shingo Kajimura.
      Choline is an essential nutrient for the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism with a critical step being its import into mitochondria. However, the underlying mechanisms and biological significance remain poorly understood. Here, we report that SLC25A48, a previously uncharacterized mitochondrial inner-membrane carrier protein, controls mitochondrial choline transport and the synthesis of choline-derived methyl donors. We found that SLC25A48 was required for brown fat thermogenesis, mitochondrial respiration, and mitochondrial membrane integrity. Choline uptake into the mitochondrial matrix via SLC25A48 facilitated the synthesis of betaine and purine nucleotides, whereas loss of SLC25A48 resulted in increased production of mitochondrial reactive oxygen species and imbalanced mitochondrial lipids. Notably, human cells carrying a single nucleotide polymorphism on the SLC25A48 gene and cancer cells lacking SLC25A48 exhibited decreased mitochondrial choline import, increased oxidative stress, and impaired cell proliferation. Together, this study demonstrates that SLC25A48 regulates mitochondrial choline catabolism, bioenergetics, and cell survival.
    Keywords:  bioenergetics; brown adipose tissue; cancer metabolism; choline; mitochondria; purine nucleotides
    DOI:  https://doi.org/10.1016/j.cmet.2024.07.010
  7. Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00278-X. [Epub ahead of print]
    Hierarchical tricarboxylic acid cycle regulation by hepatocyte arginase 2 links the urea cycle to oxidative metabolism.
    Yiming Zhang, Cassandra B Higgins, Stefani Tica, Joshua A Adams, Jiameng Sun, Shannon C Kelly, Xiaoyu Zong, Dennis J Dietzen, Terri Pietka, Samuel J Ballentine, Leah Shriver, Gary Patti, Yin Cao, Brian J DeBosch.
      Urea cycle impairment and its relationship to obesity and inflammation remained elusive, partly due to the dramatic clinical presentation of classical urea cycle defects. We generated mice with hepatocyte-specific arginase 2 deletion (Arg2LKO) and revealed a mild compensated urea cycle defect. Stable isotope tracing and respirometry revealed hepatocyte urea and TCA cycle flux defects, impaired mitochondrial oxidative metabolism, and glutamine anaplerosis despite normal energy and glucose homeostasis during early adulthood. Yet during middle adulthood, chow- and diet-induced obese Arg2LKO mice develop exaggerated glucose and lipid derangements, which are reversible by replacing the TCA cycle oxidative substrate nicotinamide adenine dinucleotide. Moreover, serum-based hallmarks of urea, TCA cycle, and mitochondrial derangements predict incident fibroinflammatory liver disease in 106,606 patients nearly a decade in advance. The data reveal hierarchical urea-TCA cycle control via ARG2 to drive oxidative metabolism. Moreover, perturbations in this circuit may causally link urea cycle compromise to fibroinflammatory liver disease.
    Keywords:  arginase; diabetes; fasting; metabolic dysfunction-associated steatohepatitis; metabolic dysfunction-associated steatotic liver disease; nicotinamide adenine dinucleotide; nicotinamide riboside; obesity; tricarboxylic acid cycle; urea cycle
    DOI:  https://doi.org/10.1016/j.cmet.2024.07.007
  8. FEBS Lett. 2024 Aug 08.
    Nrf2 as a regulator of energy metabolism and mitochondrial function.
    Alina Luchkova, Ana Mata, Susana Cadenas.
      Nuclear factor erythroid-2-related factor 2 (Nrf2) is essential for the control of cellular redox homeostasis. When activated, Nrf2 elicits cytoprotective effects through the expression of several genes encoding antioxidant and detoxifying enzymes. Nrf2 can also improve antioxidant defense via the pentose phosphate pathway by increasing NADPH availability to regenerate glutathione. Microarray and genome-wide localization analyses have identified many Nrf2 target genes beyond those linked to its redox-regulatory capacity. Nrf2 regulates several intermediary metabolic pathways and is involved in cancer cell metabolic reprogramming, contributing to malignant phenotypes. Nrf2 also modulates substrate utilization for mitochondrial respiration. Here we review the experimental evidence supporting the essential role of Nrf2 in the regulation of energy metabolism and mitochondrial function.
    Keywords:  Nrf2; energy metabolism; metabolic reprogramming; mitochondria; oxidative stress; redox signaling
    DOI:  https://doi.org/10.1002/1873-3468.14993
  9. Nat Commun. 2024 Aug 05. 15(1): 6649
    Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation.
    Alejandro Moreno-Domínguez, Olalla Colinas, Ignacio Arias-Mayenco, José M Cabeza, Juan L López-Ogayar, Navdeep S Chandel, Norbert Weissmann, Natascha Sommer, Alberto Pascual, José López-Barneo.
      Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.
    DOI:  https://doi.org/10.1038/s41467-024-51023-3
  10. J Biol Chem. 2024 Aug 02. pii: S0021-9258(24)02121-5. [Epub ahead of print] 107620
    Purinosomes spatially co-localize with mitochondrial transporters.
    Zhou Sha, Stephen J Benkovic.
      In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay (PLA) and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.
    Keywords:  APEX; Purinosomes; de novo purine biosynthesis; mitochondrial transporter; proximity ligation assay (PLA)
    DOI:  https://doi.org/10.1016/j.jbc.2024.107620
  11. Biochim Biophys Acta Rev Cancer. 2024 Aug 05. pii: S0304-419X(24)00097-0. [Epub ahead of print] 189166
    Unlocking the potential: Targeting metabolic pathways in the tumor microenvironment for Cancer therapy.
    Siyuan Jia, Ann M Bode, Xue Chen, Xiangjian Luo.
      Cancer incidence and mortality are increasing and impacting global life expectancy. Metabolic reprogramming in the tumor microenvironment (TME) is intimately related to tumorigenesis, progression, metastasis and drug resistance. Tumor cells drive metabolic reprogramming of other cells in the TME through metabolic induction of cytokines and metabolites, and metabolic substrate competition. Consequently, this boosts tumor cell growth by providing metabolic support and facilitating immunosuppression and angiogenesis. The metabolic interplay in the TME presents potential therapeutic targets. Here, we focus on the metabolic reprogramming of four principal cell subsets in the TME: CAFs, TAMs, TILs and TECs, and their interaction with tumor cells. We also summarize medications and therapies targeting these cells' metabolic pathways, particularly in the context of immune checkpoint blockade therapy.
    Keywords:  Cancer-associated fibroblasts (CAFs); Immune check point blockade (ICB); Metabolic reprogramming; Tumor endothelial cells (TECs); Tumor microenvironment (TME); Tumor-associated macrophages (TAMs); Tumor-infiltrating lymphocytes (TILs) therapy
    DOI:  https://doi.org/10.1016/j.bbcan.2024.189166
  12. JCI Insight. 2024 Aug 08. pii: e182534. [Epub ahead of print]
    Organelle communication maintains mitochondrial and endosomal homeostasis during podocyte lipotoxicity.
    Sho Hasegawa, Masaomi Nangaku, Yuto Takenaka, Chigusa Kitayama, Qi Li, Madina Saipidin, Yu Ah Hong, Jin Shang, Yusuke Hirabayashi, Naoto Kubota, Takashi Kadowaki, Reiko Inagi.
      Organelle stress exacerbates podocyte injury, contributing to perturbed lipid metabolism. Simultaneous organelle stresses occur in kidney tissues; therefore, a thorough analysis of organelle communication is crucial for understanding the progression of kidney diseases. Although organelles closely interact with one another at membrane contact sites, limited studies have explored their involvement in kidney homeostasis. The endoplasmic reticulum (ER) protein, PDZ domain-containing 8 (PDZD8), is implicated in multiple organelle tethering processes and cellular lipid homeostasis. In this study, we aimed to elucidate the role of organelle communication in podocyte injury using podocyte-specific Pdzd8-knockout mice. Our findings demonstrated that Pdzd8 deletion exacerbated podocyte injury in an accelerated obesity-related kidney disease model. Proteomic analysis of isolated glomeruli revealed that Pdzd8 deletion exacerbated mitochondrial and endosomal dysfunction during podocyte lipotoxicity. Additionally, electron microscopy revealed the accumulation of "fatty abnormal endosomes" in Pdzd8-deficient podocytes during obesity-related kidney diseases. Lipidomic analysis indicated that glucosylceramide accumulated in Pdzd8-deficient podocytes, owing to accelerated production and decelerated degradation. Thus, the organelle-tethering factor, PDZD8, plays a crucial role in maintaining mitochondrial and endosomal homeostasis during podocyte lipotoxicity. Collectively, our findings highlight the importance of organelle communication at the three-way junction among the ER, mitochondria, and endosomes in preserving podocyte homeostasis.
    Keywords:  Chronic kidney disease; Mitochondria; Nephrology; Obesity
    DOI:  https://doi.org/10.1172/jci.insight.182534
  13. J Cell Biol. 2024 Oct 07. pii: e202401024. [Epub ahead of print]223(10):
    AMPK activation induces RALDH+ tolerogenic dendritic cells by rewiring glucose and lipid metabolism.
    Eline C Brombacher, Thiago A Patente, Alwin J van der Ham, Tijmen J A Moll, Frank Otto, Fenne W M Verheijen, Esther A Zaal, Arnoud H de Ru, Rayman T N Tjokrodirijo, Celia R Berkers, Peter A van Veelen, Bruno Guigas, Bart Everts.
      Dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. Yet the contribution of AMP-activated kinase (AMPK), a central energy sensor promoting catabolism, to DC tolerogenicity remains unknown. Here, we show that AMPK activation renders human monocyte-derived DCs tolerogenic as evidenced by an enhanced ability to drive differentiation of regulatory T cells, a process dependent on increased RALDH activity. This is accompanied by several metabolic changes, including increased breakdown of glycerophospholipids, enhanced mitochondrial fission-dependent fatty acid oxidation, and upregulated glucose catabolism. This metabolic rewiring is functionally important as we found interference with these metabolic processes to reduce to various degrees AMPK-induced RALDH activity as well as the tolerogenic capacity of moDCs. Altogether, our findings reveal a key role for AMPK signaling in shaping DC tolerogenicity and suggest AMPK as a target to direct DC-driven tolerogenic responses in therapeutic settings.
    DOI:  https://doi.org/10.1083/jcb.202401024
  14. Methods Mol Biol. 2024 ;2845 79-93
    Analysis of Mitophagy Reporters in Drosophila.
    Alvaro Sanchez-Martinez, Aitor Martinez, Alexander J Whitworth.
      Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.
    Keywords:  Brain; Drosophila; Light microscopy; Mitochondria; Mitophagy; Muscle; Neurodegeneration; Reporter; mito-QC; mtx-QC
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_7
  15. Nat Microbiol. 2024 Aug;9(8): 1940-1953
    Quantitative principles of microbial metabolism shared across scales.
    Daniel Sher, Daniel Segrè, Michael J Follows.
      Metabolism is the complex network of chemical reactions occurring within every cell and organism, maintaining life, mediating ecosystem processes and affecting Earth's climate. Experiments and models of microbial metabolism often focus on one specific scale, overlooking the connectivity between molecules, cells and ecosystems. Here we highlight quantitative metabolic principles that exhibit commonalities across scales, which we argue could help to achieve an integrated perspective on microbial life. Mass, electron and energy balance provide quantitative constraints on their flow within metabolic networks, organisms and ecosystems, shaping how each responds to its environment. The mechanisms underlying these flows, such as enzyme-substrate interactions, often involve encounter and handling stages that are represented by equations similar to those for cells and resources, or predators and prey. We propose that these formal similarities reflect shared principles and discuss how their investigation through experiments and models may contribute to a common language for studying microbial metabolism across scales.
    DOI:  https://doi.org/10.1038/s41564-024-01764-0
  16. Methods Mol Biol. 2024 ;2845 67-77
    In Vivo Monitoring of Nucleophagy in Caenorhabditis elegans.
    Georgios Konstantinidis, Nektarios Tavernarakis.
      The autophagy-lysosomal pathway enables the controlled degradation of cellular contents. Nucleophagy is the selective autophagic recycling of nuclear components upon delivery to the lysosome. Although methods to monitor and quantify autophagy as well as selective types of autophagy have been developed and implemented in cells and in vivo, methods monitoring nucleophagy remain scarce. Here, we describe a procedure to monitor the autophagic engagement of an endogenous nuclear envelope component, i.e., ANC-1, the nematode homologue of the mammalian Nesprins in vivo, utilizing super-resolution microscopy.
    Keywords:  ANC-1; Autophagy; Caenorhabditis elegans; LGG-1; Nesprin; Nucleophagy; Nucleus; Super-resolution microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_6
  17. Biochim Biophys Acta Mol Basis Dis. 2024 Aug 05. pii: S0925-4439(24)00444-7. [Epub ahead of print]1870(8): 167451
    Aspartate in tumor microenvironment and beyond: Metabolic interactions and therapeutic perspectives.
    Julian Wong Soon, Maria Antonietta Manca, Agnieszka Laskowska, Julia Starkova, Katerina Rohlenova, Jakub Rohlena.
      Aspartate is a proteinogenic non-essential amino acid with several essential functions in proliferating cells. It is mostly produced in a cell autonomous manner from oxalacetate via glutamate oxalacetate transaminases 1 or 2 (GOT1 or GOT2), but in some cases it can also be salvaged from the microenvironment via transporters such as SLC1A3 or by macropinocytosis. In this review we provide an overview of biosynthetic pathways that produce aspartate endogenously during proliferation. We discuss conditions that favor aspartate uptake as well as possible sources of exogenous aspartate in the microenvironment of tumors and bone marrow, where most available data have been generated. We highlight metabolic fates of aspartate, its various functions, and possible approaches to target aspartate metabolism for cancer therapy.
    Keywords:  Amino acid; Biosynthesis; Cancer; Leukemia; Malate-aspartate shuttle; Respiratory chain; Tumor microenvironment
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167451
  18. Methods Mol Biol. 2024 ;2845 151-160
    Methods for Monitoring Mitophagy Using mt-Keima.
    Alexandra J Gilsrud, Derek P Narendra.
      Mitochondria-targeted Keima (mt-Keima) is a pH-sensitive, acid-stable fluorescent protein used for the quantification of mitophagy. Mt-Keima contains a mitochondrial matrix targeting sequence and has bimodal excitation with peaks at 440 nM in neutral environments and 586 nM in acidic environments. From this bimodal excitation, a ratiometric signal may be calculated to quantify mitophagy in live cells. This chapter describes procedures for measuring mitophagy by flow cytometry and live cell confocal microscopy with mt-Keima.
    Keywords:  Mitochondria; Mitophagy; PINK1; Parkin; Selective autophagy
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_12
  19. Nat Commun. 2024 Aug 03. 15(1): 6587
    Targeting fatty acid oxidation enhances response to HER2-targeted therapy.
    Ipshita Nandi, Linjia Ji, Harvey W Smith, Daina Avizonis, Vasilios Papavasiliou, Cynthia Lavoie, Alain Pacis, Sherif Attalla, Virginie Sanguin-Gendreau, William J Muller.
      Metabolic reprogramming, a hallmark of tumorigenesis, involves alterations in glucose and fatty acid metabolism. Here, we investigate the role of Carnitine palmitoyl transferase 1a (Cpt1a), a key enzyme in long-chain fatty acid (LCFA) oxidation, in ErbB2-driven breast cancers. In ErbB2+ breast cancer models, ablation of Cpt1a delays tumor onset, growth, and metastasis. However, Cpt1a-deficient cells exhibit increased glucose dependency that enables survival and eventual tumor progression. Consequently, these cells exhibit heightened oxidative stress and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Inhibiting Nrf2 or silencing its expression reduces proliferation and glucose consumption in Cpt1a-deficient cells. Combining the ketogenic diet, composed of LCFAs, or an anti-ErbB2 monoclonal antibody (mAb) with Cpt1a deficiency significantly perturbs tumor growth, enhances apoptosis, and reduces lung metastasis. Using an immunocompetent model, we show that Cpt1a inhibition promotes an antitumor immune microenvironment, thereby enhancing the efficacy of anti-ErbB2 mAbs. Our findings underscore the importance of targeting fatty acid oxidation alongside HER2-targeted therapies to combat resistance in HER2+ breast cancer patients.
    DOI:  https://doi.org/10.1038/s41467-024-50998-3
  20. EMBO J. 2024 Aug 05.
    Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.
    Rodaria Roussou, Dirk Metzler, Francesco Padovani, Felix Thoma, Rebecca Schwarz, Boris Shraiman, Kurt M Schmoller, Christof Osman.
      Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA.
    Keywords:  Heteroplasmy; Mathematical Modeling; Mitochondria; Mitochondrial Fission; mtDNA
    DOI:  https://doi.org/10.1038/s44318-024-00183-5
  21. Methods Mol Biol. 2024 ;2845 15-25
    Methods to Monitor Nucleophagy in Yeast.
    Ziyang Li, Hitoshi Nakatogawa.
      The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.
    Keywords:  Fluorescence microscopy; Immunoblotting; Nucleophagy; Nucleus; Vacuole; Yeast
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_2
  22. Methods Mol Biol. 2024 ;2845 161-175
    Monitoring Mitophagy Dynamics in Live Cells.
    Christina Karantanou, Sofia-Iris Bibli.
      The purpose of this protocol is to provide a comprehensive, stepwise guide for assessing mitophagy flux utilizing a live-cell mt-KEIMA approach. The proposed protocol is sensitive, reproducible, quantitative, and easy to perform. While mitophagy has been extensively studied, current methodologies primarily focus on terminal measurements, neglecting the dynamic aspect of this process. Hence, the introduction of this straightforward live-cell mitophagy tracing protocol enables real-time monitoring of the dynamics of mitochondrial selective autophagy, thereby enhancing the ability to draw conclusions regarding key regulators and the reversibility of the process. The assay employs a lentiviral approach to induce mt-KEIMA expression in primary or immortalized cell lines. Subsequently, the respective mitophagy reporter cells are observed using a live-cell imaging system at specific time intervals, and further quantification allows the detection of mitophagy flux. This protocol has proven efficacious in investigating mitophagy flux, including responses to chemical inducers or genetically modified cells over time. Notably, this approach is well-suited for large throughput screening of chemicals or appropriate gene-editing libraries that may influence mitophagy responses in cells.
    Keywords:  Flux; Live-cell imaging; Mitophagy; Screening; mt-Keima
    DOI:  https://doi.org/10.1007/978-1-0716-4067-8_13
  23. STAR Protoc. 2024 Aug 07. pii: S2666-1667(24)00405-2. [Epub ahead of print]5(3): 103240
    Protocol for the production and reconstitution of VDAC1 for functional assays.
    Grace I Dearden, Varun Ravishankar, Ken-Taro Sakata, Anant K Menon, Lucie Bergdoll.
      The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.
    Keywords:  Metabolism; Protein Biochemistry; Protein expression and purification; Single-molecule Assays
    DOI:  https://doi.org/10.1016/j.xpro.2024.103240
  24. Nat Commun. 2024 Aug 06. 15(1): 6654
    Structural basis of adenine nucleotides regulation and neurodegenerative pathology in ClC-3 exchanger.
    Yangzhuoqun Wan, Shuangshuang Guo, Wenxuan Zhen, Lizhen Xu, Xiaoying Chen, Fangyue Liu, Yi Shen, Shuangshuang Liu, Lidan Hu, Xinyan Wang, Fengcan Ye, Qinrui Wang, Han Wen, Fan Yang.
      The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s41467-024-50975-w
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