bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021–06–13
sixty-one papers selected by
Kelsey Fisher-Wellman, East Carolina University
  1. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.
  2. An Update on Isolation of Functional Mitochondria from Cells for Bioenergetics Studies.
  3. Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.
  4. Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics.
  5. Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice.
  6. Liposomal Delivery of Cyclocreatine Impairs Cancer Cell Bioenergetics Mediating Apoptosis.
  7. Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.
  8. Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.
  9. Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.
  10. Dual covalent inhibition of PKM and IMPDH targets metabolism in cutaneous metastatic melanoma.
  11. Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.
  12. DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance.
  13. The Use of Reactive Oxygen Species Production by Succinate-Driven Reverse Electron Flow as an Index of Complex 1 Activity in Isolated Brown Adipose Tissue Mitochondria.
  14. Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
  15. Cancer cell-selective modulation of mitochondrial respiration and metabolism by potent organogold(iii) dithiocarbamates.
  16. Imaging Mitochondrial Hydrogen Peroxide in Living Cells.
  17. Insights on Targeting Small Molecules to the Mitochondrial Matrix and the Preparation of MitoB and MitoP as Exomarkers of Mitochondrial Hydrogen Peroxide.
  18. Targeting the Mitochondrial Genome Via a MITO-Porter : Evaluation of mtDNA and mtRNA Levels and Mitochondrial Function.
  19. DDIT3 Directs a Dual Mechanism to Balance Glycolysis and Oxidative Phosphorylation during Glutamine Deprivation.
  20. Determination of Oxidative Phosphorylation Complexes Activities.
  21. Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.
  22. Maintenance of complex I and its super-complexes by NDUF-11 is essential for mitochondrial structure, function and health.
  23. Cancer stem cell-immune cell crosstalk in tumour progression.
  24. Multilevel regulation of Wnt signaling by Zic2 in colon cancer due to mutation of β-catenin.
  25. Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.
  26. Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis.
  27. Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting and its associated metabolism defects.
  28. SDHB Suppresses the Tumorigenesis and Development of ccRCC by Inhibiting Glycolysis.
  29. PKM1 Exerts Critical Roles in Cardiac Remodeling Under Pressure Overload in the Heart.
  30. BN-PAGE-Based Approach to Study Thyroid Hormones and Mitochondrial Function.
  31. Increased glycolysis is an early consequence of palmitate lipotoxicity mediated by redox signaling.
  32. Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS.
  33. Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues.
  34. Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53.
  35. Modeling the Energy Landscape of Side Reactions in the Cytochrome bc1 Complex.
  36. An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial membrane.
  37. Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs).
  38. Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-ΙΙ.
  39. Targeting PKM2 promotes chemosensitivity of breast cancer cells in vitro and in vivo.
  40. Quantitative single-cell proteomics as a tool to characterize cellular hierarchies.
  41. A HIF1α-GPD1 feedforward loop inhibits the progression of renal clear cell carcinoma via mitochondrial function and lipid metabolism.
  42. High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity.
  43. Measuring PGC-1α and Its Acetylation Status in Mouse Primary Myotubes.
  44. The Role of Mitophagy in Regulating Cell Death.
  45. Following the Dynamism of the Mitochondrial Network in T Cells.
  46. Novel Mitochondria-Targeted Triphenylphosphonium Conjugates of Linear β-Phosphorylated Nitrones : Preparation, 31P NMR Mitochondrial Distribution, EPR Spin Trapping Reporting, and Site-Directed Antiapoptotic Properties.
  47. Mitochondrial Coenzyme Q10 Determination Via Isotope Dilution Liquid Chromatography -Tandem Mass Spectrometry.
  48. Targeting glucose metabolism sensitizes pancreatic cancer to MEK inhibition.
  49. Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.
  50. mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.
  51. Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury.
  52. PCR-Based Determination of Mitochondrial DNA Copy Number in Multiple Species.
  53. Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine.
  54. Mitochondrial structural alterations in ovarian cancer patient-derived xenografts resistant to cisplatin.
  55. Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.
  56. Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.
  57. Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability.
  58. The allosteric protein interactions in the proton-motive function of mammalian redox enzymes of the respiratory chain.
  59. A hitchhiker's guide to mitochondrial quantification.
  60. Identification of Peroxynitrite by Profiling Oxidation and Nitration Products from Mitochondria-Targeted Arylboronic Acid.
  61. Mitochondrial Regulation Assessment by 13C-NMR Isotopomer Analysis.
  1. J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00525-1. [Epub ahead of print] 100736
    The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.
    Victor Vitvitsky, Roshan Kumar, Marouane Libiad, Allison Maebius, Aaron Landry, Ruma Banerjee.
      Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain (ETC) at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3-4 min at 37°C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool, blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and therefore, cellular energy metabolism, particularly during cell proliferation.
    Keywords:  Hydrogen sulfide; aerobic glycolysis; electron transport chain; sulfide quinone oxidoreductase
    DOI:  https://doi.org/10.1016/j.jbc.2021.100736
  2. Methods Mol Biol. 2021 ;2310 79-89
    An Update on Isolation of Functional Mitochondria from Cells for Bioenergetics Studies.
    Magdalena Lebiedzinska-Arciszewska, Lech Wojtczak, Mariusz R Wieckowski.
      Mitochondria are the organelles where the most fundamental processes of energy transformation within the cell are located. They are also involved in several processes like apoptosis and autophagy, reactive oxygen species formation, and calcium signaling, which are crucial for proper cell functioning. In addition, mitochondrial genome hosts genes encoding important proteins incorporated in respiratory chain complexes and indispensable for the oxidative phosphorylation. Studying isolated mitochondria is, therefore, crucial for better understanding of cell physiology. The presented protocol describes a relatively simple and handy method for crude mitochondrial fraction isolation from different mammalian cell lines. It includes mechanical cells disruption (homogenization) and differential centrifugation. In addition, this chapter presents two basic ways to assess mitochondrial functionality: by measuring mitochondrial inner membrane potential and coupled respiration.
    Keywords:  Cell cultures; Mitochondria isolation; Mitochondrial membrane potential; Oxidative phosphorylation; Oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_7
  3. Redox Biol. 2021 Jun 01. pii: S2213-2317(21)00179-8. [Epub ahead of print]45 102021
    Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.
    Sehwan Jang, Xavier R Chapa-Dubocq, Yulia Y Tyurina, Claudette M St Croix, Alexandr A Kapralov, Vladimir A Tyurin, Hülya Bayır, Valerian E Kagan, Sabzali Javadov.
      Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.
    Keywords:  Ferroptosis; Glutathione; Heart; Ischemia-reperfusion; Mitochondria; Oxidized phosphatidylethanolamine
    DOI:  https://doi.org/10.1016/j.redox.2021.102021
  4. Sci Signal. 2021 Jun 08. pii: eabc7405. [Epub ahead of print]14(686):
    Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics.
    Veerle W Daniels, Jason J Zoeller, Nick van Gastel, Kelley E McQueeney, Salma Parvin, Danielle S Potter, Geoffrey G Fell, Vinícius G Ferreira, Binyam Yilma, Rajat Gupta, Johan Spetz, Patrick D Bhola, Jennifer E Endress, Isaac S Harris, Emanuel Carrilho, Kristopher A Sarosiek, David T Scadden, Joan S Brugge, Anthony Letai.
      Cancer cells have differential metabolic dependencies compared to their nonmalignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), particularly those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize antiapoptotic proteins). We used high-throughput dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in nonmalignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific antiapoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD+ concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.
    DOI:  https://doi.org/10.1126/scisignal.abc7405
  5. Mol Metab. 2021 Jun 08. pii: S2212-8778(21)00114-9. [Epub ahead of print] 101269
    Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice.
    David B Stagg, Jacob R Gillingham, Alisa B Nelson, Justin E Lengfeld, D André d'Avignon, Patrycja Puchalska, Peter A Crawford.
       OBJECTIVE: Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB).
    METHODS: Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies.
    RESULTS: Exogenously administered AcAc is reduced to D-βOHB, and increases hepatic NAD+/NADH ratio, reflecting hepatic BDH1 activity. Livers of hepatocyte-specific BDH1 deficient mice produced no D-βOHB, but due to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte specific BDH1 deficient mice showed diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal overall hepatic energy charge. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia.
    CONCLUSIONS: Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.
    Keywords:  Glucose Metabolism; Hepatic ketogenesis; Liver oxidative metabolism; Mitochondrial redox homeostasis; metabolic Flux; metabolomics
    DOI:  https://doi.org/10.1016/j.molmet.2021.101269
  6. Methods Mol Biol. 2021 ;2275 173-186
    Liposomal Delivery of Cyclocreatine Impairs Cancer Cell Bioenergetics Mediating Apoptosis.
    Samayita Ganguly, Tamer Elbayoumi.
      Creatine kinase (CK) enzyme overexpression has been suggested to play a role in the process of tumorigenesis and metastasis. Cyclocreatine (CCR) is a substrate analog of creatine kinase (CK), where its phosphorylated form is a poor phosphate donor in comparison with native bioenergetic molecule, creatine phosphate (Cr-P). The compound CCR has been shown to markedly inhibit the growth of a broad spectrum of cancers, both in vitro and in vivo. Intracellularly, CCR is phosphorylated by CK to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCR ~P)], with thermodynamic and kinetic properties distinct from those of creatine phosphate (Cr-P). Distinct inhibition of tumor growth and metastasis has been attributed to CCR accumulation as CCR ~P in tumor cells, especially in those expressing a high level of CK protein, with minimal adverse effects. Unfortunately, the clinical use of CCR against malignancies is quite limited due to its amphoteric nature, which accounts for most of its extremely low membrane permeability, as well as limited oral bioavailability (BA) and poor systemic pharmacokinetics (PK).Our current work describes the encapsulation of CCR , utilizing freeze and thaw vesicles (FTV )-composed mostly of saturated PC, DOPE, and Chol-into stealth™ liposomes , postcoated with 4.5 M% PEG-PE. Following physicochemical characterization, in vitro release and cellular uptake kinetics confirmed efficient delivery of liposomal CCR (CCR-Lip), leading to intracellular accumulation of its CC-P metabolic product. Successful delivery of CCR to cancer cell effectively depleted low energetic cancer cells of ATP significantly mediating myc-induced metabolic changes. CCR-Lip showed significant antimetastatic and anticancer effectiveness against both MCF-7 and PC-3 human carcinoma models (p < 0.05-0.01), with 4- to 6-fold lower IC50 values vs. closest drug control. Such shift in bioenergetics was coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak , thus inducing a cell-intrinsic mechanism to counteract uncontrolled neoplastic proliferation, in target cancer cells. Our novel liposomal delivery system of the CCR substrate analog demonstrated strong inhibition of malignant cell bioenergetics, leading to significant antineoplastic and proapoptotic actions, against different cancers.
    Keywords:  Cell bioenergetics; Creatine kinase; phosphagen; Creatine phosphate; Cyclocreatine; Freeze and thaw nanovesicles; Proapoptotic; Stealth™ liposomes
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_11
  7. Aging Cell. 2021 Jun 07. e13408
    Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.
    Tara R Richman, Judith A Ermer, Stefan J Siira, Irina Kuznetsova, Christopher A Brosnan, Giulia Rossetti, Jessica Baker, Kara L Perks, Henrietta Cserne Szappanos, Helena M Viola, Nicola Gray, Mark Larance, Livia C Hool, Steven Zuryn, Oliver Rackham, Aleksandra Filipovska.
      Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12ep / ep ) or hyper-accurate (Mrps12ha / ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep / ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha / ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging.
    Keywords:  ageing; metabolism; mitochondria; protein synthesis
    DOI:  https://doi.org/10.1111/acel.13408
  8. Methods Mol Biol. 2021 ;2275 415-432
    Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.
    B Spurlock, K Mitra.
      The cross talk between mitochondrial dynamic structure, determined primarily by mitochondrial fission and fusion events, and mitochondrial function of energetics, primarily ATP and ROS production, is widely appreciated. Understanding the mechanistic details of such cross talk between mitochondrial structure and function needs integrated quantitative analyses between mitochondrial dynamics and energetics. Here we describe our recently designed approach of mito-SinCe2 that involves high resolution confocal microscopy of genetically expressed ratiometric fluorescent probes targeted to mitochondria, and its quantitative analyses. Mito-SinCe2 analyses allows for quantitative analyses of mitochondrial structure-function relationship in single cells toward understanding the role of mitochondria and their heterogeneity in various physiological and pathological conditions.
    Keywords:  ATP; Confocal Microscopy; Fission; Fusion; Mitochondrial Dynamics; Mitochondrial Energetics; Quantitative analyses; Ratiometric Probes; Redox State; Single Cell; Structure–Function Relationships
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_27
  9. Metabolism. 2021 Jun 03. pii: S0026-0495(21)00103-7. [Epub ahead of print] 154803
    Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.
    Christopher L Axelrod, Ciaran E Fealy, Melissa L Erickson, Gangarao Davuluri, Hisashi Fujioka, Wagner S Dantas, Emily Huang, Kathryn Pergola, Jacob T Mey, William T King, Anny Mulya, Daniel Hsia, Bartolome Burguera, Bernard Tandler, Charles L Hoppel, John P Kirwan.
       BACKGROUND AND AIMS: A diminution in skeletal muscle mitochondrial function due to ectopic lipid accumulation and excess nutrient intake is thought to contribute to insulin resistance and the development of type 2 diabetes. However, the functional integrity of mitochondria in insulin-resistant skeletal muscle remains highly controversial.
    METHODS: 19 healthy adults (age:28.4 ± 1.7 years; BMI:22.7 ± 0.3 kg/m2) received an overnight intravenous infusion of lipid (20% Intralipid) or saline followed by a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity using a randomized crossover design. Skeletal muscle biopsies were obtained after the overnight lipid infusion to evaluate activation of mitochondrial dynamics proteins, ex-vivo mitochondrial membrane potential, ex-vivo oxidative phosphorylation and electron transfer capacity, and mitochondrial ultrastructure.
    RESULTS: Overnight lipid infusion increased dynamin related protein 1 (DRP1) phosphorylation at serine 616 and PTEN-induced kinase 1 (PINK1) expression (P = 0.003 and P = 0.008, respectively) in skeletal muscle while reducing mitochondrial membrane potential (P = 0.042). The lipid infusion also increased mitochondrial-associated lipid droplet formation (P = 0.011), the number of dilated cristae, and the presence of autophagic vesicles without altering mitochondrial number or respiratory capacity. Additionally, lipid infusion suppressed peripheral glucose disposal (P = 0.004) and hepatic insulin sensitivity (P = 0.014).
    CONCLUSIONS: These findings indicate that activation of mitochondrial fission and quality control occur early in the onset of insulin resistance in human skeletal muscle. Targeting mitochondrial dynamics and quality control represents a promising new pharmacological approach for treating insulin resistance and type 2 diabetes.
    CLINICAL TRIAL REGISTRATION: NCT02697201, ClinicalTrials.gov.
    DOI:  https://doi.org/10.1016/j.metabol.2021.154803
  10. Cancer Res. 2021 Jun 07. pii: canres.2114.2020. [Epub ahead of print]
    Dual covalent inhibition of PKM and IMPDH targets metabolism in cutaneous metastatic melanoma.
    Marwa Zerhouni, Anthony R Martin, Nathan Furstoss, Vincent S Gutierrez, Emilie Jaune, Nedra Tekaya, Guillaume E Beranger, Patricia Abbe, Claire Regazzetti, Hella Amdouni, Mohsine Driowya, Patrice Dubreuil, Frédéric Luciano, Arnaud Jacquel, Meri K Tulic, Thomas Cluzeau, Brendan P O'Hara, Issam Ben-Sahra, Thierry Passeron, Rachid Benhida, Guillaume Robert, Patrick Auberger, Stéphane Rocchi.
      Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of BRAFV600E cutaneous metastatic melanoma (CMM) patients eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming which profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafemib-sensitive and -resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-2114
  11. Analyst. 2021 Jun 09.
    Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.
    Damian Hertig, Sally Maddah, Roman Memedovski, Sandra Kurth, Aitor Moreno, Matteo Pennestri, Andrea Felser, Jean-Marc Nuoffer, Peter Vermathen.
       BACKGROUND: Because of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures.
    METHODS: We demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump.
    RESULTS & DISCUSSION: Implemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption.
    DOI:  https://doi.org/10.1039/d1an00041a
  12. iScience. 2021 May 21. 24(5): 102494
    DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance.
    Juan Zhang, Graciela Terán, Mihaela Popa, Harsha Madapura, Marcus James Graeme Watson Ladds, Danai Lianoudaki, Jacob Grünler, Marie Arsenian-Henriksson, Emmet McCormack, Martin Enrique Rottenberg, Sergiu-Bogdan Catrina, Sonia Laín, Suhas Darekar.
      Dihydroorotate dehydrogenase (DHODH) is essential for the de novo synthesis of pyrimidine ribonucleotides, and as such, its inhibitors have been long used to treat autoimmune diseases and are in clinical trials for cancer and viral infections. Interestingly, DHODH is located in the inner mitochondrial membrane and contributes to provide ubiquinol to the respiratory chain. Thus, DHODH provides the link between nucleotide metabolism and mitochondrial function. Here we show that pharmacological inhibition of DHODH reduces mitochondrial respiration, promotes glycolysis, and enhances GLUT4 translocation to the cytoplasmic membrane and that by activating tumor suppressor p53, increases the expression of GDF15, a cytokine that reduces appetite and prolongs lifespan. In addition, similar to the antidiabetic drug metformin, we observed that in db/db mice, DHODH inhibitors elevate levels of circulating GDF15 and reduce food intake. Further analysis using this model for obesity-induced diabetes revealed that DHODH inhibitors delay pancreatic β cell death and improve metabolic balance.
    Keywords:  Biological sciences; Cellular physiology; Diabetology; Endocrinology; Physiology
    DOI:  https://doi.org/10.1016/j.isci.2021.102494
  13. Methods Mol Biol. 2021 ;2310 247-258
    The Use of Reactive Oxygen Species Production by Succinate-Driven Reverse Electron Flow as an Index of Complex 1 Activity in Isolated Brown Adipose Tissue Mitochondria.
    Andrea Dlasková, Kieran J Clarke, Mary F Rooney, Richard K Porter.
      We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.
    Keywords:  Brown adipose tissue; Complex 1; Complex 2; Mitochondria; NDUFA9; Reactive oxygen species; Succinate; UCP1
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_13
  14. Methods Mol Biol. 2021 ;2310 271-285
    Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
    Yue Yang, Anthony A Sauve.
      NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.
    Keywords:  18O-NAD+; Diaphorase; HPLC; Isotopes; LC-MS; Lactate; Lactate dehydrogenase (LDH); Mitochondria isolation; NAD+; NAD+/NADH cycling assay; Resazurin; Resorufin
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_15
  15. Chem Sci. 2020 Sep 18. 11(38): 10465-10482
    Cancer cell-selective modulation of mitochondrial respiration and metabolism by potent organogold(iii) dithiocarbamates.
    Randall T Mertens, Sean Parkin, Samuel G Awuah.
      Metabolic reprogramming is a key cancer hallmark that has led to the therapeutic targeting of glycolysis. However, agents that target dysfunctional mitochondrial respiration for targeted therapy remains underexplored. We report the synthesis and characterization of ten (10) novel, highly potent organometallic gold(iii) complexes supported by dithiocarbamate ligands as selective inhibitors of mitochondrial respiration. The structure of dithiocarbamates employed dictates the biological stability and cellular cytotoxicity. Most of the compounds exhibit 50% inhibitory concentration (IC50) in the low-micromolar (0.50-2.9 μM) range when tested in a panel of aggressive cancer types with significant selectivity for cancer cells over normal cells. Consequently, there is great interest in the mechanism of action of gold chemotherapeutics, particularly, considering that DNA is not the major target of most gold complexes. We investigate the mechanism of action of representative complexes, 1a and 2a in the recalcitrant triple negative breast cancer (TNBC) cell line, MDA-MB-231. Whole-cell transcriptomics sequencing revealed genes related to three major pathways, namely: cell cycle, organelle fission, and oxidative phosphorylation. 2a irreversibly and rapidly inhibits maximal respiration in TNBC with no effect on normal epithelial cells, implicating mitochondrial OXPHOS as a potential target. Furthermore, the modulation of cyclin dependent kinases and G1 cell cycle arrest induced by these compounds is promising for the treatment of cancer. This work contributes to the need for mitochondrial respiration modulators in biomedical research and outlines a systematic approach to study the mechanism of action of metal-based agents.
    DOI:  https://doi.org/10.1039/d0sc03628e
  16. Methods Mol Biol. 2021 ;2275 127-140
    Imaging Mitochondrial Hydrogen Peroxide in Living Cells.
    Alexander R Lippert, Bryan C Dickinson, Elizabeth J New.
      Hydrogen peroxide (H2O2) produced from mitochondria is intimately involved in human health and disease, but is challenging to selectively monitor inside living systems. The fluorescent probe MitoPY1 provides a practical tool for imaging mitochondrial H2O2 and has been demonstrated to function in a variety of diverse cell types. In this chapter, we describe the synthetic preparation of the small molecule probe MitoPY1 , methods for validating this probe in vitro and in live cells, and an example procedure for measuring mitochondrial H2O2 in a cell culture model of Parkinson's disease.
    Keywords:  Boronate; Fluorescence microscopy; Fluorescent probes; Hydrogen peroxide; Mitochondria; Triphenylphosphonium cations
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_8
  17. Methods Mol Biol. 2021 ;2275 87-117
    Insights on Targeting Small Molecules to the Mitochondrial Matrix and the Preparation of MitoB and MitoP as Exomarkers of Mitochondrial Hydrogen Peroxide.
    Andrew G Cairns, Stephen J McQuaker, Michael P Murphy, Richard C Hartley.
      Small molecules can be physicochemically targeted to the mitochondrial matrix using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue, or whole organism, allow for temporal control, and can be applied in a reversible dose-dependent fashion. An example is the detection and quantification of hydrogen peroxide in mitochondria of whole living organisms by MitoB. Hydrogen peroxide produced within the mitochondrial matrix is involved in signaling and implicated in the oxidative damage associated with aging and a wide range of conditions including cardiovascular disease, neurodegeneration, and cancer. MitoB accumulates in mitochondria and is converted into the exomarker, MitoP, by hydrogen peroxide in the mitochondrial matrix. The hydrogen peroxide concentration is determined from the ratio of MitoP to MitoB after a period of incubation, and this ratio is determined by mass spectrometry using d15-MitoP and d15-MitoB as internal standards. Here we discuss the targeting of small molecules to the mitochondrial matrix using TPP, and describe the synthesis of MitoB and MitoP and the deuterated standards necessary for quantification of hydrogen peroxide in the mitochondrial matrix of whole living organisms.
    Keywords:  Chemical biology; Drug delivery; Exomarker; Hydrogen peroxide; Mass spectrometry; Mitochondria; Oxidative stress; Reactive oxygen species; Targeting
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_6
  18. Methods Mol Biol. 2021 ;2275 227-245
    Targeting the Mitochondrial Genome Via a MITO-Porter : Evaluation of mtDNA and mtRNA Levels and Mitochondrial Function.
    Yuma Yamada, Hideyoshi Harashima.
      Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.
    Keywords:  MITO-Porter; Mitochondria; Mitochondrial DNA; Mitochondrial RNA knockdown; Mitochondrial drug delivery; Mitochondrial gene therapy; Mitochondrial matrix; Nucleic acid delivery
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_14
  19. Adv Sci (Weinh). 2021 06;8(11): e2003732
    DDIT3 Directs a Dual Mechanism to Balance Glycolysis and Oxidative Phosphorylation during Glutamine Deprivation.
    Mingyue Li, Rick Francis Thorne, Ronghua Shi, Xu Dong Zhang, Jingmin Li, Jingtong Li, Qingyuan Zhang, Mian Wu, Lianxin Liu.
      Extracellular glutamine represents an important energy source for many cancer cells and its metabolism is intimately involved in maintaining redox homeostasis. The heightened metabolic activity within tumor tissues can result in glutamine deficiency, necessitating metabolic reprogramming responses. Here, dual mechanisms involving the stress-responsive transcription factor DDIT3 (DNA damage induced transcript 3) that establishes an interrelationship between glycolysis and mitochondrial respiration are revealed. DDIT3 is induced during glutamine deprivation to promote glycolysis and adenosine triphosphate production via suppression of the negative glycolytic regulator TIGAR. In concert, a proportion of the DDIT3 pool translocates to the mitochondria and suppresses oxidative phosphorylation through LONP1-mediated down-regulation of COQ9 and COX4. This in turn dampens the sustained levels of reactive oxygen species that follow glutamine withdrawal. Together these mechanisms constitute an adaptive survival mechanism permitting tumor cells to survive metabolic stress induced by glutamine starvation.
    Keywords:  COQ9; COX4; DDIT3/CHOP; electron transfer chain; glutamine deprivation; glycolysis
    DOI:  https://doi.org/10.1002/advs.202003732
  20. Methods Mol Biol. 2021 ;2310 17-31
    Determination of Oxidative Phosphorylation Complexes Activities.
    João S Teodoro, Ivo F Machado, Carlos M Palmeira, Anabela P Rolo.
      Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.
    Keywords:  Mitochondria; Mitochondrial protein complexes; Polarography; Respiratory chain; Spectrophotometry
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_2
  21. Methods Mol Biol. 2021 ;2310 113-159
    Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.
    Simone Patergnani, Massimo Bonora, Esmaa Bouhamida, Alberto Danese, Saverio Marchi, Giampaolo Morciano, Maurizio Previati, Gaia Pedriali, Alessandro Rimessi, Gabriele Anania, Carlotta Giorgi, Paolo Pinton.
      Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.
    Keywords:  Cardiovascular diseases (CVD); Homeostasis; Metabolism; Mitochondrial morphology; Mitochondrial quality control; Pathology
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_9
  22. J Cell Sci. 2021 Jun 09. pii: jcs.258399. [Epub ahead of print]
    Maintenance of complex I and its super-complexes by NDUF-11 is essential for mitochondrial structure, function and health.
    Amber Knapp-Wilson, Gonçalo C Pereira, Emma Buzzard, Holly C Ford, Andrew Richardson, Robin A Corey, Chris Neal, Paul Verkade, Andrew P Halestrap, Vicki A M Gold, Patricia Kuwabara, Ian Collinson.
      Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing (but not eliminating) expression by RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, with the potential for detrimental ROS-production. Electron cryo-tomography highlight aberrant cristae morphology and inter-membrane-space widening and cristae-junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I/ super-complex maintenance. This highlights the importance of respiratory complex integrity for health and the potential of its perturbation for mitochondrial disease.
    Keywords:  Caenorhabditis elegans; Electron-transfer chain; Mitochondria; Mitochondrial ultrastructure; electron cryo-tomography; NDUF-11; Respirasome; Respiration; Super-complexes; Worm
    DOI:  https://doi.org/10.1242/jcs.258399
  23. Nat Rev Cancer. 2021 Jun 08.
    Cancer stem cell-immune cell crosstalk in tumour progression.
    Defne Bayik, Justin D Lathia.
      Cellular heterogeneity and an immunosuppressive tumour microenvironment are independent yet synergistic drivers of tumour progression and underlie therapeutic resistance. Recent studies have highlighted the complex interaction between these cell-intrinsic and cell-extrinsic mechanisms. The reciprocal communication between cancer stem cells (CSCs) and infiltrating immune cell populations in the tumour microenvironment is a paradigm for these interactions. In this Perspective, we discuss the signalling programmes that simultaneously induce CSCs and reprogramme the immune response to facilitate tumour immune evasion, metastasis and recurrence. We further highlight biological factors that can impact the nature of CSC-immune cell communication. Finally, we discuss targeting opportunities for simultaneous regulation of the CSC niche and immunosurveillance.
    DOI:  https://doi.org/10.1038/s41568-021-00366-w
  24. Cell Death Dis. 2021 Jun 07. 12(6): 584
    Multilevel regulation of Wnt signaling by Zic2 in colon cancer due to mutation of β-catenin.
    Zhengshui Xu, Jianbao Zheng, Zilu Chen, Jing Guo, Xiaopeng Li, Xingjie Wang, Chao Qu, Liyue Yuan, Chen Cheng, Xuejun Sun, Junhui Yu.
      Zinc-finger of the cerebellum 2 (Zic2) is widely implicated in cancers, but the role of Zic2 in tumorigenesis is bilateral. A recent study indicated that Zic2 could render colon cancer cells more resistant to low glucose-induced apoptosis. However, the functional roles of Zic2 in colon cancer and the underlying molecular mechanism remain elusive. Herein, we demonstrated that Zic2 was highly expressed in colon cancer tissues and correlated with poor survival. Knockdown of Zic2 inhibited colon cancer cell growth, arrested the cell cycle transition from G0/G1 to S phase, and suppressed tumor sphere formation in vitro; in addition, silencing Zic2 retarded xenograft tumor formation in vivo. Consistently, ectopic expression of Zic2 had the opposite effects. Mechanistically, Zic2 executed its oncogenic role in colon cancer by enhancing Wnt/β-catenin signaling. Zic2 directly binds to the promoter of Axin2 and transcriptionally represses Axin2 expression and subsequently promotes the accumulation and nuclear translocation of β-catenin. Meanwhile, Zic2 could activate Wnt signaling by interacting with β-catenin. Intriguingly, in HCT116 cells with intrinsic Ser45 mutation of β-catenin, which blocks the degradation-related phosphorylation of β-catenin by CK1, modified Zic2 expression did not affect the protein level of β-catenin. Altogether, our findings uncover a novel multilevel mechanism for the oncogenic activity of Zic2 in colon cancer and suggest Zic2 as a potential therapeutic target for colon cancer patients.
    DOI:  https://doi.org/10.1038/s41419-021-03863-w
  25. Methods Mol Biol. 2021 ;2310 69-77
    Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.
    Mariusz R Wieckowski, Maciej Pronicki, Agnieszka Karkucinska-Wieckowska.
      Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.
    Keywords:  Frozen sections; Histoenzymatic methods; Mitochondrial respiratory chain complexes
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_6
  26. Cell Metab. 2021 May 31. pii: S1550-4131(21)00226-6. [Epub ahead of print]
    Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis.
    Stéphanie Torrino, Eloise M Grasset, Stephane Audebert, Ilyes Belhadj, Caroline Lacoux, Meagan Haynes, Sabrina Pisano, Sophie Abélanet, Frederic Brau, Stephen Y Chan, Bernard Mari, William M Oldham, Andrew J Ewald, Thomas Bertero.
      Mechanical signals from the tumor microenvironment modulate cell mechanics and influence cell metabolism to promote cancer aggressiveness. Cells withstand external forces by adjusting the stiffness of their cytoskeleton. Microtubules (MTs) act as compression-bearing elements. Yet how cancer cells regulate MT dynamic in response to the locally constrained environment has remained unclear. Using breast cancer as a model of a disease in which mechanical signaling promotes disease progression, we show that matrix stiffening rewires glutamine metabolism to promote MT glutamylation and force MT stabilization, thereby promoting cell invasion. Pharmacologic inhibition of glutamine metabolism decreased MT glutamylation and affected their mechanical stabilization. Similarly, decreased MT glutamylation by overexpressing tubulin mutants lacking glutamylation site(s) decreased MT stability, thereby hampering cancer aggressiveness in vitro and in vivo. Together, our results decipher part of the enigmatic tubulin code that coordinates the fine-tunable properties of MT and link cell metabolism to MT dynamics and cancer aggressiveness.
    Keywords:  breast cancer; cancer cell metabolism; glutamine metabolism; glutamylation; mechanobiology; microtubules; posttranslational modifications
    DOI:  https://doi.org/10.1016/j.cmet.2021.05.009
  27. EMBO Mol Med. 2021 Jun 07. e13591
    Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting and its associated metabolism defects.
    Jason Sadek, Derek T Hall, Bianca Colalillo, Amr Omer, Anne-Marie K Tremblay, Virginie Sanguin-Gendreau, William Muller, Sergio Di Marco, Marco Emilio Bianchi, Imed-Eddine Gallouzi.
      Cachexia syndrome develops in patients with diseases such as cancer and sepsis and is characterized by progressive muscle wasting. While iNOS is one of the main effectors of cachexia, its mechanism of action and whether it could be targeted for therapy remains unexplored. Here, we show that iNOS knockout mice and mice treated with the clinically tested iNOS inhibitor GW274150 are protected against muscle wasting in models of both septic and cancer cachexia. We demonstrate that iNOS triggers muscle wasting by disrupting mitochondrial content, morphology, and energy production processes such as the TCA cycle and acylcarnitine transport. Notably, iNOS inhibits oxidative phosphorylation through impairment of complexes II and IV of the electron transport chain and reduces ATP production, leading to energetic stress, activation of AMPK, suppression of mTOR, and, ultimately, muscle atrophy. Importantly, all these effects were reversed by GW274150. Therefore, our data establish how iNOS induces muscle wasting under cachectic conditions and provide a proof of principle for the repurposing of iNOS inhibitors, such as GW274150 for the treatment of cachexia.
    Keywords:  cachexia; cancer; iNOS; inflammation; metabolism
    DOI:  https://doi.org/10.15252/emmm.202013591
  28. Front Oncol. 2021 ;11 639408
    SDHB Suppresses the Tumorigenesis and Development of ccRCC by Inhibiting Glycolysis.
    Zhiyu Fang, Qiang Sun, Huihui Yang, Junfang Zheng.
      Metabolic reprogramming is the prominent feature of clear cell renal cell carcinoma (ccRCC). Succinate dehydrogenase subunit B (SDHB) is one of subunits of mitochondrial respiratory chain complex II. The loss of SDHB function is closely related with metabolic changes in kidney cancer cells. However, the role and molecular mechanism of SDHB in ccRCC occurrence and progression are still unclear. In this study, the results of bioinformatics analyses on GEO, TCGA and oncomine databases and immunohistochemistry showed that the expression level of SDHB was downregulated in ccRCC tissues. SDHB level was gradually downregulated as ccRCC stage and grade progressed. The low level of SDHB was associated with poor prognosis of ccRCC patients, especially for advanced ccRCC patients. Increased methylation levels in SDHB gene promoter led to the downregulation of SDHB level in ccRCC tissues. SDHB was correlated with many metabolism related genes and its interacting proteins were enriched in metabolic pathways. SDHB overexpression suppressed the proliferation, colony formation and migration of ccRCC cells by inhibiting aerobic glycolysis. SDHB may be a potential prognostic marker and therapeutic target for ccRCC.
    Keywords:  SDHB; glycolysis; methylation; prognosis; renal cell carcinoma
    DOI:  https://doi.org/10.3389/fonc.2021.639408
  29. Circulation. 2021 Jun 09.
    PKM1 Exerts Critical Roles in Cardiac Remodeling Under Pressure Overload in the Heart.
    Qinfeng Li, Chao Li, Abdallah Elnwasany, Gaurav Sharma, Yu A An, Guangyu Zhang, Waleed M Elhelaly, Jun Lin, Yingchao Gong, Guihao Chen, Meihui Hui Wang, Shangang Zhao, Chongshan Dai, Charles D Smart, Juan Liu, Xiang Luo, Yingfeng Deng, Lin Tan, Shuang-Jie Lv, Shawn M Davidson, Jason W Locasale, Philip L Lorenzi, Craig R Malloy, Thomas G Gillette, Matthew G Vander Heiden, Philipp E Scherer, Luke I Szweda, Guosheng Fu, Zhao V Wang.
      Background: Metabolic remodeling precedes most alterations during cardiac hypertrophic growth under hemodynamic stress. The elevation of glucose utilization has been recognized as a hallmark of metabolic remodeling. However, its role in cardiac hypertrophic growth and heart failure in response to pressure overload remains to be fully illustrated. Here, we aimed to dissect the role of cardiac PKM1 (pyruvate kinase muscle isozyme 1) in glucose metabolic regulation and cardiac response under pressure overload. Methods: Cardiac specific deletion of PKM1 was achieved by crossing the floxed PKM1 mouse model with the cardiomyocyte-specific Cre transgenic mouse. PKM1 transgenic mice were generated under the control of tetracycline response elements, and cardiac specific overexpression of PKM1 was induced by doxycycline administration in adult mice. Pressure overload was triggered by transverse aortic constriction (TAC). Primary neonatal rat ventricular myocytes were used to dissect molecular mechanisms. Moreover, metabolomics and NMR spectroscopy analyses were conducted to determine cardiac metabolic flux in response to pressure overload. Results: We found that PKM1 expression is reduced in failing human and mouse hearts. Importantly, cardiomyocyte-specific deletion of PKM1 exacerbates cardiac dysfunction and fibrosis in response to pressure overload. Inducible overexpression of PKM1 in cardiomyocytes protects the heart against TAC-induced cardiomyopathy and heart failure. At the mechanistic level, PKM1 is required for the augmentation of glycolytic flux, mitochondrial respiration, and ATP production under pressure overload. Furthermore, deficiency of PKM1 causes a defect in cardiomyocyte growth and a decrease in pyruvate dehydrogenase complex activity at both in vitro and in vivo levels. Conclusions: These findings suggest that PKM1 plays an essential role in maintaining a homeostatic response in the heart under hemodynamic stress.
    Keywords:  PDH; PKM1; glucose oxidation
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.121.054885
  30. Methods Mol Biol. 2021 ;2310 33-45
    BN-PAGE-Based Approach to Study Thyroid Hormones and Mitochondrial Function.
    Elena Silvestri, Assunta Lombardi, Federica Cioffi, Fernando Goglia.
      In recent years, a number of advancements have been made in the study of entire mitochondrial proteomes in both physiological and pathological conditions. Naturally occurring iodothyronines (i.e., T3 and T2) greatly influence mitochondrial oxidative capacity, directly or indirectly affecting the structure and function of the respiratory chain components. Blue native PAGE (BN-PAGE) can be used to isolate enzymatically active oxidative phosphorylation (OXPHOS) complexes in one step, allowing the clinical diagnosis of mitochondrial metabolism by monitoring OXPHOS catalytic and/or structural features. Protocols for isolating mammalian liver mitochondria and subsequent one-dimensional (1D) BN-PAGE will be described in relation to the impact of thyroid hormones on mitochondrial bioenergetics.
    Keywords:  BN-PAGE; Iodothyronine; Mitochondrion; Respiratory chain; Thyroid hormone
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_3
  31. Redox Biol. 2021 Jun 01. pii: S2213-2317(21)00184-1. [Epub ahead of print]45 102026
    Increased glycolysis is an early consequence of palmitate lipotoxicity mediated by redox signaling.
    Pamela A Kakimoto, Julian David C Serna, Vitor de Miranda Ramos, Antonio Zorzano, Alicia J Kowaltowski.
      Exposure to toxic levels of fatty acids (lipotoxicity) leads to cell damage and death and is involved in the pathogenesis of the metabolic syndrome. Since the metabolic consequences of lipotoxicity are still poorly understood, we studied the bioenergetic effects of the saturated fatty acid palmitate, quantifying changes in mitochondrial morphology, real-time oxygen consumption, ATP production sources, and extracellular acidification in hepatoma cells. Surprisingly, glycolysis was enhanced by the presence of palmitate as soon as 1 h after stimulus, while oxygen consumption and oxidative phosphorylation were unchanged, despite overt mitochondrial fragmentation. Palmitate only induced mitochondrial fragmentation if glucose and glutamine were available, while glycolytic enhancement did not require glutamine, showing it is independent of mitochondrial morphological changes. Redox state was altered by palmitate, as indicated by NAD(P)H quantification. Furthermore, the mitochondrial antioxidant mitoquinone, or a selective inhibitor of complex I electron leakage (S1QEL) further enhanced palmitate-induced glycolysis. Our results demonstrate that palmitate overload and lipotoxicity involves an unexpected and early increase in glycolytic flux, while, surprisingly, no changes in oxidative phosphorylation are observed. Interestingly, enhanced glycolysis involves signaling by mitochondrially-generated oxidants, uncovering a novel regulatory mechanism for this pathway.
    Keywords:  Glycolysis; Mitochondria; Oxidative stress; Palmitic acid; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.redox.2021.102026
  32. Methods Mol Biol. 2021 ;2275 379-391
    Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS.
    Irina G Stavrovskaya, Rose M Gathungu, Susan S Bird, Bruce S Kristal.
      Untargeted lipidomics profiling by liquid chromatography -mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high resolution accurate mass (HRAM) MS detection . Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all ion fragmentation can be used to identify species of lower abundances, often missed by data dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.
    Keywords:  Cardiolipins; HCD; LC-MS; Lipidomics; Lysophospholipids; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_24
  33. Chem Sci. 2020 Aug 28. 11(36): 9875-9883
    Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues.
    Soo Yeon Kim, Arup Podder, Hyunseung Lee, Youn-Joo Cho, Eun Hee Han, Sabina Khatun, Jonathan L Sessler, Kwan Soo Hong, Sankarprasad Bhuniya.
      Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson's correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.
    DOI:  https://doi.org/10.1039/d0sc03795h
  34. Oncogene. 2021 Jun 08.
    Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53.
    Patrizia Malkomes, Ilaria Lunger, Elsie Oppermann, Khalil Abou-El-Ardat, Thomas Oellerich, Stefan Günther, Can Canbulat, Sabrina Bothur, Frank Schnütgen, Weijia Yu, Susanne Wingert, Nadine Haetscher, Claudia Catapano, Marina S Dietz, Mike Heilemann, Hans-Michael Kvasnicka, Katharina Holzer, Hubert Serve, Wolf Otto Bechstein, Michael A Rieger.
      Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.
    DOI:  https://doi.org/10.1038/s41388-021-01847-w
  35. Front Chem. 2021 ;9 643796
    Modeling the Energy Landscape of Side Reactions in the Cytochrome bc1 Complex.
    Peter Husen, Ilia A Solov'yov.
      Much of the metabolic molecular machinery responsible for energy transduction processes in living organisms revolves around a series of electron and proton transfer processes. The highly redox active enzymes can, however, also pose a risk of unwanted side reactions leading to reactive oxygen species, which are harmful to cells and are a factor in aging and age-related diseases. Using extensive quantum and classical computational modeling, we here show evidence of a particular superoxide production mechanism through stray reactions between molecular oxygen and a semiquinone reaction intermediate bound in the mitochondrial complex III of the electron transport chain, also known as the cytochrome bc1 complex. Free energy calculations indicate a favorable electron transfer from semiquinone occurring at low rates under normal circumstances. Furthermore, simulations of the product state reveal that superoxide formed at the Q o -site exclusively leaves the bc1 complex at the positive side of the membrane and escapes into the intermembrane space of mitochondria, providing a critical clue in further studies of the harmful effects of mitochondrial superoxide production.
    Keywords:  computational biophysics; electron transfer; enzymes; free energy perturbation; molecular dynamics; proteins; quantum chemical modeling; superoxide
    DOI:  https://doi.org/10.3389/fchem.2021.643796
  36. EMBO J. 2021 Jun 08. e106438
    An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial membrane.
    Fujiao Lv, Fei Qi, Zhi Zhang, Maorong Wen, Justin Kale, Alessandro Piai, Lingyu Du, Shuqing Wang, Liujuan Zhou, Yaqing Yang, Bin Wu, Zhijun Liu, Juan Del Rosario, Justin Pogmore, James J Chou, David W Andrews, Jialing Lin, Bo OuYang.
      Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.
    Keywords:  NMR structure; bax core dimer; functional mutagenesis; membrane lipid bilayer; pore formation
    DOI:  https://doi.org/10.15252/embj.2020106438
  37. Methods Mol Biol. 2021 ;2310 47-56
    Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs).
    Bibiana Correia, Maria Inês Sousa, Ana F Branco, João Ramalho-Santos.
      Mouse embryonic stem cells (mESCs) can be grown in culture, recapitulating the different states of pluripotency of their in vivo counterparts, with notably different metabolic profiles. mESCs in a naïve pluripotent state present an ambivalent metabolism, using both glycolysis and oxidative phosphorylation as energy sources. Here, we describe a method to evaluate the oxidative function of naïve mESCs using the Seahorse Extracellular Flux Analyzer coupled to flow cytometry analysis of mitochondrial transmembrane potential using the TMRM fluorescence probe, thus assessing both oxygen consumption and mitochondrial membrane potential. This may be a useful protocol for understanding how mitochondrial oxidative function and potential of mESCs change in certain circumstances, and how is it related with various pluripotency/differentiation phenotypes.
    Keywords:  Metabolism; Mitochondrial Transmembrane potential; Pluripotency; Seahorse; Stem cells
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_4
  38. J Exp Clin Cancer Res. 2021 Jun 09. 40(1): 190
    Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-ΙΙ.
    Jing Cai, Mei Yi, Yixin Tan, Xiaoling Li, Guiyuan Li, Zhaoyang Zeng, Wei Xiong, Bo Xiang.
       BACKGROUND: Pyroptosis is a lytic cell death form executed by gasdermins family proteins. Induction of tumor pyroptosis promotes anti-tumor immunity and is a potential cancer treatment strategy. Triptolide (TPL) is a natural product isolated from the traditional Chinese herb which possesses potent anti-tumor activity in human cancers. However, its role in pyroptosis remains to be elucidated.
    METHODS: Cell survival was measured by colony formation assay. Cell apoptosis was determined by Annexin V assay. Pyroptosis was evaluated by morphological features and release of interleukin 1β and lactate dehydrogenase A (LDHA). Immunofluorescence staining was employed to measure subcellular localization of proteins. Tumorigenicity was assessed by a xenograft tumor model. Expression levels of mRNAs or proteins were determined by qPCR or western blot assay, respectively.
    RESULTS: Triptolide eliminates head and neck cancer cells through inducing gasdermin E (GSDME) mediated pyroptosis. Silencing GSDME attenuates the cytotoxicity of TPL against cancer cells. TPL treatment suppresses expression of c-myc and mitochondrial hexokinase II (HK-II) in cancer cells, leading to activation of the BAD/BAX-caspase 3 cascade and cleavage of GSDME by active caspase 3. Silencing HK-II sensitizes cancer cells to TPL induced pyroptosis, whereas enforced expression of HK-II prevents TPL induced pyroptosis. Mechanistically, HK-II prevents mitochondrial translocation of BAD, BAX proteins and activation of caspase 3, thus attenuating cleavage of GSDME and pyroptosis upon TPL treatment. Furthermore, TPL treatment suppresses NRF2/SLC7A11 (also known as xCT) axis and induces reactive oxygen species (ROS) accumulation, regardless of the status of GSDME. Combination of TPL with erastin, an inhibitor of SLC7A11, exerts robust synergistic effect in suppression of tumor survival in vitro and in a nude mice model.
    CONCLUSIONS: This study not only provides a new paradigm of TPL in cancer therapy, but also highlights a crucial role of mitochondrial HK-II in linking glucose metabolism with pyroptosis.
    Keywords:  Ferroptosis; Gasdermins; Head and neck squamous cell carcinoma; Nasopharyngeal carcinoma; X(c)(−) cysteine/glutamate antiporter
    DOI:  https://doi.org/10.1186/s13046-021-01995-7
  39. Cancer Biomark. 2021 Jun 05.
    Targeting PKM2 promotes chemosensitivity of breast cancer cells in vitro and in vivo.
    Yu Wang, Han Zhao, Ping Zhao, Xingang Wang.
       BACKGROUND: Pyruvate kinase M2 (PKM2) was overexpressed in many cancers, and high PKM2 expression was related with poor prognosis and chemoresistance.
    OBJECTIVE: We investigated the expression of PKM2 in breast cancer and analyzed the relation of PKM2 expression with chemotherapy resistance to the neoadjuvant chemotherapy (NAC). We also investigated whether PKM2 could reverse chemoresistance in breast cancer cells in vitro and in vivo.
    METHODS: Immunohistochemistry (IHC) was performed in 130 surgical resected breast cancer tissues. 78 core needle biopsies were collected from breast cancer patients before neoadjuvant chemotherapy. The relation of PKM2 expression and multi-drug resistance to NAC was compared. The effect of PKM2 silencing or overexpression on Doxorubicin (DOX) sensitivity in the MCF-7 cells in vitro and in vivo was compared.
    RESULTS: PKM2 was intensively expressed in breast cancer tissues compared to adjacent normal tissues. In addition, high expression of PKM2 was associated with poor prognosis in breast cancer patients. The NAC patients with high PKM2 expression had short survival. PKM2 was an independent prognostic predictor for surgical resected breast cancer and NAC patients. High PKM2 expression was correlated with neoadjuvant treatment resistance. High PKM2 expression significantly distinguished chemoresistant patients from chemosensitive patients. In vitro and in vivo knockdown of PKM2 expression decreases the resistance to DOX in breast cancer cells in vitro and tumors in vivo.
    CONCLUSION: PKM2 expression was associated with chemoresistance of breast cancers, and could be used to predict the chemosensitivity. Furthermore, targeting PKM2 could reverse chemoresistance, which provides an effective treatment methods for patients with breast cancer.
    Keywords:  Breast cancer; chemotherapy; pyruvate kinase M2
    DOI:  https://doi.org/10.3233/CBM-210111
  40. Nat Commun. 2021 06 07. 12(1): 3341
    Quantitative single-cell proteomics as a tool to characterize cellular hierarchies.
    Erwin M Schoof, Benjamin Furtwängler, Nil Üresin, Nicolas Rapin, Simonas Savickas, Coline Gentil, Eric Lechman, Ulrich Auf dem Keller, John E Dick, Bo T Porse.
      Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.
    DOI:  https://doi.org/10.1038/s41467-021-23667-y
  41. J Exp Clin Cancer Res. 2021 Jun 07. 40(1): 188
    A HIF1α-GPD1 feedforward loop inhibits the progression of renal clear cell carcinoma via mitochondrial function and lipid metabolism.
    Ren Liu, Yuanfa Feng, Yulin Deng, Zhihao Zou, Jianheng Ye, Zhiduan Cai, Xuejin Zhu, Yingke Liang, Jianming Lu, Hui Zhang, Yong Luo, Zhaodong Han, Yangjia Zhuo, Qingling Xie, Chi Tin Hon, Yuxiang Liang, Chin-Lee Wu, Weide Zhong.
       BACKGROUND: Hypoxia signaling, especially the hypoxia inducible factor (HIF) pathway, is a major player in clear cell renal cell carcinoma (ccRCC), which is characterized by disorders in lipid and glycogen metabolism. However, the interaction between hypoxia and lipid metabolism in ccRCC progression is still poorly understood.
    METHODS: We used bioinformatic analysis and discovered that glycerol-3-phosphate dehydrogenase 1 (GPD1) may play a key role in hypoxia and lipid metabolism pathways in ccRCC. Tissue microarray, IHC staining, and survival analysis were performed to evaluate clinical function. In vitro and in vivo assays showed the biological effects of GPD1 in ccRCC progression.
    RESULTS: We found that the expression of GPD1 was downregulated in ccRCC tissues, and overexpression of GPD1 inhibited the progression of ccRCC both in vivo and in vitro. Furthermore, we demonstrated that hypoxia inducible factor-1α (HIF1α) directly regulates GPD1 at the transcriptional level, which leads to the inhibition of mitochondrial function and lipid metabolism. Additionally, GPD1 was shown to inhibit prolyl hydroxylase 3 (PHD3), which blocks prolyl-hydroxylation of HIF1α and subsequent proteasomal degradation, and thus reinforces the inhibition of mitochondrial function and phosphorylation of AMPK via suppressing glycerol-3-phosphate dehydrogenase 2 (GPD2).
    CONCLUSIONS: This study not only demonstrated that HIF1α-GPD1 forms a positive feedforward loop inhibiting mitochondrial function and lipid metabolism in ccRCC, but also discovered a new mechanism for the molecular basis of HIF1α to inhibit tumor activity, thus providing novel insights into hypoxia-lipid-mediated ccRCC therapy.
    Keywords:  GPD1; HIF1α; Hypoxia; Metabolism; ccRCC
    DOI:  https://doi.org/10.1186/s13046-021-01996-6
  42. Cell Rep. 2021 Jun 08. pii: S2211-1247(21)00562-3. [Epub ahead of print]35(10): 109212
    High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity.
    Miyeko D Mana, Amanda M Hussey, Constantine N Tzouanas, Shinya Imada, Yesenia Barrera Millan, Dorukhan Bahceci, Dominic R Saiz, Anna T Webb, Caroline A Lewis, Peter Carmeliet, Maria M Mihaylova, Alex K Shalek, Ömer H Yilmaz.
      Obesity is an established risk factor for cancer in many tissues. In the mammalian intestine, a pro-obesity high-fat diet (HFD) promotes regeneration and tumorigenesis by enhancing intestinal stem cell (ISC) numbers, proliferation, and function. Although PPAR (peroxisome proliferator-activated receptor) nuclear receptor activity has been proposed to facilitate these effects, their exact role is unclear. Here we find that, in loss-of-function in vivo models, PPARα and PPARδ contribute to the HFD response in ISCs. Mechanistically, both PPARs do so by robustly inducing a downstream fatty acid oxidation (FAO) metabolic program. Pharmacologic and genetic disruption of CPT1A (the rate-controlling enzyme of mitochondrial FAO) blunts the HFD phenotype in ISCs. Furthermore, inhibition of CPT1A dampens the pro-tumorigenic consequences of a HFD on early tumor incidence and progression. These findings demonstrate that inhibition of a HFD-activated FAO program creates a therapeutic opportunity to counter the effects of a HFD on ISCs and intestinal tumorigenesis.
    Keywords:  Apc; Cpt1a; Ppar; fatty acid oxidation; high-fat diet; intestinal stem cells
    DOI:  https://doi.org/10.1016/j.celrep.2021.109212
  43. Methods Mol Biol. 2021 ;2310 301-309
    Measuring PGC-1α and Its Acetylation Status in Mouse Primary Myotubes.
    João A Amorim, David A Sinclair.
      Metabolic flexibility is vital for organisms to respond to and survive changes in energy availability. A critical metabolic flexibility regulator is peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which regulates various transcription factors and nuclear receptors that, in turn, regulate mitochondrial homeostasis and fatty acid oxidation. PGC-1α is itself regulated, with one of the significant modes of regulation being acetylation. Thus, measuring the acetylation status of PGC-1α is a critical indicator of cells' metabolic flexibility. In this chapter, we describe a method of evaluating PGC-1α acetylation in primary mouse myotubes. This method can also be used with other cell types and tissues.
    Keywords:  Acetylation; Aging; Cachexia; Diabetes; GCN5; Mitochondrial biogenesis; PGC-1α; Respiration; SIRT1; Sarcopenia; Sirtuin deacetylase
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_17
  44. Oxid Med Cell Longev. 2021 ;2021 6617256
    The Role of Mitophagy in Regulating Cell Death.
    Sunao Li, Jiaxin Zhang, Chao Liu, Qianliang Wang, Jun Yan, Li Hui, Qiufang Jia, Haiyan Shan, Luyang Tao, Mingyang Zhang.
      Mitochondria are multifaceted organelles that serve to power critical cellular functions, including act as power generators of the cell, buffer cytosolic calcium overload, production of reactive oxygen species, and modulating cell survival. The structure and the cellular location of mitochondria are critical for their function and depend on highly regulated activities such as mitochondrial quality control (MQC) mechanisms. The MQC is regulated by several sets of processes: mitochondrial biogenesis, mitochondrial fusion and fission, mitophagy, and other mitochondrial proteostasis mechanisms such as mitochondrial unfolded protein response (mtUPR) or mitochondrial-derived vesicles (MDVs). These processes are important for the maintenance of mitochondrial homeostasis, and alterations in the mitochondrial function and signaling are known to contribute to the dysregulation of cell death pathways. Recent studies have uncovered regulatory mechanisms that control the activity of the key components for mitophagy. In this review, we discuss how mitophagy is controlled and how mitophagy impinges on health and disease through regulating cell death.
    DOI:  https://doi.org/10.1155/2021/6617256
  45. Methods Mol Biol. 2021 ;2310 287-299
    Following the Dynamism of the Mitochondrial Network in T Cells.
    Arianna Di Daniele, Luca Simula, Silvia Campello.
      The dynamism of mitochondria, considered as complex and motile organelles, is brought about by mitochondria ability to undergo cycles of fission and fusion events, whose fine balance determines their morphology in a specific physiological context. A huge body of evidence makes it possible to associate mitochondrial organization to regulation of an increasing number of key cellular processes, such as biosynthetic pathways, oxidative phosphorylation and ATP production, calcium buffering, mtDNA homeostasis, autophagy, and cell death. Here, we review the recently developed imaging methods for studying mitochondrial dynamics, including live-cell imaging, by using mitochondrial-targeted fluorescent proteins. In more details, we focus our attention on two different protocols in the T cell model, an example of nonadherent cells, which present some particularities and difficulties in the analysis of mitochondrial shape. Also, we discuss some examples of mouse models carrying mitochondria-targeted fluorescent proteins, which allow to investigate the mitochondrial morphology in vivo.
    Keywords:  Dynamic network; Fission; Fusion; Imaging; Mitochondria; Morphology
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_16
  46. Methods Mol Biol. 2021 ;2275 65-85
    Novel Mitochondria-Targeted Triphenylphosphonium Conjugates of Linear β-Phosphorylated Nitrones : Preparation, 31P NMR Mitochondrial Distribution, EPR Spin Trapping Reporting, and Site-Directed Antiapoptotic Properties.
    Consuelo Petrocchi, Sophie Thétiot-Laurent, Marcel Culcasi, Sylvia Pietri.
      The mitochondrion can be considered as the metabolic powerhouse of the cell, having a key impact on energy production, cell respiration, and intrinsic cell death. Mitochondria are also the main source of endogenous reactive oxygen species , including free radicals (FR), which are physiologically involved in signaling pathways but may promote cell damage when unregulated or excessively formed in inappropriate locations. A variety of chronic pathologies have been associated with FR-induced mitochondrial dysfunctions , such as cancer, age-related neurodegenerative diseases, and metabolic syndrome.In recent years drug design based on specific mitochondria-targeted antioxidants has become a very attractive therapeutic strategy and, among target compounds, nitrones have received growing attention because of their specific affinity toward FR. Here, we describe protocols dealing with the preparation, mitochondria permeation assessment, electron paramagnetic resonance (EPR) spin trapping setting, and antiapoptotic properties evaluation of a series of new linear nitrones vectorized by a triphenylphosphonium cation and labeled with a diethoxyphosphoryl moiety as 31P nuclear magnetic resonance (NMR) reporter with antioxidant property.
    Keywords:  31P NMR; EPR spin trapping; H2O2-induced apoptosis; Mitochondria-targeted nitrones; Mitochondrial permeability
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_5
  47. Methods Mol Biol. 2021 ;2275 329-339
    Mitochondrial Coenzyme Q10 Determination Via Isotope Dilution Liquid Chromatography -Tandem Mass Spectrometry.
    Erik Hedman, Outi Itkonen.
      Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain . Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria . In the assay, mitochondrial suspensions are spiked with CoQ10-[2H9] internal standard (IS), extracted with organic solvents and CoQ10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).
    Keywords:  Coenzyme Q10; Isotope dilution; LC-MS/MS; Mitochondrial disease; Ubiquinone
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_21
  48. Cancer Res. 2021 Jun 11. pii: canres.3792.2020. [Epub ahead of print]
    Targeting glucose metabolism sensitizes pancreatic cancer to MEK inhibition.
    Liang Yan, Bo Tu, Jun Yao, Jing Gong, Alessandro Carugo, Christopher A Bristow, Qiuyun Wang, Cihui Zhu, Bingbing Dai, Ya'an Kang, Leng Han, Ningping Feng, Yanqing Jin, Jason Fleming, Timothy P Heffernan, Wantong Yao, Haoqiang Ying.
      Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D-driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D-driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal ER stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3792
  49. EMBO Rep. 2021 Jun 04. 22(6): e51323
    Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.
    Hyun Ku Yeo, Tae Hyun Park, Hee Yeon Kim, Hyonchol Jang, Jueun Lee, Geum-Sook Hwang, Seong Eon Ryu, Si Hoon Park, Hyun Kyu Song, Hyun Seung Ban, Hye-Jin Yoon, Byung Il Lee.
      In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.
    Keywords:  MAM; PTPIP51; endoplasmic reticulum; mitochondria; phospholipid
    DOI:  https://doi.org/10.15252/embr.202051323
  50. Methods Mol Biol. 2021 ;2275 247-263
    mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.
    Laurent Chatre, Miria Ricchetti.
      Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.
    Keywords:  DNA replication; FISH; Imaging; Mitochondrial DNA; Mitochondrial disease; Single-cell; Transcription; mTRIP
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_15
  51. J Lipid Atheroscler. 2021 May;10(2): 223-239
    Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury.
    Nammi Park, Jubert Marquez, Maria Victoria Faith Garcia, Ippei Shimizu, Sung Ryul Lee, Hyoung Kyu Kim, Jin Han.
       Objective: Ischemic cardiomyopathy (ICM) is the leading cause of heart failure. Proteomic and genomic studies have demonstrated ischemic preconditioning (IPC) can assert cardioprotection against ICM through mitochondrial function regulation. Considering IPC is conducted in a relatively brief period, regulation of protein expression also occurs very rapidly, highlighting the importance of protein function modulation by post-translational modifications. This study aimed to identify and analyze novel phosphorylated mitochondrial proteins that can be harnessed for therapeutic strategies for preventing ischemia/reperfusion (I/R) injury.
    Methods: Sprague-Dawley rat hearts were used in an ex vivo Langendorff system to simulate normal perfusion, I/R, and IPC condition, after which the samples were prepared for phosphoproteomic analysis. Employing human cardiomyocyte AC16 cells, we investigated the cardioprotective role of CKMT2 through overexpression and how site-directed mutagenesis of putative CKMT2 phosphorylation sites (Y159A, Y255A, and Y368A) can affect cardioprotection by measuring CKMT2 protein activity, mitochondrial function and protein expression changes.
    Results: The phosphoproteomic analysis revealed dephosphorylation of mitochondrial creatine kinase (CKMT2) during ischemia and I/R, while preserving its phosphorylated state during IPC. CKMT2 overexpression conferred cardioprotection against hypoxia/reoxygenation (H/R) by increasing cell viability and mitochondrial adenosine triphosphate level, preserving mitochondrial membrane potential, and reduced reactive oxygen species (ROS) generation, while phosphomutations, especially in Y368, nullified cardioprotection by significantly reducing cell viability and increasing ROS production during H/R. CKMT2 overexpression increased mitochondrial function by mediating the proliferator-activated receptor γ coactivator-1α/estrogen-related receptor-α pathway, and these effects were mostly inhibited by Y368A mutation.
    Conclusion: These results suggest that regulation of quantitative expression and phosphorylation site Y368 of CKMT2 offers a unique mechanism in future ICM therapeutics.
    Keywords:  Creatine kinase, mitochondrial form; Hypoxia; Mitochondria; Phosphorylation; Reoxygenation
    DOI:  https://doi.org/10.12997/jla.2021.10.2.223
  52. Methods Mol Biol. 2021 ;2310 91-111
    PCR-Based Determination of Mitochondrial DNA Copy Number in Multiple Species.
    Tess C Leuthner, Jessica H Hartman, Ian T Ryde, Joel N Meyer.
      Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter, we describe methods for simultaneous isolation of mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents. We also briefly describe factors that affect mtDNA copy number and discuss caveats to its use as a biomarker.
    Keywords:  Copy number; Mitochondrial DNA; Mitochondrial disease; Mitochondrial toxicity; QPCR; mtDNA; mtDNA depletion
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_8
  53. Methods Mol Biol. 2021 ;2275 291-299
    Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine.
    Sander Grefte, Werner J H Koopman.
      Reactive oxygen species (ROS) play an important role in cellular (patho)physiology. Empirical evidence suggests that mitochondria are an important source of ROS, especially under pathological conditions. Here, we describe a method for ROS measurement using dihydroethidium (HEt) and live-cell microscopy.
    Keywords:  Fluorescence imaging; MitoSOX Red®; Mitochondrial membrane potential
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_18
  54. Am J Cancer Res. 2021 ;11(5): 2303-2311
    Mitochondrial structural alterations in ovarian cancer patient-derived xenografts resistant to cisplatin.
    Francesca Ricci, Alessandro Corbelli, Roberta Affatato, Rosaria Chilà, Michela Chiappa, Laura Brunelli, Robert Fruscio, Roberta Pastorelli, Fabio Fiordaliso, Giovanna Damia.
      Mitochondria have attracted attention in cancer research as organelles associated with tumor development and response to therapy. We recently reported acquisition of resistance to cisplatin (DDP) associated with a metabolic rewiring in ovarian cancer patient-derived xenografts (PDXs) models. DDP-resistant PDXs models were obtained mimicking the clinical setting, treating mice bearing sensitive-DDP tumors with multiple cycles of DDP until the development of resistance. To further characterize the metabolic rewiring, the present study focused on tumor mitochondria. We analysed by transmission electron microscopy the mitochondria structure in two models of DDP-resistant and the corresponding DDP-sensitive PDXs and evaluated tumor mDNA content, the expression of genes and proteins involved in mitochondria functionality, and mitochondria fitness-related processes, such as autophagy. We observed a decrease in the number of mitochondria paralleled by an increased volume in DDP-resistant versus DDP-sensitive PDXs. DDP-resistant PDXs presented a higher percentage of damaged mitochondria, in particular of type 2 (concave-shape), and type 3 (cristolysis) damage. We found no difference in the mDNA content, and the expression of genes involved in mitochondrial biogenesis was similar between the sensitive and resistant PDXs. An upregulation of some genes involved in mitochondrial fitness in DDP-R versus DDP-S PDXs was observed. At protein level, no difference in the expression of proteins involved in mitochondrial function and biogenesis, and in autophagy/mitophagy was found. We here reported that the acquisition of DDP resistance is associated with morphological alterations in mitochondria, even if we couldn't find any dysregulation in the studied genes/proteins that could explain the observed differences.
    Keywords:  Mitochondria; platinum resistance and ovarian carcinoma
  55. Methods Mol Biol. 2021 ;2275 301-314
    Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.
    Bettina Rieger, Karin B Busch.
      Our group has previously established a strategy utilizing fluorescence lifetime probes to image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individual mitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performing SC-FLIM, including the evaluation of fluorescence lifetimes from the FLIM images. To validate the feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.
    Keywords:  FLIM; Fluorescence sensor; Live cell imaging; Mitochondria; Respiratory supercomplexes
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_19
  56. Methods Mol Biol. 2021 ;2275 217-225
    Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.
    Takuya Hidaka, Hiroshi Sugiyama, Ganesh N Pandian.
      Mitochondria possess multiple copies of mitochondrial DNA (mtDNA) that encode 37 genes and their transcription and replication get controlled by unique molecular codes different from that in the nuclear DNA. The mtDNA has been gaining increased attention as one of the critical therapeutic targets as mutations in them impair the function of mitochondria and cause mitochondrial diseases like MELAS. In this chapter, we describe artificial control of mitochondrial transcription based on mtDNA sequence information with a new type of compounds termed MITO-PIPs, which encompasses two domains: pyrrole-imidazole polyamide as DNA recognition domain and mitochondrial penetrating peptide as the mitochondria-targeting domain. Because MITO-PIPs are amenable to tunability, they can be expanded as a synthetic strategy to modulate mitochondrial gene(s) on demand.
    Keywords:  DNA binding ligand; Fmoc solid-phase synthesis; MITO-PIP; Mitochondrial DNA; Pyrrole–imidazole polyamide; Transcription
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_13
  57. Exerc Sport Sci Rev. 2021 Jun 05.
    Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability.
    Michael D Tarpey, Adam J Amorese, Elizabeth R LaFave, Everett C Minchew, Kelsey H Fisher-Wellman, Joseph M McClung, Eli G Hvastkovs, Espen E Spangenburg.
       ABSTRACT: Breast Cancer gene 1 (BRCA1) is a large, multi-functional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.
    DOI:  https://doi.org/10.1249/JES.0000000000000265
  58. Biochimie. 2021 Jun 04. pii: S0300-9084(21)00145-0. [Epub ahead of print]
    The allosteric protein interactions in the proton-motive function of mammalian redox enzymes of the respiratory chain.
    Giuseppe Capitanio, Francesco Papa, Sergio Papa.
      Insight into mammalian respiratory complexes defines the role of allosteric protein interactions in their proton-motive activity. In cytochrome c oxidase (CxIV) conformational change of subunit I, caused by O2 binding to heme a32+-CuB+ and reduction, and stereochemical transitions coupled to oxidation/reduction of heme a and CuA, combined with electrostatic effects, determine the proton pumping activity. In ubiquinone-cytochrome c oxidoreductase (CxIII) conformational movement of Fe-S protein between cytochromes b and c1 is the key element of the proton-motive activity. In NADH-ubiquinone oxidoreductase (CxI) ubiquinone binding and reduction result in conformational changes of subunits in the quinone reaction structure which initiate proton pumping.
    Keywords:  Allosteric protein interactions; Contents; Mitochondria; Proton energy conversion; Proton-motive activity; Redox enzyme complexes; Respiratory chain
    DOI:  https://doi.org/10.1016/j.biochi.2021.05.018
  59. Mitochondrion. 2021 Jun 05. pii: S1567-7249(21)00078-7. [Epub ahead of print]
    A hitchhiker's guide to mitochondrial quantification.
    Irene Mgm Hemel, Bob Ph Engelen, Nicole Luber, Mike Gerards.
      The variety of available mitochondrial quantification tools makes it difficult to select the most reliable and accurate quantification tool. Here, we performed elaborate analyses on five open source ImageJ tools. Excessive clustering of mitochondrial structures was observed in four tools, caused by the global thresholding applied by these tools. The Mitochondrial Analyzer, which uses adaptive thresholding, outperformed the other examined tools, with accurate structural segregation and identification. Additionally, we showed that the Mitochondrial Analyzer successfully identifies mitochondrial morphology differences. Based on the observed performance, we consider the Mitochondrial Analyzer the best open source tool for mitochondrial network morphology quantification.
    Keywords:  Image analysis; ImageJ; Mitochondria; Mitochondrial Dynamics; Mitochondrial quantification
    DOI:  https://doi.org/10.1016/j.mito.2021.06.005
  60. Methods Mol Biol. 2021 ;2275 315-327
    Identification of Peroxynitrite by Profiling Oxidation and Nitration Products from Mitochondria-Targeted Arylboronic Acid.
    Jacek Zielonka, Adam Sikora, Radoslaw Podsiadly, Micael Hardy, B Kalyanaraman.
      The development of boronic probes enabled reliable detection and quantitative analysis of hydrogen peroxide , other nucleophilic hydroperoxides, hypochlorite , and peroxynitrite . The major product, in which boronate moiety of the probe is replaced by the hydroxyl group, is, however, common for all those oxidants. Here, we describe how ortho-isomer of mitochondria-targeted phenylboronic acid can be used to detect and differentiate peroxynitrite-dependent and independent probe oxidation. This method highlights detection and quantification of both the major, phenolic product and the minor, peroxynitrite-specific cyclic and nitrated products of probe oxidation.
    Keywords:  Boronic probes; HPLC-MS; Hydrogen peroxide; Mitochondria-targeted probes; Peroxynitrite; o-MitoPhB(OH)2
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_20
  61. Methods Mol Biol. 2021 ;2310 259-270
    Mitochondrial Regulation Assessment by 13C-NMR Isotopomer Analysis.
    Francisco X Carvalho, Bárbara Guerra-Carvalho, Ivana Jarak, Rui A Carvalho.
      Mitochondria play a central role in metabolic reprograming that occurs in numerous disease conditions. A precise evaluation of the extent of mitochondrial involvement in the metabolic alterations is essential for a better definition of metabolically based therapeutic strategies. In this chapter, some simple protocols are presented, using carbon 13 tracers and nuclear magnetic resonance isotopomer analysis, for the evaluation of mitochondrial contributions to intermediary metabolism and the metabolic effects of the implementation of some mitochondrial regulatory mechanisms.
    Keywords:  13C isotope tracers; Intermediary metabolism; Metabolic reprograming; Mitochondria; Mitochondrial regulation; NMR isotopomer analysis; TCA cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_14
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