bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2019‒01‒27
57 papers selected by
Christian Frezza,

  1. Loss of BCAA Catabolism during Carcinogenesis Enhances mTORC1 Activity and Promotes Tumor Development and Progression.
  2. Impairments in left ventricular mitochondrial bioenergetics precede overt cardiac dysfunction and remodelling in Duchenne muscular dystrophy.
  3. Mitochondrial Proteolysis and Metabolic Control.
  4. Oncogenic Metabolism Acts as a Prerequisite Step for Induction of Cancer Metastasis and Cancer Stem Cell Phenotype.
  5. Mitochondrial Populations Exhibit Differential Dynamic Responses to Increased Energy Demand during Exocytosis In Vivo.
  6. Inhibiting neddylation modification alters mitochondrial morphology and reprograms energy metabolism in cancer cells.
  7. ER-mitochondria interactions: Both strength and weakness within cancer cells.
  8. Is there an effect of ischemic conditioning on myocardial contractile function following acute myocardial ischemia/reperfusion injury?
  9. Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy.
  10. The yeast mitochondrial proteins Rcf1 and Rcf2 support the enzymology of the cytochrome c oxidase complex and generation of the proton motive force.
  11. Assessing Calcium-Stimulated Mitochondrial Bioenergetics Using the Seahorse XF96 Analyzer.
  12. Coenzyme Q biosynthetic proteins assemble in a substrate-dependent manner into domains at ER-mitochondria contacts.
  13. Mitochondrial Stress-Initiated Aberrant Activation of the NLRP3 Inflammasome Regulates the Functional Deterioration of Hematopoietic Stem Cell Aging.
  14. Pre-clinical evaluation of cysteamine bitartrate as a therapeutic agent for mitochondrial respiratory chain disease.
  15. Splitting the functions of Rim2, a mitochondrial iron/pyrimidine carrier.
  16. Electrophysiological Characterization of Calcium-Permeable Channels Using Planar Lipid Bilayer.
  17. The SEACIT complex is involved in the maintenance of vacuole-mitochondria contact sites and controls mitophagy.
  18. Mitochondrial stress response triggered by defects in protein synthesis quality control.
  19. Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells.
  20. Prohibitins: A Critical Role in Mitochondrial Functions and Implication in Diseases.
  21. Mechanisms by which hydrogen sulfide attenuates muscle function following ischemia-reperfusion injury: effects on Akt signaling, mitochondrial function, and apoptosis.
  22. The Role of O-GlcNAcylation for Protection against Ischemia-Reperfusion Injury.
  23. Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.
  24. Pomegranate activates TFEB to promote autophagy-lysosomal fitness and mitophagy.
  25. Metabolic rewiring of macrophages by CpG potentiates clearance of cancer cells and overcomes tumor-expressed CD47-mediated 'don't-eat-me' signal.
  26. Mechanistic Target of Rapamycin Complex 1 Promotes the Expression of Genes Encoding Electron Transport Chain Proteins and Stimulates Oxidative Phosphorylation in Primary Human Trophoblast Cells by Regulating Mitochondrial Biogenesis.
  27. Ulk1/Rab9-mediated alternative mitophagy confers cardioprotection during energy stress.
  28. Mitochondrial dynamics and inter-mitochondrial communication in the heart.
  29. S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α.
  30. Interplay between TRAP1 and sirtuin-3 modulates mitochondrial respiration and oxidative stress to maintain stemness of glioma stem cells.
  31. The Roles of Mitochondrial SIRT4 in Cellular Metabolism.
  32. Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.
  33. Purification of Functional F-ATP Synthase from Blue Native PAGE.
  34. Glutaminase 1 expression in colorectal cancer cells is induced by hypoxia and required for tumor growth, invasion, and metastatic colonization.
  35. ACOT12-Dependent Alteration of Acetyl-CoA Drives Hepatocellular Carcinoma Metastasis by Epigenetic Induction of Epithelial-Mesenchymal Transition.
  36. p53/BNIP3-dependent mitophagy limits glycolytic shift in radioresistant cancer.
  37. Uncoupling protein 2 reprograms the tumor microenvironment to support the anti-tumor immune cycle.
  38. Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter.
  39. Autophagic cell death restricts chromosomal instability during replicative crisis.
  40. An IRAK1-PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy.
  41. In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.
  42. Lactate modulates CD4+ T-cell polarization and induces an immunosuppressive environment, which sustains prostate carcinoma progression via TLR8/miR21 axis.
  43. 3-Phosphoglycerate Transhydrogenation Instead of Dehydrogenation Alleviates the Redox State Dependency of Yeast de Novo l-Serine Synthesis.
  44. NK cell metabolism.
  45. IDH-1 deficiency induces growth defects and metabolic alterations in GSPD-1-deficient Caenorhabditis elegans.
  46. MCU Regulation in Lipid Bilayer and Electrophysiological Recording.
  47. Heme detoxification by heme oxygenase-1 reinstates proliferative and immune balances upon genotoxic tissue injury.
  48. GC-MS Metabolomics Reveals Distinct Profiles of Low- and High-Grade Bladder Cancer Cultured Cells.
  49. Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells.
  50. Formylation of Eukaryotic Cytoplasmic Proteins: Linking Stress to Degradation.
  51. Ex Vivo Measurements of Ca2+ Transients in Intracellular Compartments of Skeletal Muscle Fibers by Means of Genetically Encoded Probes.
  52. In vivo Ca2+ imaging of astrocytic microdomains reveals a critical role of the amyloid precursor protein for mitochondria.
  53. Molecular intricacies of aerobic glycolysis in cancer: current insights into the classic metabolic phenotype.
  54. Inflammation-induced glycolytic switch controls suppressivity of mesenchymal stem cells via STAT1 glycosylation.
  55. A genetically encoded near-infrared fluorescent calcium ion indicator.
  56. In Vivo Monitoring of Ca2+ Uptake into Subcellular Compartments of Mouse Skeletal Muscle.
  57. Targeting Metabolic-Redox Circuits for Cancer Therapy.
  1. Cell Metab. 2019 Jan 08. pii: S1550-4131(18)30757-5. [Epub ahead of print]
    Loss of BCAA Catabolism during Carcinogenesis Enhances mTORC1 Activity and Promotes Tumor Development and Progression.
    Russell E Ericksen, Siew Lan Lim, Eoin McDonnell, Wai Ho Shuen, Maya Vadiveloo, Phillip J White, Zhaobing Ding, Royston Kwok, Philip Lee, George K Radda, Han Chong Toh, Matthew D Hirschey, Weiping Han.
      Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers.
    Keywords:  branched-chain amino acids; cancer; cancer metabolism; dietary intake; hepatocellular carcinoma; mTORC1; metabolomics; transcriptomics
    DOI:  https://doi.org/10.1016/j.cmet.2018.12.020
  2. J Physiol. 2019 Jan 23.
    Impairments in left ventricular mitochondrial bioenergetics precede overt cardiac dysfunction and remodelling in Duchenne muscular dystrophy.
    Meghan C Hughes, Sofhia V Ramos, Patrick C Turnbull, Brittany A Edgett, Jason S Huber, Nazari Polidovitch, Uwe Schlattner, Peter H Backx, Jeremy A Simpson, Christopher G R Perry.
      KEY POINTS SUMMARY: -98% of patients with Duchenne muscular dystrophy (DMD) develop cardiomyopathy, with 40% developing heart failure -While increased propensity for mitochondrial induction of cell death has been observed in left ventricle, it remains unknown whether this is linked to impaired mitochondrial respiratory control and elevated H2 O2 emission prior to the onset of cardiomyopathy -Classic mouse models of DMD demonstrate hyper-regeneration in skeletal muscle which may mask mitochondrial abnormalities. Using a model with less regenerative capacities that is more akin to DMD patients, we observed elevated left ventricular mitochondrial H2 O2 and impaired oxidative phosphorylation in the absence of cardiac remodelling or overt cardiac dysfunction at 4 weeks -These impairments were associated with dysfunctions at Complex I, governance by ADP and creatine-dependent phosphate shuttling which results in a less efficient response to energy demands -Mitochondria may be a therapeutic target for the treatment of cardiomyopathy in DMD ABSTRACT: In Duchenne muscular dystrophy (DMD), mitochondrial dysfunction is predicted as a response to numerous cellular stressors yet the degree and contribution of mitochondria to the onset of cardiomyopathy remains unknown. To resolve this uncertainty, we designed in vitro assessments of mitochondrial bioenergetics to model mitochondrial control parameters that influence cardiac function. Both left ventricular mitochondrial responsiveness to the central bioenergetic controller ADP as well as the ability of creatine to facilitate mitochondrial-cytoplasmic phosphate shuttling were assessed. These measurements were performed in D2.B10-DMDmdx /2J mice - a model that demonstrates skeletal muscle atrophy and weakness due to limited regenerative capacities and cardiomyopathy more akin to people with DMD than classic models. At 4 weeks of age, there was no evidence of cardiac remodelling or cardiac dysfunction despite impairments in ADP-stimulated respiration and ADP- attenuation of H2 O2 emission. These impairments were seen at both sub-maximal and maximal ADP concentrations despite no reductions in mitochondrial content markers. The ability of creatine to enhance ADP's control of mitochondrial bioenergetics was also impaired, suggesting an impairment in mitochondrial creatine kinase-dependent phosphate shuttling. Susceptibly to permeability transition pore opening and the subsequent activation of cell death pathways remained unchanged. Mitochondrial H2 O2 emission was elevated despite no change in markers of irreversible oxidative damage, suggesting alternative redox signalling mechanisms should be explored. These findings demonstrate that selective mitochondrial dysfunction precedes the onset of overt cardiomyopathy in D2.mdx mice, suggesting that improving mitochondrial bioenergetics by restoring ADP, creatine-dependent phosphate shuttling and Complex I should be considered for treating DMD patients. This article is protected by copyright. All rights reserved.
    Keywords:  Duchenne muscular dystrophy; cardiomyopathy; mitochondria; reactive oxygen species; respiration
    DOI:  https://doi.org/10.1113/JP277306
  3. Cold Spring Harb Perspect Biol. 2019 Jan 22. pii: a033936. [Epub ahead of print]
    Mitochondrial Proteolysis and Metabolic Control.
    Sofia Ahola, Thomas Langer, Thomas MacVicar.
      Mitochondria are metabolic hubs that use multiple proteases to maintain proteostasis and to preserve their overall quality. A decline of mitochondrial proteolysis promotes cellular stress and may contribute to the aging process. Mitochondrial proteases have also emerged as tightly regulated enzymes required to support the remarkable mitochondrial plasticity necessary for metabolic adaptation in a number of physiological scenarios. Indeed, the mutation and dysfunction of several mitochondrial proteases can cause specific human diseases with severe metabolic phenotypes. Here, we present an overview of the proteolytic regulation of key mitochondrial functions such as respiration, lipid biosynthesis, and mitochondrial dynamics, all of which are required for metabolic control. We also pay attention to how mitochondrial proteases are acutely regulated in response to cellular stressors or changes in growth conditions, a greater understanding of which may one day uncover their therapeutic potential.
    DOI:  https://doi.org/10.1101/cshperspect.a033936
  4. Oxid Med Cell Longev. 2018 ;2018 1027453
    Oncogenic Metabolism Acts as a Prerequisite Step for Induction of Cancer Metastasis and Cancer Stem Cell Phenotype.
    Su Yeon Lee, Min Kyung Ju, Hyun Min Jeon, Yig Ji Lee, Cho Hee Kim, Hye Gyeong Park, Song Iy Han, Ho Sung Kang.
      Metastasis is a major obstacle to the efficient and successful treatment of cancer. Initiation of metastasis requires epithelial-mesenchymal transition (EMT) that is regulated by several transcription factors, including Snail and ZEB1/2. EMT is closely linked to the acquisition of cancer stem cell (CSC) properties and chemoresistance, which contribute to tumor malignancy. Tumor suppressor p53 inhibits EMT and metastasis by negatively regulating several EMT-inducing transcription factors and regulatory molecules; thus, its inhibition is crucial in EMT, invasion, metastasis, and stemness. Metabolic alterations are another hallmark of cancer. Most cancer cells are more dependent on glycolysis than on mitochondrial oxidative phosphorylation for their energy production, even in the presence of oxygen. Cancer cells enhance other oncogenic metabolic pathways, such as glutamine metabolism, pentose phosphate pathway, and the synthesis of fatty acids and cholesterol. Metabolic reprogramming in cancer is regulated by the activation of oncogenes or loss of tumor suppressors that contribute to tumor progression. Oncogenic metabolism has been recently linked closely with the induction of EMT or CSC phenotypes by the induction of several metabolic enzyme genes. In addition, several transcription factors and molecules involved in EMT or CSCs, including Snail, Dlx-2, HIF-1α, STAT3, TGF-β, Wnt, and Akt, regulate oncogenic metabolism. Moreover, p53 induces metabolic change by directly regulating several metabolic enzymes. The collective data indicate the importance of oncogenic metabolism in the regulation of EMT, cell invasion and metastasis, and adoption of the CSC phenotype, which all contribute to malignant transformation and tumor development. In this review, we highlight the oncogenic metabolism as a key regulator of EMT and CSC, which is related with tumor progression involving metastasis and chemoresistance. Targeting oncometabolism might be a promising strategy for the development of effective anticancer therapy.
    DOI:  https://doi.org/10.1155/2018/1027453
  5. iScience. 2019 Jan 04. pii: S2589-0042(18)30266-9. [Epub ahead of print]11 440-449
    Mitochondrial Populations Exhibit Differential Dynamic Responses to Increased Energy Demand during Exocytosis In Vivo.
    Natalie Porat-Shliom, Olivia J Harding, Lenka Malec, Kedar Narayan, Roberto Weigert.
      Mitochondria are dynamic organelles undergoing fission, fusion, and translocation. These processes have been studied in cultured cells; however, little is known about their regulation in cells within tissues in vivo. We applied four-dimensional intravital microscopy to address this in secretory cells of the salivary gland. We found that mitochondria are organized in two populations: one juxtaposed to the basolateral plasma membrane and the other dispersed in the cytosol. Under basal conditions, central mitochondria exhibit microtubule-dependent motility and low fusion rate, whereas basolateral mitochondria are static and display high fusion rate. Increasing cellular energy demand by β-adrenergic stimulation of regulated exocytosis selectively enhanced motility and fusion of central mitochondria. Inhibition of microtubule polymerization led to inhibition of central mitochondrial motility and fusion and a marked reduction in exocytosis. This study reveals a conserved heterogeneity in mitochondrial positioning and dynamics in exocrine tissues that may have fundamental implications in organ pathophysiology.
    Keywords:  Cell Biology; Functional Aspects of Cell Biology; Optical Imaging
    DOI:  https://doi.org/10.1016/j.isci.2018.12.036
  6. JCI Insight. 2019 Jan 22. pii: 121582. [Epub ahead of print]
    Inhibiting neddylation modification alters mitochondrial morphology and reprograms energy metabolism in cancer cells.
    Qiyin Zhou, Hua Li, Yuanyuan Li, Mingjia Tan, Shaohua Fan, Cong Cao, Feilong Meng, Ling Zhu, Lili Zhao, Min-Xin Guan, Hongchuan Jin, Yi Sun.
      Abnormal activation of neddylation modification and dysregulated energy metabolism are frequently seen in many types of cancer cells. Whether and how neddylation modification affects cellular metabolism remains largely unknown. Here we showed that MLN4924, a small molecule inhibitor of neddylation modification, induces mitochondrial fission-to-fusion conversion in breast cancer cells via inhibiting ubiquitylation and degradation of fusion-promoting protein mitofusin (MFN1) by SCFβ-TrCP E3 ligase and blocking the mitochondrial translocation of fusion-inhibiting protein DRP1. Importantly, MLN4924-induced mitochondrial fusion is independent of cell cycle progression, but confers cellular survival. The Mass-Spectrometry-based metabolic profiling and mitochondrial functional assays reveal that MLN4924 inhibits TCA cycle, but promotes mitochondrial OXPHOS. MLN4924 also increases glycolysis by activating PKM2 via promoting its tetramerization. Biologically, MLN4924 coupled with OXPHOS inhibitor metformin, or glycolysis inhibitor shikonin, significantly inhibits cancer cell growth both in vitro and in vivo. Together, our study links neddylation modification and energy metabolism, and provides sound strategies for effective combinational cancer therapies.
    Keywords:  Bioenergetics; Cancer; Cell Biology; Glucose metabolism; Metabolism
    DOI:  https://doi.org/10.1172/jci.insight.121582
  7. Biochim Biophys Acta Mol Cell Res. 2019 Jan 19. pii: S0167-4889(19)30006-0. [Epub ahead of print]
    ER-mitochondria interactions: Both strength and weakness within cancer cells.
    Mabrouka Doghman-Bouguerra, Enzo Lalli.
      ER-mitochondria contact sites represent hubs for signaling that control mitochondrial biology related to several aspects of cellular survival, metabolism, cell death sensitivity and metastasis, which all contribute to tumorigenesis. Altered ER-mitochondria contacts can deregulate Ca2+ homeostasis, phospholipid metabolism, mitochondrial morphology and dynamics. MAM represent both a hot spot in cancer onset and progression and an Achilles' heel of cancer cells that can be exploited for therapeutic perspectives. Over the past years, an increasing number of cancer-related proteins, including oncogenes and tumor suppressors, have been localized in MAM and exert their pro- or antiapoptotic functions through the regulation of Ca2+ transfer and signaling between the two organelles. In this review, we highlight the central role of ER-mitochondria contact sites in tumorigenesis and focus on chemotherapeutic drugs or potential targets that act on MAM properties for new therapeutic approaches in cancer.
    Keywords:  Apoptosis; Calcium signaling; Cancer; MAM; Mitochondria; Therapeutic targets
    DOI:  https://doi.org/10.1016/j.bbamcr.2019.01.009
  8. Biochim Biophys Acta Mol Basis Dis. 2019 Jan 17. pii: S0925-4439(18)30506-4. [Epub ahead of print]
    Is there an effect of ischemic conditioning on myocardial contractile function following acute myocardial ischemia/reperfusion injury?
    Leanda Stoian, Martina Krüger, Joachim Schmitt, Petra Kleinbongard.
      Ischemic conditioning induces cardioprotection; the final infarct size following a myocardial ischemic event is reduced. However, whether ischemic conditioning has long-term beneficial effects on myocardial contractile function following such an ischemic event needs further elucidation. To date, ex vivo studies have shown that ischemic conditioning improves the contractile recovery of isolated ventricular papillary muscle or atrial trabeculae following simulated ischemia. However, in vivo animal studies and studies in patients undergoing elective cardiac surgery show conflicting results. At the subcellular level, it is known that ischemic conditioning improved energy metabolism, preserved mitochondrial respiration, ATP production, and Ca2+ homeostasis in isolated mitochondria from the myocardium. Ischemic conditioning also presents with post-translational modifications of proteins in the contractile machinery of the myocardium. The beneficial effects on myocardial contractile function need further elucidation. This article is part of a Special Issue entitled: The power of metabolism: Linking energy supply and demand to contractile function edited by Torsten Doenst, Michael Schwarzer and Christine Des Rosiers.
    Keywords:  Contractile function; Mitochondria; Myocardial ischemic conditioning; Post-translational modifications
    DOI:  https://doi.org/10.1016/j.bbadis.2018.12.020
  9. J Neurochem. 2019 Jan 21.
    Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy.
    Yea Seul Shin, James G Ryall, Joanne M Britto, Chew L Lau, Rodney J Devenish, Phillip Nagley, Philip M Beart.
      Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A and potassium cyanide, targeting complexes I, II, III and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨm ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by CCCP. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨm , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨm by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨm , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria. This article is protected by copyright. All rights reserved.
    Keywords:  Bioenergetics; Depolarization; Lysosome; Mitophagy; Neuron; PINK1
    DOI:  https://doi.org/10.1111/jnc.14667
  10. J Biol Chem. 2019 Jan 25. pii: jbc.RA118.006888. [Epub ahead of print]
    The yeast mitochondrial proteins Rcf1 and Rcf2 support the enzymology of the cytochrome c oxidase complex and generation of the proton motive force.
    Vera Strogolova, Ngoc H Hoang, Jonathan Hosler, Rosemary A Stuart.
      The yeast mitochondrial proteins Rcf1 and Rcf2 are associated with a subpopulation of the cytochrome bc 1-cytochrome c oxidase supercomplex and have been proposed to play a role in the assembly and/or modulating the activity of the cytochrome c oxidase (complex IV, CIV). Yeast mutants deficient in either Rcf1 or Rcf2 proteins can use aerobic respiration-based metabolism for growth, but the absence of both proteins results in a strong growth defect. In the present study, using assorted biochemical and biophysical analyses of Rcf1/Rcf2 single and double null-mutant yeast cells and mitochondria, we further explored how Rcf1 and Rcf2 support aerobic respiration and growth. We show that the absence of Rcf1 physically reduces the levels of CIV and diminishes the  ability of the CIV that is present to maintain a normal mitochondrial proton motive force (PMF). Although the absence of Rcf2 did not noticeably affect the physical content of CIV, the PMF generated by CIV was also lower than normal. Our results indicate that the detrimental effects of the absence of Rcf1 and Rcf2 proteins on the CIV complex are distinct in terms of CIV assembly/accumulation and additive in terms of the ability of CIV to generate PMF. Thus, the combined absence of Rcf1 and Rcf2 alters both CIV physiology and assembly. We conclude that the slow aerobic growth of the Rcf1/Rcf2 double null mutant results from diminished generation of mitochondrial PMF by CIV, and limits the level of CIV activity required for maintenance of the PMF and growth in aerobic conditions.
    Keywords:  OXPHOS; Rcf1/Rcf2; bioenergetics; cytochrome oxidase; mitochondria; mitochondrial respiratory chain complex; proton motive force
    DOI:  https://doi.org/10.1074/jbc.RA118.006888
  11. Methods Mol Biol. 2019 ;1925 197-222
    Assessing Calcium-Stimulated Mitochondrial Bioenergetics Using the Seahorse XF96 Analyzer.
    Jennifer Wettmarshausen, Fabiana Perocchi.
      The development of fluorescence-based oxygen sensors coupled with microplate-based assays for quantitative bioenergetics analyses enables screening multiple experimental conditions at once with small biological material and in a timely manner. In this chapter, we outline detailed protocols and practical tips to design and perform controlled measurements of (a) respiratory and glycolytic metabolism of intact cells, (b) substrate-dependent respiration in permeabilized cells and isolated mitochondria, and (c) calcium-dependent regulation of mitochondrial bioenergetics with Seahorse XF Flux Analyzers.
    Keywords:  Bioenergetics; Calcium; Intact cells; Mitochondria; Oxygen consumption; Permeabilized cells; Respiration; Seahorse XF analyzer
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_18
  12. J Cell Biol. 2019 Jan 23. pii: jcb.201808044. [Epub ahead of print]
    Coenzyme Q biosynthetic proteins assemble in a substrate-dependent manner into domains at ER-mitochondria contacts.
    Kelly Subramanian, Adam Jochem, Maxence Le Vasseur, Samantha Lewis, Brett R Paulson, Thiruchelvi R Reddy, Jason D Russell, Joshua J Coon, David J Pagliarini, Jodi Nunnari.
      Coenzyme Q (CoQ) lipids are ancient electron carriers that, in eukaryotes, function in the mitochondrial respiratory chain. In mitochondria, CoQ lipids are built by an inner membrane-associated, multicomponent, biosynthetic pathway via successive steps of isoprenyl tail polymerization, 4-hydroxybenzoate head-to-tail attachment, and head modification, resulting in the production of CoQ. In yeast, we discovered that head-modifying CoQ pathway components selectively colocalize to multiple resolvable domains in vivo, representing supramolecular assemblies. In cells engineered with conditional ON or OFF CoQ pathways, domains were strictly correlated with CoQ production and substrate flux, respectively, indicating that CoQ lipid intermediates are required for domain formation. Mitochondrial CoQ domains were also observed in human cells, underscoring their conserved functional importance. CoQ domains within cells were highly enriched adjacent to ER-mitochondria contact sites. Together, our data suggest that CoQ domains function to facilitate substrate accessibility for processive and efficient CoQ production and distribution in cells.
    DOI:  https://doi.org/10.1083/jcb.201808044
  13. Cell Rep. 2019 Jan 22. pii: S2211-1247(18)32076-X. [Epub ahead of print]26(4): 945-954.e4
    Mitochondrial Stress-Initiated Aberrant Activation of the NLRP3 Inflammasome Regulates the Functional Deterioration of Hematopoietic Stem Cell Aging.
    Hanzhi Luo, Wei-Chieh Mu, Rajendra Karki, Hou-Hsien Chiang, Mary Mohrin, Jiyung J Shin, Rika Ohkubo, Keisuke Ito, Thirumala-Devi Kanneganti, Danica Chen.
      Aging-associated defects in hematopoietic stem cells (HSCs) can manifest in their progeny, leading to aberrant activation of the NLRP3 inflammasome in macrophages and affecting distant tissues and organismal health span. Whether the NLRP3 inflammasome is aberrantly activated in HSCs during physiological aging is unknown. We show here that SIRT2, a cytosolic NAD+-dependent deacetylase, is required for HSC maintenance and regenerative capacity at an old age by repressing the activation of the NLRP3 inflammasome in HSCs cell autonomously. With age, reduced SIRT2 expression and increased mitochondrial stress lead to aberrant activation of the NLRP3 inflammasome in HSCs. SIRT2 overexpression, NLRP3 inactivation, or caspase 1 inactivation improves the maintenance and regenerative capacity of aged HSCs. These results suggest that mitochondrial stress-initiated aberrant activation of the NLRP3 inflammasome is a reversible driver of the functional decline of HSC aging and highlight the importance of inflammatory signaling in regulating HSC aging.
    Keywords:  NLRP3; SIRT2; SIRT3; SIRT7; aging; clonal hematopoiesis; hematopoietic stem cell; inflammasome; mitochondrial UPR; oxidative stress
    DOI:  https://doi.org/10.1016/j.celrep.2018.12.101
  14. Hum Mol Genet. 2019 Jan 22.
    Pre-clinical evaluation of cysteamine bitartrate as a therapeutic agent for mitochondrial respiratory chain disease.
    Sujay Guha, Chigoziri Konkwo, Manuela Lavorato, Neal D Mathew, Min Peng, Julian Ostrovsky, Young-Joon Kwon, Erzsebet Polyak, Richard Lightfoot, Christoph Seiler, Rui Xiao, Michael Bennett, Zhe Zhang, Eiko Nakamaru-Ogiso, Marni J Falk.
      Cysteamine bitartrate is an FDA-approved therapy for nephropathic cystinosis postulated to enhance glutathione biosynthesis. We hypothesized this antioxidant effect may reduce oxidative stress in primary mitochondrial respiratory chain (RC) disease, improving cellular viability and organismal health. Here, we systematically evaluated the therapeutic potential of cysteamine bitartrate in RC disease models spanning three evolutionary species. These pre-clinical studies demonstrated the narrow therapeutic window of cysteamine bitartrate, with toxicity at millimolar levels directly correlating with marked induction of hydrogen peroxide production. Micromolar range cysteamine bitartrate treatment in C. elegans gas-1(fc21) RC complex I (NDUFS2-/-) disease invertebrate worms significantly improved mitochondrial membrane potential and oxidative stress, with corresponding modest improvement in fecundity but not lifespan. At 10 to 100 μM concentrations, cysteamine bitartrate improved multiple RC complex disease FBXL4 human fibroblast survival, and protected both complex I (rotenone) and complex IV (azide) D. rerio vertebrate zebrafish models from brain death. Mechanistic profiling of cysteamine bitartrate effects showed it increases aspartate levels and flux, without increasing total glutathione levels. Transcriptional normalization was greater in RC disease human cells than C. elegans of broadly dysregulated intermediary metabolic, glutathione, cell defense, DNA, and immune pathways, with similar rescue in both models of downregulated ribosome and proteasome pathway expression. Overall, these data suggest cysteamine bitartrate may hold therapeutic potential in RC disease, although not through obvious modulation of total glutathione levels. Careful consideration is required to determine safe and effective cysteamine bitartrate concentrations to further evaluate in clinical trials of human subjects with primary mitochondrial RC disease.
    DOI:  https://doi.org/10.1093/hmg/ddz023
  15. Mitochondrion. 2019 Jan 17. pii: S1567-7249(18)30209-5. [Epub ahead of print]
    Splitting the functions of Rim2, a mitochondrial iron/pyrimidine carrier.
    Simon A B Knight, Heeyong Yoon, Ashutosh K Pandey, Jayashree Pain, Debkumar Pain, Andrew Dancis.
      Rim2 is an unusual mitochondrial carrier protein capable of transporting both iron and pyrimidine nucleotides. Here we characterize two point mutations generated in the predicted substrate-binding site, finding that they yield disparate effects on iron and pyrimidine transport. The Rim2 (E248A) mutant was deficient in mitochondrial iron transport activity. By contrast, the Rim2 (K299A) mutant specifically abrogated pyrimidine nucleotide transport and exchange, while leaving iron transport activity largely unaffected. Strikingly, E248A preserved TTP/TTP homo exchange but interfered with TTP/TMP hetero exchange, perhaps because proton coupling was dependent on the E248 acidic residue. Rim2-dependent iron transport was unaffected by pyrimidine nucleotides. Rim2-dependent pyrimidine transport was competed by Zn2+ but not by Fe2+, Fe3+ or Cu2+. The iron and pyrimidine nucleotide transport processes displayed different salt requirements; pyrimidine transport was dependent on the salt content of the buffer whereas iron transport was salt independent. In mitochondria containing Rim2 (E248A), iron proteins were decreased, including aconitase (FeS), pyruvate dehydrogenase (lipoic acid containing) and cytochrome c (heme protein). Additionally, the rate of FeS cluster synthesis in isolated and intact mitochondria was decreased compared with the K299A mutant, consistent with the impairment of iron-dependent functions in that mutant. In summary, mitochondrial iron transport and pyrimidine transport by Rim2 occur separately and independently. Rim2 could be a bifunctional carrier protein.
    Keywords:  Heme; Iron; Iron-sulfur clusters; Mitochondria; Mitochondrial carrier protein; Pyrimidine; Yeast
    DOI:  https://doi.org/10.1016/j.mito.2018.12.005
  16. Methods Mol Biol. 2019 ;1925 65-73
    Electrophysiological Characterization of Calcium-Permeable Channels Using Planar Lipid Bilayer.
    Vanessa Checchetto, Ildikò Szabò.
      Numerous researchers tried to identify the key players of calcium signaling in mitochondria using molecular and cell biology techniques for more than five decades. However, only an integrated approach involving also electrophysiological techniques has finally allowed to define the components of the protein complex responsible for the uptake of this ion into mitochondria.Here we describe the protocol used for the electrophysiological characterization of the mitochondrial calcium uniporter (MCU) complex: the following outline indicates step-by-step the setup of planar lipid bilayer experiments.
    Keywords:  Calcium channels; Membrane protein synthesis in cell-free system; Planar lipid bilayer
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_6
  17. Cell Mol Life Sci. 2019 Jan 23.
    The SEACIT complex is involved in the maintenance of vacuole-mitochondria contact sites and controls mitophagy.
    Yinxing Ma, Alexis Moors, Nadine Camougrand, Svetlana Dokudovskaya.
      The major signaling pathway that regulates cell growth and metabolism is under the control of the target of rapamycin complex 1 (TORC1). In Saccharomyces cerevisiae the SEA complex is one of the TORC1 upstream regulators involved in amino acid sensing and autophagy. Here, we performed analysis of the expression, interactions and localization of SEA complex proteins under different conditions, varying parameters such as sugar source, nitrogen availability and growth phase. Our results show that the SEA complex promotes mitochondria degradation either by mitophagy or by general autophagy. In addition, the SEACIT subcomplex is involved in the maintenance of the vacuole-mitochondria contact sites. Thus, the SEA complex appears to be an important link between the TORC1 pathway and regulation of mitochondria quality control.
    Keywords:  Autophagy; Membrane contact sites; Mitochondria; Mitophagy; SEA complex; TORC1; Vacuole
    DOI:  https://doi.org/10.1007/s00018-019-03015-6
  18. Life Sci Alliance. 2019 Feb;pii: e201800219. [Epub ahead of print]2(1):
    Mitochondrial stress response triggered by defects in protein synthesis quality control.
    Uwe Richter, Kah Ying Ng, Fumi Suomi, Paula Marttinen, Taina Turunen, Christopher Jackson, Anu Suomalainen, Helena Vihinen, Eija Jokitalo, Tuula A Nyman, Marita A Isokallio, James B Stewart, Cecilia Mancini, Alfredo Brusco, Sara Seneca, Anne Lombès, Robert W Taylor, Brendan J Battersby.
      Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.
    DOI:  https://doi.org/10.26508/lsa.201800219
  19. J Mol Biol. 2019 Jan 22. pii: S0022-2836(19)30031-2. [Epub ahead of print]
    Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells.
    Shivali Rawat, Valpadashi Anusha, Manoranjan Jha, K Sreedurgalakshmi, Swasti Raychaudhuri.
      Proteostasis is maintained by optimal expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers protein aggregation and is implicated in many age-related pathologies. In this study, using quantitative proteomics and microscopy, we show that aggregation of many nuclear-encoded mitochondrial proteins is an early protein-destabilization event during short-term proteasome inhibition. Among these, Respiratory Chain Complex (RCC) subunits represent a group of functionally related proteins consistently forming aggregates under multiple proteostasis-stresses with varying aggregation-propensities. Sequence analysis reveals that several RCC subunits, irrespective of the cleavable mitochondrial targeting sequence (MTS), contain low complexity regions (LCR) at the N-terminus. Using different chimeric and mutant constructs, we show that these low complexity regions partially contribute to the intrinsic instability of multiple RCC subunits. Taken together, we propose that physicochemically driven aggregation of unassembled RCC subunits destabilizes their functional assembly inside mitochondria. This eventually deregulates the biogenesis of respiratory complexes and marks the onset of mitochondrial dysfunction.
    Keywords:  Low complexity region; Mitochondria; Mitochondrial respiratory chain complex; Protein aggregation; Protein misfolding; Protein turnover; Proteomics; Proteostasis
    DOI:  https://doi.org/10.1016/j.jmb.2019.01.022
  20. Cells. 2019 Jan 18. pii: E71. [Epub ahead of print]8(1):
    Prohibitins: A Critical Role in Mitochondrial Functions and Implication in Diseases.
    Anna Signorile, Giuseppe Sgaramella, Francesco Bellomo, Domenico De Rasmo.
      Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are proteins that are ubiquitously expressed, and are present in the nucleus, cytosol, and mitochondria. Depending on the cellular localization, PHB1 and PHB2 have distinctive functions, but more evidence suggests a critical role within mitochondria. In fact, PHB proteins are highly expressed in cells that heavily depend on mitochondrial function. In mitochondria, these two proteins assemble at the inner membrane to form a supra-macromolecular structure, which works as a scaffold for proteins and lipids regulating mitochondrial metabolism, including bioenergetics, biogenesis, and dynamics in order to determine the cell fate, death, or life. PHB alterations have been found in aging and cancer, as well as neurodegenerative, cardiac, and kidney diseases, in which significant mitochondrial impairments have been observed. The molecular mechanisms by which prohibitins regulate mitochondrial function and their role in pathology are reviewed and discussed herein.
    Keywords:  apoptosis; mitochondria; mitochondrial dynamics; oxidative phosphorylation; prohibitins
    DOI:  https://doi.org/10.3390/cells8010071
  21. J Transl Med. 2019 Jan 21. 17(1): 33
    Mechanisms by which hydrogen sulfide attenuates muscle function following ischemia-reperfusion injury: effects on Akt signaling, mitochondrial function, and apoptosis.
    Michael D Wetzel, Joseph C Wenke.
      Ischemia-reperfusion injury is caused by a period of ischemia followed by massive blood flow into a tissue that had experienced restricted blood flow. The severity of the injury is dependent on the time the tissue was restricted from blood flow, becoming more severe after longer ischemia times. This can lead to many complications such as tissue necrosis, cellular apoptosis, inflammation, metabolic and mitochondrial dysfunction, and even organ failure. One of the emerging therapies to combat ischemic reperfusion injury complications is hydrogen sulfide, which is a gasotransmitter that diffuses across cell membranes to exert effects on various signaling pathways regulating cell survival such as Akt, mitochondrial activity, and apoptosis. Although commonly thought of as a toxic gas, low concentrations of hydrogen sulfide have been shown to be beneficial in promoting tissue survival post-ischemia, and modulate a wide variety of cellular responses. This review will detail the mechanisms of hydrogen sulfide in affecting the Akt signaling pathway, mitochondrial function, and apoptosis, particularly in regards to ischemic reperfusion injury in muscle tissue. It will conclude with potential clinical applications of hydrogen sulfide, combinations with other therapies, and perspectives for future studies.
    Keywords:  Akt; Apoptosis; Hydrogen sulfide; Ischemia reperfusion injury; Mitochondria; Muscle; eNOS
    DOI:  https://doi.org/10.1186/s12967-018-1753-7
  22. Int J Mol Sci. 2019 Jan 18. pii: E404. [Epub ahead of print]20(2):
    The Role of O-GlcNAcylation for Protection against Ischemia-Reperfusion Injury.
    Rebekka Vibjerg Jensen, Ioanna Andreadou, Derek J Hausenloy, Hans Erik Bøtker.
      Ischemia reperfusion injury (IR injury) associated with ischemic heart disease contributes significantly to morbidity and mortality. O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification that plays an important role in numerous biological processes, both in normal cell functions and disease. O-GlcNAc increases in response to stress. This increase mediates stress tolerance and cell survival, and is protective. Increasing O-GlcNAc is protective against IR injury. Experimental cellular and animal models, and also human studies, have demonstrated that protection against IR injury by ischemic preconditioning, and the more clinically applicable remote ischemic preconditioning, is associated with increases in O-GlcNAc levels. In this review we discuss how the principal mechanisms underlying tissue protection against IR injury and the associated immediate elevation of O-GlcNAc may involve attenuation of calcium overload, attenuation of mitochondrial permeability transition pore opening, reduction of endoplasmic reticulum stress, modification of inflammatory and heat shock responses, and interference with established cardioprotective pathways. O-GlcNAcylation seems to be an inherent adaptive cytoprotective response to IR injury that is activated by mechanical conditioning strategies.
    Keywords:  O-GlcNAc; cardioprotection; ischemia-reperfusion injury
    DOI:  https://doi.org/10.3390/ijms20020404
  23. Nat Protoc. 2019 Jan 25.
    Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.
    Min Yuan, Daniel M Kremer, He Huang, Susanne B Breitkopf, Issam Ben-Sahra, Brendan D Manning, Costas A Lyssiotis, John M Asara.
      Targeted tandem mass spectrometry (LC-MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6-7 h for a single sample from experimental labelling and metabolite extraction to peak integration.
    DOI:  https://doi.org/10.1038/s41596-018-0102-x
  24. Sci Rep. 2019 Jan 24. 9(1): 727
    Pomegranate activates TFEB to promote autophagy-lysosomal fitness and mitophagy.
    Sijie Tan, Chye Yun Yu, Zhi Wei Sim, Zun Siong Low, Brianna Lee, Faith See, Nyo Min, Archana Gautam, Justin Jang Hann Chu, Kee Woei Ng, Esther Wong.
      Mitochondrial dysfunction underscores aging and diseases. Mitophagy (mitochondria + autophagy) is a quality control pathway that preserves mitochondrial health by targeting damaged mitochondria for autophagic degradation. Hence, molecules or compounds that can augment mitophagy are therapeutic candidates to mitigate mitochondrial-related diseases. However, mitochondrial stress remains the most effective inducer of mitophagy. Thus, identification of mitophagy-inducing regimes that are clinically relevant is favorable. In this study, pomegranate extract (PE) supplementation is shown to stimulate mitophagy. PE activates transcription factor EB (TFEB) to upregulate the expression of autophagy and lysosomal genes for mitochondrial quality control under basal and stress conditions. Basally, PE alters mitochondrial morphology and promotes recruitment of autophagosomes to the mitochondria (mitophagosome formation). Upon onset of mitochondrial stress, PE further augments mitophagosome formation, and engages PINK1 and Parkin to the mitochondria to potentiate mitophagy. This cellular phenomenon of PE-induced mitophagy helps to negate superfluous mitochondrial reactive oxygen species (ROS) production and mitochondrial impairment. Overall, our study highlights the potential of PE supplementation as a physiological therapy to modulate TFEB activity to alleviate mitochondrial dysfunction in aging and mitochondrial-related diseases.
    DOI:  https://doi.org/10.1038/s41598-018-37400-1
  25. Nat Immunol. 2019 Jan 21.
    Metabolic rewiring of macrophages by CpG potentiates clearance of cancer cells and overcomes tumor-expressed CD47-mediated 'don't-eat-me' signal.
    Mingen Liu, Roddy S O'Connor, Sophie Trefely, Kathleen Graham, Nathaniel W Snyder, Gregory L Beatty.
      Macrophages enforce antitumor immunity by engulfing and killing tumor cells. Although these functions are determined by a balance of stimulatory and inhibitory signals, the role of macrophage metabolism is unknown. Here, we study the capacity of macrophages to circumvent inhibitory activity mediated by CD47 on cancer cells. We show that stimulation with a CpG oligodeoxynucleotide, a Toll-like receptor 9 agonist, evokes changes in the central carbon metabolism of macrophages that enable antitumor activity, including engulfment of CD47+ cancer cells. CpG activation engenders a metabolic state that requires fatty acid oxidation and shunting of tricarboxylic acid cycle intermediates for de novo lipid biosynthesis. This integration of metabolic inputs is underpinned by carnitine palmitoyltransferase 1A and adenosine tri-phosphate citrate lyase, which, together, impart macrophages with antitumor potential capable of overcoming inhibitory CD47 on cancer cells. Our findings identify central carbon metabolism to be a novel determinant and potential therapeutic target for stimulating antitumor activity by macrophages.
    DOI:  https://doi.org/10.1038/s41590-018-0292-y
  26. Sci Rep. 2019 Jan 22. 9(1): 246
    Mechanistic Target of Rapamycin Complex 1 Promotes the Expression of Genes Encoding Electron Transport Chain Proteins and Stimulates Oxidative Phosphorylation in Primary Human Trophoblast Cells by Regulating Mitochondrial Biogenesis.
    Fredrick J Rosario, Madhulika B Gupta, Leslie Myatt, Theresa L Powell, Jeremy P Glenn, Laura Cox, Thomas Jansson.
      Trophoblast oxidative phosphorylation provides energy for active transport and protein synthesis, which are critical placental functions influencing fetal growth and long-term health. The molecular mechanisms regulating trophoblast mitochondrial oxidative phosphorylation are largely unknown. We hypothesized that mechanistic Target of Rapamycin Complex 1 (mTORC1) is a positive regulator of key genes encoding Electron Transport Chain (ETC) proteins and stimulates oxidative phosphorylation in trophoblast and that ETC protein expression is down-regulated in placentas of infants with intrauterine growth restriction (IUGR). We silenced raptor (mTORC1 inhibition), rictor (mTORC2 inhibition) or DEPTOR (mTORC1/2 activation) in cultured term primary human trophoblast (PHT) cells. mTORC1 inhibition caused a coordinated down-regulation of 18 genes encoding ETC proteins representing all ETC complexes. Inhibition of mTORC1, but not mTORC2, decreased protein expression of ETC complexes I-IV, mitochondrial basal, ATP coupled and maximal respiration, reserve capacity and proton leak, whereas activation of mTORC1 had the opposite effects. Moreover, placental protein expression of ETC complexes was decreased and positively correlated to mTOR signaling activity in IUGR. By controlling trophoblast ATP production, mTORC1 links nutrient and O2 availability and growth factor signaling to placental function and fetal growth. Reduced placental mTOR activity may impair mitochondrial respiration and contribute to placental insufficiency in IUGR pregnancies.
    DOI:  https://doi.org/10.1038/s41598-018-36265-8
  27. J Clin Invest. 2019 Jan 22. pii: 125980. [Epub ahead of print]
    Ulk1/Rab9-mediated alternative mitophagy confers cardioprotection during energy stress.
    Rimpy Dhingra, Inna Rabinovich-Nikitin, Lorrie A Kirshenbaum.
      The heart relies on mitochondria-derived energy production for continuous contraction and relaxation; therefore, the maintenance of a pool of healthy mitochondria is essential for sustaining normal cardiac performance. Mitophagy serves as a critical process for maintaining mitochondrial quality control and involves the PTEN-induced kinase 1/Parkin (Pink1/Parkin) pathway and autophagosomes labeled with the autophagy proteins autophagy-related 7 (ATG) and light chain 3 (LC3). In this issue of the JCI, Saito and colleagues identify an alternative pathway for mitophagy that utilizes the serine/threonine protein kinase Unc-51-like kinase 1 (Ulk1) and the small GTPase Rab9 to clear damaged mitochondria independently of conventional autophagy proteins. Together, the results of this study reveal that Ulk1 phosphorylation of Rab9 at serine 179 is critical for alternative mitophagy and cardioprotection under energy stress conditions.
    DOI:  https://doi.org/10.1172/JCI125980
  28. Arch Biochem Biophys. 2019 Jan 18. pii: S0003-9861(18)30677-5. [Epub ahead of print]663 214-219
    Mitochondrial dynamics and inter-mitochondrial communication in the heart.
    Yang-Po Cao, Ming Zheng.
      Mitochondria are organelles highly dynamic in most types of cells, constantly changing morphology and forming dynamically continuous networks. Defects of mitochondrial dynamics are associated with various human diseases including neurodegenerative diseases and cardiovascular diseases. In the heart, mitochondria are rigidly organized between myofilaments into a crystal-like lattice pattern, the apparently limited mitochondrial movement raises the question of whether mitochondria communicate with each other dynamically. A large body of evidence reveals abnormal mitochondrial morphology in cardiac diseases and that deficiencies of mitochondrial dynamic related proteins lead to cardiac dysfunctions, indicating an essential role of mitochondrial dynamics in regulating the cardiac function. Here we will review mitochondrial dynamics in the heart with the focus on recent findings of the direct inter-mitochondrial communication in adult cardiomyocytes, and will discuss the possible regulating mechanisms.
    Keywords:  Communication; Dynamic; Heart; Mitochondria
    DOI:  https://doi.org/10.1016/j.abb.2019.01.017
  29. Aging (Albany NY). 2019 Jan 23.
    S100A13 promotes senescence-associated secretory phenotype and cellular senescence via modulation of non-classical secretion of IL-1α.
    Yuanyuan Su, Chenzhong Xu, Zhaomeng Sun, Yao Liang, Guodong Li, Tanjun Tong, Jun Chen.
      Senescent cells display the senescence-associated secretory phenotype (SASP) which plays important roles in cancer, aging, etc. Cell surface-bound IL-1α is a crucial SASP factor and acts as an upstream regulator to induce NF-κB activity and subsequent SASP genes transcription. IL-1α exports to cell surface via S100A13 protein-dependent non-classical secretory pathway. However, the status of this secretory pathway during cellular senescence and its role in cellular senescence remain unknown. Here, we show that S100A13 is up-regulated in various types of cellular senescence. S100A13 overexpression increases cell surface-associated IL-1α level, NF-κB activity and subsequent multiple SASP genes induction, whereas S100A13 knockdown has an opposite role. We also exhibit that Cu2+ level is elevated during cellular senescence. Lowering Cu2+ level decreases cell surface-bound IL-1α level, NF-κB activity and SASP production. Moreover, S100A13 overexpression promotes oncogene Ras-induced cell senescence (Ras OIS), Doxorubicin-induced cancer cell senescence (TIS) and replicative senescence, while impairment of non-classical secretory pathway of IL-1α delays cellular senescence. In addition, intervention of S100A13 affects multiple SASP and cellular senescence mediators including p38, γ-H2AX, and mTORC1. Taken together, our findings unveil a critical role of the non-classical secretory pathway of IL-1α in cellular senescence and SASP regulation.
    Keywords:  Cu 2+; IL-1α; S100A13; SASP; cell senescence; non-classical protein secretory pathway
    DOI:  https://doi.org/10.18632/aging.101760
  30. Cancer Res. 2019 Jan 25. pii: canres.2558.2018. [Epub ahead of print]
    Interplay between TRAP1 and sirtuin-3 modulates mitochondrial respiration and oxidative stress to maintain stemness of glioma stem cells.
    Hye-Kyung Park, Jun-Hee Hong, Young Taek Oh, Sung Soo Kim, Jinlong Yin, An-Jung Lee, Young Chan Chae, Jong Heon Kim, Sung-Hye Park, Chul-Kee Park, Myung-Jin Park, Jong Bae Park, Byoung Heon Kang.
      Glioblastoma (GBM) cancer stem cells (CSC) are primarily responsible for metastatic dissemination, resistance to therapy, and relapse of GBM, the most common and aggressive brain tumor. Development and maintenance of CSC require orchestrated metabolic rewiring and metabolic adaptation to a changing microenvironment. Here we show that cooperative interplay between the mitochondrial chaperone TRAP1 and the major mitochondria deacetylase sirtuin-3 (SIRT3) in glioma stem cells (GSC) increases mitochondrial respiratory capacity and reduces production of reactive oxygen species. This metabolic regulation endowed GSC with metabolic plasticity, facilitated adaptation to stress (particularly reduced nutrient supply), and maintained "stemness." Inactivation of TRAP1 or SIRT3 compromised their interdependent regulatory mechanisms, leading to metabolic alterations, loss of stemness, and suppression of tumor formation by GSC in vivo. Thus, targeting the metabolic mechanisms regulating interplay between TRAP1 and SIRT3 may provide a novel therapeutic option for intractable GBM patients.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-18-2558
  31. Front Endocrinol (Lausanne). 2018 ;9 783
    The Roles of Mitochondrial SIRT4 in Cellular Metabolism.
    Zheying Min, Jiangman Gao, Yang Yu.
      Sirtuins comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases that regulate the life span, aging, and metabolism. Seven sirtuin family members (SIRT1-7) have been identified in mammals, including humans. Despite the indispensable role of mitochondrial sirtuin 4 (SIRT4) in metabolic regulation, the primary enzymatic activity of SIRT4 remains enigmatic. SIRT4 possesses ADP-ribosyltransferase, lipoamidase and deacylase activities. Interestingly, the enzymatic activities and substrates of SIRT4 vary in different tissues and cells. SIRT4 inhibits insulin secretion in pancreatic β cells and regulates insulin sensitivity as a deacylase in the pancreas. SIRT4 represses fatty acid oxidation (FAO) in muscle and liver cells differently. SIRT4 has also been identified as a mitochondrial-localized tumor suppressor. A comprehensive understanding of the enzymology of SIRT4 in metabolism is essential for developing novel therapeutic agents for human metabolic diseases. This review will update the roles of SIRT4 in cellular and organismal metabolic homeostasis.
    Keywords:  SIRT4; enzymatic activities; fatty acid metabolism; insulin secretion; mitochondrial; tumor suppressor
    DOI:  https://doi.org/10.3389/fendo.2018.00783
  32. Biochem J. 2019 Jan 22. pii: BCJ20180435. [Epub ahead of print]
    Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.
    Isabelle Gerin, Marina Bury, Francesca Baldin, Julie Graff, Emile Van Schaftingen, Guido T Bommer.
      Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects.Phosphoglycolate concentrations were below 5 µM in wild type U2OS and HCT116 cells, and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side-activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki of < 10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances.In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.
    Keywords:  DNA damage response; glycolysis; metabolic regulation
    DOI:  https://doi.org/10.1042/BCJ20180435
  33. Methods Mol Biol. 2019 ;1925 233-243
    Purification of Functional F-ATP Synthase from Blue Native PAGE.
    Chiara Galber, Giulia Valente, Sophia von Stockum, Valentina Giorgio.
      In the presence of Ca2+, F-ATP synthase preparations eluted from Blue Native gels generate electrophysiological currents that are typical of an inner mitochondrial membrane mega-channel, the permeability transition pore. Here we describe an experimental protocol for purification of F-ATP synthase that allows to maintain the enzyme assembly and activity that are essential for catalysis and channel formation.
    Keywords:  Blue Native PAGE; F-ATP synthase; Gel purification; Lipid bilayer; Permeability transition
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_20
  34. Cell Death Dis. 2019 Jan 17. 10(2): 40
    Glutaminase 1 expression in colorectal cancer cells is induced by hypoxia and required for tumor growth, invasion, and metastatic colonization.
    Lisha Xiang, Jun Mou, Bin Shao, Yuquan Wei, Houjie Liang, Naoharu Takano, Gregg L Semenza, Ganfeng Xie.
      Cancer cells re-program their metabolic machinery to meet the requirements of malignant transformation and progression. Glutaminase 1 (GLS1) was traditionally known as a mitochondrial enzyme that hydrolyzes glutamine into glutamate and fuels rapid proliferation of cancer cells. However, emerging evidence has now revealed that GLS1 might be a novel oncogene involved in tumorigenesis and progression of human cancers. In this study, we sought to determine whether GLS1 implicated in invasion and metastasis of colorectal carcinoma, and its underlying molecular mechanism. By analyzing a large set of clinical data from online datasets, we found that GLS1 is overexpressed in cancers compared with adjacent normal tissues, and associated with increased patient mortality. Immunohistochemical analysis of GLS1 staining showed that high GLS1 expression is significantly correlated with lymph node metastasis and advanced clinical stage in colorectal cancer patients. To investigate the underlying mechanism, we analyzed the Cancer Genome Atlas database and found that GLS1 mRNA expression is associated with a hypoxia signature, which is correlated with an increased risk of metastasis and mortality. Furthermore, reduced oxygen availability increases GLS1 mRNA and protein expression, due to transcriptional activation by hypoxia-inducible factor 1. GLS1 expression in colorectal cancer cells is required for hypoxia-induced migration and invasion in vitro and for tumor growth and metastatic colonization in vivo.
    DOI:  https://doi.org/10.1038/s41419-018-1291-5
  35. Cell Metab. 2019 Jan 16. pii: S1550-4131(18)30756-3. [Epub ahead of print]
    ACOT12-Dependent Alteration of Acetyl-CoA Drives Hepatocellular Carcinoma Metastasis by Epigenetic Induction of Epithelial-Mesenchymal Transition.
    Ming Lu, Wen-Wei Zhu, Xuan Wang, Jing-Jie Tang, Kai-Li Zhang, Guang-Yang Yu, Wei-Qing Shao, Zhi-Fei Lin, Sheng-Hao Wang, Lu Lu, Jian Zhou, Lian-Xin Wang, Hu-Liang Jia, Qiong-Zhu Dong, Jin-Hong Chen, Jian-Quan Lu, Lun-Xiu Qin.
      Metabolic reprogramming plays an important role in supporting tumor growth. However, little is known about the metabolic alterations that promote cancer metastasis. In this study, we identify acyl-CoA thioesterase 12 (ACOT12) as a key player in hepatocellular carcinoma (HCC) metastasis. The expression of ACOT12 is significantly down-regulated in HCC tissues and is closely associated with HCC metastasis and poor survival of HCC patients. Gain- and loss-of-function studies demonstrate that ACOT12 suppresses HCC metastasis both in vitro and in vivo. Further mechanistic studies reveal that ACOT12 regulates the cellular acetyl-CoA levels and histone acetylation in HCC cells and that down-regulation of ACOT12 promotes HCC metastasis by epigenetically inducing TWIST2 expression and the promotion of epithelial-mesenchymal transition. Taken together, our findings link the alteration of acetyl-CoA with HCC metastasis and imply that ACOT12 could be a prognostic marker and a potential therapeutic target for combating HCC metastasis.
    Keywords:  ACOT12; TWIST2; acetyl-CoA; cancer metastasis; epithelial-mesenchymal transition; hepatocellular carcinoma; histone acetylation
    DOI:  https://doi.org/10.1016/j.cmet.2018.12.019
  36. Oncogene. 2019 Jan 21.
    p53/BNIP3-dependent mitophagy limits glycolytic shift in radioresistant cancer.
    Hyo Won Chang, Mi Ra Kim, Hyang Ju Lee, Hye Min Lee, Gui Chul Kim, Yoon Sun Lee, Hae Yun Nam, Myungjin Lee, Hye Jin Jang, Kyung Eun Lee, Jong Cheol Lee, Youngro Byun, Seong Who Kim, Sang Yoon Kim.
      The role of p53 in genotoxic therapy-induced metabolic shift in cancers is not yet known. In this study, we investigated the role of p53 in the glycolytic shift in head and neck squamous cell carcinoma cell lines following irradiation. Isogenic p53-null radioresistant cancer cells established through cumulative irradiation showed decreased oxygen consumption and increased glycolysis with compromised mitochondria, corresponding with their enhanced sensitivity to drugs that target glycolysis. In contrast, radioresistant cancer cells with wild-type p53 preserved their primary metabolic profile with intact mitophagic processes and maintained their mitochondrial integrity. Moreover, we identified a previously unappreciated link between p53 and mitophagy, which limited the glycolytic shift through the BNIP3-dependent clearance of abnormal mitochondria. Thus, drugs targeting glycolysis could be used as an alternative strategy for overcoming radioresistant cancers, and the p53 status could be used as a biomarker for selecting participants for clinical trials.
    DOI:  https://doi.org/10.1038/s41388-019-0697-6
  37. Nat Immunol. 2019 Feb;20(2): 206-217
    Uncoupling protein 2 reprograms the tumor microenvironment to support the anti-tumor immune cycle.
    Wan-Chen Cheng, Yao-Chen Tsui, Simone Ragusa, Viktor H Koelzer, Marco Mina, Fabien Franco, Heinz Läubli, Benjamin Tschumi, Daniel Speiser, Pedro Romero, Alfred Zippelius, Tatiana V Petrova, Kirsten Mertz, Giovanni Ciriello, Ping-Chih Ho.
      Immune checkpoint blockade therapy has shifted the paradigm for cancer treatment. However, the majority of patients lack effective responses due to insufficient T cell infiltration in tumors. Here we show that expression of mitochondrial uncoupling protein 2 (UCP2) in tumor cells determines the immunostimulatory feature of the tumor microenvironment (TME) and is positively associated with prolonged survival. UCP2 reprograms the immune state of the TME by altering its cytokine milieu in an interferon regulatory factor 5-dependent manner. Consequently, UCP2 boosts the conventional type 1 dendritic cell- and CD8+ T cell-dependent anti-tumor immune cycle and normalizes the tumor vasculature. Finally we show, using either a genetic or pharmacological approach, that induction of UCP2 sensitizes melanomas to programmed cell death protein-1 blockade treatment and elicits effective anti-tumor responses. Together, this study demonstrates that targeting the UCP2 pathway is a potent strategy for alleviating the immunosuppressive TME and overcoming the primary resistance of programmed cell death protein-1 blockade.
    DOI:  https://doi.org/10.1038/s41590-018-0290-0
  38. Methods Mol Biol. 2019 ;1925 75-86
    Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter.
    Vivek Garg, Yuriy Y Kirichok.
      Mitochondria accumulate significant amounts of calcium when cytosolic calcium is elevated above the resting levels of 50-100 nM during signaling events. This calcium uptake is primarily mediated by a macromolecular protein assembly called mitochondrial calcium uniporter (MCU) that resides in the mitochondrial inner membrane. In 2004, we applied patch-clamp technique for the first time to record calcium currents from the mitochondrial inner membrane and proved unequivocally that MCU is a highly selective calcium channel. This chapter describes how patch-clamp technique can be applied to record the Ca2+ uniporter currents from the mitochondrial inner membrane, isolation of mitochondria from the heart tissue, and preparation of mitoplast using French Press. We also discuss advantages of patch-clamp technique as compared to other methods of determining mitochondrial uniporter activity and important considerations in applying patch-clamp technique to such a small subcellular organelle. With small variations in the bath and pipette solution composition, the same methodology can be applied to study any other currents (e.g., H+ or Cl-) from the mitochondrial inner membrane.
    Keywords:  Calcium channel; MCU; Mitochondria; Patch clamp; Uniporter
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_7
  39. Nature. 2019 Jan 23.
    Autophagic cell death restricts chromosomal instability during replicative crisis.
    Joe Nassour, Robert Radford, Adriana Correia, Javier Miralles Fusté, Brigitte Schoell, Anna Jauch, Reuben J Shaw, Jan Karlseder.
      Replicative crisis is a senescence-independent process that acts as a final barrier against oncogenic transformation by eliminating pre-cancerous cells with disrupted cell cycle checkpoints1. It functions as a potent tumour suppressor and culminates in extensive cell death. Cells rarely evade elimination and evolve towards malignancy, but the mechanisms that underlie cell death in crisis are not well understood. Here we show that macroautophagy has a dominant role in the death of fibroblasts and epithelial cells during crisis. Activation of autophagy is critical for cell death, as its suppression promoted bypass of crisis, continued proliferation and accumulation of genome instability. Telomere dysfunction specifically triggers autophagy, implicating a telomere-driven autophagy pathway that is not induced by intrachromosomal breaks. Telomeric DNA damage generates cytosolic DNA species with fragile nuclear envelopes that undergo spontaneous disruption. The cytosolic chromatin fragments activate the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and engage the autophagy machinery. Our data suggest that autophagy is an integral component of the tumour suppressive crisis mechanism and that loss of autophagy function is required for the initiation of cancer.
    DOI:  https://doi.org/10.1038/s41586-019-0885-0
  40. Nat Cell Biol. 2019 Jan 21.
    An IRAK1-PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy.
    Peter H Liu, Richa B Shah, Yuanyuan Li, Arshi Arora, Peter Man-Un Ung, Renuka Raman, Andrej Gorbatenko, Shingo Kozono, Xiao Zhen Zhou, Vincent Brechin, John M Barbaro, Ruth Thompson, Richard M White, Julio A Aguirre-Ghiso, John V Heymach, Kun Ping Lu, Jose M Silva, Katherine S Panageas, Avner Schlessinger, Robert G Maki, Heath D Skinner, Elisa de Stanchina, Samuel Sidi.
      Drug-based strategies to overcome tumour resistance to radiotherapy (R-RT) remain limited by the single-agent toxicity of traditional radiosensitizers (for example, platinums) and a lack of targeted alternatives. In a screen for compounds that restore radiosensitivity in p53 mutant zebrafish while tolerated in non-irradiated wild-type animals, we identified the benzimidazole anthelmintic oxfendazole. Surprisingly, oxfendazole acts via the inhibition of IRAK1, a kinase thus far implicated in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) immune responses. IRAK1 drives R-RT in a pathway involving IRAK4 and TRAF6 but not the IL-1R/TLR-IRAK adaptor MyD88. Rather than stimulating nuclear factor-κB, radiation-activated IRAK1 prevented apoptosis mediated by the PIDDosome complex (comprising PIDD, RAIDD and caspase-2). Countering this pathway with IRAK1 inhibitors suppressed R-RT in tumour models derived from cancers in which TP53 mutations predict R-RT. Moreover, IRAK1 inhibitors synergized with inhibitors of PIN1 a prolyl isomerase essential for IRAK1 activation, in response to pathogens and as shown here, ionizing radiation. These data identify an IRAK1 radiation-response pathway as a rational chemoradiation therapy target.
    DOI:  https://doi.org/10.1038/s41556-018-0260-7
  41. Sci Rep. 2019 Jan 23. 9(1): 340
    In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.
    Eul Hyun Suh, Edward P Hackett, R Max Wynn, David T Chuang, Bo Zhang, Weibo Luo, A Dean Sherry, Jae Mo Park.
      Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo 13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.
    DOI:  https://doi.org/10.1038/s41598-018-37390-0
  42. Oncogene. 2019 Jan 21.
    Lactate modulates CD4+ T-cell polarization and induces an immunosuppressive environment, which sustains prostate carcinoma progression via TLR8/miR21 axis.
    G Comito, A Iscaro, M Bacci, A Morandi, L Ippolito, M Parri, I Montagnani, M R Raspollini, S Serni, L Simeoni, E Giannoni, P Chiarugi.
      Leukocyte infiltration plays an active role in controlling tumor development. In the early stages of carcinogenesis, T cells counteract tumor growth. However, in advanced stages, cancer cells and infiltrating stromal components interfere with the immune control and instruct immune cells to support, rather than counteract, tumor malignancy, via cell-cell contact or soluble mediators. In particular, metabolites are emerging as active players in driving immunosuppression. Here we demonstrate that in a prostate cancer model lactate released by glycolytic cancer-associated fibroblasts (CAFs) acts on CD4+ T cells, shaping T-cell polarization. In particular, CAFs exposure (i) reduces the percentage of the antitumoral Th1 subset, inducing a lactate-dependent, SIRT1-mediated deacetylation/degradation of T-bet transcription factor; (ii) increases Treg cells, driving naive T cells polarization, through a lactate-based NF-kB activation and FoxP3 expression. In turn, this metabolic-based CAF-immunomodulated environment exerts a pro-invasive effect on prostate cancer cells, by activating a previously unexplored miR21/TLR8 axis that sustains cancer malignancy.
    DOI:  https://doi.org/10.1038/s41388-019-0688-7
  43. Biochemistry. 2019 Jan 22.
    3-Phosphoglycerate Transhydrogenation Instead of Dehydrogenation Alleviates the Redox State Dependency of Yeast de Novo l-Serine Synthesis.
    Nicole Paczia, Julia Becker-Kettern, Jean-François Conrotte, Javier O Cifuente, Marcelo E Guerin, Carole L Linster.
      The enzymatic mechanism of 3-phosphoglycerate to 3-phosphohydroxypyruvate oxidation, which forms the first step of the main conserved de novo serine synthesis pathway, has been revisited recently in certain microorganisms. While this step is classically considered to be catalyzed by an NAD-dependent dehydrogenase (e.g., PHGDH in mammals), evidence has shown that in Pseudomonas, Escherichia coli, and Saccharomyces cerevisiae, the PHGDH homologues act as transhydrogenases. As such, they use α-ketoglutarate, rather than NAD+, as the final electron acceptor, thereby producing D-2-hydroxyglutarate in addition to 3-phosphohydroxypyruvate during 3-phosphoglycerate oxidation. Here, we provide a detailed biochemical and sequence-structure relationship characterization of the yeast PHGDH homologues, encoded by the paralogous SER3 and SER33 genes, in comparison to the human and other PHGDH enzymes. Using in vitro assays with purified recombinant enzymes as well as in vivo growth phenotyping and metabolome analyses of yeast strains engineered to depend on either Ser3, Ser33, or human PHGDH for serine synthesis, we confirmed that both yeast enzymes act as transhydrogenases, while the human enzyme is a dehydrogenase. In addition, we show that the yeast paralogs differ from the human enzyme in their sensitivity to inhibition by serine as well as hydrated NADH derivatives. Importantly, our in vivo data support the idea that a 3PGA transhydrogenase instead of dehydrogenase activity confers a growth advantage under conditions where the NAD+:NADH ratio is low. The results will help to elucidate why different species evolved different reaction mechanisms to carry out a widely conserved metabolic step in central carbon metabolism.
    DOI:  https://doi.org/10.1021/acs.biochem.8b00990
  44. J Leukoc Biol. 2019 Jan 24.
    NK cell metabolism.
    Clair M Gardiner.
      Natural Killer (NK) cells are important antiviral and anticancer effector cells. They have excellent potential for immunotherapy although impaired functions during cancer limit their effectiveness. The discovery that cellular metabolism can impact on and regulate immune functions has led to an explosion of articles in this new area of immunometabolism. Metabolism has recently been shown to impact both murine and human NK cell biology. This review is targeted for newcomers to the field; it will introduce basic concepts in the area of immunometabolism including key aspects of glucose metabolism and mitochondrial function. It will review our current understanding of how metabolism of NK cells is differentially impacted in a variety of important situations. This is a rapidly expanding and exciting area of research that holds great potential for improving NK cell-based immunotherapies.
    Keywords:  NK; Natural Killer; Oxphos; glycolysis; metabolism
    DOI:  https://doi.org/10.1002/JLB.MR0718-260R
  45. J Mol Med (Berl). 2019 Jan 19.
    IDH-1 deficiency induces growth defects and metabolic alterations in GSPD-1-deficient Caenorhabditis elegans.
    Hung-Chi Yang, Hsiang Yu, You-Cheng Liu, Tzu-Ling Chen, Arnold Stern, Szecheng J Lo, Daniel Tsun-Yee Chiu.
      NADPH is a reducing equivalent that maintains redox homeostasis and supports reductive biosynthesis. Lack of major NADPH-producing enzymes predisposes cells to growth retardation and demise. It was hypothesized that double deficiency of the NADPH-generating enzymes, GSPD-1 (Glucose-6-phosphate 1-dehydrogenase), a functional homolog of human glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and IDH-1 (isocitrate dehydrogenase-1) affect growth and development in the nematode, Caenorhabditis elegans (C. elegans). The idh-1;gspd-1(RNAi) double-deficient C. elegans model displayed shrinkage of body size, growth retardation, slowed locomotion, and impaired molting. Global metabolomic analysis was employed to address whether or not metabolic pathways were altered by severe NADPH insufficiency by the idh-1;gspd-1(RNAi) double-deficiency. The principal component analysis (PCA) points to a distinct metabolomic profile of idh-1;gspd-1(RNAi) double-deficiency. Further metabolomic analysis revealed that NADPH-dependent and glutamate-dependent amino acid biosynthesis were significantly affected. The reduced pool of amino acids may affect protein synthesis, as indicated by the absence of NAS-37 expression during the molting process. In short, double deficiency of GSPD-1 and IDH-1 causes growth retardation and molting defects, which are, in part, attributed to defective protein synthesis, possibly mediated by altered amino acid biosynthesis and metabolism in C. elegans.
    Keywords:  Amino acid; C. elegans; Development; GSPD-1; IDH-1; Metabolomic; Molting
    DOI:  https://doi.org/10.1007/s00109-018-01740-2
  46. Methods Mol Biol. 2019 ;1925 59-63
    MCU Regulation in Lipid Bilayer and Electrophysiological Recording.
    Vanessa Checchetto, Ildikò Szabò.
      The mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uniporter dominant negative b- subunit (MCUb) are pore-forming components of the uniporter complex. We expressed these MCU subunits in cell-free transcription/translation systems, and we studied them, at the single molecule level, using the electrophysiological technique of planar lipid bilayer.We showed that MCU gives rise to single-channel Ca2+ currents. In contrast, MCUb alone does not display calcium-permeable channel activity, while the co-expression of MCUb:MCU drastically alters the calcium permeation mediated by MCU subunit.
    Keywords:  Calcium signaling; Ion channel; Planar lipid bilayer
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_5
  47. Cell Death Dis. 2019 Jan 25. 10(2): 72
    Heme detoxification by heme oxygenase-1 reinstates proliferative and immune balances upon genotoxic tissue injury.
    Andreas Hedblom, Seyed M Hejazi, Giacomo Canesin, Reeham Choudhury, Khalid A Hanafy, Eva Csizmadia, Jenny L Persson, Barbara Wegiel.
      Phenotypic changes of myeloid cells are critical to the regulation of premature aging, development of cancer, and responses to infection. Heme metabolism has a fundamental role in the regulation of myeloid cell function and activity. Here, we show that deletion of heme oxygenase-1 (HO-1), an enzyme that removes heme, results in an impaired DNA damage response (DDR), reduced cell proliferation, and increased cellular senescence. We detected increased levels of p16INK4a, H2AXγ, and senescence-associated-β-galactosidase (SA-β-Gal) in cells and tissues isolated from HO-1-deficient mice. Importantly, deficiency of HO-1 in residential macrophages in chimeric mice results in elevated DNA damage and senescence upon radiation-induced injury. Mechanistically, we found that mammalian target of rapamycin (mTOR)/S6 protein signaling is critical for heme and HO-1-regulated phenotype of macrophages. Collectively, our data indicate that HO-1, by detoxifying heme, blocks p16INK4a expression in macrophages, preventing DNA damage and cellular senescence.
    DOI:  https://doi.org/10.1038/s41419-019-1342-6
  48. Metabolites. 2019 Jan 18. pii: E18. [Epub ahead of print]9(1):
    GC-MS Metabolomics Reveals Distinct Profiles of Low- and High-Grade Bladder Cancer Cultured Cells.
    Daniela Rodrigues, Joana Pinto, Ana Margarida Araújo, Carmen Jerónimo, Rui Henrique, Maria de Lourdes Bastos, Paula Guedes de Pinho, Márcia Carvalho.
      Previous studies have shown that metabolomics can be a useful tool to better understand the mechanisms of carcinogenesis; however, alterations in biochemical pathways that lead to bladder cancer (BC) development have hitherto not been fully investigated. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to unveil the metabolic alterations between low-grade and high-grade BC cultured cell lines. Multivariable analysis revealed a panel of metabolites responsible for the separation between the two tumorigenic cell lines. Significantly lower levels of fatty acids, including myristic, palmitic, and palmitoleic acids, were found in high-grade versus low-grade BC cells. Furthermore, significantly altered levels of some amino acids were observed between low- and high-grade BC, namely glycine, leucine, methionine, valine, and aspartic acid. This study successfully demonstrated the potential of metabolomic analysis to discriminate BC cells according to tumor aggressiveness. Moreover, these findings suggest that bladder tumorigenic cell lines of different grades disclose distinct metabolic profiles, mainly affecting fatty acid biosynthesis and amino acid metabolism to compensate for higher energetic needs.
    Keywords:  GC-MS; bladder cancer; cancer progression; endometabolome; in vitro; metabolic pathways; metabolomic signatures
    DOI:  https://doi.org/10.3390/metabo9010018
  49. Sci Immunol. 2019 Jan 25. pii: eaap9520. [Epub ahead of print]4(31):
    Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells.
    Lelisa F Gemta, Peter J Siska, Marin E Nelson, Xia Gao, Xiaojing Liu, Jason W Locasale, Hideo Yagita, Craig L Slingluff, Kyle L Hoehn, Jeffrey C Rathmell, Timothy N J Bullock.
      In the context of solid tumors, there is a positive correlation between the accumulation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TILs) and favorable clinical outcomes. However, CD8+ TILs often exhibit a state of functional exhaustion, limiting their activity, and the underlying molecular basis of this dysfunction is not fully understood. Here, we show that TILs found in human and murine CD8+ melanomas are metabolically compromised with deficits in both glycolytic and oxidative metabolism. Although several studies have shown that tumors can outcompete T cells for glucose, thus limiting T cell metabolic activity, we report that a down-regulation in the activity of ENOLASE 1, a critical enzyme in the glycolytic pathway, represses glycolytic activity in CD8+ TILs. Provision of pyruvate, a downstream product of ENOLASE 1, bypasses this inactivity and promotes both glycolysis and oxidative phosphorylation, resulting in improved effector function of CD8+ TILs. We found high expression of both enolase 1 mRNA and protein in CD8+ TILs, indicating that the enzymatic activity of ENOLASE 1 is regulated posttranslationally. These studies provide a critical insight into the biochemical basis of CD8+ TIL dysfunction.
    DOI:  https://doi.org/10.1126/sciimmunol.aap9520
  50. Trends Biochem Sci. 2019 Jan 17. pii: S0968-0004(18)30274-3. [Epub ahead of print]
    Formylation of Eukaryotic Cytoplasmic Proteins: Linking Stress to Degradation.
    Mohamed A Eldeeb, Richard P Fahlman, Mansoore Esmaili, Edward A Fon.
      Unlike prokaryotes, N-terminal formylation has been confined to a handful of mitochondrial proteins in eukaryotes. A recent study unveils a new role for eukaryotic cytoplasmic N-terminal formylation linking diverse cellular stresses to N-terminal-dependent protein degradation. These findings suggest broad cellular implications in higher eukaryotes for N-terminal methionine formylation.
    Keywords:  Gcn2; N-end rule; N-terminal modification; amino acid limitation; formylation; protein degradation; ubiquitin
    DOI:  https://doi.org/10.1016/j.tibs.2018.12.008
  51. Methods Mol Biol. 2019 ;1925 103-109
    Ex Vivo Measurements of Ca2+ Transients in Intracellular Compartments of Skeletal Muscle Fibers by Means of Genetically Encoded Probes.
    Gaia Gherardi, Cristina Mammucari.
      We report a method for ex vivo measurements of Ca2+ transients in skeletal muscle fibers, both in the sarcoplasma and into the mitochondria. These measurements are based on the use of genetically encoded probes. Addition of targeting DNA sequences, in frame with the probe encoding sequence, ensures protein expression in specific compartments. The use of probes with different excitation spectra allows the simultaneous determination of cytosolic and mitochondrial Ca2+ transients in the same fiber. Probe encoding plasmids are expressed in flexor digitorum brevis (FDB) muscles by means of the in vivo electroporation technique. Measurements are then performed ex vivo in isolated single myofibers.
    Keywords:  Ca2+ measurements; Cytosol; Genetically encoded probes; Mitochondria; Skeletal muscle fibers
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_9
  52. Glia. 2019 Jan 22.
    In vivo Ca2+ imaging of astrocytic microdomains reveals a critical role of the amyloid precursor protein for mitochondria.
    Elena Montagna, Sophie Crux, Manja Luckner, Julia Herber, Alessio V Colombo, Petar Marinković, Sabina Tahirovic, Stefan F Lichtenthaler, Gerhard Wanner, Ulrike C Müller, Carmelo Sgobio, Jochen Herms.
      The investigation of amyloid precursor protein (APP) has been mainly confined to its neuronal functions, whereas very little is known about its physiological role in astrocytes. Astrocytes exhibit a particular morphology with slender extensions protruding from somata and primary branches. Along these fine extensions, spontaneous calcium transients occur in spatially restricted microdomains. Within these microdomains mitochondria are responsible for local energy supply and Ca2+ buffering. Using two-photon in vivo Ca2+ imaging, we report a significant decrease in the density of active microdomains, frequency of spontaneous Ca2+ transients and slower Ca2+ kinetics in mice lacking APP. Mechanistically, these changes could be potentially linked to mitochondrial malfunction as our in vivo and in vitro data revealed severe, APP-dependent structural mitochondrial fragmentation in astrocytes. Functionally, such mitochondria exhibited prolonged kinetics and morphology dependent signal size of ATP-induced Ca2+ transients. Our results highlight a prominent role of APP in the modulation of Ca2+ activity in astrocytic microdomains whose precise functioning is crucial for the reinforcement and modulation of synaptic function. This study provides novel insights in APP physiological functions which are important for the understanding of the effects of drugs validated in Alzheimer's disease treatment that affect the function of APP.
    Keywords:   in vivo Ca2+ imaging; amyloid precursor protein; astrocyte; microdomain; mitochondria fragmentation
    DOI:  https://doi.org/10.1002/glia.23584
  53. Crit Rev Biochem Mol Biol. 2019 Jan 22. 1-16
    Molecular intricacies of aerobic glycolysis in cancer: current insights into the classic metabolic phenotype.
    Shanmugasundaram Ganapathy-Kanniappan.
      Aerobic glycolysis is the process of oxidation of glucose into pyruvate followed by lactate production under normoxic condition. Distinctive from its anaerobic counterpart (i.e. glycolysis that occurs under hypoxia), aerobic glycolysis is frequently witnessed in cancers, popularly known as the "Warburg effect", and it is one of the earliest known evidences of metabolic alteration in neoplasms. Intracellularly, aerobic glycolysis circumvents mitochondrial oxidative phosphorylation (OxPhos), facilitating an increased rate of glucose hydrolysis. This in turn enables cancer cells to successfully compete with normal cells for glucose uptake in order to maintain uninterrupted growth. In addition, evading OxPhos mitigates excessive generation/accumulation of reactive oxygen species that otherwise may be deleterious to cells. Emerging data indicate that aerobic glycolysis in cancer also promotes glutaminolysis to satisfy the precursor requirements of certain biosynthetic processes (e.g. nucleic acids). Next, the metabolic intermediates of aerobic glycolysis also feed the pentose phosphate pathway (PPP) to facilitate macromolecular biosynthesis necessary for cancer cell growth and proliferation. Extracellularly, the extrusion of the end-product of aerobic glycolysis, i.e. lactate, alters the tumor microenvironment, and impacts cancer-associated cells. Collectively, accumulating data unequivocally demonstrate that aerobic glycolysis implicates myriad of molecular and functional processes to support cancer progression. This review, in the light of recent research, dissects the molecular intricacies of its regulation, and also deliberates the emerging paradigms to target aerobic glycolysis in cancer therapy.
    Keywords:  Aerobic glycolysis; Warburg effect; lactate pH; mitochondrial OxPhos; monocarboxylate transporters; tumor metabolism
    DOI:  https://doi.org/10.1080/10409238.2018.1556578
  54. Leukemia. 2019 Jan 24.
    Inflammation-induced glycolytic switch controls suppressivity of mesenchymal stem cells via STAT1 glycosylation.
    R Jitschin, M Böttcher, D Saul, S Lukassen, H Bruns, R Loschinski, A B Ekici, A Reis, A Mackensen, D Mougiakakos.
      Mesenchymal stem cells (MSCs) represent key contributors to tissue homeostasis and promising therapeutics for hyperinflammatory conditions including graft-versus-host disease. Their immunomodulatory effects are controlled by microenvironmental signals. The MSCs' functional response towards inflammatory cues is known as MSC-"licensing" and includes indoleamine 2,3-dioxygenase (IDO) upregulation. MSCs use tryptophan-depleting IDO to suppress T-cells. Increasing evidence suggests that several functions are (co-)determined by the cells' metabolic commitment. MSCs are capable of both, high levels of glycolysis and of oxidative phosphorylation. Although several studies have addressed alterations of the immune regulatory phenotype elicited by inflammatory priming metabolic mechanisms controlling this process remain unknown. We demonstrate that inflammatory MSC-licensing causes metabolic shifts including enhanced glycolysis and increased fatty acid oxidation. Yet, only interfering with glycolysis impacts IDO upregulation and impedes T-cell-suppressivity. We identified the Janus kinase (JAK)/signal transducer and activator of transcription (STAT)1 pathway as a regulator of both glycolysis and IDO, and show that enhanced glucose turnover is linked to abundant STAT1 glycosylation. Inhibiting the responsible O-acetylglucosamine (O-GlcNAc) transferase abolishes STAT1 activity together with IDO upregulation. Our data suggest that STAT1-O-GlcNAcylation increases its stability towards degradation thus sustaining downstream effects. This pathway could represent a target for interventions aiming to enhance the MSCs' immunoregulatory potency.
    DOI:  https://doi.org/10.1038/s41375-018-0376-6
  55. Nat Methods. 2019 Jan 21.
    A genetically encoded near-infrared fluorescent calcium ion indicator.
    Yong Qian, Kiryl D Piatkevich, Benedict Mc Larney, Ahmed S Abdelfattah, Sohum Mehta, Mitchell H Murdock, Sven Gottschalk, Rosana S Molina, Wei Zhang, Yingche Chen, Jiahui Wu, Mikhail Drobizhev, Thomas E Hughes, Jin Zhang, Eric R Schreiter, Shy Shoham, Daniel Razansky, Edward S Boyden, Robert E Campbell.
      We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.
    DOI:  https://doi.org/10.1038/s41592-018-0294-6
  56. Methods Mol Biol. 2019 ;1925 127-142
    In Vivo Monitoring of Ca2+ Uptake into Subcellular Compartments of Mouse Skeletal Muscle.
    Rüdiger Rudolf, Sofie Trajanovska, David Grant Allen, Tullio Pozzan.
      Ca2+ regulates many functions of skeletal muscle, including excitation-contraction coupling, energy homeostasis, and fiber-type-specific gene expression. However, microscopic observation of Ca2+ signalling in live skeletal muscle tissue has been hampered, in particular, by the combination of the high speed of Ca2+ transients and the contractile properties that are inherent to muscle. The present chapter describes methods to visualize Ca2+ signals during relaxation-contraction cycles in different subcellular compartments at high spatiotemporal resolution or at the global muscle level in combination with simultaneous measurements of muscle force. These protocols employ transfection of genetically encoded ratiometric Ca2+ sensors and two-photon microscopy as well as force transducers and associated hardware for data acquisition. Information on how to determine subcellular localization of the genetically encoded Ca2+ sensors and on how to calibrate the ratiometric data in a semiquantitative manner is given in the final paragraphs.
    Keywords:  Cameleon; FRET; Force transducer; Mitochondria; Myoplasm; Ratiometric measurement; Sarcoplasmic reticulum; Two-photon microscopy
    DOI:  https://doi.org/10.1007/978-1-4939-9018-4_11
  57. Trends Biochem Sci. 2019 Jan 21. pii: S0968-0004(19)30001-5. [Epub ahead of print]
    Targeting Metabolic-Redox Circuits for Cancer Therapy.
    Kui Wang, Jingwen Jiang, Yunlong Lei, Shengtao Zhou, Yuquan Wei, Canhua Huang.
      Metabolic alterations and elevated levels of reactive oxygen species (ROS) are two characteristics of cancer. The metabolic patterns of cancer cells are elaborately reprogrammed to fulfill the high biomass demands of rapid propagation. ROS, the byproducts of metabolic processes, are accumulated in cancer cells partially due to metabolic abnormalities or oncogenic mutations. To prevent oxidative damage, cancer cells can orchestrate metabolic adaptation to maintain reduction-oxidation (redox) balance by producing reducing equivalents. ROS, acting as second messengers, can in turn manipulate metabolic pathways by directly or indirectly affecting the function of metabolic enzymes. In this review we discuss how cancer cell metabolism and redox signaling are intertwined, with an emphasis on the perspective of targeting metabolic-redox circuits for cancer therapy.
    Keywords:  ROS; cancer therapy; metabolism; redox modifications; redox signaling
    DOI:  https://doi.org/10.1016/j.tibs.2019.01.001
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