bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2018‒12‒16
77 papers selected by
Christian Frezza,

  1. Cytosolic lipid excess-induced mitochondrial dysfunction is the cause or effect of high fat diet-induced skeletal muscle insulin resistance: a molecular insight.
  2. MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo.
  3. Drp1/Fis1-mediated mitochondrial fragmentation leads to lysosomal dysfunction in cardiac models of Huntington's disease.
  4. Mitochondrial membrane transporters and metabolic switch in heart failure.
  5. Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity.
  6. Epithelial-Mesenchymal Transition Directs Stem Cell Polarity via Regulation of Mitofusin.
  7. Epigenetic Factors and Mitochondrial Biology in Yeast: A New Paradigm for the Study of Cancer Metabolism?
  8. A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.
  9. Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage.
  10. PDK4 Augments ER-Mitochondria Contact to Dampen Skeletal Muscle Insulin Signaling During Obesity.
  11. Restricting mitochondrial GRK2 post-ischemia confers cardioprotection by reducing myocyte death and maintaining glucose oxidation.
  12. Mitoproteomics: Tackling Mitochondrial Dysfunction in Human Disease.
  13. Proton leak regulates mitochondrial reactive oxygen species generation in endothelial cell activation and inflammation - A novel concept.
  14. Acetaminophen cytotoxicity in HepG2 cells is associated with a decoupling of glycolysis from the TCA cycle, loss of NADPH production, and suppression of anabolism.
  15. NDUFV2 pseudogene (NDUFV2P1) contributes to mitochondrial complex I deficits in schizophrenia.
  16. Integrated multi-omics characterization reveals a distinctive metabolic signature and the role of NDUFA4L2 in promoting angiogenesis, chemoresistance, and mitochondrial dysfunction in clear cell renal cell carcinoma.
  17. Mitochondrial Fission Is Required for Blue Light-Induced Apoptosis and Mitophagy in Retinal Neuronal R28 Cells.
  18. Preadipocytes of obese humans display gender-specific bioenergetic responses to glucose and insulin.
  19. Evolving and Expanding the Roles of Mitophagy as a Homeostatic and Pathogenic Process.
  20. Defective respiration and one-carbon metabolism contribute to impaired naïve T cell activation in aged mice.
  21. APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS.
  22. P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.
  23. ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties.
  24. Differential regulation of cysteine oxidative post-translational modifications in high and low aerobic capacity.
  25. PUMA induction mediates acetaminophen-induced necrosis and liver injury.
  26. Bcl-2 inhibitors enhance FGFR inhibitor-induced mitochondrial-dependent cell death in FGFR2 mutant endometrial cancer.
  27. Chronic energy depletion due to iron deficiency impairs dendritic mitochondrial motility during hippocampal neuron development.
  28. Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria.
  29. Application of Mito-Priming to Generate BCL-2 Addicted Cells.
  30. Mitochondria and aging.
  31. Cancer stem-like properties and gefitinib resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2.
  32. Protein S-glutathionylation: The linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria.
  33. Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice.
  34. Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.
  35. Prohibitin 2-mediated mitophagy attenuates renal tubular epithelial cells injury by regulating mitochondrial dysfunction and NLRP3 inflammasome activation.
  36. Toll-like receptor 2 regulates metabolic reprogramming in gastric cancer via superoxide dismutase 2.
  37. Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity.
  38. Insulin-like growth factor-1 activates AMPK to augment mitochondrial function and correct neuronal metabolism in sensory neurons in type 1 diabetes.
  39. Quantification of Mitochondrial Genome Transcription in Male Reproductive Cells by Real-Time PCR.
  40. Roles of the mitochondrial genetics in cancer metastasis: not to be ignored any longer.
  41. Rapid Assessment of Mitochondrial Complex I Activity and Metabolic Phenotyping of Breast Cancer Cells by NAD(p)H Cytometry.
  42. Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP).
  43. Developmental regulation of an organelle tether coordinates mitochondrial remodeling in meiosis.
  44. Activation of Keap1-Nrf2 signaling by 4-octyl itaconate protects human umbilical vein endothelial cells from high glucose.
  45. Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission.
  46. p53-mediated adaptation to serine starvation is retained by a common tumour-derived mutant.
  47. Upregulation of tryptophanyl-tRNA synthethase adapts human cancer cells to nutritional stress caused by tryptophan degradation.
  48. Hyperactivation of Nrf2 increases stress tolerance at the cost of aging acceleration due to metabolic deregulation.
  49. TGF-β Upregulated Mitochondria Mass through the SMAD2/3→C/EBPβ→PRMT1 Signal Pathway in Primary Human Lung Fibroblasts.
  50. Dissecting the regulation and function of ATP at the single-cell level.
  51. GABA-modulating bacteria of the human gut microbiota.
  52. Loss of Mitochondrial AAA Proteases AFG3L2 and YME1L Impairs Mitochondrial Structure and Respiratory Chain Biogenesis.
  53. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.
  54. Multi-scale computational study of the Warburg effect, reverse Warburg effect and glutamine addiction in solid tumors.
  55. RagC phosphorylation autoregulates mTOR complex 1.
  56. Mitochondrial Permeability Transition: A Molecular Lesion with Multiple Drug Targets.
  57. Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase.
  58. STAT3 inhibition reduces macrophage number and tumor growth in neurofibroma.
  59. Schizophrenia-associated mt-DNA SNPs exhibit highly variable haplogroup affiliation and nuclear ancestry: Bi-genomic dependence raises major concerns for link to disease.
  60. O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity.
  61. Mitochondrial Dynamics Is Critical for the Full Pluripotency and Embryonic Developmental Potential of Pluripotent Stem Cells.
  62. Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy.
  63. Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming.
  64. Irisin Mediates Effects on Bone and Fat via αV Integrin Receptors.
  65. Dysregulated autophagy contributes to caspase-dependent neuronal apoptosis.
  66. Potent immunosuppressive effects of the oncometabolite R-2-hydroxyglutarate.
  67. The mitochondrial chaperone Prohibitin 1 negatively regulates interleukin-8 in human liver cancers.
  68. Lysine methylation by the mitochondrial methyltransferase FAM173B optimizes the function of mitochondrial ATP synthase.
  69. ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosome-lysosome fusion.
  70. Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.
  71. Methods to Probe Calcium Regulation by BCL-2 Family Members.
  72. Elucidating the contribution of ETC complexes I and II to the respirasome formation in cardiac mitochondria.
  73. Metabolic features and regulation in cell senescence.
  74. The emerging link between cancer, metabolism, and circadian rhythms.
  75. Dimethyl fumarate: Regulatory effects on the immune system in the treatment of multiple sclerosis.
  76. Germline mutations of renal cancer predisposition genes and clinical relevance in Chinese patients with sporadic, early-onset disease.
  77. Succinate dehydrogenase mutations: paraganglioma imaging and at-risk population screening.
  1. Mol Biol Rep. 2018 Dec 08.
    Cytosolic lipid excess-induced mitochondrial dysfunction is the cause or effect of high fat diet-induced skeletal muscle insulin resistance: a molecular insight.
    Baishali Alok Jana, Pavan Kumar Chintamaneni, Praveen Thaggikuppe Krishnamurthy, Ashish Wadhwani, Suresh Kumar Mohankumar.
      Mitochondria play a central role in the energy homeostasis in eukaryotic cells by generating ATP via oxidative metabolism of nutrients. Excess lipid accumulation and impairments in mitochondrial function have been considered as putative mechanisms for the pathogenesis of skeletal muscle insulin resistance. Accumulation of lipids in tissues occurs due to either excessive fatty acid uptake, decreased fatty acid utilization or both. Consequently, elevated levels cytosolic lipid metabolites, triglycerides, diacylglycerol and ceramides have been demonstrated to adversely affect glucose homeostasis. Several recent studies indicate that reduced insulin-stimulated ATP synthesis and reduced expression of mitochondrial enzymes and PPAR-γ coactivator, in high fat feeding (lipid overload) are associated with insulin resistance. Despite the fact, few notable studies suggest mitochondrial dysfunction is prevalent in type 2 diabetes mellitus; it is still not clear whether the defects in mitochondrial function are the cause of insulin resistance or the consequential effects of insulin resistance itself. Thus, there is a growing interest in understanding the intricacies of mitochondrial function and its association with cytosolic lipid excess. This review therefore critically examines the molecular cascades linking cytosolic lipid excess and mitochondrial dysfunction in the pathogenesis of high fat diet-induced insulin resistance in skeletal muscle. The sequential processes following the excess intake of high fat diet in skeletal muscle includes, accumulation of cytosolic fatty acids, increased production of reactive oxygen species, mutations and ageing, and decreased mitochondrial biogenesis. The consequent mitochondrial dysfunction is then leading to decreased β-oxidation, respiratory functions and glycolysis and increased glucolipotoxicity. These events collectively induce the insulin resistance in skeletal muscle.
    Keywords:  Cytosolic lipids; High fat diet; Insulin resistance; Mitochondria; Type 2 diabetes mellitus
    DOI:  https://doi.org/10.1007/s11033-018-4551-7
  2. Proc Natl Acad Sci U S A. 2018 Dec 12. pii: 201816656. [Epub ahead of print]
    MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo.
    Erol C Bayraktar, Lou Baudrier, Ceren Özerdem, Caroline A Lewis, Sze Ham Chan, Tenzin Kunchok, Monther Abu-Remaileh, Andrew L Cangelosi, David M Sabatini, Kıvanç Birsoy, Walter W Chen.
      Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.
    Keywords:  MITO-Tag Mice; lipidomics; metabolomics; mitochondria; proteomics
    DOI:  https://doi.org/10.1073/pnas.1816656115
  3. J Mol Cell Cardiol. 2018 Dec 11. pii: S0022-2828(18)30795-8. [Epub ahead of print]
    Drp1/Fis1-mediated mitochondrial fragmentation leads to lysosomal dysfunction in cardiac models of Huntington's disease.
    A U Joshi, A E Ebert, B Haileselassie, D Mochly-Rosen.
      Huntington's disease (HD) is a fatal hereditary neurodegenerative disorder, best known for its clinical triad of progressive motor impairment, cognitive deficits and psychiatric disturbances, is caused by CAG-repeat expansion in exon 1 of Huntingtin (HTT). However, in addition to the neurological disease, mutant HTT (mHTT), which is ubiquitously expressed in all tissues, impairs other organ systems. Not surprisingly, cardiovascular dysautonomia as well as the deterioration of circadian rhythms are among the earliest detectable pathophysiological changes in individuals with HD. Mitochondrial dysfunction in the brain and skeletal muscle in HD has been well documented, as the disease progresses. However, not much is known about mitochondrial abnormalities in the heart. In this study, we describe a role for Drp1/Fis1-mediated excessive mitochondrial fission and dysfunction, associated with lysosomal dysfunction in H9C2 expressing long polyglutamine repeat (Q73) and in human iPSC-derived cardiomyocytes transfected with Q77. Expression of long polyglutamine repeat led to reduced ATP production and mitochondrial fragmentation. We observed an increased accumulation of damaged mitochondria in the lysosome that was coupled with lysosomal dysfunction. Importantly, reducing Drp1/Fis1-mediated mitochondrial damage significantly improved mitochondrial function and cell survival. Finally, reducing Fis1-mediated Drp1 recruitment to the mitochondria, using the selective inhibitor of this interaction, P110, improved mitochondrial structure in the cardiac tissue of R6/2 mice. We suggest that drugs focusing on the central nervous system will not address mitochondrial function across all organs, and therefore will not be a sufficient strategy to treat or slow down HD disease progression.
    Keywords:  Bioenergetics; Drp1; Fis1; Heart; Huntington's disease; Mitochondria; P110
    DOI:  https://doi.org/10.1016/j.yjmcc.2018.12.004
  4. Heart Fail Rev. 2018 Dec 10.
    Mitochondrial membrane transporters and metabolic switch in heart failure.
    Vikas Kumar, T R Santhosh Kumar, C C Kartha.
      Mitochondrial dysfunction is widely recognized as a major factor for the progression of cardiac failure. Mitochondrial uptake of metabolic substrates and their utilization for ATP synthesis, electron transport chain activity, reactive oxygen species levels, ion homeostasis, mitochondrial biogenesis, and dynamics as well as levels of reactive oxygen species in the mitochondria are key factors which regulate mitochondrial function in the normal heart. Alterations in these functions contribute to adverse outcomes in heart failure. Iron imbalance and oxidative stress are also major factors for the evolution of cardiac hypertrophy, heart failure, and aging-associated pathological changes in the heart. Mitochondrial ATP-binding cassette (ABC) transporters have a key role in regulating iron metabolism and maintenance of redox status in cells. Deficiency of mitochondrial ABC transporters is associated with an impaired mitochondrial electron transport chain complex activity, iron overload, and increased levels of reactive oxygen species, all of which can result in mitochondrial dysfunction. In this review, we discuss the role of mitochondrial ABC transporters in mitochondrial metabolism and metabolic switch, alterations in the functioning of ABC transporters in heart failure, and mitochondrial ABC transporters as possible targets for therapeutic intervention in cardiac failure.
    Keywords:  Cardiac hypertrophy; Heart failure; Metabolic shift; Mitochondrial ABC transporters; Mitochondrial dysfunction
    DOI:  https://doi.org/10.1007/s10741-018-9756-2
  5. FASEB J. 2018 Dec 14. fj201801292R
    Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity.
    Essam A Assali, Dovi Shlomo, Jialiu Zeng, Evan P Taddeo, Kyle M Trudeau, Karel A Erion, Aaron H Colby, Mark W Grinstaff, Marc Liesa, Guy Las, Orian S Shirihai.
      Chronic exposure of pancreatic β cells to high concentrations of free fatty acids leads to lipotoxicity (LT)-mediated suppression of glucose-stimulated insulin secretion. This effect is in part caused by a decline in mitochondrial function as well as by a reduction in lysosomal acidification. Because both mitochondria and lysosomes can alter one another's function, it remains unclear which initiating dysfunction sets off the detrimental cascade of LT, ultimately leading to β-cell failure. Here, we investigated the effects of restoring lysosomal acidity on mitochondrial function under LT. Our results show that LT induces a dose-dependent lysosomal alkalization accompanied by an increase in mitochondrial mass. This increase is due to a reduction in mitochondrial turnover as analyzed by MitoTimer, a fluorescent protein for which the emission is regulated by mitochondrial clearance rate. Mitochondrial oxygen consumption rate, citrate synthase activity, and ATP content are all reduced by LT. Restoration of lysosomal acidity using lysosome-targeted nanoparticles is accompanied by stimulation of mitochondrial turnover as revealed by mitophagy measurements and the recovery of mitochondrial mass. Remarkably, re-acidification restores citrate synthase activity and ATP content in an insulin secreting β-cell line (INS-1). Furthermore, nanoparticle-mediated lysosomal reacidification rescues mitochondrial maximal respiratory capacity in both INS-1 cells and primary mouse islets. Therefore, our results indicate that mitochondrial dysfunction is downstream of lysosomal alkalization under lipotoxic conditions and that recovery of lysosomal acidity is sufficient to restore the bioenergetic defects.-Assali, E. A., Shlomo, D., Zeng, J., Taddeo, E. P., Trudeau, K. M., Erion, K. A., Colby, A. H., Grinstaff, M. W., Liesa, M., Las, G., Shirihai, O. S. Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity.
    Keywords:  autophagy; bioenergetics; free fatty acids; islets; photoactivated nanoparticles
    DOI:  https://doi.org/10.1096/fj.201801292R
  6. Cell Metab. 2018 Nov 27. pii: S1550-4131(18)30681-8. [Epub ahead of print]
    Epithelial-Mesenchymal Transition Directs Stem Cell Polarity via Regulation of Mitofusin.
    Meng-Ju Wu, Yu-Syuan Chen, Mi Ran Kim, Chao-Ching Chang, Silpa Gampala, Yingsheng Zhang, Yueyang Wang, Chih-Yu Chang, Jer-Yen Yang, Chun-Ju Chang.
      Mitochondria are dynamic organelles that have been linked to stem cell homeostasis. However, the mechanisms involved in mitochondrial regulation of stem cell fate determination remain elusive. Here we discover that epithelial-mesenchymal transition (EMT), a key process in cancer progression, induces mitochondrial fusion through regulation of the miR200c-PGC1α-MFN1 pathway. EMT-activated MFN1 forms a complex with PKCζ and is required for PKCζ-mediated NUMB phosphorylation and dissociation from the cortical membrane to direct asymmetric division of mammary stem cells, where fused mitochondria are tethered by MFN1-PKCζ to the cortical membrane and asymmetrically segregated to the stem cell-like progeny with enhanced glutathione synthesis and reactive oxygen species scavenging capacities, allowing sustaining of a self-renewing stem cell pool. Suppression of MFN1 expression leads to equal distribution of the fragmented mitochondria in both progenies that undergo symmetric luminal cell differentiation. Together, this study elucidates an essential role of mitofusin in stem cell fate determination to mediate EMT-associated stemness.
    Keywords:  EMT; cell plasticity; mammary stem cell; mitochondrial dynamics; mitofusin; stem cell fate; stem cell polarity
    DOI:  https://doi.org/10.1016/j.cmet.2018.11.004
  7. Front Pharmacol. 2018 ;9 1349
    Epigenetic Factors and Mitochondrial Biology in Yeast: A New Paradigm for the Study of Cancer Metabolism?
    Antonella Stoppacciaro, Serena Di Vito, Patrizia Filetici.
      Bidirectional cross-talk between nuclear and mitochondrial DNA is fundamental for cell homeostasis. Epigenetic mechanisms regulate the inter-organelle communication between nucleus and mitochondria. Recent research highlights not only the retrograde activation of nuclear gene transcription in case of mitochondria dysfunction, but also the role of post-translational modifications of mitochondrial proteins in respiratory metabolism. Here we discuss some aspects and novel findings in Saccharomyces cerevisiae. In yeast, KAT-Gcn5 and DUB-Ubp8 have a role in respiration and are localized, as single proteins, into mitochondria. These findings, beside the canonical and widely known nuclear activity of SAGA complex in chromatin regulation, provide novel clues on promising aspects linking evolutionary conserved epigenetic factors to the re-programmed metabolism of cancer cells.
    Keywords:  cancer; epigenetic; mitochondria; reprogramming; yeast
    DOI:  https://doi.org/10.3389/fphar.2018.01349
  8. Nucleic Acids Res. 2018 Dec 12.
    A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.
    Zidong Jia, Ye Zhang, Qiang Li, Zhenzhen Ye, Yuqi Liu, Changzhu Fu, Xiaohui Cang, Meng Wang, Min-Xin Guan.
      The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations.
    DOI:  https://doi.org/10.1093/nar/gky1241
  9. Methods Mol Biol. 2019 ;1877 201-216
    Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage.
    Sweta Iyer, Rachel T Uren, Ruth M Kluck.
      Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization. Here we describe biochemical protocols that can be used on most cell types to gain a population overview of BAK and BAX status.
    Keywords:  Apoptosis; BAK; BAX; BCL-2 proteins; Cytochrome c; Disulfide linkage; Mitochondria; Oligomerization; Pore formation
    DOI:  https://doi.org/10.1007/978-1-4939-8861-7_14
  10. Diabetes. 2018 Dec 06. pii: db180363. [Epub ahead of print]
    PDK4 Augments ER-Mitochondria Contact to Dampen Skeletal Muscle Insulin Signaling During Obesity.
    Themis Thoudam, Chae-Myeong Ha, Jaechan Leem, Dipanjan Chanda, Jong-Seok Park, Hyo-Jeong Kim, Jae-Han Jeon, Yeon-Kyung Choi, Suthat Liangpunsakul, Yang Hoon Huh, Tae-Hwan Kwon, Keun-Gyu Park, Robert A Harris, Kyu-Sang Park, Hyun-Woo Rhee, In-Kyu Lee.
      Mitochondria-associated ER membrane (MAM) is a structural link between mitochondria and endoplasmic reticulum (ER). MAM regulates Ca2+ transport from the ER to mitochondria via an IP3R1-GRP75-VDAC1 complex-dependent mechanism. Excessive MAM formation may cause mitochondrial Ca2+ overload and mitochondrial dysfunction. However, the exact implication of MAM formation in metabolic syndromes remains debatable. Here, we demonstrate that pyruvate dehydrogenase kinase 4 (PDK4) interacts with and stabilizes IP3R1-GRP75-VDAC1 complex at the MAM interface. Obesity-induced increase in PDK4 activity augments MAM formation and suppresses insulin signaling. Conversely, PDK4 inhibition dampens MAM formation and improves insulin signaling by preventing MAM-induced mitochondrial Ca2+ accumulation, mitochondrial dysfunction and ER stress. Furthermore, Pdk4-/- mice exhibit reduced MAM formation and are protected against diet-induced skeletal muscle insulin resistance. Finally, forced formation and stabilization of MAMs with synthetic ER-mitochondria linker prevented the beneficial effects of PDK4 deficiency on insulin signaling. Overall, our findings demonstrate a critical mediatory role of PDK4 in the development of skeletal muscle insulin resistance via enhancement of MAM formation.
    DOI:  https://doi.org/10.2337/db18-0363
  11. Sci Signal. 2018 Dec 11. pii: eaau0144. [Epub ahead of print]11(560):
    Restricting mitochondrial GRK2 post-ischemia confers cardioprotection by reducing myocyte death and maintaining glucose oxidation.
    Priscila Y Sato, J Kurt Chuprun, Laurel A Grisanti, Meryl C Woodall, Brett R Brown, Rajika Roy, Christopher J Traynham, Jessica Ibetti, Anna M Lucchese, Ancai Yuan, Konstantinos Drosatos, Doug G Tilley, Erhe Gao, Walter J Koch.
      Increased abundance of GRK2 [G protein-coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.
    DOI:  https://doi.org/10.1126/scisignal.aau0144
  12. Oxid Med Cell Longev. 2018 ;2018 1435934
    Mitoproteomics: Tackling Mitochondrial Dysfunction in Human Disease.
    María Gómez-Serrano, Emilio Camafeita, Marta Loureiro, Belén Peral.
      Mitochondria are highly dynamic and regulated organelles that historically have been defined based on their crucial role in cell metabolism. However, they are implicated in a variety of other important functions, making mitochondrial dysfunction an important axis in several pathological contexts. Despite that conventional biochemical and molecular biology approaches have provided significant insight into mitochondrial functionality, innovative techniques that provide a global view of the mitochondrion are still necessary. Proteomics fulfils this need by enabling accurate, systems-wide quantitative analysis of protein abundance. More importantly, redox proteomics approaches offer unique opportunities to tackle oxidative stress, a phenomenon that is intimately linked to aging, cardiovascular disease, and cancer. In addition, cutting-edge proteomics approaches reveal how proteins exert their functions in complex interaction networks where even subtle alterations stemming from early pathological states can be monitored. Here, we describe the proteomics approaches that will help to deepen the role of mitochondria in health and disease by assessing not only changes to mitochondrial protein composition but also alterations to their redox state and how protein interaction networks regulate mitochondrial function and dynamics. This review is aimed at showing the reader how the application of proteomics approaches during the last 20 years has revealed crucial mitochondrial roles in the context of aging, neurodegenerative disorders, metabolic disease, and cancer.
    DOI:  https://doi.org/10.1155/2018/1435934
  13. Arch Biochem Biophys. 2018 Dec 03. pii: S0003-9861(18)30688-X. [Epub ahead of print]662 68-74
    Proton leak regulates mitochondrial reactive oxygen species generation in endothelial cell activation and inflammation - A novel concept.
    Gayani K Nanayakkara, Hong Wang, Xiaofeng Yang.
      Mitochondria are capable of detecting cellular insults and orchestrating inflammatory responses. Mitochondrial reactive oxygen species (mtROS) are intermediates that trigger inflammatory signaling cascades in response to our newly proposed conditional damage associated molecular patterns (DAMP). We recently reported that increased proton leak regulates mtROS generation and thereby exert physiological and pathological activation of endothelial cells. Herein, we report the recent progress in determining the roles of proton leak in regulating mtROS, and highlight several important findings: 1) The majority of mtROS are generated in the complexes I and III of electron transport chain (ETC); 2) Inducible proton leak and mtROS production are mutually regulated; 3) ATP synthase-uncoupled ETC activity and mtROS regulate both physiological and pathological endothelial cell activation and inflammation initiation; 4) Mitochondrial Ca2+ uniporter and exchanger proteins have an impact on proton leak and mtROS generation; 5) MtROS connect signaling pathways between conditional DAMP-regulated immunometabolism and histone post-translational modifications (PTM) and gene expression. Continuous improvement of our understanding in this aspect of mitochondrial function would provide novel insights and generate novel therapeutic targets for the treatment of sterile inflammatory disorders such as metabolic diseases, cardiovascular diseases and cancers.
    Keywords:  Cardiovascular diseases; Electron transport chain (ETC) uncoupling; Endothelial cell activation; Mitochondrial reactive oxygen species (mtROS); Proton leak
    DOI:  https://doi.org/10.1016/j.abb.2018.12.002
  14. Arch Toxicol. 2018 Dec 14.
    Acetaminophen cytotoxicity in HepG2 cells is associated with a decoupling of glycolysis from the TCA cycle, loss of NADPH production, and suppression of anabolism.
    Volker Behrends, Guro F Giskeødegård, Natalia Bravo-Santano, Michal Letek, Hector C Keun.
      Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. We found that APAP exposure leads to limited cellular death and substantial growth inhibition. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. Co-treatment with a specific inhibitor of the lactate/H+ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth.
    Keywords:  Acetaminophen; GC–MS; HepG2; Isotopomer spectral analysis; Metabolomics; NMR
    DOI:  https://doi.org/10.1007/s00204-018-2371-0
  15. Mol Psychiatry. 2018 Dec 10.
    NDUFV2 pseudogene (NDUFV2P1) contributes to mitochondrial complex I deficits in schizophrenia.
    Oded Bergman, Rachel Karry, Jumana Milhem, Dorit Ben-Shachar.
      Mitochondria together with other cellular components maintain a constant crosstalk, modulating transcriptional and posttranslational processes. We and others demonstrated mitochondrial multifaceted dysfunction in schizophrenia, with aberrant complex I (CoI) as a major cause. Here we show deficits in CoI activity and homeostasis in schizophrenia-derived cell lines. Focusing on a core CoI subunit, NDUFV2, one of the most severely affected subunits in schizophrenia, we observed reduced protein level and functioning, with no change in mRNA transcripts. We further show that NDUFV2 pseudogene (NDUFV2P1) expression is increased in schizophrenia-derived cells and in postmortem brain specimens. In schizophrenia and controls pooled samples, NDUFV2P1 level demonstrated a significant inverse correlation with NDUFV2 pre- and matured protein level and with CoI-driven cellular respiration. Our data suggest a role for a pseudogene in its parent-gene regulation and possibly in CoI dysfunction in schizophrenia. The abnormal expression of the pseudogene may be one element of a vicious circle in which CoI deficits lead to mitochondrial dysfunction potentially affecting genome-wide regulation of gene expression, including the expression of pseudogenes.
    DOI:  https://doi.org/10.1038/s41380-018-0309-9
  16. Aging (Albany NY). 2018 12 11.
    Integrated multi-omics characterization reveals a distinctive metabolic signature and the role of NDUFA4L2 in promoting angiogenesis, chemoresistance, and mitochondrial dysfunction in clear cell renal cell carcinoma.
    Giuseppe Lucarelli, Monica Rutigliano, Fabio Sallustio, Domenico Ribatti, Andrea Giglio, Martina Lepore Signorile, Valentina Grossi, Paola Sanese, Anna Napoli, Eugenio Maiorano, Cristina Bianchi, Roberto A Perego, Matteo Ferro, Elena Ranieri, Grazia Serino, Lauren N Bell, Pasquale Ditonno, Cristiano Simone, Michele Battaglia.
      An altered metabolism is involved in the development of clear cell - renal cell carcinoma (ccRCC), and in this tumor many altered genes play a fundamental role in controlling cell metabolic activities. We delineated a large-scale metabolomic profile of human ccRCC, and integrated it with transcriptomic data to connect the variations in cancer metabolism with gene expression changes. Moreover, to better analyze the specific contribution of metabolic gene alterations potentially associated with tumorigenesis and tumor progression, we evaluated the transcription profile of primary renal tumor cells. Untargeted metabolomic analysis revealed a signature of an increased glucose uptake and utilization in ccRCC. In addition, metabolites related to pentose phosphate pathway were also altered in the tumor samples in association with changes in Krebs cycle intermediates and related metabolites. We identified NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) as the most highly expressed gene in renal cancer cells and evaluated its role in sustaining angiogenesis, chemoresistance, and mitochondrial dysfunction. Finally, we showed that silencing of NDUFA4L2 affects cell viability, increases mitochondrial mass, and induces ROS generation in hypoxia.
    Keywords:  NDUFA4L2; metabolomics; mitochondria; renal cell carcinoma; transcriptome
    DOI:  https://doi.org/10.18632/aging.101685
  17. Front Mol Neurosci. 2018 ;11 432
    Mitochondrial Fission Is Required for Blue Light-Induced Apoptosis and Mitophagy in Retinal Neuronal R28 Cells.
    Jia-Yu Li, Kun Zhang, Dan Xu, Wen-Tian Zhou, Wen-Qing Fang, Yu-Ying Wan, Dan-Dan Yan, Miao-Yu Guo, Jin-Xin Tao, Wen-Chuan Zhou, Fan Yang, Li-Ping Jiang, Xiao-Jian Han.
      Light emitting diodes (LEDs) are widely used to provide illumination due to their low energy requirements and high brightness. However, the LED spectrum contains an intense blue light component which is phototoxic to the retina. Recently, it has been reported that blue light may directly impinge on mitochondrial function in retinal ganglion cells (RGCs). Mitochondria are high dynamic organelles that undergo frequent fission and fusion events. The aim of our study was to elucidate the role of mitochondrial dynamics in blue light-induced damage in retinal neuronal R28 cells. We found that exposure to blue light (450 nm, 1000 lx) for up to 12 h significantly up-regulated the expression of mitochondrial fission protein Drp1, while down-regulating the expression of mitochondrial fusion protein Mfn2 in cells. Mitochondrial fission was simultaneously stimulated by blue light irradiation. In addition, exposure to blue light increased the production of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP), and induced apoptosis in R28 cells. Notably, Drp1 inhibitor Mdivi-1 and Drp1 RNAi not only attenuated blue light-induced mitochondrial fission, but also alleviated blue light-induced ROS production, MMP disruption and apoptosis in cells. Compared with Mdivi-1 and Drp1 RNAi, the antioxidant N-acetyl-L-cysteine (NAC) only slightly inhibited mitochondrial fission, while significantly alleviating apoptosis after blue light exposure. Moreover, we examined markers for mitophagy, which is responsible for the clearance of dysfunctional mitochondria. It was found that blue light stimulated the conversion of LC3B-I to LC3B-II as well as the expression of PINK1 in R28 cells. Mdivi-1 or Drp1 RNAi efficiently inhibited the blue light-induced expression of PINK1 and co-localization of LC3 with mitochondria. Thus, our data suggest that mitochondrial fission is required for blue light-induced mitochondrial dysfunction and apoptosis in RGCs.
    Keywords:  apoptosis; blue light; mitochondrial fission; mitophagy; retinal neuronal cells
    DOI:  https://doi.org/10.3389/fnmol.2018.00432
  18. Mol Metab. 2018 Nov 26. pii: S2212-8778(18)31046-9. [Epub ahead of print]
    Preadipocytes of obese humans display gender-specific bioenergetic responses to glucose and insulin.
    Michaela Keuper, Lucia Berti, Bernhard Raedle, Stephan Sachs, Anja Böhm, Louise Fritsche, Andreas Fritsche, Hans-Ulrich Häring, Martin Hrabě de Angelis, Martin Jastroch, Susanna M Hofmann, Harald Staiger.
      BACKGROUND/OBJECTIVES: Although the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age- and BMI-matched) donors must exist.SUBJECTS/METHODS: Using Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined.
    RESULTS: Dissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity.
    CONCLUSIONS: Collectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose.
    Keywords:  Cellular metabolism; Glycolysis; Oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.molmet.2018.11.006
  19. Physiol Rev. 2019 Jan 01. 99(1): 853-892
    Evolving and Expanding the Roles of Mitophagy as a Homeostatic and Pathogenic Process.
    Åsa B Gustafsson, Gerald W Dorn.
      The central functions fulfilled by mitochondria as both energy generators essential for tissue homeostasis and gateways to programmed apoptotic and necrotic cell death mandate tight control over the quality and quantity of these ubiquitous endosymbiotic organelles. Mitophagy, the targeted engulfment and destruction of mitochondria by the cellular autophagy apparatus, has conventionally been considered as the mechanism primarily responsible for mitochondrial quality control. However, our understanding of how, why, and under what specific conditions mitophagy is activated has grown tremendously over the past decade. Evidence is accumulating that nonmitophagic mitochondrial quality control mechanisms are more important to maintaining normal tissue homeostasis whereas mitophagy is an acute tissue stress response. Moreover, previously unrecognized mitophagic regulation of mitochondrial quantity control, metabolic reprogramming, and cell differentiation suggests that the mechanisms linking genetic or acquired defects in mitophagy to neurodegenerative and cardiovascular diseases or cancer are more complex than simple failure of normal mitochondrial quality control. Here, we provide a comprehensive overview of mitophagy in cellular homeostasis and disease and examine the most revolutionary concepts in these areas. In this context, we discuss evidence that atypical mitophagy and nonmitophagic pathways play central roles in mitochondrial quality control, functioning that was previously considered to be the primary domain of mitophagy.
    DOI:  https://doi.org/10.1152/physrev.00005.2018
  20. Proc Natl Acad Sci U S A. 2018 Dec 10. pii: 201804149. [Epub ahead of print]
    Defective respiration and one-carbon metabolism contribute to impaired naïve T cell activation in aged mice.
    Noga Ron-Harel, Giulia Notarangelo, Jonathan M Ghergurovich, Joao A Paulo, Peter T Sage, Daniel Santos, F Kyle Satterstrom, Steven P Gygi, Joshua D Rabinowitz, Arlene H Sharpe, Marcia C Haigis.
      T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naïve T cell activation. We observed a decrease in the number and activation of naïve T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naïve T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.
    Keywords:  T cells; aging; metabolism; mitochondria; one-carbon metabolism
    DOI:  https://doi.org/10.1073/pnas.1804149115
  21. EMBO Mol Med. 2018 Dec 14. pii: e9582. [Epub ahead of print]
    APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS.
    Alba Signes, Raffaele Cerutti, Anna S Dickson, Cristiane Benincá, Elizabeth C Hinchy, Daniele Ghezzi, Rosalba Carrozzo, Enrico Bertini, Michael P Murphy, James A Nathan, Carlo Viscomi, Erika Fernandez-Vizarra, Massimo Zeviani.
      Loss-of-function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild-type APOPT1 in control and patient-derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady-state levels were shown to be controlled by the ubiquitination-proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.
    Keywords:  APOPT1‐COA8; cytochrome c oxidase; mitochondrial encephalopathy; proteasome–ubiquitin system; reactive oxygen species
    DOI:  https://doi.org/10.15252/emmm.201809582
  22. FASEB J. 2018 Dec 10. fj201800788R
    P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.
    Matteo Savoia, Chiara Cencioni, Mattia Mori, Sandra Atlante, Germana Zaccagnini, Paolo Devanna, Lucia Di Marcotullio, Bruno Botta, Fabio Martelli, Andreas M Zeiher, Alfredo Pontecorvi, Antonella Farsetti, Francesco Spallotta, Carlo Gaetano.
      The epigenetic enzyme p300/CBP-associated factor (PCAF) belongs to the GCN5-related N-acetyltransferase (GNAT) family together with GCN5. Although its transcriptional and post-translational function is well characterized, little is known about its properties as regulator of cell metabolism. Here, we report the mitochondrial localization of PCAF conferred by an 85 aa mitochondrial targeting sequence (MTS) at the N terminus region of the protein. In mitochondria, one of the PCAF targets is the isocitrate dehydrogenase 2 (IDH2) acetylated at lysine 180. This PCAF-regulated post-translational modification might reduce IDH2 affinity for isocitrate as a result of a conformational shift involving predictively the tyrosine at position 179. Site-directed mutagenesis and functional studies indicate that PCAF regulates IDH2, acting at dual level during myoblast differentiation: at a transcriptional level together with MyoD, and at a post-translational level by direct modification of lysine acetylation in mitochondria. The latter event determines a decrease in IDH2 function with negative consequences on muscle fiber formation in C2C12 cells. Indeed, a MTS-deprived PCAF does not localize into mitochondria, remains enriched into the nucleus, and contributes to a significant increase of muscle-specific gene expression enhancing muscle differentiation. The role of PCAF in mitochondria is a novel finding shedding light on metabolic processes relevant to early muscle precursor differentiation.-Savoia, M., Cencioni, C., Mori, M., Atlante, S., Zaccagnini, G., Devanna, P., Di Marcotullio, L., Botta, B., Martelli, F., Zeiher, A. M., Pontecorvi, A., Farsetti, A., Spallotta, F., Gaetano, C. P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.
    Keywords:  IDH2; PCAF; metabolism; mitochondrion; α-ketoglutarate
    DOI:  https://doi.org/10.1096/fj.201800788R
  23. Oxid Med Cell Longev. 2018 ;2018 9765027
    ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties.
    G Nannelli, E Terzuoli, V Giorgio, S Donnini, P Lupetti, A Giachetti, P Bernardi, M Ziche.
      Endothelial cells (ECs) are dynamic cells that turn from growth into senescence, the latter being associated with cellular dysfunction, altered metabolism, and age-related cardiovascular diseases. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme metabolizing acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal (4-HNE). In conditions in which lipid peroxidation products and reactive oxygen species (ROS) are accumulated, ECs become dysfunctional and significantly contribute to the progression of vascular-dependent diseases. The aim of the present study has been to investigate whether inhibition of ALDH2 alters endothelial functions together with the impairment of bioenergetic functions, accelerating the acquisition of a senescent phenotype. HUVECs transfected with siRNA targeting ALDH2 or treated with daidzin, an ALDH2 inhibitor, were used in this study. We observed an alteration in cell morphology associated with endothelial dysfunctions. Loss of ALDH2 reduced cell proliferation and migration and increased paracellular permeability. To assess bioenergetic function in intact ECs, extracellular flux analysis was carried out to establish oxygen consumption rates (OCR). We observed a decrease in mitochondrial respiration and reserve capacity that coincided with SA-β-Gal accumulation and an increase in p21 and p53 expression in siALDH2 or daidzin-treated HUVECs. Treatment with N-acetyl-L-cysteine (NAC) reduced endothelial dysfunctions mediated by siALDH2, indicating that oxidative stress downstream to siALDH2 plays an instrumental role. Our results highlight that ALDH2 impairment accelerates the acquisition of a premature senescent phenotype, a change likely to be associated with the observed reduction of mitochondrial respiration and reserve capacity.
    DOI:  https://doi.org/10.1155/2018/9765027
  24. Sci Rep. 2018 Dec 11. 8(1): 17772
    Differential regulation of cysteine oxidative post-translational modifications in high and low aerobic capacity.
    Rodrigo W A Souza, Christiano R R Alves, Alessandra Medeiros, Natale Rolim, Gustavo J J Silva, José B N Moreira, Marcia N Alves, Martin Wohlwend, Mohammed Gebriel, Lars Hagen, Animesh Sharma, Lauren G Koch, Steven L Britton, Geir Slupphaug, Ulrik Wisløff, Patricia C Brum.
      Given the association between high aerobic capacity and the prevention of metabolic diseases, elucidating the mechanisms by which high aerobic capacity regulates whole-body metabolic homeostasis is a major research challenge. Oxidative post-translational modifications (Ox-PTMs) of proteins can regulate cellular homeostasis in skeletal and cardiac muscles, but the relationship between Ox-PTMs and intrinsic components of oxidative energy metabolism is still unclear. Here, we evaluated the Ox-PTM profile in cardiac and skeletal muscles of rats bred for low (LCR) and high (HCR) intrinsic aerobic capacity. Redox proteomics screening revealed different cysteine (Cys) Ox-PTM profile between HCR and LCR rats. HCR showed a higher number of oxidized Cys residues in skeletal muscle compared to LCR, while the opposite was observed in the heart. Most proteins with differentially oxidized Cys residues in the skeletal muscle are important regulators of oxidative metabolism. The most oxidized protein in the skeletal muscle of HCR rats was malate dehydrogenase (MDH1). HCR showed higher MDH1 activity compared to LCR in skeletal, but not cardiac muscle. These novel findings indicate a clear association between Cys Ox-PTMs and aerobic capacity, leading to novel insights into the role of Ox-PTMs as an essential signal to maintain metabolic homeostasis.
    DOI:  https://doi.org/10.1038/s41598-018-35728-2
  25. Hepatology. 2018 Dec 14.
    PUMA induction mediates acetaminophen-induced necrosis and liver injury.
    Dongshi Chen, Hong-Min Ni, Lei Wang, Xiaowen Ma, Jian Yu, Wen-Xing Ding, Lin Zhang.
      Acetaminophen (APAP) overdose is one of the leading causes of hepatotoxicity and acute liver failure in the United States. Accumulating evidence suggests that hepatocyte necrosis plays a critical role in APAP-induced liver injury. However, the mechanisms of APAP-induced necrosis and liver injury are not fully understood. In this study, we found that p53 up-regulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family member, was markedly induced by APAP in the mouse livers and in isolated human and mouse hepatocytes. PUMA deficiency suppressed APAP-induced mitochondrial dysfunction and release of cell death factors from mitochondria, and protected against APAP-induced hepatocyte necrosis and liver injury in mice. PUMA induction by APAP was p53-independent, and required RIP1 and JNK via transcriptional activation. Furthermore, a small-molecule PUMA inhibitor, administered after APAP treatment, mitigated APAP-induced hepatocyte necrosis and liver injury. Conclusions: Our results demonstrate that RIP1/JNK-dependent PUMA induction mediates APAP-induced liver injury by promoting hepatocyte mitochondrial dysfunction and necrosis, and suggest that PUMA inhibition is useful for alleviating acute hepatotoxicity due to APAP overdose. This article is protected by copyright. All rights reserved.
    Keywords:   PUMA ; acetaminophen; liver injury; mitochondria; necrosis
    DOI:  https://doi.org/10.1002/hep.30422
  26. Mol Oncol. 2018 Dec 08.
    Bcl-2 inhibitors enhance FGFR inhibitor-induced mitochondrial-dependent cell death in FGFR2 mutant endometrial cancer.
    Leisl M Packer, Samantha J Stehbens, Vanessa F Bonazzi, Jennifer Gunter, Robert J Ju, Micheal Ward, Michael Gartside, Sara Byron, Pamela M Pollock.
      Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release, and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; furthermore, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with Bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.
    Keywords:  ABT263; BGJ398; Endometrial cancer; FGFR2 inhibitor; cell death
    DOI:  https://doi.org/10.1002/1878-0261.12422
  27. J Neurosci. 2018 Dec 06. pii: 1504-18. [Epub ahead of print]
    Chronic energy depletion due to iron deficiency impairs dendritic mitochondrial motility during hippocampal neuron development.
    Thomas W Bastian, William C von Hohenberg, Michael K Georgieff, Lorene M Lanier.
      During development, neurons require highly integrated metabolic machinery to meet the large energy demands of growth, differentiation, and synaptic activity within their complex cellular architecture. Dendrites/axons require anterograde trafficking of mitochondria for local ATP synthesis to support these processes. Acute energy depletion impairs mitochondrial dynamics, but how chronic energy insufficiency affects mitochondrial trafficking and quality control during neuronal development is unknown. Since iron deficiency (ID) impairs mitochondrial respiration/ATP production, we treated mixed-sex embryonic mouse hippocampal neuron cultures with the iron chelator deferoxamine (DFO) to model chronic energetic insufficiency and its effects on mitochondrial dynamics during neuronal development. At 11 days in vitro (DIV), DFO reduced average mitochondrial speed by increasing the pause frequency of individual dendritic mitochondria. Time spent in anterograde motion was reduced; retrograde motion was spared. The average size of moving mitochondria was reduced and the expression of fusion and fission genes was altered, indicating impaired mitochondrial quality control. Mitochondrial density was not altered suggesting that respiratory capacity and not location is the key factor for mitochondrial regulation of early dendritic growth/branching. At 18DIV, the overall density of mitochondria within terminal dendritic branches was reduced in DFO-treated neurons, which may contribute to the long-term deficits in connectivity and synaptic function following early-life ID. The study provides new insights into the cross-regulation between energy production and dendritic mitochondrial dynamics during neuronal development and may be particularly relevant to neuropsychiatric and neurodegenerative diseases, many of which are characterized by impaired brain iron homeostasis, energy metabolism and mitochondrial trafficking.SIGNIFICANCE STATEMENTThis study uses a primary neuronal culture model of iron deficiency to address a gap in understanding of how dendritic mitochondrial dynamics are regulated when energy depletion occurs during a critical period of neuronal maturation. At the beginning of peak dendritic growth/branching, iron deficiency reduces mitochondrial speed through increased pause frequency, decreases mitochondrial size, and alters fusion/fission gene expression. At this stage, mitochondrial density in terminal dendrites is not altered, suggesting that total mitochondrial oxidative capacity and not trafficking is the main mechanism underlying dendritic complexity deficits in iron-deficient neurons. Our findings provide foundational support for future studies exploring the mechanistic role of developmental mitochondrial dysfunction in neurodevelopmental, psychiatric, and neurodegenerative disorders characterized by mitochondrial energy production and trafficking deficits.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1504-18.2018
  28. EMBO J. 2018 Dec 11. pii: e99360. [Epub ahead of print]
    Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria.
    Nadia Cummins, Andrea Tweedie, Steven Zuryn, Jesus Bertran-Gonzalez, Jürgen Götz.
      Accumulation of the protein tau characterises Alzheimer's disease and other tauopathies, including familial forms of frontotemporal dementia (FTD) that carry pathogenic tau mutations. Another hallmark feature of these diseases is the accumulation of dysfunctional mitochondria. Although disease-associated tau is known to impair several aspects of mitochondrial function, it is still unclear whether it also directly impinges on mitochondrial quality control, specifically Parkin-dependent mitophagy. Using the mito-QC mitophagy reporter, we found that both human wild-type (hTau) and FTD mutant tau (hP301L) inhibited mitophagy in neuroblastoma cells, by reducing mitochondrial translocation of Parkin. In the Caenorhabditis elegans nervous system, hTau expression reduced mitophagy, whereas hP301L expression completely inhibited it. These effects were not due to changes in the mitochondrial membrane potential or the cytoskeleton, as tau specifically impaired Parkin recruitment to defective mitochondria by sequestering it in the cytosol. This sequestration was mediated by aberrant interactions of Parkin with the projection domain of tau. As mitochondria are dysfunctional in neurodegenerative conditions, these data suggest a vicious cycle, with tau also inhibiting the degradation of damaged mitochondria.
    Keywords:   C. elegans ; Alzheimer's disease; autophagy; mitochondria
    DOI:  https://doi.org/10.15252/embj.201899360
  29. Methods Mol Biol. 2019 ;1877 45-60
    Application of Mito-Priming to Generate BCL-2 Addicted Cells.
    Jonathan Lopez, Stephen W G Tait.
      The majority of apoptotic stimuli trigger cell death through the mitochondrial pathway of apoptosis. Invariably, mitochondrial apoptosis requires engagement of mitochondrial outer membrane permeabilization or MOMP to initiate cell death. We have developed a new method, called mito-priming, that allows for rapid and synchronous induction of mitochondrial apoptosis in an on-target manner. Mito-priming uses coexpression of pro- and antiapoptotic Bcl-2 proteins to render cells sensitive to the addition of Bcl-2 targeting BH3-mimetic drugs. This chapter describes how to design mito-priming constructs and apply them to generate mito-primed lines. Second, we describe how to validate cell death sensitivity of mito-primed lines using different methods. Finally, we describe how to generate MOMP-resistant cell lines, using CRISPR-Cas9 mediated deletion of BAX and BAK. Facilitating the investigation of mitochondrial apoptosis, mito-priming provides a clean, robust way to induce mitochondrial apoptosis both in vitro and in vivo.
    Keywords:  Apoptosis; BCL-2; BH3 mimetic mito-priming; BH3-only; MOMP; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-4939-8861-7_3
  30. BMB Rep. 2018 Dec 14. pii: 4455. [Epub ahead of print]
    Mitochondria and aging.
    Jyung Mean Son, Changhan Lee.
      Aging is accompanied by a time-dependent progressive deterioration of multiple factors of the cellular system. The past several decades have witnessed major leaps in our understanding of the biological mechanism of aging using dietary, genetic, pharmacological, and physical interventions. Metabolic processes, including nutrient sensing pathways and mitochondrial function, have emerged as prominent regulators of aging. Mitochondria have been considered to play a key role largely due to their production of reactive oxygen species (ROS), resulting in DNA damage that accumulates over time and ultimately causes cellular failure. This theory, known as the mitochondrial free radical theory of aging (MFRTA), was favored by the aging field, but increasing inconsistent evidence has led to criticism and rejection of this idea. However, MFRTA should not be hastily rejected in its entirety because we now understand that ROS is not simply an undesired toxic metabolic byproduct, but also an important signaling molecule that is vital to cellular fitness. Notably, mitochondrial function, a term traditionally referred to bioenergetics and apoptosis, has since expanded considerably. It encompasses numerous other key biological processes, including the following: (i) complex metabolic processes, (ii) intracellular and endocrine signaling/communication, and (iii) immunity/inflammation. Here, we will discuss shortcomings of previous concepts regarding mitochondria in aging and their emerging roles based on recent advances. We will also discuss how the mitochondrial genome integrates with major theories on the evolution of aging.
  31. Oncogene. 2018 Dec 07.
    Cancer stem-like properties and gefitinib resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2.
    Tatsunori Nishimura, Asuka Nakata, Xiaoxi Chen, Kurumi Nishi, Makiko Meguro-Horike, Soichiro Sasaki, Kenji Kita, Shin-Ichi Horike, Kaori Saitoh, Keiko Kato, Kaori Igarashi, Takahiko Murayama, Susumu Kohno, Chiaki Takahashi, Naofumi Mukaida, Seiji Yano, Tomoyoshi Soga, Arinobu Tojo, Noriko Gotoh.
      Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinib-sensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.
    DOI:  https://doi.org/10.1038/s41388-018-0589-1
  32. Chem Biol Interact. 2018 Dec 08. pii: S0009-2797(18)31097-4. [Epub ahead of print]
    Protein S-glutathionylation: The linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria.
    Adrian Young, Robert Gill, Ryan J Mailloux.
      Protein S-glutathionylation reactions are a ubiquitous oxidative modification required to control protein function in response to changes in redox buffering capacity. These reactions are rapid and reversible and are, for the most part, enzymatically mediated by glutaredoxins (GRX) and glutathione S-transferases (GST). Protein S-glutathionylation has been found to control a range of cell functions in response to different physiological cues. Although these reactions occur throughout the cell, mitochondrial proteins seem to be highly susceptible to reversible S-glutathionylation, a feature attributed to the unique physical properties of this organelle. Indeed, mitochondria contain a number of S-glutathionylation targets which includes proteins involved in energy metabolism, solute transport, reactive oxygen species (ROS) production, proton leaks, apoptosis, antioxidant defense, and mitochondrial fission and fusion. Moreover, it has been found that conjugation and removal of glutathione from proteins in mitochondria fulfills a number of important physiological roles and defects in these reactions can have some dire pathological consequences. Here, we provide an updated overview on mitochondrial protein S-glutathionylation reactions and their importance in cell functions and physiology.
    DOI:  https://doi.org/10.1016/j.cbi.2018.12.003
  33. Toxicol Appl Pharmacol. 2018 Dec 05. pii: S0041-008X(18)30540-4. [Epub ahead of print]
    Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice.
    Hye-Youn Cho, Laura Miller-DeGraff, Terry Blankenship-Paris, Xuting Wang, Douglas A Bell, Fred Lih, Leesa Deterding, Vijayalakshmi Panduri, Daniel L Morgan, Masayuki Yamamoto, Anita J Reddy, Paul Talalay, Steven R Kleeberger.
      Nrf2 is essential to antioxidant response element (ARE)-mediated host defense. Sulforaphane (SFN) is a phytochemical antioxidant known to affect multiple cellular targets including Nrf2-ARE pathway in chemoprevention. However, the role of SFN in non-malignant airway disorders remain unclear. To test if pre-activation of Nrf2-ARE signaling protects lungs from oxidant-induced acute injury, wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) mice were given SFN orally or as standardized broccoli sprout extract diet (SBE) before hyperoxia or air exposure. Hyperoxia-induced pulmonary injury and oxidation indices were significantly reduced by SFN or SBE in Nrf2+/+ mice but not in Nrf2-/- mice. SFN upregulated a large cluster of basal lung genes that are involved in mitochondrial oxidative phosphorylation, energy metabolism, and cardiovascular protection only in Nrf2+/+ mice. Bioinformatic analysis elucidated ARE-like motifs on these genes. Transcript abundance of the mitochondrial machinery genes remained significantly higher after hyperoxia exposure in SFN-treated Nrf2+/+ mice than in SFN-treated Nrf2-/- mice. Nuclear factor-κB was suggested to be a central molecule in transcriptome networks affected by SFN. Minor improvement of hyperoxia-caused lung histopathology and neutrophilia by SFN in Nrf2-/- mice implies Nrf2-independent or alternate effector mechanisms. SFN is suggested to be as a preventive intervention in a preclinical model of acute lung injury by linking mitochondria and Nrf2. Administration of SFN alleviated acute lung injury-like pathogenesis in a Nrf2-dependent manner. Potential AREs in the SFN-inducible transcriptome for mitochondria bioenergetics provided a new insight into the downstream mechanisms of Nrf2-mediated pulmonary protection.
    Keywords:  Antioxidant response element; Broccoli; Hyperoxia; Lung; Microarray
    DOI:  https://doi.org/10.1016/j.taap.2018.12.004
  34. Cell Death Differ. 2018 Dec 13.
    Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.
    Sandra M Hill, Lidia Wrobel, David C Rubinsztein.
      Autophagy is a conserved intracellular degradation pathway essential for protein homeostasis, survival and development. Defects in autophagic pathways have been connected to a variety of human diseases, including cancer and neurodegeneration. In the process of macroautophagy, cytoplasmic cargo is enclosed in a double-membrane structure and fused to the lysosome to allow for digestion and recycling of material. Autophagosome formation is primed by the ULK complex, which enables the downstream production of PI(3)P, a key lipid signalling molecule, on the phagophore membrane. The PI(3)P is generated by the PI3 kinase (PI3K) complex, consisting of the core components VPS34, VPS15 and Beclin 1. Beclin 1 is a central player in autophagy and constitutes a molecular platform for the regulation of autophagosome formation and maturation. Post-translational modifications of Beclin 1 affect its stability, interactions and ability to regulate PI3K activity, providing the cell with a plethora of strategies to fine-tune the levels of autophagy. Being such an important regulator, Beclin 1 is a potential target for therapeutic intervention and interfering with the post-translational regulation of Beclin 1 could be one way of manipulating the levels of autophagy. In this review, we provide an overview of the known post-translational modifications of Beclin 1 that govern its role in autophagy and how these modifications are maintained by input from several upstream signalling pathways. ▓.
    DOI:  https://doi.org/10.1038/s41418-018-0254-9
  35. Am J Physiol Renal Physiol. 2018 Dec 12.
    Prohibitin 2-mediated mitophagy attenuates renal tubular epithelial cells injury by regulating mitochondrial dysfunction and NLRP3 inflammasome activation.
    Yao Xu, Jingjing Wang, Wangjie Xu, Feng Ding, Wei Ding.
      Accumulating evidence demonstrates that mitochondrial dysfunction and inflammasome activation play a critical role in the pathogenesis of renal tubular injury through the production of reactive oxygen species and cytokines. Prohibitin 2 (PHB2) is a newly identified intracellular receptor of mitophagy (a type of autophagy) that mediates selective removal of damaged mitochondria, and it could possibly play a renoprotective role in kidney disease. In this study, we confirmed that autophagy is activated in tubular epithelial cells treated with angiotensin II, and that inhibition of autophagy results in tubular cell injury. Strikingly, PHB2 knockdown reduced the level of mitophagy and augmented cell death, while overexpression of PHB2 provided protection against NLRP3-induced inflammatory pathways through amelioration of mitochondrial dysfunction. Our research is the first to experimentally demonstrate the role of PHB2 in kidney disease, and thereby, to provide a better understanding of how autophagy modulates inflammation in renal tubules. These data highlight PHB2 as a therapeutic target in the future treatment of CKD.
    Keywords:  CKD; Prohibitin 2; inflammation; mitochondrial dysfunction; mitophagy
    DOI:  https://doi.org/10.1152/ajprenal.00420.2018
  36. Int J Cancer. 2018 Dec 11.
    Toll-like receptor 2 regulates metabolic reprogramming in gastric cancer via superoxide dismutase 2.
    You Dong Liu, Liang Yu, Le Ying, Jesse Balic, Hugh Gao, Nian Tao Deng, Alison West, Feng Yan, Cheng Bo Ji, Daniel Gough, Patrick Tan, Brendan J Jenkins, Ji Kun Li.
      Toll-like receptors (TLRs) play critical roles in host defence following recognition of conserved microbial- and host-derived components, and their dysregulation is a common feature of various inflammation-associated cancers, including gastric cancer (GC). Despite the recent recognition that metabolic reprogramming is a hallmark of cancer, the molecular effectors of altered metabolism during tumorigenesis remain unclear. Here, using bioenergetics function assays on human GC cells, we reveal that ligand-induced activation of TLR2, predominantly through TLR1/2 heterodimer, augments both oxidative phosphorylation (OXPHOS) and glycolysis, with a bias towards glycolytic activity. Notably, DNA microarray-based expression profiling of human cancer cells stimulated with TLR2 ligands demonstrated significant enrichment of gene-sets for oncogenic pathways previously implicated in metabolic regulation, including reactive oxygen species (ROS), p53 and Myc. Moreover, the redox gene encoding the manganese-dependent mitochondrial enzyme, superoxide dismutase (SOD)2, was strongly induced at the mRNA and protein levels by multiple signalling pathways downstream of TLR2, namely JAK-STAT3, JNK MAPK and NF-κB. Furthermore, siRNA-mediated suppression of SOD2 ameliorated the TLR2-induced metabolic shift in human GC cancer cells. Importantly, patient-derived tissue microarrays and bioinformatics interrogation of clinical datasets indicated that upregulated expression of TLR2 and SOD2 were significantly correlated in human GC, and the TLR2-SOD2 axis was associated with multiple clinical parameters of advanced stage disease, including distant metastasis, microvascular invasion and stage, as well as poor survival. Collectively, our findings reveal a novel TLR2-SOD2 axis as a potential biomarker for therapy and prognosis in cancer. This article is protected by copyright. All rights reserved.
    Keywords:  Gastric cancer; Toll-like receptor 2; metabolism; superoxide dismutase 2
    DOI:  https://doi.org/10.1002/ijc.32060
  37. Cell Death Dis. 2018 Dec 13. 9(12): 1191
    Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity.
    Benny Zhitomirsky, Anna Yunaev, Roman Kreiserman, Ariel Kaplan, Michal Stark, Yehuda G Assaraf.
      Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca2+, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.
    DOI:  https://doi.org/10.1038/s41419-018-1227-0
  38. Mol Metab. 2018 Nov 28. pii: S2212-8778(18)30977-3. [Epub ahead of print]
    Insulin-like growth factor-1 activates AMPK to augment mitochondrial function and correct neuronal metabolism in sensory neurons in type 1 diabetes.
    Mohamad-Reza Aghanoori, Darrell R Smith, Shiva Shariati-Ievari, Andrew Ajisebutu, Annee Nguyen, Fiona Desmond, Carlos H A Jesus, Xiajun Zhou, Nigel A Calcutt, Michel Aliani, Paul Fernyhough.
      OBJECTIVE: Diabetic sensorimotor polyneuropathy (DSPN) affects approximately half of diabetic patients leading to significant morbidity. There is impaired neurotrophic growth factor signaling, AMP-activated protein kinase (AMPK) activity and mitochondrial function in dorsal root ganglia (DRG) of animal models of type 1 and type 2 diabetes. We hypothesized that sub-optimal insulin-like growth factor 1 (IGF-1) signaling in diabetes drives loss of AMPK activity and mitochondrial function, both contributing to development of DSPN.METHODS: Age-matched control Sprague-Dawley rats and streptozotocin (STZ)-induced type 1 diabetic rats with/without IGF-1 therapy were used for in vivo studies. For in vitro studies, DRG neurons from control and STZ-diabetic rats were cultured and treated with/without IGF-1 in the presence or absence of inhibitors or siRNAs.
    RESULTS: Dysregulation of mRNAs for IGF-1, AMPKα2, ATP5a1 (subunit of ATPase), and PGC-1β occurred in DRG of diabetic vs. control rats. IGF-1 up-regulated mRNA levels of these genes in cultured DRGs from control or diabetic rats. IGF-1 treatment of DRG cultures significantly (P < 0.05) increased phosphorylation of Akt, P70S6K, AMPK and acetyl-CoA carboxylase (ACC). Mitochondrial gene expression and oxygen consumption rate (spare respiratory capacity), ATP production, mtDNA/nDNA ratio and neurite outgrowth were augmented (P < 0.05). AMPK inhibitor, Compound C, or AMPKα1-specific siRNA suppressed IGF-1 elevation of mitochondrial function, mtDNA and neurite outgrowth. Diabetic rats treated with IGF-1 exhibited reversal of thermal hypoalgesia and, in a separate study, reversed the deficit in corneal nerve profiles. In diabetic rats, IGF-1 elevated the levels of AMPK and P70S6K phosphorylation, raised Complex IV-MTCO1 and Complex V-ATP5a protein expression, and restored the enzyme activities of Complex IV and I in the DRG. IGF-1 prevented TCA metabolite build-up in nerve.
    CONCLUSIONS: In DRG neuron cultures IGF-1 signals via AMPK to elevate mitochondrial function and drive axonal outgrowth. We propose that this signaling axis mediates IGF-1-dependent protection from distal dying-back of fibers in diabetic neuropathy.
    Keywords:  AMPK; Axon regeneration; Diabetic neuropathy; IGF-1; Oxygen consumption rate
    DOI:  https://doi.org/10.1016/j.molmet.2018.11.008
  39. Methods Mol Biol. 2019 ;1894 171-180
    Quantification of Mitochondrial Genome Transcription in Male Reproductive Cells by Real-Time PCR.
    Cheng Xu, Qian Liu, Jin Xu, Aihua Gu.
      Mitochondria are organelles that play a key role in the regulation of cell energy metabolism, biosynthesis, and cell death. Mitochondria are also involved in important physiological processes, such as the three-carboxylic acid cycle, the oxidation of fatty acids and amino acids, and calcium ion homeostasis. The energy demands of male germ cells during mitosis and meiosis are higher than those of somatic cells, suggesting that mitochondria play a critical role in sperm. Mitochondria are nanotoxicity targets in male germ cells. The level of mitochondrial genome transcription is reflective of mitochondrial function and can therefore be used as a quantitative index for evaluating nanotoxicity. This study describes how to use real-time PCR to evaluate the effects of nanomaterials on mitochondrial genome transcription in male reproductive cells.
    Keywords:  Germ cell; Mitochondria; Nanotoxicity; Real-time PCR; Transcription
    DOI:  https://doi.org/10.1007/978-1-4939-8916-4_11
  40. Cancer Metastasis Rev. 2018 Dec 12.
    Roles of the mitochondrial genetics in cancer metastasis: not to be ignored any longer.
    Thomas C Beadnell, Adam D Scheid, Carolyn J Vivian, Danny R Welch.
      Mitochondrial DNA (mtDNA) encodes for only a fraction of the proteins that are encoded within the nucleus, and therefore has typically been regarded as a lesser player in cancer biology and metastasis. Accumulating evidence, however, supports an increased role for mtDNA impacting tumor progression and metastatic susceptibility. Unfortunately, due to this delay, there is a dearth of data defining the relative contributions of specific mtDNA polymorphisms (SNP), which leads to an inability to effectively use these polymorphisms to guide and enhance therapeutic strategies and diagnosis. In addition, evidence also suggests that differences in mtDNA impact not only the cancer cells but also the cells within the surrounding tumor microenvironment, suggesting a broad encompassing role for mtDNA polymorphisms in regulating the disease progression. mtDNA may have profound implications in the regulation of cancer biology and metastasis. However, there are still great lengths to go to understand fully its contributions. Thus, herein, we discuss the recent advances in our understanding of mtDNA in cancer and metastasis, providing a framework for future functional validation and discovery.
    Keywords:  Metabolism; Metastasis; Mitochondrial genetics; Polymorphism; Tumor progression
    DOI:  https://doi.org/10.1007/s10555-018-9772-7
  41. Cytometry A. 2018 Dec 11.
    Rapid Assessment of Mitochondrial Complex I Activity and Metabolic Phenotyping of Breast Cancer Cells by NAD(p)H Cytometry.
    V Krishnan Ramanujan.
      Cancer cells are known to display a variety of metabolic reprogramming strategies to fulfill their own growth and proliferative agenda. With the advent of high resolution imaging strategies, metabolomics techniques, and so forth, there is an increasing appreciation of critical role that tumor cell metabolism plays in the overall breast cancer (BC) growth. In this report, we demonstrate a sensitive, flow-cytometry-based assay for rapidly assessing the metabolic phenotypes in isolated suspensions of breast cancer cells. By measuring the temporal variation of NAD(p)H signals in unlabeled, living cancer cells, and by measuring mitochondrial membrane potential {Δψm } in fluorescently labeled cells, we demonstrate that these signals can reliably distinguish the metabolic phenotype of human breast cancer cells and can track the cellular sensitivity to drug candidates. We further show the utility of this metabolic ratio {Δψm /NAD(p)H} in monitoring mitochondrial functional improvement as well as metabolic heterogeneity in primary murine tumor cells isolated from tumor biopsies. Together, these results demonstrate a novel possibility for rapid metabolic functional screening applications as well as a metabolic phenotyping tool for determining drug sensitivity in living cancer cells. © 2018 International Society for Advancement of Cytometry.
    Keywords:  NAD(p)H; breast cancer; metabolic plasticity; metabolism; mitochondria; tumor heterogeneity
    DOI:  https://doi.org/10.1002/cyto.a.23681
  42. Methods Mol Biol. 2019 ;1877 77-91
    Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP).
    Lindsey M Ludwig, Katrina L Maxcy, James L LaBelle.
      The BCL-2 family of proteins orchestrates a complex signaling network that governs the balance between cellular survival and death. A comprehensive understanding of the mechanistic interactions between these proteins continues to evolve in normal and malignant cells. The functional variation by individual BCL-2 proteins in different cell types has driven clinical therapeutic development in targeting individual BCL-2 members with the goal of fine-tuning cell death in diseased cells. Given the importance of understanding and validating the effect of activating or inhibiting BCL-2 protein interactions in individual cells, the methods used to measure apoptotic cell death have undergone increased scrutiny. Here, we describe two in vitro flow cytometry-based methods that are useful in measuring BCL-2 proteins and mitochondrial-based cell death in complex cell populations.
    Keywords:  Apoptosis; BCL-2 proteins; Flow cytometry; Intracellular staining; Mitochondria outer membrane permeabilization (MOMP)
    DOI:  https://doi.org/10.1007/978-1-4939-8861-7_5
  43. J Cell Biol. 2018 Dec 11. pii: jcb.201807097. [Epub ahead of print]
    Developmental regulation of an organelle tether coordinates mitochondrial remodeling in meiosis.
    Eric M Sawyer, Pallavi R Joshi, Victoria Jorgensen, Julius Yunus, Luke E Berchowitz, Elçin Ünal.
      Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation and organelle architecture are known, but the interface between them remains unexplored. We analyzed the regulation of mitochondrial detachment from the cell cortex, a known meiotic alteration to mitochondrial morphology. We found that mitochondrial detachment is enabled by the programmed destruction of the mitochondria-endoplasmic reticulum-cortex anchor (MECA), an organelle tether that bridges mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We further present evidence for Ime2-dependent phosphorylation and degradation of MECA in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.
    DOI:  https://doi.org/10.1083/jcb.201807097
  44. Biochem Biophys Res Commun. 2018 Dec 10. pii: S0006-291X(18)32675-5. [Epub ahead of print]
    Activation of Keap1-Nrf2 signaling by 4-octyl itaconate protects human umbilical vein endothelial cells from high glucose.
    Chun Tang, Shengyu Tan, Yiqing Zhang, Lini Dong, Yan Xu.
      High glucose (HG) induces oxidative injury to cultured human umbilical vein endothelial cells (HUVECs). Recent studies have discovered 4-octyl itaconate (OI) as a novel and cell permeable Nrf2 (nuclear-factor-E2-related factor 2) activator. Its potential activity in HG-treated HUVECs was tested here. In HUVECs OI disrupted Keap1-Nrf2 association, causing Nrf2 protein accumulation and nuclear translocation, as well as transcription and expression of Nrf2-ARE-dependent genes, including HO1, NQO1 and GCLM. Significantly, pretreatment with OI potently inhibited HG (40 mM glucose)-induced death and apoptosis of HUVECs. Moreover, OI potently inhibited HG-induced reactive oxygen species (ROS) production, lipid peroxidation, superoxide accumulation and mitochondrial depolarization in HUVECs. Activation of Nrf2 is required for OI-induced cytoprotection in HUVECs. Nrf2 shRNA or knockout (by CRISPR/Cas9 method) reversed OI-mediated HUVEC protection against HG. Further studies showed that Keap1 silencing or Cys151S mutation mimicked and nullified OI-induced activity in HUVECs. Taken together, we conclude that OI activates Keap1-Nrf2 signaling to protect HUVECs from HG.
    Keywords:  4-octyl itaconate; HUVECs; High glucose; Nrf2 signaling; Oxidative injury
    DOI:  https://doi.org/10.1016/j.bbrc.2018.12.032
  45. Nat Commun. 2018 12 07. 9(1): 5239
    Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission.
    Sukrut C Kamerkar, Felix Kraus, Alice J Sharpe, Thomas J Pucadyil, Michael T Ryan.
      Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission.
    DOI:  https://doi.org/10.1038/s41467-018-07543-w
  46. Cancer Metab. 2018 ;6 18
    p53-mediated adaptation to serine starvation is retained by a common tumour-derived mutant.
    Timothy J Humpton, Andreas K Hock, Oliver D K Maddocks, Karen H Vousden.
      Background: In response to oncogenic stress, the tumour suppressor protein p53 can induce the elimination of cells through induction of cell death or senescence, helping to restrain malignant progression. Conversely, under nutrient stress, p53 can protect cells by supporting metabolic adaptation. Many cancers express mutant p53 proteins that have lost the cell-elimination properties of wild-type p53. However, a previous report showed that a tumour-derived mutant can retain the ability to support cells under glutamine starvation.Results: We show that a commonly occurring p53 mutant, R248W, retains wild-type ability to support survival under serine starvation. R248W, but not R175H, can engage p21 and MDM2, which both function to limit oxidative stress and facilitate the switch to de novo serine synthesis. In vivo, the growth of R248W-expressing tumours is resistant to dietary depletion of serine and glycine, correlating with an increased capacity to limit ROS compared to tumours expressing R175H. Human cancers expressing this p53 mutant show a worse outcome.
    Conclusion: Our work shows that mutant p53s can selectively retain wild-type p53 functions that allow adaptation to serine starvation through the activation of antioxidant defence pathways. Tumours containing this p53 mutation are resistant to serine-limited conditions and less responsive to therapy.
    Keywords:  Antioxidant; MDM2; Serine starvation; p53
    DOI:  https://doi.org/10.1186/s40170-018-0191-6
  47. Oncoimmunology. 2018 ;7(12): e1486353
    Upregulation of tryptophanyl-tRNA synthethase adapts human cancer cells to nutritional stress caused by tryptophan degradation.
    Isabell Adam, Dyah L Dewi, Joram Mooiweer, Ahmed Sadik, Soumya R Mohapatra, Bianca Berdel, Melanie Keil, Jana K Sonner, Kathrin Thedieck, Adam J Rose, Michael Platten, Ines Heiland, Saskia Trump, Christiane A Opitz.
      Tryptophan (Trp) metabolism is an important target in immuno-oncology as it represents a powerful immunosuppressive mechanism hijacked by tumors for protection against immune destruction. However, it remains unclear how tumor cells can proliferate while degrading the essential amino acid Trp. Trp is incorporated into proteins after it is attached to its tRNA by tryptophanyl-tRNA synthestases. As the tryptophanyl-tRNA synthestases compete for Trp with the Trp-catabolizing enzymes, the balance between these enzymes will determine whether Trp is used for protein synthesis or is degraded. In human cancers expression of the Trp-degrading enzymes indoleamine-2,3-dioxygenase-1 (IDO1) and tryptophan-2,3-dioxygenase (TDO2) was positively associated with the expression of the tryptophanyl-tRNA synthestase WARS. One mechanism underlying the association between IDO1 and WARS identified in this study is their joint induction by IFNγ released from tumor-infiltrating T cells. Moreover, we show here that IDO1- and TDO2-mediated Trp deprivation upregulates WARS expression by activating the general control non-derepressible-2 (GCN2) kinase, leading to phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) and induction of activating transcription factor 4 (ATF4). Trp deprivation induced cytoplasmic WARS expression but did not increase nuclear or extracellular WARS levels. GCN2 protected the cells against the effects of Trp starvation and enabled them to quickly make use of Trp for proliferation once it was replenished. Computational modeling of Trp metabolism revealed that Trp deficiency shifted Trp flux towards WARS and protein synthesis. Our data therefore suggest that the upregulation of WARS via IFNγ and/or GCN2-peIF2α-ATF4 signaling protects Trp-degrading cancer cells from excessive intracellular Trp depletion.
    Keywords:  3-dioxygenase; Indoleamine-2, 3-dioxygenase; cancer metabolism; immunosuppression; immunosurveillance; inflammation and cancer; nutrients; proliferation; starvation; tRNA synthetase; tryptophan; tryptophan-2; tumor
    DOI:  https://doi.org/10.1080/2162402X.2018.1486353
  48. Aging Cell. 2018 Dec 10. e12845
    Hyperactivation of Nrf2 increases stress tolerance at the cost of aging acceleration due to metabolic deregulation.
    Eleni N Tsakiri, Sentiljana Gumeni, Kalliopi K Iliaki, Dimitra Benaki, Konstantinos Vougas, Gerasimos P Sykiotis, Vassilis G Gorgoulis, Emmanuel Mikros, Luca Scorrano, Ioannis P Trougakos.
      Metazoans viability depends on their ability to regulate metabolic processes and also to respond to harmful challenges by mounting anti-stress responses; these adaptations were fundamental forces during evolution. Central to anti-stress responses are a number of short-lived transcription factors that by functioning as stress sensors mobilize genomic responses aiming to eliminate stressors. We show here that increased expression of nuclear factor erythroid 2-related factor (Nrf2) in Drosophila activated cytoprotective modules and enhanced stress tolerance. However, while mild Nrf2 activation extended lifespan, high Nrf2 expression levels resulted in developmental lethality or, after inducible activation in adult flies, in altered mitochondrial bioenergetics, the appearance of Diabetes Type 1 hallmarks and aging acceleration. Genetic or dietary suppression of Insulin/IGF-like signaling (IIS) titrated Nrf2 activity to lower levels, largely normalized metabolic pathways signaling, and extended flies' lifespan. Thus, prolonged stress signaling by otherwise cytoprotective short-lived stress sensors perturbs IIS resulting in re-allocation of resources from growth and longevity to somatic preservation and stress tolerance. These findings provide a reasonable explanation of why most (if not all) cytoprotective stress sensors are short-lived proteins, and it also explains the build-in negative feedback loops (shown here for Nrf2); the low basal levels of these proteins, and why their suppressors were favored by evolution.
    Keywords:  Nrf2; aging; insulin/IGF-like; metabolism; mitostasis; proteostasis
    DOI:  https://doi.org/10.1111/acel.12845
  49. J Immunol. 2018 Dec 10. pii: ji1800782. [Epub ahead of print]
    TGF-β Upregulated Mitochondria Mass through the SMAD2/3→C/EBPβ→PRMT1 Signal Pathway in Primary Human Lung Fibroblasts.
    Qingzhu Sun, Lei Fang, Xuemei Tang, Shemin Lu, Michael Tamm, Daiana Stolz, Michael Roth.
      Tissue remodeling of subepithelial mesenchymal cells is a major pathologic condition of chronic obstructive pulmonary disease and asthma. Fibroblasts contribute to fibrotic events and inflammation in both airway diseases. Recent mechanistic studies established a link between mitochondrial dysfunction or aberrant biogenesis leading to tissue remodeling of the airway wall in asthma. Protein arginine methyltransferase-1 (PRMT1) participated in airway wall remodeling in pulmonary inflammation. This study investigated the mechanism by which PRMT1 regulates mitochondrial mass in primary human airway wall fibroblasts. Fibroblasts from control or asthma patients were stimulated with TGF-β for up to 48 h, and the signaling pathways controlling PRMT1 expression and mitochondrial mass were analyzed. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI-1. The SMAD2/3 pathway was blocked by SB203580 and C/EBPβ by small interference RNA treatment. The data obtained from unstimulated cells showed a significantly higher basal expression of PRMT1 and mitochondrial markers in asthmatic compared with control fibroblasts. In all cells, TGF-β significantly increased the expression of PRMT1 through SMAD2/3 and C/EBPβ. Subsequently, PRMT1 upregulated the expression of the mitochondria regulators PGC-1α and heat shock protein 60. Both the inhibition of the SAMD2/3 pathway or PRMT1 attenuated TGF-β-induced mitochondrial mass and C/EBPβ and α-SMA expression. These findings suggest that the signaling sequence controlling mitochondria in primary human lung fibroblasts is as follows: TGF-β→SMAD2/3→C/EBPβ→PRMT1→PGC-1α. Therefore, PRMT1 and C/EBPβ present a novel therapeutic and diagnostic target for airway wall remodeling in chronic lung diseases.
    DOI:  https://doi.org/10.4049/jimmunol.1800782
  50. PLoS Biol. 2018 Dec 14. 16(12): e3000095
    Dissecting the regulation and function of ATP at the single-cell level.
    Jianhan Zhang, Xu Han, Yihan Lin.
      Regulation of cellular ATP level is critical for diverse biological processes and may be defective in diseases such as cancer and mitochondrial disorders. While mitochondria play critical roles in ATP level regulation, we still lack a systematic and quantitative picture of how individual mitochondrial-related genes contribute to cellular ATP level and how dysregulated ATP levels may affect downstream cellular processes. Advances in genetically encoded ATP biosensors have provided new opportunities for addressing these issues. ATP biosensors allow researchers to quantify the changes of ATP levels in real time at the single-cell level and characterize corresponding effects at the cellular, tissue, and organismal level. Along this direction, several recent single-cell studies using ATP biosensors, including the work by Mendelsohn and colleagues, have started to uncover the principles for how genetic and nongenetic parameters may modulate ATP levels to affect cellular functions and human health.
    DOI:  https://doi.org/10.1371/journal.pbio.3000095
  51. Nat Microbiol. 2018 Dec 10.
    GABA-modulating bacteria of the human gut microbiota.
    Philip Strandwitz, Ki Hyun Kim, Darya Terekhova, Joanne K Liu, Anukriti Sharma, Jennifer Levering, Daniel McDonald, David Dietrich, Timothy R Ramadhar, Asama Lekbua, Nader Mroue, Conor Liston, Eric J Stewart, Marc J Dubin, Karsten Zengler, Rob Knight, Jack A Gilbert, Jon Clardy, Kim Lewis.
      The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23-65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression.
    DOI:  https://doi.org/10.1038/s41564-018-0307-3
  52. Int J Mol Sci. 2018 Dec 07. pii: E3930. [Epub ahead of print]19(12):
    Loss of Mitochondrial AAA Proteases AFG3L2 and YME1L Impairs Mitochondrial Structure and Respiratory Chain Biogenesis.
    Jana Cesnekova, Marie Rodinova, Hana Hansikova, Jiri Zeman, Lukas Stiburek.
      Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.
    Keywords:  AAA complex; AFG3L2; YME1L; mitochondria; protease
    DOI:  https://doi.org/10.3390/ijms19123930
  53. FASEB J. 2018 Dec 12. fj201801498R
    Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.
    Sara Violante, Nihad Achetib, Carlo W T van Roermund, Jacob Hagen, Tetyana Dodatko, Frédéric M Vaz, Hans R Waterham, Hongjie Chen, Myriam Baes, Chunli Yu, Carmen A Argmann, Sander M Houten.
      Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid β-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.
    Keywords:  CPT2 deficiency; acylcarnitine; fatty acid oxidation; mitochondria; organellar crosstalk
    DOI:  https://doi.org/10.1096/fj.201801498R
  54. PLoS Comput Biol. 2018 Dec;14(12): e1006584
    Multi-scale computational study of the Warburg effect, reverse Warburg effect and glutamine addiction in solid tumors.
    Mengrou Shan, David Dai, Arunodai Vudem, Jeffrey D Varner, Abraham D Stroock.
      Cancer metabolism has received renewed interest as a potential target for cancer therapy. In this study, we use a multi-scale modeling approach to interrogate the implications of three metabolic scenarios of potential clinical relevance: the Warburg effect, the reverse Warburg effect and glutamine addiction. At the intracellular level, we construct a network of central metabolism and perform flux balance analysis (FBA) to estimate metabolic fluxes; at the cellular level, we exploit this metabolic network to calculate parameters for a coarse-grained description of cellular growth kinetics; and at the multicellular level, we incorporate these kinetic schemes into the cellular automata of an agent-based model (ABM), iDynoMiCS. This ABM evaluates the reaction-diffusion of the metabolites, cellular division and motion over a simulation domain. Our multi-scale simulations suggest that the Warburg effect provides a growth advantage to the tumor cells under resource limitation. However, we identify a non-monotonic dependence of growth rate on the strength of glycolytic pathway. On the other hand, the reverse Warburg scenario provides an initial growth advantage in tumors that originate deeper in the tissue. The metabolic profile of stromal cells considered in this scenario allows more oxygen to reach the tumor cells in the deeper tissue and thus promotes tumor growth at earlier stages. Lastly, we suggest that glutamine addiction does not confer a selective advantage to tumor growth with glutamine acting as a carbon source in the tricarboxylic acid (TCA) cycle, any advantage of glutamine uptake must come through other pathways not included in our model (e.g., as a nitrogen donor). Our analysis illustrates the importance of accounting explicitly for spatial and temporal evolution of tumor microenvironment in the interpretation of metabolic scenarios and hence provides a basis for further studies, including evaluation of specific therapeutic strategies that target metabolism.
    DOI:  https://doi.org/10.1371/journal.pcbi.1006584
  55. EMBO J. 2018 Dec 14. pii: e99548. [Epub ahead of print]
    RagC phosphorylation autoregulates mTOR complex 1.
    Guang Yang, Sean J Humphrey, Danielle S Murashige, Deanne Francis, Qiao-Ping Wang, Kristen C Cooke, Greg Neely, David E James.
      The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. Here, we identify and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. We find that RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, we identify mTORC1 as the upstream kinase of RagC on S21. Our data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity.
    Keywords:  RagC; autophagy; cell growth; mTORC1; phosphorylation
    DOI:  https://doi.org/10.15252/embj.201899548
  56. Trends Pharmacol Sci. 2018 Dec 06. pii: S0165-6147(18)30210-4. [Epub ahead of print]
    Mitochondrial Permeability Transition: A Molecular Lesion with Multiple Drug Targets.
    Thomas Briston, David L Selwood, Gyorgy Szabadkai, Michael R Duchen.
      Mitochondrial permeability transition, as the consequence of opening of a mitochondrial permeability transition pore (mPTP), is a cellular catastrophe. Initiating bioenergetic collapse and cell death, it has been implicated in the pathophysiology of major human diseases, including neuromuscular diseases of childhood, ischaemia-reperfusion injury, and age-related neurodegenerative disease. Opening of the mPTP represents a major therapeutic target, as it can be mitigated by a number of compounds. However, clinical studies have so far been disappointing. We therefore address the prospects and challenges faced in translating in vitro findings to clinical benefit. We review the role of mPTP opening in disease, discuss recent findings defining the putative structure of the mPTP, and explore strategies to identify novel, clinically useful mPTP inhibitors, highlighting key considerations in the drug discovery process.
    Keywords:  calcium; cyclophilin D; drug discovery; mPTP; mitochondria
    DOI:  https://doi.org/10.1016/j.tips.2018.11.004
  57. Elife. 2018 Dec 10. pii: e42179. [Epub ahead of print]7
    Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase.
    Jeremy G Bird, Urmimala Basu, David Kuster, Aparna Ramachandran, Ewa Grudzien-Nogalska, Atif Towheed, Douglas C Wallace, Megerditch Kiledjian, Dmitry Temiakov, Smita S Patel, Richard H Ebright, Bryce E Nickels.
      Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non‑canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.
    Keywords:  E. coli; S. cerevisiae; biochemistry; chemical biology; chromosomes; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.42179
  58. Oncogene. 2018 Dec 12.
    STAT3 inhibition reduces macrophage number and tumor growth in neurofibroma.
    Jonathan S Fletcher, Mitchell G Springer, Kwangmin Choi, Edwin Jousma, Tilat A Rizvi, Eva Dombi, Mi-Ok Kim, Jianqiang Wu, Nancy Ratner.
      Plexiform neurofibroma, a benign peripheral nerve tumor, is associated with the biallelic loss of function of the NF1 tumor suppressor in Schwann cells. Here, we show that FLLL32, a small molecule inhibitor of JAK2/STAT3 signaling, reduces neurofibroma growth in mice with conditional, biallelic deletion of Nf1 in the Schwann cell lineage. FLLL32 treatment or Stat3 deletion in tumor cells reduced inflammatory cytokine expression and tumor macrophage numbers in neurofibroma. Although STAT3 inhibition downregulated the chemokines CCL2 and CCL12, which can signal through CCR2 to recruit macrophages to peripheral nerves, deletion of Ccr2 did not improve survival or reduce macrophage numbers in neurofibroma-bearing mice. Interestingly, Iba1+; F4/80+;CD11b+ macrophages accounted for ~20-40% of proliferating cells in untreated tumors. FLLL32 suppressed macrophage proliferation, implicating STAT3-dependent, local proliferation in neurofibroma macrophage accumulation, and decreased Schwann cell proliferation and increased Schwann cell death. The functions of STAT3 signaling in neurofibroma Schwann cells and macrophages, and its relevance as a therapeutic target in neurofibroma, merit further investigation.
    DOI:  https://doi.org/10.1038/s41388-018-0600-x
  59. PLoS One. 2018 ;13(12): e0208828
    Schizophrenia-associated mt-DNA SNPs exhibit highly variable haplogroup affiliation and nuclear ancestry: Bi-genomic dependence raises major concerns for link to disease.
    Christian M Hagen, Vanessa F Gonçalves, Paula L Hedley, Jonas Bybjerg-Grauholm, Marie Bækvad-Hansen, Christine S Hansen, Jørgen K Kanters, Jimmi Nielsen, Ole Mors, Alfonso B Demur, Thomas D Als, Merete Nordentoft, Anders Børglum, Preben B Mortensen, James Kennedy, Thomas M Werge, David M Hougaard, Michael Christiansen.
      Mitochondria play a significant role in human diseases. However, disease associations with mitochondrial DNA (mtDNA) SNPs have proven difficult to replicate. An analysis of eight schizophrenia-associated mtDNA SNPs, in 23,743 Danes without a psychiatric diagnosis and 2,538 schizophrenia patients, revealed marked inter-allelic differences in mitochondrial haplogroup affiliation and nuclear ancestry. This bi-genomic dependence could entail population stratification. Only two mitochondrial SNPs, m.15043A and m.15218G, were significantly associated with schizophrenia. However, these associations disappeared when corrected for haplogroup affiliation and nuclear ancestry. The extensive bi-genomic dependence documented here is a major concern when interpreting historic, as well as designing future, mtDNA association studies.
    DOI:  https://doi.org/10.1371/journal.pone.0208828
  60. Cell Host Microbe. 2018 Dec 12. pii: S1931-3128(18)30558-4. [Epub ahead of print]24(6): 791-803.e6
    O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity.
    Tianliang Li, Xinghui Li, Kuldeep S Attri, Changhong Liu, Lupeng Li, Laura E Herring, John M Asara, Yu L Lei, Pankaj K Singh, Chengjiang Gao, Haitao Wen.
      Increased glucose metabolism in immune cells not only serves as a hallmark feature of acute inflammation but also profoundly affects disease outcome following bacterial infection and tissue damage. However, the role of individual glucose metabolic pathways during viral infection remains largely unknown. Here we demonstrate an essential function of the hexosamine biosynthesis pathway (HBP)-associated O-linked β-N-acetylglucosamine (O-GlcNAc) signaling in promoting antiviral innate immunity. Challenge of macrophages with vesicular stomatitis viruses (VSVs) enhances HBP activity and downstream protein O-GlcNAcylation. Human and murine cells deficient of O-GlcNAc transferase, a key enzyme for protein O-GlcNAcylation, show defective antiviral immune responses upon VSV challenge. Mechanistically, O-GlcNAc transferase-mediated O-GlcNAcylation of the signaling adaptor MAVS on serine 366 is required for K63-linked ubiquitination of MAVS and subsequent downstream retinoic-acid inducible gene-like receptor -antiviral signaling activation. Thus, our study identifies a molecular mechanism by which HBP-mediated O-GlcNAcylation regulates MAVS function and highlights the importance of glucose metabolism in antiviral innate immunity.
    Keywords:  MAVS; O-GlcNAc transferase; antiviral immunity; glucose metabolism; hexosamine biosynthesis pathway (HBP)
    DOI:  https://doi.org/10.1016/j.chom.2018.11.001
  61. Cell Metab. 2018 Dec 01. pii: S1550-4131(18)30684-3. [Epub ahead of print]
    Mitochondrial Dynamics Is Critical for the Full Pluripotency and Embryonic Developmental Potential of Pluripotent Stem Cells.
    Xiuying Zhong, Peng Cui, Yongping Cai, Lihua Wang, Xiaoping He, Peipei Long, Kangyang Lu, Ronghui Yan, Ying Zhang, Xin Pan, Xiaoyang Zhao, Wei Li, Huafeng Zhang, Qi Zhou, Ping Gao.
      While the pluripotency of stem cells is known to determine the fate of embryonic development, the mechanisms underlying the acquisition and maintenance of full pluripotency largely remain elusive. Here, we show that the balance between mitochondrial fission and fusion is critical for the full pluripotency of stem cells. By analyzing induced pluripotent stem cells with differential developmental potential, we found that excess mitochondrial fission is associated with an impaired embryonic developmental potential. We further uncover that the disruption of mitochondrial dynamics impairs the differentiation and embryonic development of pluripotent stem cells; most notably, pluripotent stem cells that display excess mitochondrial fission fail to produce live-born offspring by tetraploid complementation. Mechanistically, excess mitochondrial fission increases cytosolic Ca2+ entry and CaMKII activity, leading to ubiquitin-mediated proteasomal degradation of β-Catenin protein. Our results reveal a previously unappreciated fundamental role for mitochondrial dynamics in determining the full pluripotency and embryonic developmental potential of pluripotent stem cells.
    Keywords:  CaMKII; full pluripotency; mitochondrial dynamics; pluripotent stem cells; β-Catenin
    DOI:  https://doi.org/10.1016/j.cmet.2018.11.007
  62. EMBO Mol Med. 2018 Dec 10. pii: e9456. [Epub ahead of print]
    Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy.
    Jayasimman Rajendran, Janne Purhonen, Saara Tegelberg, Olli-Pekka Smolander, Matthias Mörgelin, Jan Rozman, Valerie Gailus-Durner, Helmut Fuchs, Martin Hrabe de Angelis, Petri Auvinen, Eero Mervaala, Howard T Jacobs, Marten Szibor, Vineta Fellman, Jukka Kallijärvi.
      Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l p.S78G knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.
    Keywords:  BCS1L; GRACILE syndrome; complex III; mitochondrial disorder; respiratory chain
    DOI:  https://doi.org/10.15252/emmm.201809456
  63. Cell Rep. 2018 Dec 11. pii: S2211-1247(18)31764-9. [Epub ahead of print]25(11): 3059-3073.e10
    Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming.
    Luca Simula, Ilenia Pacella, Alessandra Colamatteo, Claudio Procaccini, Valeria Cancila, Matteo Bordi, Claudia Tregnago, Mauro Corrado, Martina Pigazzi, Vincenzo Barnaba, Claudio Tripodo, Giuseppe Matarese, Silvia Piconese, Silvia Campello.
      Mitochondria are key players in the regulation of T cell biology by dynamically responding to cell needs, but how these dynamics integrate in T cells is still poorly understood. We show here that the mitochondrial pro-fission protein Drp1 fosters migration and expansion of developing thymocytes both in vitro and in vivo. In addition, we find that Drp1 sustains in vitro clonal expansion and cMyc-dependent metabolic reprogramming upon activation, also regulating effector T cell numbers in vivo. Migration and extravasation defects are also exhibited in Drp1-deficient mature T cells, unveiling its crucial role in controlling both T cell recirculation in secondary lymphoid organs and accumulation at tumor sites. Moreover, the observed Drp1-dependent imbalance toward a memory-like phenotype favors T cell exhaustion in the tumor microenvironment. All of these findings support a crucial role for Drp1 in several processes during T cell development and in anti-tumor immune-surveillance.
    Keywords:  Drp1; T cells; cMyc; cell migration; cell proliferation; exhaustion; metabolic reprogramming; mitochondrial dynamics; thymocytes; tumor immune-surveillance
    DOI:  https://doi.org/10.1016/j.celrep.2018.11.018
  64. Cell. 2018 Dec 13. pii: S0092-8674(18)31334-5. [Epub ahead of print]175(7): 1756-1768.e17
    Irisin Mediates Effects on Bone and Fat via αV Integrin Receptors.
    Hyeonwoo Kim, Christiane D Wrann, Mark Jedrychowski, Sara Vidoni, Yukiko Kitase, Kenichi Nagano, Chenhe Zhou, Joshua Chou, Virginia-Jeni A Parkman, Scott J Novick, Timothy S Strutzenberg, Bruce D Pascal, Phuong T Le, Daniel J Brooks, Alexander M Roche, Kaitlyn K Gerber, Laura Mattheis, Wenjing Chen, Hua Tu, Mary L Bouxsein, Patrick R Griffin, Roland Baron, Clifford J Rosen, Lynda F Bonewald, Bruce M Spiegelman.
      Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.
    Keywords:  Irisin receptor; bone resorption; integrin αV; irisin; osteocyte; sclerostin; subcutaneous (inguinal) adipose tissues; ucp1
    DOI:  https://doi.org/10.1016/j.cell.2018.10.025
  65. Cell Death Dis. 2018 Dec 11. 9(12): 1189
    Dysregulated autophagy contributes to caspase-dependent neuronal apoptosis.
    Yuhyun Chung, Juhyung Lee, Shinae Jung, Yangsin Lee, Jin Won Cho, Young J Oh.
      Autophagy is a regulated, intracellular degradation process that delivers unnecessary or dysfunctional cargo to the lysosome. Autophagy has been viewed as an adaptive survival response to various stresses, whereas in other cases, it promotes cell death. Therefore, both deficient and excessive autophagy may lead to cell death. In this study, we specifically attempted to explore whether and how dysregulated autophagy contributes to caspase-dependent neuronal cell death induced by the neurotoxin 6-hydroxydopamine (6-OHDA). Ultrastructural and biochemical analyses indicated that MN9D neuronal cells and primary cultures of cortical neurons challenged with 6-OHDA displayed typical features of autophagy. Cotreatment with chloroquine and monitoring autophagic flux by a tandem mRFP-EGFP-tagged LC3 probe indicated that the autophagic phenomena were primarily caused by dysregulated autophagic flux. Consequently, cotreatment with an antioxidant but not with a pan-caspase inhibitor significantly blocked 6-OHDA-stimulated dysregulated autophagy. These results indicated that 6-OHDA-induced generation of reactive oxygen species (ROS) played a critical role in triggering neuronal death by causing dysregulated autophagy and subsequent caspase-dependent apoptosis. The results of the MTT reduction, caspase-3 activation, and TUNEL assays indicated that pharmacological inhibition of autophagy using 3-methyladenine or deletion of the autophagy-related gene Atg5 significantly inhibited 6-OHDA-induced cell death. Taken together, our results suggest that abnormal induction of autophagic flux promotes apoptotic neuronal cell death, and that the treatments limiting dysregulated autophagy may have a strong neuroprotective potential.
    DOI:  https://doi.org/10.1038/s41419-018-1229-y
  66. Oncoimmunology. 2018 ;7(12): e1528815
    Potent immunosuppressive effects of the oncometabolite R-2-hydroxyglutarate.
    Lorenzo Galluzzi, Guido Kroemer.
      Somatic gain-of-function mutations in isocitrate dehydrogenase (NADP(+)) 1, cytosolic (IDH1) or isocitrate dehydrogenase (NADP(+)) 2, mitochondrial (IDH2) are bona fide oncogenic drivers of acute myeloid leukemia and glioma because the neomorphic enzymes catalyze the synthesis of R-2-hydroxylutarate (R-2-HG), an oncometabolite with robust epigenetic effects. Recent data indicate that R-2-HG released by malignant cells can accumulate in the extracellular space and be taken up by T lymphocytes, ultimately compromising their capacity to mediate anticancer immune responses. Thus, R-2-HG drives oncogenesis and tumor progression not only as a cancer cell-autonomous epigenetic modifier, but also as an immunosuppressive metabolite. Chemical inhibitors of mutant IDH1 and IDH2, which currently are under clinical evaluation, may therefore mediate dual anticancer effects by targeting cancer cells and, at the same time, relieving R-2-HG-mediated immunosuppression.
    Keywords:  HIF-1α; cancer-associated fibroblasts; cytotoxic T lymphocytes; immunosurveillance; immunotherapy; ivosidenib
    DOI:  https://doi.org/10.1080/2162402X.2018.1528815
  67. J Biol Chem. 2018 Dec 06. pii: jbc.RA118.004863. [Epub ahead of print]
    The mitochondrial chaperone Prohibitin 1 negatively regulates interleukin-8 in human liver cancers.
    Jin Won Yang, Ben Murray, Lucia Barbier-Torres, Ting Liu, Zhenqiu Liu, Heping Yang, Wei Fan, Jiaohong Wang, Yuan Li, Ekihiro Seki, José M Mato, Shelly C Lu.
      Prohibitin 1 (PHB1) is a mitochondrial chaperone whose expression is dysregulated in cancers. In liver cancer, PHB1 acts as a tumor suppressor but the mechanisms of tumor suppression are incompletely understood. Here we aimed to determine PHB1 target genes to better understand how PHB1 influences liver tumorigenesis. Using RNA-seq analysis, we found interleukin-8 (IL-8) to be one of the most highly upregulated genes following PHB1 silencing in HepG2 cells. Induction of IL-8 expression also occurred in multiple liver and non-liver cancer cell lines. We examined samples from 178 patients with hepatocellular carcinoma (HCC) and found that IL-8 mRNA levels were increased, while PHB1 mRNA levels were decreased, in the tumors compared to adjacent non-tumorous tissues. Notably, HCC patients with high IL-8 expression have significantly reduced survival. An inverse correlation between PHB1 and IL-8 mRNA levels is found in HCCs with reduced PHB1 expression. To understand the molecular basis for these observations, we altered PHB1 levels in liver cancer cells. Overexpression of PHB1 resulted in lowered IL-8 expression and secretion. Silencing PHB1 increased JNK and NF-κB activity, induced nuclear accumulation of c-JUN and p65 and enhanced their binding to the IL-8 promoter containing AP-1 and NF-κB elements. Conditioned medium from PHB1-silenced HepG2 cells increased migration and invasion of parental HepG2 and SK-hep-1 cells, and this was blocked by co-treatment with neutralizing IL-8 antibody. In summary, our findings show that reduced PHB1 expression induces IL-8 transcription by activating NF-κB and AP-1, resulting in enhanced IL-8 expression and release to promote tumorigenesis.
    Keywords:  NF-kappa B (NF-KB); c-Jun N-terminal kinase (JNK); hepatocellular carcinoma; interleukin-8; invasion; migration; prohibitin 1
    DOI:  https://doi.org/10.1074/jbc.RA118.004863
  68. J Biol Chem. 2018 Dec 10. pii: jbc.RA118.005473. [Epub ahead of print]
    Lysine methylation by the mitochondrial methyltransferase FAM173B optimizes the function of mitochondrial ATP synthase.
    Jędrzej M Małecki, Hanneke L D M Willemen, Rita Pinto, Angela Y Y Ho, Anders Moen, Ingrid F Kjønstad, Boudewijn M T Burgering, Fried Zwartkruis, Niels Eijkelkamp, Pål Ø Falnes.
      Lysine methylation is an important post-translational modification that is also present on mitochondrial proteins, but the mitochondrial lysine-specific methyltransferases (KMTs) responsible for modification are in most cases unknown. Here, we set out to determine the function of human family with sequence similarity 173 member B (FAM173B), a mitochondrial methyltransferase (MTase) reported to promote chronic pain. Using bioinformatics analyses and biochemical assays, we found that FAM173B contains an atypical, non-cleavable mitochondrial targeting sequence responsible for its localization to mitochondria. Interestingly, CRISPR/Cas9-mediated knock-out (KO) of FAM173B in mammalian cells abrogated trimethylation of Lys-43 in ATP synthase c-subunit (ATPSc), a modification previously reported as ubiquitous among metazoans. ATPSc methylation was restored by complementing the KO cells with enzymatically active human FAM173B or with a putative FAM173B orthologue from the nematode Caenorhabditis elegans. Interestingly, lack of Lys-43 methylation caused aberrant incorporation of ATPSc into the ATP synthase complex, and resulted in decreased ATP-generating ability of the complex, as well as decreased mitochondrial respiration. In summary, we have identified FAM173B as the long-sought KMT responsible for methylation of ATPSc, a key protein in cellular ATP production, and have demonstrated functional significance of ATPSc methylation. We suggest renaming FAM173B to ATPSc-KMT (gene name ATPSCKMT).
    Keywords:  ATP synthase; ATP synthase c-subunit; F1FO-ATPase; FAM173B; metabolic regulation; methyltransferase; mitochondria; mitochondrial respiratory chain complex; oxidative phosphorylation; post-translational modification (PTM); protein lysine methylation; protein methylation
    DOI:  https://doi.org/10.1074/jbc.RA118.005473
  69. J Cell Biol. 2018 Dec 11. pii: jcb.201804165. [Epub ahead of print]
    ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosome-lysosome fusion.
    Ruoxi Wang, Jieqiong Tan, Tingting Chen, Hailong Han, Runyi Tian, Ya Tan, Yiming Wu, Jingyi Cui, Fang Chen, Jie Li, Lu Lv, Xinjie Guan, Shuai Shang, Jiahong Lu, Zhuohua Zhang.
      Mutations in ATP13A2 cause Kufor-Rakeb syndrome, an autosomal recessive form of juvenile-onset atypical Parkinson's disease (PD). Recent work tied ATP13A2 to autophagy and other cellular features of neurodegeneration, but how ATP13A2 governs numerous cellular functions in PD pathogenesis is not understood. In this study, the ATP13A2-deficient mouse developed into aging-dependent phenotypes resembling those of autophagy impairment. ATP13A2 deficiency impaired autophagosome-lysosome fusion in cultured cells and in in vitro reconstitution assays. In ATP13A2-deficient cells or Drosophila melanogaster or mouse tissues, lysosomal localization and activity of HDAC6 were reduced, with increased acetylation of tubulin and cortactin. Wild-type HDAC6, but not a deacetylase-inactive mutant, restored autophagosome-lysosome fusion, antagonized cortactin hyperacetylation, and promoted lysosomal localization of cortactin in ATP13A2-deficient cells. Mechanistically, ATP13A2 facilitated recruitment of HDAC6 and cortactin to lysosomes. Cortactin overexpression in cultured cells reversed ATP13A2 deficiency-associated impairment of autophagosome-lysosome fusion. PD-causing ATP13A2 mutants failed to rescue autophagosome-lysosome fusion or to promote degradation of protein aggregates and damaged mitochondria. These results suggest that ATP13A2 recruits HDAC6 to lysosomes to deacetylate cortactin and promotes autophagosome-lysosome fusion and autophagy. This study identifies ATP13A2 as an essential molecular component for normal autophagy flux in vivo and implies potential treatments targeting HDAC6-mediated autophagy for PD.
    DOI:  https://doi.org/10.1083/jcb.201804165
  70. Cytometry A. 2018 Dec 11.
    Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.
    Ning Ma, Gopakumar Kamalakshakurup, Mohammad Aghaamoo, Abraham P Lee, Michelle A Digman.
      Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry.
    Keywords:  circulating tumor cells; droplet microfluidics; fluorescence lifetime imaging microscopy; metabolism; phasor analysis; quiescent stage; single cell analysis
    DOI:  https://doi.org/10.1002/cyto.a.23673
  71. Methods Mol Biol. 2019 ;1877 173-183
    Methods to Probe Calcium Regulation by BCL-2 Family Members.
    Marcos A Carpio, Samuel G Katz.
      BCL-2 family members have additional roles beyond direct regulation of mitochondrial outer membrane permeabilization (MOMP) in apoptosis. One such important function is the release of calcium from the endoplasmic reticulum (ER), which critically contributes to the process of apoptosis. Here, we describe a protocol to measure calcium levels in the ER, mitochondria, and cytosol, with specific consideration of BCL-2 family biology.
    Keywords:  Apoptosis; BCL-2 Family; Calcium; Endoplasmic reticulum; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-4939-8861-7_12
  72. Sci Rep. 2018 Dec 07. 8(1): 17732
    Elucidating the contribution of ETC complexes I and II to the respirasome formation in cardiac mitochondria.
    Sehwan Jang, Sabzali Javadov.
      Mitochondrial electron transport chain (ETC) plays a central role in ATP synthesis, and its dysfunction is associated with human diseases. Recent studies revealed that individual ETC complexes are assembled into supercomplexes. The main supercomplex, respirasome composed of complexes I, III, and IV has been suggested to improve electron channeling and control ROS production, maintain the structural integrity of ETC complexes and prevent protein aggregation in the inner mitochondrial membrane. However, many questions related to the structural organization of the respirasome, particularly, a possible role of complexes I and II in respirasome formation remain unclear. Here, we investigated whether genetic and pharmacological inhibition of complexes I and II affect respirasome assembly in cardioblast cells and isolated cardiac mitochondria. Pharmacological inhibition of the enzymatic activity of complexes I and II stimulated disruption of the respirasome. Likewise, knockdown of the complex I subunit NDUFA11 stimulated dissociation of respirasome and reduced the activity of complexes I, III, and IV. However, silencing of the membrane-anchored SDHC subunit of complex II had no effect on the respirasome assembly but reduced the activity of complexes II and IV. Downregulation of NDUFA11 or SDHC reduced ATP production and increased mitochondrial ROS production. Overall, these studies, for the first time, provide biochemical evidence that the complex I activity, and the NDUFA11 subunit are important for assembly and stability of the respirasome. The SDHC subunit of complex II is not involved in the respirasome however the complex may play a regulatory role in respirasome formation.
    DOI:  https://doi.org/10.1038/s41598-018-36040-9
  73. BMB Rep. 2018 Dec 11. pii: 4444. [Epub ahead of print]
    Metabolic features and regulation in cell senescence.
    So Mee Kwon, Sun Mi Hong, Young-Kyoung Lee, Seongki Min, Gyesoon Yoon.
      Organismal aging is accompanied by a host of progressive metabolic alterations and an accumulation of senescent cells, along with functional decline and the appearance of multiple diseases. This implies that the metabolic features of cell senescence may contribute to the organism's metabolic changes and be closely linked to age-associated diseases, especially metabolic syndromes. However, there is no clear understanding of senescent metabolic characteristics. Here, we review key metabolic features and regulators of cellular senescence, focusing on mitochondrial dysfunction and anabolic deregulation, and their link to other senescence phenotypes and aging. We further discuss the mechanistic involvement of the metabolic regulators mTOR, AMPK, and GSK3, proposing them as key metabolic switches for modulating senescence.
  74. Nat Med. 2018 Dec;24(12): 1795-1803
    The emerging link between cancer, metabolism, and circadian rhythms.
    Selma Masri, Paolo Sassone-Corsi.
      The circadian clock is a complex cellular mechanism that, through the control of diverse metabolic and gene expression pathways, governs a large array of cyclic physiological processes. Epidemiological and clinical data reveal a connection between the disruption of circadian rhythms and cancer that is supported by recent preclinical data. In addition, results from animal models and molecular studies underscore emerging links between cancer metabolism and the circadian clock. This has implications for therapeutic approaches, and we discuss the possible design of chronopharmacological strategies.
    DOI:  https://doi.org/10.1038/s41591-018-0271-8
  75. J Cell Physiol. 2018 Dec 07.
    Dimethyl fumarate: Regulatory effects on the immune system in the treatment of multiple sclerosis.
    Arezoo Hosseini, Ali Masjedi, Behzad Baradaran, Mohammad Hojjat-Farsangi, Ghasem Ghalamfarsa, Enayat Anvari, Farhad Jadidi-Niaragh.
      Dimethyl fumarate (DMF) is an important oral treatment option for various autoimmune diseases, such as multiple sclerosis (MS) and psoriasis. DMF and its dynamic metabolite, monomethyl fumarate (MMF) are the major compounds that exert therapeutic effects on several pathologic conditions in part, through downregulation of immune responses. The exact mechanism of DMF is yet to be fully understood even though its beneficial effects on the immune system are extensively studied. It has been shown that DMF/MMF can affect various immune cells, which can get involved in both the naive and adaptive immune systems, such as T cells, B cells, dendritic cells, macrophages, neutrophils, and natural killer cells. It is suggested that DMF/MMF may exert their effect on immune cells through inhibition of nuclear factor-κB translocation, upregulation of nuclear factor erythroid-derived 2(E2)-related factor antioxidant pathway, and activation of hydroxyl carboxylic acid receptor 2. In this review, the mechanisms underlying the modulatory functions of DMF or MMF on the main immune cell populations involved in the immunopathogenesis of MS are discussed.
    Keywords:  dimethyl fumarate; immune system; multiple sclerosis; treatment
    DOI:  https://doi.org/10.1002/jcp.27930
  76. Cancer. 2018 Dec 12.
    Germline mutations of renal cancer predisposition genes and clinical relevance in Chinese patients with sporadic, early-onset disease.
    Junlong Wu, Hongkai Wang, Christopher J Ricketts, Youfeng Yang, Maria J Merino, Hailiang Zhang, Guohai Shi, Hualei Gan, W Marston Linehan, Yao Zhu, Dingwei Ye.
      BACKGROUND: An inherited susceptibility to renal cancers is associated with multiple predisposing genes, but most screening tests are limited to patients with a family history. Next-generation sequencing (NGS)-based multigene panels provide an efficient and adaptable tool for investigating pathogenic germline mutations on a larger scale. This study investigated the frequency of pathogenic germline mutations in renal cancer predisposition genes in patients with sporadic, early-onset disease.METHODS: An NGS-based panel of 23 known and potential renal cancer predisposition genes was used to analyze germline mutations in 190 unrelated Chinese patients under the age of 45 years who presented with renal tumors. The detected variants were filtered for pathogenicity, and then their frequencies were calculated and correlated with clinical features. Germline variants of the fumarate hydratase (FH) and BRCA1-associated protein 1 (BAP1) genes were comprehensively analyzed because of their aggressive potential.
    RESULTS: In total, 18 patients (9.5%) had germline mutations in 10 genes. Twelve of these 18 patients had alterations in renal cancer predisposition genes (6.3%), and 6 patients had mutations in potential predisposition genes such as BRCA1/2. Notably, pathogenic mutation carriers had a significant family history in second-degree relatives in comparison with those without pathogenic mutations (P < .001). Variants of unknown clinical significance in FH and BAP1 demonstrated evidence of additional somatic loss in tumors.
    CONCLUSIONS: In patients with early-onset disease, a multigene panel identified a high pathogenic germline mutation rate in renal cancer predisposition genes. This study emphasizes the importance of screening patients with early-onset disease for mutations in cancer predisposition genes. Germline screening should be encouraged in early-onset patients to provide personalized medicine and improve patient outcomes.
    Keywords:  BRCA1-associated protein 1 (BAP1); cancer predisposition; early onset; fumarate hydratase (FH); next-generation sequencing; renal tumor
    DOI:  https://doi.org/10.1002/cncr.31908
  77. Clin Radiol. 2018 Dec 11. pii: S0009-9260(18)30587-7. [Epub ahead of print]
    Succinate dehydrogenase mutations: paraganglioma imaging and at-risk population screening.
    T Cavenagh, J Patel, N Nakhla, A Elstob, M Ingram, B Barber, K Snape, G Bano, I Vlahos.
      Paragangliomas are rare vascular tumours of the autonomic nervous system. They can be classified as sympathetic or parasympathetic. Sympathetic paragangliomas, which include phaeochromocytomas, tend to be functional and symptomatic. Parasympathetic paragangliomas are usually non-functional and may present with mass effect. Forty percent of paragangliomas are linked to genetic syndromes, most commonly due to mutations of the succinate dehydrogenase (SDH) enzyme complex and are collectively known as paraganglioma syndromes, of which five are described. Genetic testing is recommended for all patients, and their first-degree relatives, diagnosed with paragangliomas. When SDH mutations are discovered, biochemical screening and imaging surveillance is indicated. There is currently no consensus on imaging surveillance protocols. Most advocate full-body imaging, but the choice of technique and frequency varies. If paragangliomas are demonstrated, functional imaging to look for synchronous tumours or metastases is indicated. 2-[18F]-fluoro-2-deoxy-d-glucose (18F-FDG) positron-emission tomography (PET)-computed tomography (CT) is the technique of choice for metastatic evaluation, but [123I]-metaiodobenzylguanidine or [111In]-DTPA-octreotide scintigraphy are also utilised. Current research into emerging positron-emitting radiolabelled somatostatin analogues have yielded promising results, which is likely to be reflected in future guidelines. As genetic testing becomes increasingly prevalent, the need to answer the remaining questions regarding surveillance imaging is paramount.
    DOI:  https://doi.org/10.1016/j.crad.2018.11.004
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