bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2019‒05‒26
forty-five papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit


  1. Cell Metab. 2019 Apr 30. pii: S1550-4131(19)30197-4. [Epub ahead of print]
    Dumesic PA, Egan DF, Gut P, Tran MT, Parisi A, Chatterjee N, Jedrychowski M, Paschini M, Kazak L, Wilensky SE, Dou F, Bogoslavski D, Cartier JA, Perrimon N, Kajimura S, Parikh SM, Spiegelman BM.
      Mitochondrial abundance and function are tightly controlled during metabolic adaptation but dysregulated in pathological states such as diabetes, neurodegeneration, cancer, and kidney disease. We show here that translation of PGC1α, a key governor of mitochondrial biogenesis and oxidative metabolism, is negatively regulated by an upstream open reading frame (uORF) in the 5' untranslated region of its gene (PPARGC1A). We find that uORF-mediated translational repression is a feature of PPARGC1A orthologs from human to fly. Strikingly, whereas multiple inhibitory uORFs are broadly present in fish PPARGC1A orthologs, they are completely absent in the Atlantic bluefin tuna, an animal with exceptionally high mitochondrial content. In mice, an engineered mutation disrupting the PPARGC1A uORF increases PGC1α protein levels and oxidative metabolism and confers protection from acute kidney injury. These studies identify a translational regulatory element governing oxidative metabolism and highlight its potential contribution to the evolution of organismal mitochondrial function.
    Keywords:  5’ untranslated region; PGC1α; bluefin tuna; evolution; ischemic kidney injury; metabolism; mitochondria; oxidative phosphorylation; translational regulation; upstream open reading frame
    DOI:  https://doi.org/10.1016/j.cmet.2019.04.013
  2. Biochim Biophys Acta Mol Basis Dis. 2019 May 20. pii: S0925-4439(19)30171-1. [Epub ahead of print]
    Desquiret-Dumas V, Leman G, Wetterwald C, Chupin S, Lebert A, Khiati S, Le Mao M, Geffroy G, Kane MS, Chevrollier A, Goudenege D, Gadras C, Tessier L, Barth M, Leruez S, Amati-Bonneau P, Henrion D, Bonneau D, Procaccio V, Reynier P, Lenaers G, Gueguen N.
      Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.
    Keywords:  Complex I assembly; Complex I deficiency; Metabolic reprogramming; Mitochondrial metabolism; ROS production
    DOI:  https://doi.org/10.1016/j.bbadis.2019.05.011
  3. Elife. 2019 May 20. pii: e45572. [Epub ahead of print]8
    Kang YP, Torrente L, Falzone A, Elkins CM, Liu M, Asara JM, Dibble CC, DeNicola G.
      NRF2 is emerging as a major regulator of cellular metabolism. However, most studies have been performed in cancer cells, where co-occurring mutations and tumor selective pressures complicate the influence of NRF2 on metabolism. Here we use genetically engineered, non-transformed primary murine cells to isolate the most immediate effects of NRF2 on cellular metabolism. We find that NRF2 promotes the accumulation of intracellular cysteine and engages the cysteine homeostatic control mechanism mediated by cysteine dioxygenase 1 (CDO1), which catalyzes the irreversible metabolism of cysteine to cysteine sulfinic acid (CSA). Notably, CDO1 is preferentially silenced by promoter methylation in human non-small cell lung cancers (NSCLC) harboring mutations in KEAP1, the negative regulator of NRF2. CDO1 silencing promotes proliferation of NSCLC by limiting the futile metabolism of cysteine to the wasteful and toxic byproducts CSA and sulfite (SO32-), and depletion of cellular NADPH. Thus, CDO1 is a metabolic liability for NSCLC cells with high intracellular cysteine, particularly NRF2/KEAP1 mutant cells.
    Keywords:  biochemistry; cancer biology; chemical biology; human; mouse
    DOI:  https://doi.org/10.7554/eLife.45572
  4. J Am Soc Nephrol. 2019 May 24. pii: ASN.2018111117. [Epub ahead of print]
    Cho SG, Xiao X, Wang S, Gao H, Rafikov R, Black S, Huang S, Ding HF, Yoon Y, Kirken RA, Yin XM, Wang HG, Dong Z.
      BACKGROUND: Mitochondria are dynamic organelles that undergo fission and fusion. During cell stress, mitochondrial dynamics shift to fission, leading to mitochondrial fragmentation, membrane leakage, and apoptosis. Mitochondrial fragmentation requires the cleavage of both outer and inner membranes, but the mechanism of inner membrane cleavage is unclear. Bif-1 and prohibitin-2 may regulate mitochondrial dynamics.METHODS: We used azide-induced ATP depletion to incite cell stress in mouse embryonic fibroblasts and renal proximal tubular cells, and renal ischemia-reperfusion to induce stress in mice. We also used knockout cells and mice to determine the role of Bif-1, and used multiple techniques to analyze the molecular interaction between Bif-1 and prohibitin-2.
    RESULTS: Upon cell stress, Bif-1 translocated to mitochondria to bind prohibitin-2, resulting in the disruption of prohibitin complex and proteolytic inactivation of the inner membrane fusion protein OPA1. Bif-1-deficiency inhibited prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis. Domain deletion analysis indicated that Bif-1 interacted with prohibitin-2 via its C-terminus. Notably, mutation of Bif-1 at its C-terminal tryptophan-344 not only prevented Bif-1/prohibitin-2 interaction but also reduced prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis, supporting a pathogenic role of Bif-1/prohibitin-2 interaction. In mice, Bif-1 bound prohibitin-2 during renal ischemia/reperfusion injury, and Bif-1-deficiency protected against OPA1 proteolysis, mitochondrial fragmentation, apoptosis and kidney injury.
    CONCLUSIONS: These findings suggest that during cell stress, Bif-1 regulates mitochondrial inner membrane by interacting with prohibitin-2 to disrupt prohibitin complexes and induce OPA1 proteolysis and inactivation.
    Keywords:  apoptosis; mitochondria; renal ischemia
    DOI:  https://doi.org/10.1681/ASN.2018111117
  5. Nat Metab. 2019 Jan;1(1): 158-171
    Diebold LP, Gil HJ, Gao P, Martinez CA, Weinberg SE, Chandel NS.
      Endothelial cells (ECs) require glycolysis for proliferation and migration during angiogenesis; however, the necessity for the mitochondrial respiratory chain during angiogenesis is not known. Here we report that inhibition of respiratory chain complex III impairs proliferation, but not migration of ECs in vitro by decreasing the NAD+/NADH ratio. To determine whether mitochondrial respiration is necessary for angiogenesis in vivo, we conditionally ablate a subunit of the respiratory chain complex III (QPC) in ECs. Loss of QPC decreases respiration, resulting in diminished EC proliferation, and impairment in retinal and tumor angiogenesis. Loss of QPC does not decrease genes associated with anabolism or nucleotides levels in ECs, but diminishes amino acid levels. Our findings indicate that mitochondrial respiration is necessary for angiogenesis, and that the primary role of mitochondria in ECs is to serve as biosynthetic organelles for cell proliferation.
    DOI:  https://doi.org/10.1038/s42255-018-0011-x
  6. Methods Mol Biol. 2019 ;1978 219-241
    Cordes T, Metallo CM.
      Metabolism plays a central role in virtually all diseases, including diabetes, cancer, and neurodegeneration. Detailed analysis is required to identify the specific metabolic pathways dysregulated in the context of a given disease or biological perturbation. Measurement of metabolite concentrations can provide some insights into altered pathway activity or enzyme function, but since most biochemicals are metabolized by various enzymes in distinct pathways within cells and tissues, these approaches are somewhat limited. By applying metabolic tracers to a biological system, one can visualize pathway-specific information depending on the tracer used and analytes measured. To this end, stable isotope tracers and mass spectrometry are emerging as important tools for the examination of metabolic pathways and fluxes in cultured mammalian cells and other systems. Here, we describe a detailed workflow for quantifying metabolic processes in mammalian cell cultures using stable isotopes and gas chromatography coupled to mass spectrometry (GC-MS). As a case study, we apply 13C isotopic labeled glucose and glutamine to a cancer cell line to quantify substrate utilization for TCA metabolism and lipogenesis. Guidelines are also provided for interpretation of data and considerations for application to other cell systems. Ultimately, this approach provides a robust and precise method for quantifying stable isotope labeling in metabolite pools that can be applied to diverse biological systems.
    Keywords:  Fragment; Gas chromatography mass spectrometry (GC-MS); Isotopologue distribution; Isotopomer spectral analysis (ISA); MTBSTFA; Metabolic flux; Metabolism; Metabolite extraction; Stable isotope tracer; TBDMS
    DOI:  https://doi.org/10.1007/978-1-4939-9236-2_14
  7. J Mol Biol. 2019 May 16. pii: S0022-2836(19)30284-0. [Epub ahead of print]
    Callegari S, Müller T, Schulz C, Lenz C, Jans DC, Wissel M, Opazo F, Rizzoli SO, Jakobs S, Urlaub H, Rehling P, Deckers M.
      Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space towards the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the Mitochondrial Contact site and Cristae Organizing System (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.
    Keywords:  MICOS; Membrane insertion; Mitochondria; Mitochondrial carrier proteins; Mitochondrial import; TIM22; TIM23; Translocase
    DOI:  https://doi.org/10.1016/j.jmb.2019.05.015
  8. Nat Cell Biol. 2019 May 20.
    Carreras-Sureda A, Jaña F, Urra H, Durand S, Mortenson DE, Sagredo A, Bustos G, Hazari Y, Ramos-Fernández E, Sassano ML, Pihán P, van Vliet AR, González-Quiroz M, Torres AK, Tapia-Rojas C, Kerkhofs M, Vicente R, Kaufman RJ, Inestrosa NC, Gonzalez-Billault C, Wiseman RL, Agostinis P, Bultynck G, Court FA, Kroemer G, Cárdenas JC, Hetz C.
      Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.
    DOI:  https://doi.org/10.1038/s41556-019-0329-y
  9. Nature. 2019 May 22.
    Mårtensson CU, Priesnitz C, Song J, Ellenrieder L, Doan KN, Boos F, Floerchinger A, Zufall N, Oeljeklaus S, Warscheid B, Becker T.
      Mitochondrial biogenesis and functions depend on the import of precursor proteins via the 'translocase of the outer membrane' (TOM complex). Defects in protein import lead to an accumulation of mitochondrial precursor proteins that induces a range of cellular stress responses. However, constitutive quality-control mechanisms that clear trapped precursor proteins from the TOM channel under non-stress conditions have remained unknown. Here we report that in Saccharomyces cerevisiae Ubx2, which functions in endoplasmic reticulum-associated degradation, is crucial for this quality-control process. A pool of Ubx2 binds to the TOM complex to recruit the AAA ATPase Cdc48 for removal of arrested precursor proteins from the TOM channel. This mitochondrial protein translocation-associated degradation (mitoTAD) pathway continuously monitors the TOM complex under non-stress conditions to prevent clogging of the TOM channel with precursor proteins. The mitoTAD pathway ensures that mitochondria maintain their full protein-import capacity, and protects cells against proteotoxic stress induced by impaired transport of proteins into mitochondria.
    DOI:  https://doi.org/10.1038/s41586-019-1227-y
  10. EMBO J. 2019 May 24. pii: e101347. [Epub ahead of print]
    Pastore N, Vainshtein A, Herz NJ, Huynh T, Brunetti L, Klisch TJ, Mutarelli M, Annunziata P, Kinouchi K, Brunetti-Pierri N, Sassone-Corsi P, Ballabio A.
      Autophagy and energy metabolism are known to follow a circadian pattern. However, it is unclear whether autophagy and the circadian clock are coordinated by common control mechanisms. Here, we show that the oscillation of autophagy genes is dependent on the nutrient-sensitive activation of TFEB and TFE3, key regulators of autophagy, lysosomal biogenesis, and cell homeostasis. TFEB and TFE3 display a circadian activation over the 24-h cycle and are responsible for the rhythmic induction of genes involved in autophagy during the light phase. Genetic ablation of TFEB and TFE3 in mice results in deregulated autophagy over the diurnal cycle and altered gene expression causing abnormal circadian wheel-running behavior. In addition, TFEB and TFE3 directly regulate the expression of Rev-erbα (Nr1d1), a transcriptional repressor component of the core clock machinery also involved in the regulation of whole-body metabolism and autophagy. Comparative analysis of the cistromes of TFEB/TFE3 and REV-ERBα showed an extensive overlap of their binding sites, particularly in genes involved in autophagy and metabolic functions. These data reveal a direct link between nutrient and clock-dependent regulation of gene expression shedding a new light on the crosstalk between autophagy, metabolism, and circadian cycles.
    Keywords:  MiT‐TFE; REV‐ERBα; circadian rhythm; gene oscillation
    DOI:  https://doi.org/10.15252/embj.2018101347
  11. FEBS Open Bio. 2019 May 24.
    Hayashishita M, Kawahara H, Yokota N.
      Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin- and p62/sequestosome 1-mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m-chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2-associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference-mediated depletion of endogenous BAG6 prevented Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/sequestosome 1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.
    Keywords:   CCCP ; BAG6; Mitochondria; PINK1; Parkin; p62
    DOI:  https://doi.org/10.1002/2211-5463.12677
  12. Oncotarget. 2019 Apr 12. 10(28): 2675-2692
    Kotredes KP, Razmpour R, Lutton E, Alfonso-Prieto M, Ramirez SH, Gamero AM.
      The family of isocitrate dehydrogenase (IDH) enzymes is vital for cellular metabolism, as IDH1 and IDH2 are required for the decarboxylation of isocitrate to α-ketoglutarate. Heterozygous somatic mutations in IDH1 or IDH2 genes have been detected in many cancers. They share the neomorphic production of the oncometabolite (R)-2-hydroxyglutarate [(R)-2-HG]. With respect to IDH2, it is unclear whether all IDH2 mutations display the same or differ in tumorigenic properties and degrees of chemosensitivity. Here, we evaluated the three most frequent IDH2 mutations occurring in cancer. The predicted changes to the enzyme structure introduced by these individual mutations are supported by the observed production of (R)-2-HG. However, their tumorigenic properties, response to chemotherapeutic agents, and baseline activation of STAT3 differed. Paradoxically, the varying levels of endogenous (R)-2-HG produced by each IDH2 mutant inversely correlated with their respective growth rates. Interestingly, while we found that (R)-2-HG stimulated the growth of non-transformed cells, (R)-2-HG also displayed antitumor activity by suppressing the growth of tumors harboring wild type IDH2. The mitogenic effect of (R)-2-HG in immortalized cells could be switched to antiproliferative by transformation with oncogenic RAS. Thus, our findings show that despite their shared (R)-2-HG production, IDH2 mutations are not alike and differ in shaping tumor cell behavior and response to chemotherapeutic agents. Our study also reveals that under certain conditions, (R)-2-HG has antitumor properties.
    Keywords:  IDH2; biomarker; chemotherapy; glioblastoma; tumorigenesis
    DOI:  https://doi.org/10.18632/oncotarget.26848
  13. Endocr J. 2019 May 18.
    Hashimoto N, Nagano H, Tanaka T.
      The tumor suppressor gene p53 is mutated in approximately more than 50% of human cancers. p53 is also referred to as the "cellular gatekeeper" or "guardian of the genome" because it protects the body from spreading mutated genome induced by various stress. When the cells receives stimuli such as DNA damage, oncogene activation, oxidative stress or undernutrition, p53 gives rise to a number of cellular responses, including cell cycle arrest, apoptosis, cellular senescence and metabolic adaptation. Related to energy metabolisms, it has been reported that p53 reduces glycolysis and enhances mitochondrial respiration. p53 is also involved in the regulation of other cellular metabolism and energy production systems: amino acid metabolism, fatty acid metabolism, nucleic acid metabolism, anti-oxidation, mitochondrial quality control, and autophagy. Moreover, recent studies have shown that p53 gene polymorphisms affect life expectancy and lifestyle-related disease such as type 2 diabetes, suggesting that there is a certain relationship between p53 function and metabolic disorders. In addition, mutant p53 protein does not only lose the tumor suppressor function, but it also gains novel oncogenic function and contributes to tumor development, involving cellular metabolism modification. Therefore, the importance of multifunctionality of p53, particularly with regard to intracellular metabolisms, arouses therapeutic interest and calls attention as the key molecule among cancer, lifestyle-related diseases and life expectancy.
    Keywords:  Cancer; Diabetes; Lifestyle-related diseases; Metabolism; Tumor suppressor p53
    DOI:  https://doi.org/10.1507/endocrj.EJ18-0565
  14. EBioMedicine. 2019 May 16. pii: S2352-3964(19)30316-0. [Epub ahead of print]
    Peng W, Huang W, Ge X, Xue L, Zhao W, Xue J.
      BACKGROUND: Emerging evidence suggests that metabolic alterations are a hallmark of cancer cells and contribute to tumor initiation and development. Cancer cells primarily utilize aerobic glycolysis (the Warburg effect) to produce energy and support anabolic growth. The type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) is profoundly implicated in tumorigenesis, however, little is known about its role in reprogrammed energy metabolism.METHODS: Loss- and gain-of-function studies were applied to determine the oncogenic roles of PIPKIγ in colorectal cancer. Transcriptome analysis, real-time qPCR, immunohistochemical staining, Western blotting, and metabolic analysis were carried out to uncover the cellular mechanism of PIPKIγ.
    FINDINGS: In this study, we showed that PIPKIγ was frequently upregulated in colorectal cancer and predicted a poor prognosis. Genetic silencing of pan-PIPKIγ suppressed cell proliferation and aerobic glycolysis of colorectal cancer. In contrast, the opposite effects were observed by overexpression of PIPKIγ_i2. Importantly, PIPKIγ-induced prolific effect was largely glycolysis-dependent. Mechanistically, PIPKIγ facilitated activation of PI3K/Akt/mTOR signaling pathways to upregulate c-Myc and HIF1α levels, which regulate expression of glycolytic enzymes to enhance glycolysis. Moreover, pharmacological inhibition by PIPKIγ activity with the specific inhibitor UNC3230 significantly inhibited colorectal cancer glycolysis and tumor growth.
    INTERPRETATION: Our findings reveal a new regulatory role of PIPKIγ in Warburg effect and provide a key contributor in colorectal cancer metabolism with potential therapeutic potentials. FUND: National Key Research and Development Program of China, Outstanding Clinical Discipline Project of Shanghai Pudong, Natural Science Foundation of China, and Science and Technology Commission of Shanghai Municipality.
    Keywords:  Colorectal cancer; PIPKIγ; Phosphatidylinositol kinase; Tumor growth; Warburg effect
    DOI:  https://doi.org/10.1016/j.ebiom.2019.05.015
  15. Immunol Cell Biol. 2019 May 25.
    O'Sullivan D.
      The development and persistence of memory T cells are integral to effective host defenses. Cell metabolism has a central role in the maintenance of these populations and engagement of specific metabolic pathways can influence T cell fate. Nuances in memory T cell metabolism are both context dependent, and exist, on a spectrum that complements and supports the activity of specific memory T cell subsets. This review explores how metabolic pathways and substrate choice can influence phenotype and function in memory T cells. This article is protected by copyright. All rights reserved.
    Keywords:  Immunometabolism; Memory T cell metabolism; Mitochondria
    DOI:  https://doi.org/10.1111/imcb.12274
  16. Semin Cell Dev Biol. 2019 May 17. pii: S1084-9521(18)30182-4. [Epub ahead of print]
    Rimessi A, Pedriali G, Vezzani B, Tarocco A, Marchi S, Wieckowski MR, Giorgi C, Pinton P.
      Organelles were originally considered to be individual cellular compartments with a defined organization and function. However, recent studies revealed that organelles deeply communicate within each other via Ca2+ exchange. This communication, mediated by specialized membrane regions in close apposition between two organelles, regulate cellular functions, including metabolism and cell fate decisions. Advances in microscopy techniques, molecular biology and biochemistry have increased our understanding of these interorganelle platforms. Research findings suggest that interorganellar Ca2+ signaling, which is altered in cancer, influences tumorigenesis and tumor progression by controlling cell death programs and metabolism. Here, we summarize the available data on the existence and composition of interorganelle platforms connecting the endoplasmic reticulum with mitochondria, the plasma membrane, or endolysosomes. Finally, we provide a timely overview of the potential function of interorganellar Ca2+ signaling in maintaining cellular homeostasis.
    DOI:  https://doi.org/10.1016/j.semcdb.2019.05.015
  17. Genetics. 2019 May 13. pii: genetics.302238.2019. [Epub ahead of print]
    Saatchi F, Kirchmaier AL.
      Fumarase is a well-characterized TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate. In mammals, fumarase acts as a tumor suppressor, and loss-of-function mutations in the FH gene in hereditary leiomyomatosis and renal cell cancer result in the accumulation of intracellular fumarate, an inhibitor of α-ketoglutarate-dependent dioxygenases. Fumarase promotes DNA repair by non-homologous end joining in mammalian cells through interaction with the histone variant H2A.Z, and inhibition of KDM2B, a H3 K36-specific histone demethylase. Here, we report that Saccharomyces cerevisiae fumarase, Fum1p, acts as a response factor during DNA replication stress, and fumarate enhances survival of yeast lacking Htz1p (H2A.Z in mammals). We observed that exposure to DNA replication stress led to upregulation as well as nuclear enrichment of Fum1p, and raising levels of fumarate in cells via deletion of FUM1 or addition of exogenous fumarate suppressed the sensitivity to DNA replication stress of htz1Δ mutants. This suppression was independent of modulating nucleotide pool levels. Rather, our results are consistent with fumarate conferring resistance to DNA replication stress in htz1Δ mutants by inhibiting the H3 K4-specific histone demethylase Jhd2p, and increasing H3 K4 methylation. Although the timing of checkpoint activation and deactivation remained largely unaffected by fumarate, sensors and mediators of the DNA replication checkpoint were required for fumarate-dependent resistance to replication stress in the htz1Δ mutants. Together, our findings imply metabolic enzymes and metabolites aid in processing replicative intermediates by affecting chromatin modification states, thereby promoting genome integrity.
    Keywords:  DNA replication stress; HTZ1; JHD2; fumarate; histone methylation
    DOI:  https://doi.org/10.1534/genetics.119.302238
  18. Methods Mol Biol. 2019 ;1978 155-165
    Li H, Tennessen JM.
      The fruit fly Drosophila melanogaster has emerged as an ideal system in which to study 2-hydroxyglutarate (2HG) metabolism. Unlike many mammalian tissues and cell lines, which primarily accumulate D- or L-2HG as the result of genetic mutations or metabolic stress, Drosophila larvae accumulate high concentrations of L-2HG during normal larval growth. As a result, flies represent one of the few model systems that allows for studies of endogenous L-2HG metabolism. Moreover, the Drosophila genome not only encodes key enzymes involved in the synthesis and degradation of D-2HG, but the fly has also been used as to investigate the in vivo effects of oncogenic isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations. All of these studies, however, rely on mass spectrometry-based methods to distinguish between the D- and L-2HG enantiomers. While such approaches are common among labs studying mammalian cell culture, few Drosophila studies have attempted to resolve and measure the individual 2HG enantiomers. Here we describe a highly reproducible gas chromatography-mass spectrometry (GC-MS)-based protocol that allows for quantitative measurements of both 2HG enantiomers in Drosophila homogenates.
    Keywords:  2-Hydroxyglutarate; Drosophila; Gas chromatography-mass spectrometry; Metabolomics; Oncometabolite
    DOI:  https://doi.org/10.1007/978-1-4939-9236-2_10
  19. Integr Comp Biol. 2019 May 23. pii: icz047. [Epub ahead of print]
    Sokolova IM, Sokolov EP, Haider F.
      Oxygen (O2) is essential for most metazoan life due to its central role in in mitochondrial oxidative phosphorylation (OXPHOS), which generates >90% of the cellular ATP. Oxygen fluctuations are an ultimate mitochondrial stressor resulting in mitochondrial damage, energy deficiency and cell death. This work provides an overview of the known and putative mechanisms involved in mitochondrial tolerance to fluctuating oxygen conditions in hypoxia-tolerant organisms including aquatic and terrestrial vertebrates and invertebrates. Mechanisms of regulation of the mitochondrial OXPHOS and electron transport system (ETS) (including alternative oxidases), sulfide tolerance, regulation of redox status and mitochondrial quality control, and the potential role of hypoxia-inducible factor (HIF) in mitochondrial tolerance to hypoxia are discussed. Mitochondrial phenotypes of distantly related animal species reveal common features including conservation and/or anticipatory upregulation of ETS capacity, suppression of ROS-producing electron flux through ubiquinone, reversible suppression of OXPHOS activity, and investment into the mitochondrial quality control mechanisms. Despite the putative importance of oxidative stress in adaptations to hypoxia, establishing the link between hypoxia tolerance and mitochondrial redox mechanisms is complicated by the difficulties of establishing the species-specific concentration thresholds above which the damaging effects of ROS outweigh their potentially adaptive signaling function. The key gaps in our knowledge about the potential mechanisms of mitochondrial tolerance to hypoxia include regulation of mitochondrial biogenesis and fusion/fission dynamics, and HIF-dependent metabolic regulation that require further investigation in hypoxia-tolerant species. Future physiological, molecular and genetic studies of mitochondrial responses to hypoxia and reoxygenation in phylogenetically diverse hypoxia-tolerant species could reveal novel solutions to the ubiquitous and metabolically severe problem of O2 deficiency and would have important implications for understanding the evolution of hypoxia tolerance and the potential mitigation of pathological states caused by O2 fluctuations.
    DOI:  https://doi.org/10.1093/icb/icz047
  20. Cancers (Basel). 2019 May 17. pii: E688. [Epub ahead of print]11(5):
    Villa E, Ali ES, Sahu U, Ben-Sahra I.
      Cancer cells exhibit a dynamic metabolic landscape and require a sufficient supply of nucleotides and other macromolecules to grow and proliferate. To meet the metabolic requirements for cell growth, cancer cells must stimulate de novo nucleotide synthesis to obtain adequate nucleotide pools to support nucleic acid and protein synthesis along with energy preservation, signaling activity, glycosylation mechanisms, and cytoskeletal function. Both oncogenes and tumor suppressors have recently been identified as key molecular determinants for de novo nucleotide synthesis that contribute to the maintenance of homeostasis and the proliferation of cancer cells. Inactivation of tumor suppressors such as TP53 and LKB1 and hyperactivation of the mTOR pathway and of oncogenes such as MYC, RAS, and AKT have been shown to fuel nucleotide synthesis in tumor cells. The molecular mechanisms by which these signaling hubs influence metabolism, especially the metabolic pathways for nucleotide synthesis, continue to emerge. Here, we focus on the current understanding of the molecular mechanisms by which oncogenes and tumor suppressors modulate nucleotide synthesis in cancer cells and, based on these insights, discuss potential strategies to target cancer cell proliferation.
    Keywords:  AKT; MYC; RAS; cancer metabolism; de novo nucleotide synthesis; mTORC1; metabolic vulnerability; oncogenes; short term and long-term regulation; tumor suppressors
    DOI:  https://doi.org/10.3390/cancers11050688
  21. Methods Mol Biol. 2019 ;1978 259-268
    Kalucka J, Ghesquière B, Fendt SM, Carmeliet P.
      Blood vessels are lined by a streamlined monolayer of quiescent endothelial cells (ECs). Although these cells can remain quiescent for years, different stimuli (ischemia, inflammation) and growth factors can activate them and drive a process of new vessel formation (angiogenesis). Emerging evidence reveals that cellular metabolism is a key determinant of the EC subtype specification. The use of stable isotope tracing and mass spectrometry analysis has been essential for the discovery that fatty acid metabolism contributes to EC proliferation and lymphatic EC differentiation. This chapter describes the methodology for setting up palmitate-based tracer metabolomics and the subsequent liquid chromatography-mass spectrometry (LC-MS)-based analysis. As such, tracer metabolomics can be used: (1) to identify the different metabolic pathways relying on carbons provided by fatty acid oxidation and (2) to quantify the relative contributions of palmitate-derived carbons. We begin by providing a background and general principles regarding the use of stable isotopes to study fatty acid metabolism. We then proceed with detailed procedures for the labeling conditions, sample preparation, and subsequent LC-MS analysis.
    Keywords:  Angiogenesis; EC metabolism; Fatty acids; Mass spectrometry; Stable isotopes
    DOI:  https://doi.org/10.1007/978-1-4939-9236-2_16
  22. PLoS One. 2019 ;14(5): e0213116
    Lee L, Seager R, Nakamura Y, Wilkinson KA, Henley JM.
      The mitochondrial outer membrane protein Mitochondrial Fission Factor (Mff) plays a key role in both physiological and pathological fission. It is well established that at stressed or functionally impaired mitochondria, PINK1 recruits the ubiquitin ligase Parkin which ubiquitinates Mff and other mitochondrial outer membrane proteins to facilitate the removal of defective mitochondria and maintain the integrity of the mitochondrial network. Here we show that, in addition to this clearance pathway, Parkin also ubiquitinates Mff in a PINK1-dependent manner under non-stressed conditions to regulate constitutive Mff turnover. We further show that removing Parkin via shRNA-mediated knockdown does not completely prevent Mff ubiquitination under these conditions, indicating that at least one other ubiquitin ligase contributes to Mff proteostasis. These data suggest that that Parkin plays a role in physiological maintenance of mitochondrial membrane protein composition in unstressed cells through constitutive low-level activation.
    DOI:  https://doi.org/10.1371/journal.pone.0213116
  23. Br J Cancer. 2019 May 22.
    Sellers K, Allen TD, Bousamra M, Tan J, Méndez-Lucas A, Lin W, Bah N, Chernyavskaya Y, MacRae JI, Higashi RM, Lane AN, Fan TW, Yuneva MO.
      BACKGROUND: Previous studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking.METHODS: Publicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices.
    RESULTS: Analysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture.
    CONCLUSIONS: Our data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.
    DOI:  https://doi.org/10.1038/s41416-019-0464-z
  24. Semin Cancer Biol. 2019 May 15. pii: S1044-579X(19)30034-3. [Epub ahead of print]
    Lawlor K, Pérez-Montero S, Lima A, Rodríguez TA.
      The maintenance of tissue homeostasis and health relies on the efficient removal of damaged or otherwise suboptimal cells. One way this is achieved is through cell competition, a fitness quality control mechanism that eliminates cells that are less fit than their neighbours. Through this process, cell competition has been shown to play diverse roles in development and in the adult, including in homeostasis and tumour suppression. However, over the last few years it has also become apparent that certain oncogenic mutations can provide cells with a competitive advantage that promotes their expansion via the elimination of surrounding wild-type cells. Thus, understanding how this process is initiated and regulated will provide important insights with relevance to a number of different research areas. A key question in cell competition is what determines the competitive fitness of a cell. Here, we will review what is known about this question by focussing on two non-mutually exclusive possibilities; first, that the activity of a subset of transcription factors determines competitive fitness, and second, that the outcome of cell competition is determined by the relative cellular metabolic status.
    DOI:  https://doi.org/10.1016/j.semcancer.2019.05.010
  25. Methods Mol Biol. 2019 ;1978 269-283
    Violante S, Berisa M, Thomas TH, Cross JR.
      Stable isotope tracing allows a metabolic substrate to be followed through downstream biochemical reactions, thereby providing unparalleled insights into the metabolic wiring of cells. This approach stops short of modeling absolute fluxes but is relatively straightforward and has become increasingly accessible due to the widespread adoption of high-resolution mass spectrometers. Analysis of both dynamic and steady-state labeling patterns in downstream metabolites provides valuable qualitative information as to their origin and relative rates of production. Stable isotope tracing is, therefore, a powerful way to understand the impact of genetic alterations and defined perturbations on metabolism. In this chapter, we describe a liquid chromatography-mass spectrometry (LC-MS) protocol for stable isotope tracing using 13C-L-arginine in a macrophage cell line. A similar approach can be used to follow other stable isotope tracers, and notes are provided with advice on how this protocol can be generalized for use in other settings.
    Keywords:  Fluxomics; Metabolomics; Stable isotope; Tracing experiments
    DOI:  https://doi.org/10.1007/978-1-4939-9236-2_17
  26. JCI Insight. 2019 May 23. pii: 128248. [Epub ahead of print]5
    Braganza AC, Corey CG, Santanasto AJ, Distefano G, Coen PM, Glynn NW, Nouraie SM, Goodpaster BH, Newman AB, Shiva S.
      BACKGROUND: Physical function decreases with age, and though bioenergetic alterations contribute to this decline, the mechanisms by which mitochondrial function changes with age remains unclear. This is partially because human mitochondrial studies require highly invasive procedures, such as muscle biopsies, to obtain live tissue with functional mitochondria. However, recent studies demonstrate that circulating blood cells are potentially informative in identifying systemic bioenergetic changes. Here, we hypothesize that human platelet bioenergetics reflect bioenergetics measured in muscle biopsies.METHODS & RESULTS: We demonstrate that maximal and ATP-linked respiratory rate measured in isolated platelets from older adults (86-93 years) correlates significantly with maximal respiration (r = 0.595; P = 0.003) measured by muscle biopsy respirometry and maximal ATP production (r = 0.643; P = 0.004) measured by 31P-MRS respectively, in the same individuals. Comparison of platelet bioenergetics in this aged cohort to platelets from younger adults (18-35 years) shows aged adults demonstrate lower basal and ATP-linked respiration. Platelets from older adults also show enhanced proton leak, which is likely due to increased protein levels of uncoupling protein 2, and correlates with increased gate speed in this cohort (r = 0.58; P = 0.0019). While no significant difference in glycolysis was observed in older adults compared to younger adults, platelet glycolytic rate correlated with fatigability (r = 0.44; P = 0.016).
    CONCLUSIONS: These data advance the mechanistic understanding of age-related changes in mitochondrial function. Further, they suggest that measuring platelet bioenergetics provides a potential supplement or surrogate for muscle biopsy measurement and may be a valuable tool to study mitochondrial involvement in age-related decline of physical function.
    Keywords:  Bioenergetics; Muscle Biology; Platelets; Skeletal muscle; Vascular Biology
    DOI:  https://doi.org/10.1172/jci.insight.128248
  27. Sci Signal. 2019 May 21. pii: eaav3048. [Epub ahead of print]12(582):
    Selvarajah B, Azuelos I, Platé M, Guillotin D, Forty EJ, Contento G, Woodcock HV, Redding M, Taylor A, Brunori G, Durrenberger PF, Ronzoni R, Blanchard AD, Mercer PF, Anastasiou D, Chambers RC.
      The differentiation of fibroblasts into a transient population of highly activated, extracellular matrix (ECM)-producing myofibroblasts at sites of tissue injury is critical for normal tissue repair. Excessive myofibroblast accumulation and persistence, often as a result of a failure to undergo apoptosis when tissue repair is complete, lead to pathological fibrosis and are also features of the stromal response in cancer. Myofibroblast differentiation is accompanied by changes in cellular metabolism, including increased glycolysis, to meet the biosynthetic demands of enhanced ECM production. Here, we showed that transforming growth factor-β1 (TGF-β1), the key pro-fibrotic cytokine implicated in multiple fibrotic conditions, increased the production of activating transcription factor 4 (ATF4), the transcriptional master regulator of amino acid metabolism, to supply glucose-derived glycine to meet the amino acid requirements associated with enhanced collagen production in response to myofibroblast differentiation. We further delineated the signaling pathways involved and showed that TGF-β1-induced ATF4 production depended on cooperation between canonical TGF-β1 signaling through Smad3 and activation of mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). ATF4, in turn, promoted the transcription of genes encoding enzymes of the de novo serine-glycine biosynthetic pathway and glucose transporter 1 (GLUT1). Our findings suggest that targeting the TGF-β1-mTORC1-ATF4 axis may represent a novel therapeutic strategy for interfering with myofibroblast function in fibrosis and potentially in other conditions, including cancer.
    DOI:  https://doi.org/10.1126/scisignal.aav3048
  28. J Biol Chem. 2019 May 19. pii: jbc.RA119.007983. [Epub ahead of print]
    Yam M, Engel AL, Wang Y, Zhu S, Hauer A, Zhang R, Lohner D, Huang J, Dinterman M, Zhao C, Chao JR, Du J.
      The retinal pigment epithelium (RPE) is a monolayer of pigmented cells between the choroid and the retina. RPE dysfunction underlies many retinal degenerative diseases, including age-related macular degeneration, the leading cause of age-related blindness. To perform its various functions in nutrient transport, phagocytosis of the outer segment, and cytokine secretion, the RPE relies on an active energy metabolism. We previously reported that human RPE cells prefer proline as a nutrient and transport proline-derived metabolites to the apical, or retinal, side. In this study, we investigated how RPE utilizes proline in vivo and why proline is a preferred substrate. By using 13C proline labeling both ex vivo and in vivo, we found that the retina rarely uses proline directly, whereas the RPE utilizes it at a high rate, exporting proline-derived mitochondrial intermediates for use by the retina. We observed that in primary human RPE cell culture, proline is the only amino acid whose uptake increases with cellular maturity. In human RPE, proline was sufficient to stimulate de novo serine synthesis, increase reductive carboxylation, and protect against oxidative damage. Blocking proline catabolism in RPE impaired glucose metabolism and glutathione production. Notably, in an acute model of RPE-induced retinal degeneration, dietary proline improved visual function. In conclusion, proline is an important nutrient that supports RPE metabolism and the metabolic demand of the retina.
    Keywords:  age-related macular degeneration (AMD); amino acid; cell metabolism; glucose metabolism; mitochondrial metabolism; oxidative stress; retinal metabolism; tricarboxylic acid cycle (TCA cycle) (Krebs cycle); visual function
    DOI:  https://doi.org/10.1074/jbc.RA119.007983
  29. Sci Rep. 2019 May 20. 9(1): 7623
    Ait-Aissa K, Blaszak SC, Beutner G, Tsaih SW, Morgan G, Santos JH, Flister MJ, Joyce DL, Camara AKS, Gutterman DD, Donato AJ, Porter GA, Beyer AM.
      Coronary artery disease (CAD) is a leading cause of death worldwide and frequently associated with mitochondrial dysfunction. Detailed understanding of abnormalities in mitochondrial function that occur in patients with CAD is lacking. We evaluated mitochondrial damage, energy production, and mitochondrial complex activity in human non-CAD and CAD hearts. Fresh and frozen human heart tissue was used. Cell lysate or mitochondria were isolated using standard techniques. Mitochondrial DNA (mtDNA), NAD + and ATP levels, and mitochondrial oxidative phosphorylation capacity were evaluated. Proteins critical to the regulation of mitochondrial metabolism and function were also evaluated in tissue lysates. PCR analysis revealed an increase in mtDNA lesions and the frequency of mitochondrial common deletion, both established markers for impaired mitochondrial integrity in CAD compared to non-CAD patient samples. NAD+ and ATP levels were significantly decreased in CAD subjects compared to Non-CAD (NAD+ fold change: non-CAD 1.00 ± 0.17 vs. CAD 0.32 ± 0.12* and ATP fold change: non-CAD 1.00 ± 0.294 vs. CAD 0.01 ± 0.001*; N = 15, P < 0.005). We observed decreased respiration control index in CAD tissue and decreased activity of complexes I, II, and III. Expression of ETC complex subunits and respirasome formation were increased; however, elevations in the de-active form of complex I were observed in CAD. We observed a corresponding increase in glycolytic flux, indicated by a rise in pyruvate kinase and lactate dehydrogenase activity, indicating a compensatory increase in glycolysis for cellular energetics. Together, these results indicate a shift in mitochondrial metabolism from oxidative phosphorylation to glycolysis in human hearts subjects with CAD.
    DOI:  https://doi.org/10.1038/s41598-019-43761-y
  30. Semin Cell Dev Biol. 2019 May 21. pii: S1084-9521(18)30166-6. [Epub ahead of print]
    Vander Linden C, Corbet C.
      Precision oncology is the practice of matching one therapy to one specific patient, based on particular genetic tumor alterations, in order to achieve the best clinical response. Despite an expanding arsenal of targeted therapies, many patients still have a poor outcome because tumor cells show a remarkable capacity to develop drug resistance, thereby leading to tumor relapse. Besides genotype-driven resistance mechanisms, tumor microenvironment (TME) peculiarities strongly contribute to generate an intratumoral phenotypic heterogeneity that affects disease progression and treatment outcome. In this Review, we describe how TME-mediated metabolic heterogeneities actively participate to therapeutic failure. We report how a lactate-based metabolic symbiosis acts as a mechanism of adaptive resistance to targeted therapies and we describe the role of mitochondrial metabolism, in particular oxidative phosphorylation (OXPHOS), to support the growth and survival of therapy-resistant tumor cells in a variety of cancers. Finally, we detail potential metabolism-interfering therapeutic strategies aiming to eradicate OXPHOS-dependent relapse-sustaining malignant cells and we discuss relevant (pre)clinical models that may help integrate TME-driven metabolic heterogeneity in precision oncology.
    Keywords:  Cancer; Drug resistance; Metabolism; Organoid; Oxidative phosphorylation; Precision oncology; Targeted therapy
    DOI:  https://doi.org/10.1016/j.semcdb.2019.05.016
  31. Semin Cell Dev Biol. 2019 May 21. pii: S1084-9521(18)30218-0. [Epub ahead of print]
    Bacci M, Ippolito L, Magnelli L, Giannoni E, Chiarugi P.
      Metabolic reprogramming as well as the flexible utilisation of fuel sources by tumour cells has been considered not only intrinsic to malignant cells but also sustained by resident and/or recruited stromal cells. The complexity of tumour-stroma cross-talk is experienced by neoplastic cells through profound changes in the own metabolic machinery. In such context, mitochondria are dynamic organelles that receive, orchestrate and exchange a multiplicity of stromal cues within the tumour cells to finely regulate key metabolic and signalling pathways, allowing malignant cells to adapt and thrive in an ever-changing environment. In this review, we focus on how tumour mitochondria are coached by stromal metabolic supply and how this re-education sustains tumour malignant traits.
    Keywords:  Epithelial-to-mesenchymal transition; Mitochondrial metabolism; Tumour metabolism; Tumour microenvironment
    DOI:  https://doi.org/10.1016/j.semcdb.2019.05.009
  32. Sci Adv. 2019 May;5(5): eaau8857
    Di Rienzo M, Antonioli M, Fusco C, Liu Y, Mari M, Orhon I, Refolo G, Germani F, Corazzari M, Romagnoli A, Ciccosanti F, Mandriani B, Pellico MT, De La Torre R, Ding H, Dentice M, Neri M, Ferlini A, Reggiori F, Kulesz-Martin M, Piacentini M, Merla G, Fimia GM.
      Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32's ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.
    DOI:  https://doi.org/10.1126/sciadv.aau8857
  33. FASEB J. 2019 May 23. fj201900073R
    Kim YY, Um JH, Yoon JH, Kim H, Lee DY, Lee YJ, Jee HJ, Kim YM, Jang JS, Jang YG, Chung J, Park HT, Finkel T, Koh H, Yun J.
      Mitophagy has been implicated in mitochondrial quality control and in various human diseases. However, the study of in vivo mitophagy remains limited. We previously explored in vivo mitophagy using a transgenic mouse expressing the mitochondria-targeted fluorescent protein Keima (mt-Keima). Here, we generated mt-Keima Drosophila to extend our efforts to study mitophagy in vivo. A series of experiments confirmed that mitophagy can be faithfully and quantitatively measured in mt-Keima Drosophila. We also showed that alterations in mitophagy upon environmental and genetic perturbation can be measured in mt-Keima Drosophila. Analysis of different tissues revealed a variation in basal mitophagy levels in Drosophila tissues. In addition, we found a significant increase in mitophagy levels during Drosophila embryogenesis. Importantly, loss-of-function genetic analysis demonstrated that the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)-Parkin pathway is essential for the induction of mitophagy in vivo in response to hypoxic exposure and rotenone treatment. These studies showed that the mt-Keima Drosophila system is a useful tool for understanding the role and molecular mechanism of mitophagy in vivo. In addition, we demonstrated the essential role of the PINK1-Parkin pathway in mitophagy induction in response to mitochondrial dysfunction.-Kim, Y. Y., Um, J.-H., Yoon, J.-H., Kim, H., Lee, D.-Y., Lee, Y. J., Jee, H. J., Kim, Y. M., Jang, J. S., Jang, Y.-G., Chung, J., Park, H. T., Finkel, T., Koh, H., Yun, J. Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.
    Keywords:  Drosophila model; confocal microscopy; in vivo mitophagy; quantitation of mitophagy
    DOI:  https://doi.org/10.1096/fj.201900073R
  34. Autophagy. 2019 May 23. 1-3
    Losier TT, Russell RC.
      Macroautophagy/autophagy is a conserved degradative pathway that host cells use to deal with invading pathogens. Despite significant overlap with starvation-induced autophagy, the early signaling that potentiates anti-bacterial autophagy is still unclear. Here we report AMPK, an upstream kinase regulating starvation-mediated autophagy induction, is activated in response to bacterial infection. AMPK inhibits MTORC1, an autophagy repressor, and activates autophagic ULK1 and PIK3C3/VPS34 complexes. Although AMPK-mediated inhibition of MTORC1 is not accompanied by the induction of bulk autophagy, AMPK regulation is critical for selectively targeting the bacteria for degradation. Moreover, AMPK signaling is triggered by the detection of bacteria-derived outer membrane vesicles and does not require bacterial invasion. Together, these data characterize and highlight the significance of AMPK signaling in priming the autophagic response to bacterial infection. Abbreviations: AMPK: AMP-activated protein kinase; MTORC1: MTOR complex 1; ULK1: Unc-51 like kinase 1; PIK3C3/VPS34: Phosphatidylinositol 3-kinase catalytic subunit type 3.
    Keywords:  AMPK; MTORC1; ULK1; Xenophagy; autophagy; outer membrane vesicles
    DOI:  https://doi.org/10.1080/15548627.2019.1618640
  35. Elife. 2019 May 21. pii: e43038. [Epub ahead of print]8
    Jewell JL, Fu V, Hong AW, Yu FX, Meng D, Melick CH, Wang H, Lam WM, Yuan HX, Taylor SS, Guan KL.
      The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.
    Keywords:  cancer biology; cancer cells; cell biology; cells; human; mouse
    DOI:  https://doi.org/10.7554/eLife.43038
  36. Biochim Biophys Acta Mol Basis Dis. 2019 May 20. pii: S0925-4439(19)30172-3. [Epub ahead of print]
    Kim H, Lee JH, Park JW.
      Acetaminophen (APAP)-induced hepatotoxicity is a major factor in liver failure and its toxicity is associated with the generation of reactive oxygen species (ROS), decreased levels of reduced glutathione (GSH) and overall oxidative stress. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) was demonstrated as an essential enzyme for mitochondria to maintain their antioxidant system by generating NADPH, which is an essential reducing equivalent for GSH turnover in mitochondria. Here, we investigated the role of IDH2 in APAP-induced liver injury with IDH2 deficient (idh2-/-) mice. Hepatotoxicity was promoted through apoptotic cell death following APAP administration in IDH2 deficient hepatocytes compared to that in wild-type hepatocytes. Apoptosis was found to result from the induction of ER stress and mitochondrial dysfunction as shown by the blocking the effect of phenylbutyrate and Mdivi1, respectively. In addition, mito-TEMPO, a scavenger of mitochondrial ROS, was seen to ameliorate APAP-induced hepatotoxicity in idh2-/- mice. In conclusion, IDH2 deficiency leads to a fundamental shortage of GSH that increases susceptibility to ROS generation and oxidative stress. This leads to excessive mitochondrial dysfunction and ER stress induction in response to APAP administration. Our study provides further evidence that IDH2 has a protective role against APAP-induced liver injury and emphasizes the importance of the elaborate linkages and functions of the antioxidant system in liver health.
    Keywords:  Acetaminophen; ER stress; IDH2; Mitochondrial dysfunction; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.bbadis.2019.05.012
  37. Mol Cancer Res. 2019 May 20. pii: molcanres.1233.2018. [Epub ahead of print]
    Dahl ES, Buj R, Leon KE, Newell JM, Imamura Y, Bitler BG, Snyder NW, Aird KM.
      Epithelial ovarian cancer (EOC) is the deadliest gynecological cancer. High-grade serous carcinoma (HGSC) is the most frequently diagnosed and lethal histosubtype of EOC. A significant proportion of HGSC patients relapse with chemoresistant disease. Therefore, there is an urgent need for novel therapeutic strategies for HGSC. Metabolic reprogramming is a hallmark of cancer cells, and targeting metabolism for cancer therapy may be beneficial. Here, we found that in comparison to normal fallopian tube epithelial cells, HGSC cells preferentially utilize glucose in the TCA cycle and not for aerobic glycolysis. This correlated with universally increased TCA cycle enzyme expression in HGSC cells under adherent conditions. HGSC disseminates as tumor cell spheroids within the peritoneal cavity. We found that wildtype isocitrate dehydrogenase I (IDH1) is the only TCA cycle enzyme upregulated in both adherent and spheroid conditions and is associated with reduced progression-free survival. IDH1 protein expression is also increased in primary HGSC patient tumors. Pharmacological inhibition or knockdown of IDH1 decreased proliferation of multiple HGSC cell lines by inducing senescence. Mechanistically, suppression of IDH1 increased the repressive histone mark H3K9me2 at multiple E2F target gene loci, which led to decreased expression of these genes. Altogether, these data suggest that increased IDH1 activity is an important metabolic adaptation in HGSC and that targeting wildtype IDH1 in HGSC alters the repressive histone epigenetic landscape to induce senescence. Implications: Inhibition of IDH1 may act as a novel therapeutic approach to alter both the metabolism and epigenetics of HGSC as a pro-senescent therapy.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-18-1233
  38. Semin Cancer Biol. 2019 May 15. pii: S1044-579X(18)30172-X. [Epub ahead of print]
    Paglia S, Sollazzo M, Di Giacomo S, Strocchi S, Grifoni D.
      Cancer has long been regarded and treated as a foreign body appearing by mistake inside a living organism. However, now we know that cancer cells communicate with neighbours, thereby creating modified environments able to support their unusual need for nutrients and space. Understanding the molecular basis of these bi-directional interactions is thus mandatory to approach the complex nature of cancer. Since their discovery, MYC proteins have been showing to regulate a steadily increasing number of processes impacting cell fitness, and are consistently found upregulated in almost all human tumours. Of interest, MYC takes part in cell competition, an evolutionarily conserved fitness comparison strategy aimed at detecting weakened cells, which are then committed to death, removed from the tissue and replaced by fitter neighbours. During physiological development, MYC-mediated cell competition is engaged to eliminate cells with suboptimal MYC levels, so as to guarantee selective growth of the fittest and proper homeostasis, while transformed cells expressing high levels of MYC coopt cell competition to subvert tissue constraints, ultimately disrupting homeostasis. Therefore, the interplay between cells with different MYC levels may result in opposite functional outcomes, depending on the nature of the players. In the present review, we describe the most recent findings on the role of MYC-mediated cell competition in different contexts, with a special emphasis on its impact on cancer initiation and progression. We also discuss the relevance of competition-associated cell death to cancer disease.
    Keywords:  Cancer; Cell competition; Cell death; MYC
    DOI:  https://doi.org/10.1016/j.semcancer.2019.05.009
  39. Sci Rep. 2019 May 24. 9(1): 7824
    Schinaman JM, Rana A, Ja WW, Clark RI, Walker DW.
      The FDA approved drug rapamycin can prolong lifespan in diverse species and delay the onset of age-related disease in mammals. However, a number of fundamental questions remain unanswered regarding the mechanisms by which rapamycin modulates age-related pathophysiology and lifespan. Alterations in the gut microbiota can impact host physiology, metabolism and lifespan. While recent studies have shown that rapamycin treatment alters the gut microbiota in aged animals, the causal relationships between rapamycin treatment, microbiota dynamics and aging are not known. Here, using Drosophila as a model organism, we show that rapamycin-mediated alterations in microbiota dynamics in aged flies are associated with improved markers of intestinal and muscle aging. Critically, however, we show that the beneficial effects of rapamycin treatment on tissue aging and lifespan are not dependent upon the microbiota. Indeed, germ-free flies show delayed onset of intestinal barrier dysfunction, improved proteostasis in aged muscles and a significant lifespan extension upon rapamycin treatment. In contrast, genetic inhibition of autophagy impairs the ability of rapamycin to mediate improved gut health and proteostasis during aging. Our results indicate that rapamycin-mediated modulation of the microbiota in aged animals is not causally required to slow tissue and organismal aging.
    DOI:  https://doi.org/10.1038/s41598-019-44106-5
  40. J Biol Chem. 2019 May 20. pii: jbc.RA118.006302. [Epub ahead of print]
    Koyano F, Yamano K, Kosako H, Tanaka K, Matsuda N.
      PINK1 (PARK6) and PARKIN (PARK2) are causal genes of recessive familial Parkinson's disease. Parkin is a ubiquitin ligase E3 that conjugates ubiquitin to impaired-mitochondrial proteins for organelle degradation. PINK1, a Ser/Thr kinase that accumulates only on impaired mitochondria, phosphorylates two authentic substrates, the ubiquitin-like domain of Parkin and ubiquitin. Our group and others have revealed that both the subcellular localization and ligase activity of Parkin are regulated through interactions with phosphorylated ubiquitin. Once PINK1 localizes on impaired mitochondria, PINK1-catalyzed phospho-ubiquitin recruits and activates Parkin. Parkin then supplies a ubiquitin chain to PINK1 for phosphorylation. The amplified ubiquitin functions as a signal for the sequestration and degradation of the damaged mitochondria. Although, a bewildering variety of Parkin substrates have been reported, the basis for Parkin substrate specificity remains poorly understood. Moreover, the mechanism underlying initial activation and translocation of Parkin onto mitochondria remains unclear, as the presence of ubiquitin on impaired mitochondria is thought to be a prerequisite for the initial PINK1 phosphorylation process. Here, we show that artificial mitochondria-targeted proteins are ubiquitylated by Parkin, suggesting that substrate specificity of Parkin is not determined by its amino acid sequence. Moreover, recruitment and activation of Parkin are delayed following depletion of the mitochondrial E3, MITOL/March5. We propose a model in which the initial step in Parkin recruitment and activation requires protein ubiquitylation by MITOL/March5 with subsequent PINK1-mediated phosphorylation. As PINK1 and Parkin amplify the ubiquitin signal via a positive feedback loop, the low substrate specificity of Parkin might facilitate this amplification process.
    Keywords:  Parkinson disease; mitophagy; parkin; ubiquitin; ubiquitin ligase
    DOI:  https://doi.org/10.1074/jbc.RA118.006302
  41. Hum Mol Genet. 2019 May 25. pii: ddz103. [Epub ahead of print]
    Zhou X, Curbo S, Zhao Q, Krishnan S, Kuiper R, Karlsson A.
      Deoxyguanosine kinase (DGUOK) provides guanosine and adenosine nucleotides for mitochondrial DNA (mtDNA) replication, and its deficiency in humans leads to hepatocerebral mtDNA depletion syndrome or to isolated hepatic disease. There are poor treatment options for DGUOK deficiency and the aim of this study was to generate a model for further studies of the disease that could reveal novel treatment strategies. We report a Dguok deficient mouse strain that, similar to humans, is most severely affected in liver. The Dguok complete knockout mice (Dguok-/-) were born normal, but began to lose weight at week 6. A change of fur color from black to blueish gray started at week 16 and was complete at week 20. The movements and behavior were indistinguishable compared to wild type mice. A decrease of mtDNA copy number occurred in multiple tissues, with liver being most severely affected. The mtDNA encoded protein cytochrome c oxidase was much lower in Dguok-/- liver tissue than in the wild type, whereas the expression of the nuclear encoded succinate dehydrogenase complex subunit A was unaffected. Histopathology showed severe alterations and immunohistochemistry showed signs of both oxidative stress and regeneration in Dguok-/- liver. The subcutaneous fat layer was undetectable in Dguok-/- which, in addition to gene expression analysis, indicated an altered lipid metabolism. We conclude that Dguok has a major role for the synthesis of deoxyribonucleotides for mtDNA replication particularly in liver, similar to the human disorder. Our data also show a catabolic lipid metabolism in liver tissue of Dguok-/-.
    DOI:  https://doi.org/10.1093/hmg/ddz103
  42. Hum Mol Genet. 2019 May 25. pii: ddz109. [Epub ahead of print]
    Bender D, Kaczmarek AT, Santamaria-Araujo JA, Stueve B, Waltz S, Bartsch D, Kurian L, Cirak S, Schwarz G.
      Sulfite oxidase (SO) is encoded by the nuclear SUOX gene and catalyzes the final step in cysteine catabolism thereby oxidizing sulfite to sulfate. Oxidation of sulfite is dependent on two cofactors within SO, a heme and the molybdenum cofactor (Moco), the later forming the catalytic site of sulfite oxidation. SO localizes to the intermembrane space of mitochondria where both, pre-SO processing as well as cofactor insertion are essential steps during SO maturation. Isolated SO deficiency (iSOD) is a rare inborn error of metabolism caused by mutations in the SUOX gene that lead to non-functional SO. ISOD is characterized by rapidly progressive neurodegeneration and death in early infancy. We diagnosed an iSOD patient with homozygous mutation of SUOX at c.1084G>A replacing Gly362 to serine. To understand the mechanism of disease, we expressed patient-derived G362S SO in E. coli and surprisingly found full catalytic activity while in patient fibroblasts no SO activity was detected, suggesting differences between bacterial and human expression. Moco reconstitution of apo-G362S SO was found to be approximately 90-fold reduced in comparison to apo-WT SO in vitro. In line, levels of SO-bound Moco in cells overexpressing G362S SO were significantly reduced compared to cells expressing WT SO providing evidence for compromised maturation of G362S SO in cellulo. Addition of molybdate to culture medium partially rescued impaired Moco binding of G362S SO and restored SO activity in patient fibroblasts. Thus, this study demonstrates the importance of the orchestrated maturation of SO and provides a first case of Moco-responsive iSOD.
    DOI:  https://doi.org/10.1093/hmg/ddz109
  43. Diabetes. 2019 Jun;68(6): 1099-1108
    Bai J, Liu F.
      It has been appreciated for many years that there is a strong association between metabolism and immunity in advanced metazoan organisms. Distinct immune signatures and signaling pathways have been found not only in immune but also in metabolic cells. The newly discovered DNA-sensing cGAS-cGAMP-STING pathway mediates type I interferon inflammatory responses in immune cells to defend against viral and bacterial infections. Recent studies show that this pathway is also activated by host DNA aberrantly localized in the cytosol, contributing to increased sterile inflammation, insulin resistance, and the development of nonalcoholic fatty liver disease (NAFLD). Potential interactions of the cGAS-cGAMP-STING pathway with mTORC1 signaling, autophagy, and apoptosis have been reported, suggesting an important role of the cGAS-cGAMP-STING pathway in the networking and coordination of these important biological processes. However, the regulation, mechanism of action, and tissue-specific role of the cGAS-cGAMP-STING signaling pathway in metabolic disorders remain largely elusive. It is also unclear whether targeting this signaling pathway is effective for the prevention and treatment of obesity-induced metabolic diseases. Answers to these questions would provide new insights for developing effective therapeutic interventions for metabolic diseases such as insulin resistance, NAFLD, and type 2 diabetes.
    DOI:  https://doi.org/10.2337/dbi18-0052
  44. Methods Mol Biol. 2019 ;1978 13-26
    Nemkov T, Reisz JA, Gehrke S, Hansen KC, D'Alessandro A.
      Metabolomics has emerged in the past decade as a highly attractive and impactful technique for phenotype-level profiling in diverse biological applications. Most recently, the dual developments of high-throughput analytical techniques along with dramatically increased sensitivity of high-resolution mass spectrometers have enabled the routine analysis of hundreds of unique samples per day. We have previously reported a robust 3 min isocratic metabolomics platform for the quantification of amino acids and the key pathways of central carbon and nitrogen metabolism. Building on this work, we describe here a 5 min reverse phase gradient followed by global, untargeted profiling of the hydrophilic metabolome. In addition to observing those metabolites measured in the 3 min run, the use of the longer gradient run here also allows for coverage of less polar compounds such as fatty acids and acylcarnitines, both key players in mitochondrial and lipid metabolism, without a significant sacrifice in throughput.
    Keywords:  Gradient; High-throughput; Isocratic; Mass spectrometry; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-4939-9236-2_2
  45. Proc Natl Acad Sci U S A. 2019 May 20. pii: 201900890. [Epub ahead of print]
    Xu Y, Anjaneyulu M, Donelian A, Yu W, Greenberg ML, Ren M, Owusu-Ansah E, Schlame M.
      Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.
    Keywords:  cardiolipin; lipids; membrane; mitochondria; respiration
    DOI:  https://doi.org/10.1073/pnas.1900890116