bims-camemi Biomed news
on Mitochondrial metabolism in cancer
Issue of 2019‒02‒10
seventy-two papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit


  1. Cell Rep. 2019 Feb 05. pii: S2211-1247(19)30086-5. [Epub ahead of print]26(6): 1557-1572.e8
    Fisher-Wellman KH, Draper JA, Davidson MT, Williams AS, Narowski TM, Slentz DH, Ilkayeva OR, Stevens RD, Wagner GR, Najjar R, Hirschey MD, Thompson JW, Olson DP, Kelly DP, Koves TR, Grimsrud PA, Muoio DM.
      Acyl CoA metabolites derived from the catabolism of carbon fuels can react with lysine residues of mitochondrial proteins, giving rise to a large family of post-translational modifications (PTMs). Mass spectrometry-based detection of thousands of acyl-PTMs scattered throughout the proteome has established a strong link between mitochondrial hyperacylation and cardiometabolic diseases; however, the functional consequences of these modifications remain uncertain. Here, we use a comprehensive respiratory diagnostics platform to evaluate three disparate models of mitochondrial hyperacylation in the mouse heart caused by genetic deletion of malonyl CoA decarboxylase (MCD), SIRT5 demalonylase and desuccinylase, or SIRT3 deacetylase. In each case, elevated acylation is accompanied by marginal respiratory phenotypes. Of the >60 mitochondrial energy fluxes evaluated, the only outcome consistently observed across models is a ∼15% decrease in ATP synthase activity. In sum, the findings suggest that the vast majority of mitochondrial acyl PTMs occur as stochastic events that minimally affect mitochondrial bioenergetics.
    Keywords:  ATP synthase; lysine acylation; malonylation; mitochondrial diagnostics
    DOI:  https://doi.org/10.1016/j.celrep.2019.01.057
  2. Cell Chem Biol. 2018 Dec 18. pii: S2451-9456(18)30440-9. [Epub ahead of print]
    Booty LM, Gawel JM, Cvetko F, Caldwell ST, Hall AR, Mulvey JF, James AM, Hinchy EC, Prime TA, Arndt S, Beninca C, Bright TP, Clatworthy MR, Ferdinand JR, Prag HA, Logan A, Prudent J, Krieg T, Hartley RC, Murphy MP.
      Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.
    Keywords:  glutathione; mitochondria; mitochondria targeting; redox signaling; thiol redox state; thioredoxin
    DOI:  https://doi.org/10.1016/j.chembiol.2018.12.002
  3. J Cell Commun Signal. 2019 Feb 04.
    Mohanty A, Tiwari-Pandey R, Pandey NR.
      Mitochondria, the dynamic organelles and power house of eukaryotic cells function as metabolic hubs of cells undergoing continuous cycles of fusion and fission. Recent findings have made it increasingly apparent that mitochondria essentially involved in energy production have evolved as principal intracellular signaling platforms regulating not only innate immunity but also inflammatory responses. Perturbations in mitochondrial dynamics, including fusion/fission, electron transport chain (ETC) architecture and cristae organization have now been actively correlated to modulate metabolic activity and immune function of innate and adaptive immune cells. Several newly identified mitochondrial proteins in mitochondrial outer membrane such as mitochondrial antiviral signaling protein (MAVS) and with mitochondrial DNA acting as danger-associated molecular pattern (DAMP) and mitochondrial ROS generated from mitochondrial sources have potentially established mitochondria as key signaling platforms in antiviral immunity in vertebrates and thereby orchestrating adaptive immune cell activations respectively. A thorough understanding of emerging and intervening role of mitochondria in toll-like receptor-mediated innate immune responses and NLRP3 inflammasome complex activation has gained lucidity in recent years that advocates the imposing functions of mitochondria in innate immunity. Fascinatingly, also how the signals stemming from the endoplasmic reticulum co-operate with the mitochondria to activate the NLRP3 inflammasome is now looked ahead as a stage to unravel as to how different mitochondrial and associated organelle stress responses co-operate to bring about inflammatory consequences. This has also opened avenues of research for revealing mitochondrial targets that could be exploited for development of novel therapeutics to treat various infectious, inflammatory, and autoimmune disorders. Thus, this review explores our current understanding of intricate interplay between mitochondria and other cellular processes like autophagy in controlling mitochondrial homeostasis and regulation of innate immunity and inflammatory responses.
    Keywords:  Inflammasome; Inflammation; Innate immune response; Mitochondrial dynamics; Mitophagy; NLRP3
    DOI:  https://doi.org/10.1007/s12079-019-00507-9
  4. Arch Biochem Biophys. 2019 Feb 01. pii: S0003-9861(18)30881-6. [Epub ahead of print]
    Díaz-Vegas A, Eisner V, Jaimovich E.
      Mitochondria represent the main source of ATP in skeletal muscle and mitochondria activity increases after muscle fiber depolarization. The regulation of mitochondrial function during contraction in skeletal muscle, however, is poorly understood. Skeletal muscle has a particular distribution of mitochondria where three distinct populations can be recognized. The most studied populations are the ones positioned deep into the myofibers between the myofibrils (intermyofibrillar mitochondria), and that located immediately beneath sarcolemma (subsarcolemmal mitochondria); a less studied population locates covering the myonuclei, as a continuation of the subsarcolemmal population. All mitochondria populations undergo fusion and fission events and intermyofibrillar mitochondria are interconnected; mitochondrial communication is necessary to maintain not only the energetic homeostasis of the muscle but its contractile function, as well. The mechanism supporting communication between subsarcolemmal and intermyofibrillar mitochondria is unknown. The recently described MCU complex of proteins has provided a new insight into the role of calcium as a regulator of mitochondrial function. Whether the different mitochondria populations have different calcium handling capacity and whether mitochondria Ca2+ has a role in energy transmission along the mitochondria network are intriguing issues that emerge when studying the link between electrical stimulation of the muscle fiber and the mitochondria metabolic output.
    DOI:  https://doi.org/10.1016/j.abb.2019.01.037
  5. Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Feb 04. pii: S1388-1981(18)30228-2. [Epub ahead of print]
    Raja V, Salsaa M, Joshi AS, Li Y, van Roermund CWT, Saadat N, Lazcano P, Schmidtke M, Hüttemann M, Gupta SV, Wanders RJA, Greenberg ML.
      Previous studies have shown that the cardiolipin (CL)-deficient yeast mutant, crd1Δ, has decreased levels of acetyl-CoA and decreased activities of the TCA cycle enzymes aconitase and succinate dehydrogenase. These biochemical phenotypes are expected to lead to defective TCA cycle function. In this study, we report that signaling and anaplerotic metabolic pathways that supplement defects in the TCA cycle are essential in crd1Δ mutant cells. The crd1Δ mutant is synthetically lethal with mutants in the TCA cycle, retrograde (RTG) pathway, glyoxylate cycle, and pyruvate carboxylase 1. Glutamate levels were decreased, and the mutant exhibited glutamate auxotrophy. Glyoxylate cycle genes were up-regulated, and the levels of glyoxylate metabolites succinate and citrate were increased in crd1Δ. Import of acetyl-CoA from the cytosol into mitochondria is essential in crd1Δ, as deletion of the carnitine-acetylcarnitine translocase led to lethality in the CL mutant. β-oxidation was functional in the mutant, and oleate supplementation rescued growth defects. These findings suggest that TCA cycle deficiency caused by the absence of CL necessitates activation of anaplerotic pathways to replenish acetyl-CoA and TCA cycle intermediates. Implications for Barth syndrome, a genetic disorder of CL metabolism, are discussed.
    Keywords:  Cardiolipin; Carnitine shuttle; Glyoxylate cycle; RTG pathway; TCA cycle; β-Oxidation
    DOI:  https://doi.org/10.1016/j.bbalip.2019.02.001
  6. Methods Mol Biol. 2019 ;1928 469-478
    Bentham RB, Bryson K, Szabadkai G.
      Transcription of a large set of nuclear-encoded genes underlies biogenesis of mitochondria, regulated by a complex network of transcription factors and co-regulators. A remarkable heterogeneity can be detected in the expression of these genes in different cell types and tissues, and the recent availability of large gene expression compendiums allows the quantification of specific mitochondrial biogenesis patterns. We have developed a method to effectively perform this task. Massively correlated biclustering (MCbiclust) is a novel bioinformatics method that has been successfully applied to identify co-regulation patterns in large genesets, underlying essential cellular functions and determining cell types. The method has been recently evaluated and made available as a package in Bioconductor for R. One of the potential applications of the method is to compare expression of nuclear-encoded mitochondrial genes or larger sets of metabolism-related genes between different cell types or cellular metabolic states. Here we describe the essential steps to use MCbiclust as a tool to investigate co-regulation of mitochondrial genes and metabolic pathways.
    Keywords:  Biclustering; Gene expression; MCbiclust; Metabolism; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_24
  7. Free Radic Biol Med. 2019 Feb 04. pii: S0891-5849(18)32178-6. [Epub ahead of print]
    Antonucci S, Mulvey JF, Burger N, Di Sante M, Hall AR, Hinchy EC, Caldwell ST, Gruszczyk AV, Deshwal S, Hartley RC, Kaludercic N, Murphy MP, Di Lisa F, Krieg T.
      Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca2+] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic.
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.01.034
  8. Methods Mol Biol. 2019 ;1928 125-147
    Sidoli S, Trefely S, Garcia BA, Carrer A.
      Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin-bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, falls within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation, and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt, and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4-20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation.
    Keywords:  Acetyl-CoA; Histones; Mass spectrometry; Metabolism; Proteomics
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_9
  9. Mol Med Rep. 2019 Jan 28.
    Li S, Sun C, Gu Y, Gao X, Zhao Y, Yuan Y, Zhang F, Hu P, Liang W, Cao K, Zhang J, Wang Z, Ye J.
      Increasing evidence has indicated that mutations of isocitrate dehydrogenase 1/2 (IDH1/2) contribute to the metabolic reprogramming of cancer cells; however their functions in lipid metabolism remain unknown. In the present study, the parental and IDH1 (R132H/+) mutant HCT116 cells were treated with various concentrations of oleic acid (OA) or palmitic acid (PA) in the presence or absence of glucose. The results demonstrated that mutation of IDH1 exacerbated the effects of OA and PA on cell viability and apoptosis, and consistently elevated the production of reactive oxygen species in HCT116 cells, particularly in the absence of glucose. Furthermore, mutation of IDH1 inhibited the rate of fatty acid oxidation (FAO), but elevated the glucose consumption in HCT116 cells. The results of immunoblotting and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) indicated that the expression of glucose transporter 1 was upregulated, whereas that of carnitine palmitoyl transferase 1 was downregulated in IDH1 mutant HCT116 cells. Although mitochondrial DNA quantification demonstrated that mutation of IDH1 had no effect on the quantity of mitochondria, immunoblotting and RT‑qPCR revealed that mutation of IDH1 in HCT116 cells significantly downregulated the expression of cytochrome c (CYCS) and CYCS oxidase IV, two important components in mitochondrial respiratory chain. These results indicated that mutation of IDH1 aggravated the fatty acid‑induced oxidative stress in HCT116 cells, by suppressing FAO and disrupting the mitochondrial respiratory chain. The results of the present study may provide novel insight into therapeutic strategies for the treatment of cancer types with IDH mutation.
    DOI:  https://doi.org/10.3892/mmr.2019.9903
  10. Front Mol Biosci. 2018 ;5 120
    Smolders VF, Zodda E, Quax PHA, Carini M, Barberà JA, Thomson TM, Tura-Ceide O, Cascante M.
      Cardiovascular diseases (CVD) are the leading cause of death worldwide. CVD comprise a range of diseases affecting the functionality of the heart and blood vessels, including acute myocardial infarction (AMI) and pulmonary hypertension (PH). Despite their different causative mechanisms, both AMI and PH involve narrowed or blocked blood vessels, hypoxia, and tissue infarction. The endothelium plays a pivotal role in the development of CVD. Disruption of the normal homeostasis of endothelia, alterations in the blood vessel structure, and abnormal functionality are essential factors in the onset and progression of both AMI and PH. An emerging theory proposes that pathological blood vessel responses and endothelial dysfunction develop as a result of an abnormal endothelial metabolism. It has been suggested that, in CVD, endothelial cell metabolism switches to higher glycolysis, rather than oxidative phosphorylation, as the main source of ATP, a process designated as the Warburg effect. The evidence of these alterations suggests that understanding endothelial metabolism and mitochondrial function may be central to unveiling fundamental mechanisms underlying cardiovascular pathogenesis and to identifying novel critical metabolic biomarkers and therapeutic targets. Here, we review the role of the endothelium in the regulation of vascular homeostasis and we detail key aspects of endothelial cell metabolism. We also describe recent findings concerning metabolic endothelial cell alterations in acute myocardial infarction and pulmonary hypertension, their relationship with disease pathogenesis and we discuss the future potential of pharmacological modulation of cellular metabolism in the treatment of cardiopulmonary vascular dysfunction. Although targeting endothelial cell metabolism is still in its infancy, it is a promising strategy to restore normal endothelial functions and thus forestall or revert the development of CVD in personalized multi-hit interventions at the metabolic level.
    Keywords:  acute myocardial infarction; cellular metabolism; endothelial dysfunction; glycolysis; metabolic targets; pulmonary hypertension; systems biology
    DOI:  https://doi.org/10.3389/fmolb.2018.00120
  11. Mol Cell. 2019 Jan 23. pii: S1097-2765(19)30002-4. [Epub ahead of print]
    Sekine S, Wang C, Sideris DP, Bunker E, Zhang Z, Youle RJ.
      Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.
    Keywords:  OMA1; TIM23 complex; TOM complex; mitochondrial protease; mitophagy
    DOI:  https://doi.org/10.1016/j.molcel.2019.01.002
  12. Methods Mol Biol. 2019 ;1928 55-67
    Newman AC, Labuschagne CF, Vousden KH, Maddocks ODK.
      Tracing the fate of carbon-13 (13C) labeled metabolites within cells by liquid chromatography mass spectrometry (LCMS) is a powerful analytical technique used for many years in the study of cell metabolism. Conventional experiments using LCMS and labeled nutrients tend to track the incorporation of 13C from exogenous nutrients (such as amino acids) into other, relatively proximal, cellular metabolites. Several labs have extended this technique to track transfer of 13C from the metabolite pool onto macromolecules, such as DNA, where methylation acts as an important functional modification. Here we describe a complete method that integrates previously established techniques to simultaneously track the use of 13C-serine or 13C-methionine into metabolite pools of the methionine cycle and into methylation of DNA and RNA. Given the ability to track methyl-transfer in a time-dependent way, this technique can provide temporal information about active methyl-transfer as well as quantification of total DNA/RNA methylation levels.
    Keywords:  Carbon-13; DNA; Flux; Liquid chromatography mass spectrometry; Methionine; Methyl-transfer; Methylation; One-carbon metabolism; RNA; Serine
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_4
  13. Nat Chem Biol. 2019 Feb 04.
    Kulkarni RA, Bak DW, Wei D, Bergholtz SE, Briney CA, Shrimp JH, Alpsoy A, Thorpe AL, Bavari AE, Crooks DR, Levy M, Florens L, Washburn MP, Frizzell N, Dykhuizen EC, Weerapana E, Linehan WM, Meier JL.
      Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
    DOI:  https://doi.org/10.1038/s41589-018-0217-y
  14. Nature. 2019 Feb 06.
    Vriens K, Christen S, Parik S, Broekaert D, Yoshinaga K, Talebi A, Dehairs J, Escalona-Noguero C, Schmieder R, Cornfield T, Charlton C, Romero-Pérez L, Rossi M, Rinaldi G, Orth MF, Boon R, Kerstens A, Kwan SY, Faubert B, Méndez-Lucas A, Kopitz CC, Chen T, Fernandez-Garcia J, Duarte JAG, Schmitz AA, Steigemann P, Najimi M, Hägebarth A, Van Ginderachter JA, Sokal E, Gotoh N, Wong KK, Verfaillie C, Derua R, Munck S, Yuneva M, Beretta L, DeBerardinis RJ, Swinnen JV, Hodson L, Cassiman D, Verslype C, Christian S, Grünewald S, Grünewald TGP, Fendt SM.
      Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.
    DOI:  https://doi.org/10.1038/s41586-019-0904-1
  15. Int J Oncol. 2019 Jan 28.
    Romero-Garcia S, Prado-Garcia H.
      In addition to their role in providing cellular energy, mitochondria fulfill a key function in cellular calcium management. The present review provides an integrative view of cellular and mitochondrial calcium homeostasis, and discusses how calcium regulates mitochondrial dynamics and functionality, thus affecting various cellular processes. Calcium crosstalk exists in the domain created between the endoplasmic reticulum and mitochondria, which is known as the mitochondria‑associated membrane (MAM), and controls cellular homeostasis. Calcium signaling participates in numerous biochemical and cellular processes, where calcium concentration, temporality and durability are part of a regulated, finely tuned interplay in non‑transformed cells. In addition, cancer cells modify their MAMs, which consequently affects calcium homeostasis to support mesenchymal transformation, migration, invasiveness, metastasis and autophagy. Alterations in calcium homeostasis may also support resistance to apoptosis, which is a serious problem facing current chemotherapeutic treatments. Notably, mitochondrial dynamics are also affected by mitochondrial calcium concentration to promote cancer survival responses. Dysregulated levels of mitochondrial calcium, alongside other signals, promote mitoflash generation in tumor cells, and an increased frequency of mitoflashes may induce epithelial‑to‑mesenchymal transition. Therefore, cancer cells remodel their calcium balance through numerous mechanisms that support their survival and growth.
    DOI:  https://doi.org/10.3892/ijo.2019.4696
  16. Cell Metab. 2019 Feb 05. pii: S1550-4131(19)30012-9. [Epub ahead of print]29(2): 243-245
    Das M, Alzaid F, Bayry J.
      Mitochondria, the powerhouse of the cell, known for producing energy through oxidative phosphorylation and the Krebs cycle, continue gaining notoriety for roles beyond bioenergetics. Recently in Nature, Weinberg et al. (2019) reported that mitochondrial complex III is indispensable for suppressive function of regulatory T cells, thus highlighting the importance of mitochondria in immune tolerance.
    DOI:  https://doi.org/10.1016/j.cmet.2019.01.012
  17. Methods Mol Biol. 2019 ;1928 353-363
    Zhang J, Zhang Q.
      A large amount of energy used for nutrient processing and cellular functions is essential for tumorigenesis. Total intracellular adenosine triphosphate (ATP) is mainly generated by glycolysis and mitochondrial oxidative phosphorylation. Here, we provide a protocol for measurements of energy metabolism in cancer cells by using Seahorse XF24 Extracellular Flux analyzer. Specifically, this machine measures glycolysis by analyzing the extracellular acidification rate (ECAR) and measures mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR), through real-time and live cell analysis. This protocol is provided for researchers who are unfamiliar with the method and to aid them in carrying out the technique successfully.
    Keywords:  ECAR; Energy metabolism; Glycolysis; OCR; Oxidative phosphorylation; Seahorse XF24 Extracellular Flux analyzer
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_18
  18. Dev Cell. 2019 Jan 23. pii: S1534-5807(18)31122-5. [Epub ahead of print]
    Ramachandran PV, Savini M, Folick AK, Hu K, Masand R, Graham BH, Wang MC.
      Lysosomes and mitochondria are both crucial cellular organelles for metabolic homeostasis and organism health. However, mechanisms linking their metabolic activities to promote organism longevity remain poorly understood. We discovered that the induction of specific lysosomal signaling mediated by a LIPL-4 lysosomal acid lipase and its lipid chaperone LBP-8 increases mitochondrial ß-oxidation to reduce lipid storage and promote longevity in Caenorhabditis elegans. We further discovered that increased mitochondrial ß-oxidation reduces mitochondrial electron transport chain complex II activity, contributing to the induction of reactive oxygen species in mitochondria (mtROS) and the longevity effect conferred by LIPL-4-LBP-8 signaling. Moreover, by activating the JUN-1 transcription factor downstream of mtROS, the LIPL-4-LBP-8 signaling pathway induces antioxidant targets and oxidative stress tolerance. Together, these results reveal regulatory mechanisms by which lysosomal signaling triggers adjustments in mitochondrial activity and suggest the significance of these metabolic adjustments for improving metabolic fitness, redox homeostasis, and longevity.
    Keywords:  aging; inter-organelle coordination; longevity; lysosomal signaling; metabolism; mitochondrial signaling; redox homeostasis
    DOI:  https://doi.org/10.1016/j.devcel.2018.12.022
  19. Cancer Cell. 2019 Jan 16. pii: S1535-6108(18)30584-1. [Epub ahead of print]
    Kim J, Yu L, Chen W, Xu Y, Wu M, Todorova D, Tang Q, Feng B, Jiang L, He J, Chen G, Fu X, Xu Y.
      The tumor suppressor p53 is somatically mutated in half of all human cancers. Paradoxically, the wild-type p53 (WTp53) is often retained in certain human cancers, such as hepatocarcinoma (HCC). We discovered a physiological and oncogenic role of WTp53 in suppressing pyruvate-driven oxidative phosphorylation by inducing PUMA. PUMA inhibits mitochondrial pyruvate uptake by disrupting the oligomerization and function of mitochondrial pyruvate carrier (MPC) through PUMA-MPC interaction, which depends on IκB kinase-mediated phosphorylation of PUMA at Ser96/106. High expression levels of PUMA are correlated with decreased mitochondrial pyruvate uptake and increased glycolysis in HCCs and poor prognosis of HCC patients. These findings are instrumental for cancer drug discovery aiming at activating WTp53 or restoring WTp53 activity to p53 mutants.
    Keywords:  PUMA; glycolysis; mitochondria; mitochondrial pyruvate carrier; oxidative phosphorylation; p53
    DOI:  https://doi.org/10.1016/j.ccell.2018.12.012
  20. Methods Mol Biol. 2019 ;1928 365-387
    Blacker TS, Sewell MDE, Szabadkai G, Duchen MR.
      Altered metabolism is a hallmark of cancer, both resulting from and driving oncogenesis. The NAD and NADP redox couples play a key role in a large number of the metabolic pathways involved. In their reduced forms, NADH and NADPH, these molecules are intrinsically fluorescent. As the average time for fluorescence to be emitted following excitation by a laser pulse, the fluorescence lifetime, is exquisitely sensitive to changes in the local environment of the fluorophore, imaging the fluorescence lifetime of NADH and NADPH offers the potential for label-free monitoring of metabolic changes inside living tumors. Here, we describe the biological, photophysical, and methodological considerations required to establish fluorescence lifetime imaging (FLIM) of NAD(P)H as a routine method for profiling the metabolism of living cancer cells and tissues.
    Keywords:  Autofluorescence; Cancer metabolism; Fluorescence lifetime imaging; Live-cell microscopy; NADH; NADPH
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_19
  21. Free Radic Biol Med. 2019 Feb 04. pii: S0891-5849(18)31476-X. [Epub ahead of print]134 445-457
    Tang M, Huang Z, Luo X, Liu M, Wang L, Qi Z, Huang S, Zhong J, Chen J, Li L, Wu D, Chen L.
      Excess iron accumulation and cardiac oxidative stress have been shown as important mediators of cardiac hypertrophy, whereas it remains largely elusive about the occurrence of mitochondrial iron overload and its significance during cardiac hypertrophy. In the present study, we aim to investigate the role of NCOA4-mediated ferritinophagy and SFXN1-dependent mitochondria iron overload in apelin-13-induced cardiomyocytes hypertrophy. Apelin-13 significantly promotes ferric citrate (FAC)-induced total cellular and mitochondria ion production, as well as mitochondria ROS contents. Mechanistically, apelin-13 effectively induces the expression of SFXN1, a mitochondria iron transporting protein and NCOA4, a cargo receptor of ferritinophagy in dose and time-dependent manner. Conversely, blockade of APJ by F13A abolishes these stimulatory effects. In addition, apelin-13-triggered mitochondria iron overload is reversed by the genetic inhibition of SFXN1 and NCOA4. NCOA4 deficiency via its silencing also interferes with the enhanced expression of SFXN1 evoked by apelin-13. In apelin-13-treated H9c2 cells, the promotion in cell diameter, volume as well as protein contents are obviously suppressed by the knockdown of NCOA4 and SFXN1 with their corresponding siRNAs. Remarkably, the human and murine hypertrophic hearts models, as well as apelin-13-injected mice models, present evident cardiac mitochondrial iron deposition and raised expressions of NCOA4 and SFXN1. Taken together, these results provide experimental evidences that NCOA4-mediated ferritinophagy might be defined as an essential mechanism leading to apelin-13-cardiomyocytes hypertrophy in SFXN1-dependent mitochondria iron overload manners.
    Keywords:  Apelin-13; Cardiomyocytes hypertrophy; Ferritin; Ferritinophagy; Mitochondria iron overload; ROS; sideroflexin1
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.01.052
  22. Anal Chem. 2019 Feb 06.
    Feng R, Guo L, Fang J, Jia Y, Wang X, Wei Q, Yu X.
      Mitochondria membrane potential (MMP) play significant roles during metabolism, signaling, and other important bioevents. Visualization of MMP levels is essential for many biological researches. However, fluorescent probes for monitoring MMP levels in dual emission colors are still deficient, which greatly limited the development of relative research areas. In this work, a pair of fluorescent probes have been designed and synthesized to monitor the MMP levels in dual emission colors based on Forster resonance energy transfer (FRET) mechanism. The FRET donor (FixD) is constructed by linking a benzyl chloride group to a fluorophore with bright green emission. The FixD could target mitochondria and be immobilized in mitochondria by linking to the thiol group of mitochondrial proteins. The FRET acceptor (LA) is designed with green absorption and deep-red emission. In live cells with high MMP levels, FixD and LA both target mitochondria, and deep-red (DR) emission could be detected with the excitation of 405 nm. Particularly, the spectral shift of fluorescence upon the decrease of MMP is up to 110 nm, which is greatly favorable for the clear observation of MMP levels. With the decrease of MMP, LA would be re-leased from mitochondria while FixD be still immobilized in mitochondria, and decreased DR emission and increased green fluorescence could be detected due to the absence of FRET. In this manner, the MMP levels could be monitored in dual emission colors.
    DOI:  https://doi.org/10.1021/acs.analchem.8b05822
  23. EMBO J. 2019 Feb 08. pii: e100492. [Epub ahead of print]
    Anderson R, Lagnado A, Maggiorani D, Walaszczyk A, Dookun E, Chapman J, Birch J, Salmonowicz H, Ogrodnik M, Jurk D, Proctor C, Correia-Melo C, Victorelli S, Fielder E, Berlinguer-Palmini R, Owens A, Greaves LC, Kolsky KL, Parini A, Douin-Echinard V, LeBrasseur NK, Arthur HM, Tual-Chalot S, Schafer MJ, Roos CM, Miller JD, Robertson N, Mann J, Adams PD, Tchkonia T, Kirkland JL, Mialet-Perez J, Richardson GD, Passos JF.
      Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.
    Keywords:  ageing; cardiomyocytes; senescence; senolytics; telomeres
    DOI:  https://doi.org/10.15252/embj.2018100492
  24. Methods Mol Biol. 2019 ;1928 427-439
    Qie S, He D, Sang N.
      Enhanced glutaminolysis and glycolysis are the two most remarkable biochemical features of cancer cell metabolism, reflecting increased utilization of glutamine and glucose in proliferating cells. Most solid tumors often outgrow the blood supply, resulting in a tumor microenvironment characterized by the depletion of glutamine, glucose, and oxygen. Whereas mechanisms by which cancer cells sense and metabolically adapt to hypoxia have been well characterized with a variety of cancer types, mechanisms by which different types of tumor cells respond to a dynamic change of glutamine availability and the underlying importance remains to be characterized. Here we describe the protocol, which uses cultured Hep3B cells as a model in determining glutamine-dependent proliferation, metabolite rescuing, and cellular responses to glutamine depletion. These protocols may be modified to study the metabolic roles of glutamine in other types of tumor or non-tumor cells as well.
    Keywords:  Anaplerosis; Cell proliferation; Endoplasmic reticulum stress; Glutamine depletion; Metabolism; Nitrogen anabolism
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_22
  25. Metabolites. 2019 Feb 01. pii: E23. [Epub ahead of print]9(2):
    Tronci L, Caria P, Frau DV, Liggi S, Piras C, Murgia F, Santoru ML, Pibiri M, Deiana M, Griffin JL, Vanni R, Atzori L.
      Background: Thyroid cancer is the most common endocrine malignancy, with papillary thyroid carcinoma (PTC) being the most common (85⁻90%) among all the different types of thyroid carcinomas. Cancer cells show metabolic alterations and, due to their rapid proliferation, an accumulation of reactive oxygen species, playing a fundamental role in cancer development and progression. Currently, the crosstalk among thyrocytes metabolism, redox balance and oncogenic mutations remain poorly characterized. The aim of this study was to investigate the interplay among metabolic alterations, redox homeostasis and oncogenic mutations in PTC-derived cells. Methods: Metabolic and redox profile, glutamate-cysteine ligase, glutaminase-1 and metabolic transporters were evaluated in PTC-derived cell lines with distinguished genetic background (TPC-1, K1 and B-CPAP), as well as in an immortalized thyroid cell line (Nthy-ori3-1) selected as control. Results: PTC-derived cells, particularly B-CPAP cells, harboring BRAF, TP53 and human telomerase reverse transcriptase (hTERT) mutation, displayed an increase of metabolites and transporters involved in energetic pathways. Furthermore, all PTC-derived cells showed altered redox homeostasis, as reported by the decreased antioxidant ratios, as well as the increased levels of intracellular oxidant species. Conclusion: Our findings confirmed the pivotal role of the metabolism and redox state regulation in the PTC biology. Particularly, the most perturbed metabolic phenotypes were found in B-CPAP cells, which are characterized by the most aggressive genetic background.
    Keywords:  cancer cell metabolism; metabolic profile; metabolomics; oxidative stress; papillary thyroid carcinoma
    DOI:  https://doi.org/10.3390/metabo9020023
  26. Methods Mol Biol. 2019 ;1928 69-76
    Creed S, McKenzie M.
      The mitochondrial membrane potential (Δψm) drives the generation of ATP by mitochondria. Interestingly, Δψm is higher in many cancer cells comparted to healthy noncancerous cell types, providing a unique metabolic marker. This feature has also been exploited for therapeutic use by utilizing drugs that specifically accumulate in the mitochondria of cancer cells with high Δψm. As such, the assessment of Δψm can provide very useful information as to the metabolic state of a cancer cell, as well as its potential for malignancy. In addition, the measurement of Δψm can also be used to test the ability of novel anticancer therapies to disrupt mitochondrial metabolism and cause cell death.Here, we outline two methods for assessing Δψm in cancer cells using confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM). In the first protocol, we describe a technique to quantitatively measure Δψm, which can be used to compare Δψm between different cell types. In the second protocol, we describe a technique for assessing changes to Δψm over time, which can be used to determine the effectiveness of different therapeutic compounds or drugs in modulating mitochondrial function.
    Keywords:  Cancer cells; Confocal imaging; Fluorescence; Membrane potential; Mitochondria; Osteosarcoma; TMRM
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_5
  27. Oxid Med Cell Longev. 2019 ;2019 8069583
    De Luise M, Guarnieri V, Ceccarelli C, D'Agruma L, Porcelli AM, Gasparre G.
      The Von Hippel-Lindau (VHL) syndrome has been rarely associated with renal oncocytomas, and tumors usually show HIF1α chronic stabilization. By contrast, oncocytomas mainly associated with respiratory chain (RC) defects due to severe mitochondrial DNA (mtDNA) mutations are incapable of stabilizing HIF1α, since oxygen consumption by the RC is dramatically diminished and prolylhydroxylase activity is increased by α-ketoglutarate accumulation following Krebs cycle slowdown. Here, we investigate the cooccurrence of a pseudohypoxic condition with oncocytic transformation in a case of VHL-associated renal oncocytoma. While HIF1α was abundant in nuclei concordantly with defects in VHL, negative staining of its targets carbonic anhydrase IX (CAIX) and glucose transporter GLUT1, usually overexpressed in VHL-associated neoplasms, suggested HIF1α to be present in its inactive (hydroxylated) form. MtDNA sequencing and immunohistochemistry analyses revealed a MT-CO1 stop-gain mutation and cytochrome c oxidase loss. We suggest that a mitochondrial respiration impairment may lead to hyperhydroxylation of the transcription factor, which we confirmed by specific staining of hydroxylated HIF1α. Such inactive form hence accumulated in the VHL-deficient tumor, where it may contribute to the benign nature of the neoplasm. We propose that the protumorigenic role of HIF1α in VHL cancers may be blunted through drugs inhibiting mitochondrial respiratory complexes, such as metformin.
    DOI:  https://doi.org/10.1155/2019/8069583
  28. FASEB J. 2019 Feb 06. fj201802209R
    Geng J, Wei M, Yuan X, Liu Z, Wang X, Zhang D, Luo L, Wu J, Guo W, Qin ZH.
      TP53-induced glycolysis and apoptosis regulator (TIGAR), a glycolytic inhibitor, plays vital roles in regulating cellular metabolism and oxidative stress. However, the role of highly expressed TIGAR in skeletal muscle remains unexplored. In the present study, TIGAR levels varied in different skeletal muscles and fibers. An exhaustive swimming test with a load corresponding to 5% of body weight was utilized in mice to assess the effects of TIGAR on exercise-induced fatigue and muscle damage. The running time and metabolic indicators were significantly greater in wild-type (WT) mice compared with TIGAR knockout (KO) mice. Poor exercise capacity was accompanied by decreased type IIA fibers in TIGAR KO mice. Decreased mitochondrial number and mitochondrial oxidative phosphorylation were observed more in TIGAR KO mice than in WT mice, which were involved in sirtuin 1 (SIRT1)-mediated deacetylation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and resveratrol treatment in TIGAR KO mice can increase mitochondrial content and exercise time. Much more TIGAR was also detected in mitochondria during exhaustive exercise. In addition, TIGAR, rather than mitochondria-targeted TIGAR achieved by in vitro plasmid transfection, promoted SIRT1-PGC1α pathway. Glutathione S-transferase-TIGAR pull-down assay followed by liquid chromatography mass spectrometry found that TIGAR interacted with ATP synthase F1 subunit α (ATP5A1), and its binding to ATP5A1 increased during exhaustive exercise. Overexpression of mitochondrial-TIGAR enhanced ATP generation, maintained mitochondrial membrane potential and reduced mitochondrial oxidative stress under hypoxia condition. Taken together, our results uncovered a novel role for TIGAR in mitochondrial regulation in fast-twitch oxidative skeletal muscle through SIRT1-PGC1α and translocation into mitochondria, which contribute to the increase in exercise endurance of mice.-Geng, J., Wei, M., Yuan, X., Liu, Z., Wang, X., Zhang, D., Luo, L., Wu, J., Guo, W., Qin, Z.-H. TIGAR regulates mitochondrial functions through SIRT1-PGC1α pathway and translocation of TIGAR into mitochondria in skeletal muscle.
    Keywords:  exhaustive exercise; mitochondrial content; type IIA fiber
    DOI:  https://doi.org/10.1096/fj.201802209R
  29. Methods Mol Biol. 2019 ;1928 337-352
    Hsu MY, Porporato PE, Wyart E.
      Cancer cachexia is a metabolic disease characterized by a negative energy balance associated with systemic weight loss and poor quality of life.In particular, skeletal muscle, which represents almost 50% of the total body mass, is strongly affected, and metabolic alterations therein (e.g., insulin resistance and mitochondrial dysfunction) can eventually support tumor growth by facilitating nutrient scavenging by the growing mass. Interestingly, metabolic interventions on wasting muscle have been proven to be protective, advocating for the importance of metabolic regulation in the wasting muscle.Here, we will briefly define the current knowledge of metabolic regulation in cachexia and provide a protocol to grow and differentiate in vitro myotubes for the assessment of mitochondrial metabolism during cachexia.
    Keywords:  Cachexia; Energy metabolism; Muscle wasting; Myotube differentiation; Oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_17
  30. Cell Death Dis. 2019 Feb 08. 10(2): 111
    Denton D, Xu T, Dayan S, Nicolson S, Kumar S.
      The majority of developmentally programmed cell death (PCD) is mediated by caspase-dependent apoptosis; however, additional modalities, including autophagy-dependent cell death, have important spatiotemporally restricted functions. Autophagy involves the engulfment of cytoplasmic components in a double membrane vesicle for delivery to the lysosome. An established model for autophagy-dependent PCD is Drosophila larval midgut removal during metamorphosis. Our previous work demonstrated that growth arrest is required to initiate autophagy-dependent midgut degradation and Target of rapamycin (Tor) limits autophagy induction. In further studies, we uncovered a role for Decapentaplegic (Dpp) in coordinating midgut degradation. Here, we provide new data to show that Dpp interacts with Tor during midgut degradation. Inhibiting Tor rescued the block in midgut degradation due to Dpp signaling. We propose that Dpp is upstream of Tor and down-regulation promotes growth arrest and autophagy-dependent midgut degradation. These findings underscore a relationship between Dpp and Tor signaling in the regulation of cell growth and tissue removal.
    DOI:  https://doi.org/10.1038/s41419-019-1368-9
  31. Proc Natl Acad Sci U S A. 2019 Feb 07. pii: 201816391. [Epub ahead of print]
    Jia D, Lu M, Jung KH, Park JH, Yu L, Onuchic JN, Kaipparettu BA, Levine H.
      Metabolic plasticity enables cancer cells to switch their metabolism phenotypes between glycolysis and oxidative phosphorylation (OXPHOS) during tumorigenesis and metastasis. However, it is still largely unknown how cancer cells orchestrate gene regulation to balance their glycolysis and OXPHOS activities. Previously, by modeling the gene regulation of cancer metabolism we have reported that cancer cells can acquire a stable hybrid metabolic state in which both glycolysis and OXPHOS can be used. Here, to comprehensively characterize cancer metabolic activity, we establish a theoretical framework by coupling gene regulation with metabolic pathways. Our modeling results demonstrate a direct association between the activities of AMPK and HIF-1, master regulators of OXPHOS and glycolysis, respectively, with the activities of three major metabolic pathways: glucose oxidation, glycolysis, and fatty acid oxidation. Our model further characterizes the hybrid metabolic state and a metabolically inactive state where cells have low activity of both glycolysis and OXPHOS. We verify the model prediction using metabolomics and transcriptomics data from paired tumor and adjacent benign tissue samples from a cohort of breast cancer patients and RNA-sequencing data from The Cancer Genome Atlas. We further validate the model prediction by in vitro studies of aggressive triple-negative breast cancer (TNBC) cells. The experimental results confirm that TNBC cells can maintain a hybrid metabolic phenotype and targeting both glycolysis and OXPHOS is necessary to eliminate their metabolic plasticity. In summary, our work serves as a platform to symmetrically study how tuning gene activity modulates metabolic pathway activity, and vice versa.
    Keywords:  OXPHOS; TNBC; Warburg; hybrid; metabolic reprogramming
    DOI:  https://doi.org/10.1073/pnas.1816391116
  32. FEBS Lett. 2019 Feb 07.
    Gureev AP, Shaforostova EA, Popov VN, Starkov AA.
      Methylene blue (MB) is a promising prodrug to treat mitochondrial dysfunctions that is currently being used in clinical trials for Alzheimer's disease. MB can penetrate the blood brain barrier, accumulating in brain mitochondria where it acts as a redox mediator in the electron transfer chain (ETC). Mitochondrial flavins are thought to reduce MB, which is then oxidized by cytochrome c, thereby bypassing inhibited Complex I of ETC. We found that in mouse brain mitochondria, MB fails to restore the membrane potential and respiration inhibited by antimycin. Furthermore, antimycin inhibits MB-induced H2 O2 generation. Our data suggest that the acceptor of electrons from MB is a Qo ubiquinol-binding site of Complex III; thus, MB-based drugs might not be helpful in mitochondrial dysfunctions involving Complex III inhibition. This article is protected by copyright. All rights reserved.
    Keywords:  antimycin; brain mitochondria; complex III; cytochrome c; electron transport chain; methylene blue; rotenone
    DOI:  https://doi.org/10.1002/1873-3468.13332
  33. Diabetes. 2019 Feb 06. pii: db180090. [Epub ahead of print]
    Kruger C, Nguyen TT, Breaux C, Guillory A, Mangelli M, Fridianto KT, Kovalik JP, Burk DH, Noland RC, Mynatt R, Stadler K.
      Proximal tubular epithelial cells are highly energy demanding. Their energy need is covered mostly from mitochondrial fatty acid oxidation. It is suggested, but not entirely clear whether derailments in fatty acid metabolism and mitochondrial dysfunction are forerunners of tubular damage. Here we modeled mitochondrial overload by creating mice lacking the enzyme carnitine acetyl-transferase (CrAT) in the proximal tubules, thus limiting a primary mechanism to export carbons under conditions of substrate excess. Mice developed tubular disease and interestingly, secondary glomerulosclerosis. This was accompanied by increased levels of apoptosis regulator and fibrosis markers, increased oxidative stress and abnormal profiles of acylcarnitines and organic acids suggesting profound impairments in all major forms of nutrient metabolism. When mice with CrAT deletion were placed on a high fat diet, kidney disease was more severe and developed faster. Primary proximal tubular cells isolated from the knockout mice displayed energy deficit and impaired respiration before the onset of pathology, suggesting mitochondrial respiratory abnormalities as a potential underlying mechanism. Our findings support the hypothesis that derailments of mitochondrial energy metabolism may be causative to chronic kidney disease. Our results also suggest that tubular injury may be a primary event followed by secondary glomerulosclerosis, raising the possibility that focusing on normalizing tubular cell mitochondrial function and energy balance could be an important preventative strategy.
    DOI:  https://doi.org/10.2337/db18-0090
  34. Cell Metab. 2019 Feb 05. pii: S1550-4131(19)30009-9. [Epub ahead of print]29(2): 236-238
    Venida A, Perera RM.
      Cancer cells are dependent on functional autophagy both within their cytoplasm and systemically in the host to maintain growth. How systemic autophagy directly contributes to tumor growth remains unclear. In a study published in Nature, Poillet-Perez et al. (2018) show that host autophagy helps to maintain the levels of circulating arginine that feed tumor growth.
    DOI:  https://doi.org/10.1016/j.cmet.2019.01.009
  35. Nat Commun. 2019 Feb 07. 10(1): 636
    Park MK, Yao Y, Xia W, Setijono SR, Kim JH, Vila IK, Chiu HH, Wu Y, Billalabeitia EG, Lee MG, Kalb RG, Hung MC, Pandolfi PP, Song SJ, Song MS.
      PTEN is a lipid phosphatase that antagonizes the PI3K/AKT pathway and is recognized as a major dose-dependent tumor suppressor. The cellular mechanisms that control PTEN levels therefore offer potential routes to therapy, but these are as yet poorly defined. Here we demonstrate that PTEN plays an unexpected role in regulating its own stability through the transcriptional upregulation of the deubiquitinase USP11 by the PI3K/FOXO pathway, and further show that this feedforward mechanism is implicated in its tumor-suppressive role, as mice lacking Usp11 display increased susceptibility to PTEN-dependent tumor initiation, growth and metastasis. Notably, USP11 is downregulated in cancer patients, and correlates with PTEN expression and FOXO nuclear localization. Our findings therefore demonstrate that PTEN-PI3K-FOXO-USP11 constitute the regulatory feedforward loop that improves the stability and tumor suppressive activity of PTEN.
    DOI:  https://doi.org/10.1038/s41467-019-08481-x
  36. J Mol Biol. 2019 Feb 04. pii: S0022-2836(19)30059-2. [Epub ahead of print]
    Brambley C, Marsee JD, Halper N, Miller JM.
      Oxidative stress is a common challenge to mitochondrial function where Reactive Oxygen Species are capable of significant organelle damage. The generation of mitochondrial Reactive Oxygen Species occurs in the inner membrane and matrix compartments as a consequence of subunit function in the electron transport chain and citric acid cycle, respectively. Maintenance of mitochondrial proteostasis and stress response is facilitated by compartmentalized proteases that couple the energy of ATP hydrolysis to unfolding and the regulated removal of damaged, misfolded, or aggregated proteins. The mitochondrial protease YME1L functions in the maintenance of proteostasis in the intermembrane space. YME1L is an inner membrane-anchored hexameric protease with distinct N-terminal, transmembrane, AAA+ (ATPases Associated with various cellular Activities), and C-terminal M41 zinc-dependent protease domains. The effect of oxidative stress on enzymes such as YME1L tasked with maintaining proteostasis is currently unclear. We report here that recombinant YME1L undergoes a reversible conformational change in response to oxidative stress that involves the interaction of one hydrogen peroxide molecule per YME1L monomer with affinities equal to 31 ± 2 mM and 26 ± 1 mM for conditions lacking or including nucleotide, respectively. Our data also reveal that oxidative stress does not significantly impact nucleotide binding equilibria, but does stimulate a two-fold increase in the rate constant for high-affinity ATP binding from (8.9 ± 0.2) × 105 M-1 s-1 to (1.5 ± 0.1) × 106 M-1 s-1. Taken together, these data may suggest a mechanism for the regulated processing of YME1L by other inner membrane proteases such as OMA1.
    Keywords:  AAA+ protease; ATP-dependent protease; Mitochondria; Proteostasis; Reactive oxygen species; YME1; Zinc-metalloprotease
    DOI:  https://doi.org/10.1016/j.jmb.2019.01.039
  37. Sci Rep. 2019 Feb 06. 9(1): 1461
    Lie S, Wang T, Forbes B, Proud CG, Petersen J.
      Ammonia can be utilised as an alternative nitrogen source to glutamine to support cell proliferation. However, the underlying molecular mechanisms and whether all cells have this ability is not fully understood. We find that eleven cancer and non-cancerous cell lines have opposite abilities to tolerate and utilise ammonia to support proliferation in a glutamine-depleted environment. HEK293, Huh7, T47D and MCF7 cells can use ammonia, when starved of glutamine, to support proliferation to varying degrees. Glutamine depletion reduced mTORC1 activity, while additional ammonia supplementation diminished this mTORC1 inhibition. Depletion of glutamine promoted a rapid and transient activation of AMPK, whereas, additional ammonia supplementation blocked this starvation-induced AMPK activation. As expected, drug-induced AMPK activation reduced cell proliferation in glutamine-depleted cells supplemented with ammonia. Surprisingly, mTORC1 activity was largely unchanged despite the enhanced AMPK activity, suggesting that AMPK does not inhibit mTORC1 signalling under these conditions. Finally, glutamate dehydrogenase (GDH) inhibition, a key enzyme regulating ammonia assimilation, leads to AMPK activation, mTORC1 inhibition and reduced proliferation. Ammonia provides an alternative nitrogen source that aids certain cancer cells ability to thrive in nutrient-deprived environment. The ability of cells to utilise ammonia as a nitrogen source is intricately linked to AMPK, mTORC1 and GDH.
    DOI:  https://doi.org/10.1038/s41598-018-37509-3
  38. Mol Biol Rep. 2019 Feb 06.
    Jha D, Mitra Mazumder P.
      Metabolic syndrome is an agglomeration of disorders including obesity, diabetes and cardiovascular diseases and characterized as chronic mild inflammation which elevates the circulatory inflammatory markers. This could be due to mitochondrial dysfunction, oxidative stress and hypoxia as a consequence of high fat diet (HFD) intake. The present study focuses on the effects of HFD on lactate and mitochondrial metabolism as well as tissue dependent changes in glucose transporter (GLUT) expression in liver, skeletal muscles and adipose tissue of mouse. Lactate dehydrogenase (LDH) and mitochondrial dysfunction established the link between the occurrences of metabolic stress due to HFD. In this work, it was observed that chronic HFD administration aggravated the metabolic alterations by causing reduced ATP production, imbalanced oxidative stress and altered class 1 GLUTs expression. Chronic HFD significantly reduced (p < 0.001) the superoxide dismutase (SOD), catalase (CAT) activities alongside elevated liver injury markers AST and ALT. This in turn causes decreased ATP/ADP ratio, mitochondrial dysfunction and exacerbated LDH levels. This imbalance further led to altered GLUT expression in hepatic cells, adipose tissue and skeletal muscles. HFD significantly (p < 0.001) upregulated the GLUT 1 and 3 expressions while significant downregulated (p < 0.001) GLUT 2 and 4 expression in liver, skeletal muscles and white adipose tissue. These results revealed the link between class 1 GLUTs, mitochondrial dysfunction and HFD-induced metabolic disorder. It can be concluded that HFD impacts mitochondrial metabolism and reprograms tissue-dependent glucose transporter.
    Keywords:  GLUT 1–4; High fat diet; LDH; Liver; Mitochondrial dysfunction; Skeletal muscles; White adipose tissue
    DOI:  https://doi.org/10.1007/s11033-019-04623-y
  39. Oncogene. 2019 Feb 04.
    Li Z, Jiao X, Di Sante G, Ertel A, Casimiro MC, Wang M, Katiyar S, Ju X, Klopfenstein DV, Tozeren A, Dampier W, Chepelev I, Jeltsch A, Pestell RG.
      Lysine methylation of histones and non-histone substrates by the SET domain containing protein lysine methyltransferase (KMT) G9a/EHMT2 governs transcription contributing to apoptosis, aberrant cell growth, and pluripotency. The positioning of chromosomes within the nuclear three-dimensional space involves interactions between nuclear lamina (NL) and the lamina-associated domains (LAD). Contact of individual LADs with the NL are dependent upon H3K9me2 introduced by G9a. The mechanisms governing the recruitment of G9a to distinct subcellular sites, into chromatin or to LAD, is not known. The cyclin D1 gene product encodes the regulatory subunit of the holoenzyme that phosphorylates pRB and NRF1 thereby governing cell-cycle progression and mitochondrial metabolism. Herein, we show that cyclin D1 enhanced H3K9 dimethylation though direct association with G9a. Endogenous cyclin D1 was required for the recruitment of G9a to target genes in chromatin, for G9a-induced H3K9me2 of histones, and for NL-LAD interaction. The finding that cyclin D1 is required for recruitment of G9a to target genes in chromatin and for H3K9 dimethylation, identifies a novel mechanism coordinating protein methylation.
    DOI:  https://doi.org/10.1038/s41388-019-0723-8
  40. Methods Mol Biol. 2019 ;1928 29-44
    Witney TH, Lewis DY.
      Positron emission tomography (PET) enables the noninvasive spatiotemporal analysis of cancer metabolism in vivo. Both natural and nonnatural PET tracers have been developed to assess metabolic pathways during tumorigenesis, cancer progression, and metastasis. Here we describe the dynamic in vivo PET/CT imaging of the glucose analogue [18F]fluoro-2-deoxy-D-glucose (FDG), taking into consideration the methodology for alternative metabolic PET substrates.
    Keywords:  Carbon-11; FDG; Fluorine-18; Fluorodeoxyglucose; Imaging; Mouse; PET; Positron emission tomography
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_2
  41. J Immunol. 2019 Feb 04. pii: ji1800575. [Epub ahead of print]
    Gómez-Cabañas L, López-Cotarelo P, Criado-García O, Murphy MP, Boya P, Rodríguez-Fernández JL.
      The immunological synapse (IS) is a superstructure formed during T cell activation at the zone of contact between T cells and dendritic cells (DCs). The IS includes specific molecular components in the T cell and DCs sides that may result in different functionality. Most of the studies on the IS have focused on the T cell side of this structure and, in contrast, the information available on the IS of DCs is sparse. Autophagy is a cellular process involved in the clearance of damaged proteins and organelles via lysosomal degradation. Mitophagy is the selective autophagy of damaged mitochondria. In this study, it is shown that IS formation induces clustering of mitochondria in the IS of DCs and partial depolarization of these organelles. At the IS of the DCs also accumulate autophagy and mitophagy markers, even when the kinase complex mTORC1, an inhibitor of the autophagy, is active. Together the results presented indicate that IS formation induces local clustering of mitochondria and mitophagy, which could be a homeostatic mechanism to control the quality of mitochondria in this region. The data underline the complexity of the regulatory mechanisms operating in the IS of DCs.
    DOI:  https://doi.org/10.4049/jimmunol.1800575
  42. FEBS J. 2019 Feb 06.
    Bernardino RL, Dias TR, Moreira BP, Cunha M, Barros A, Oliveira E, Sousa M, Alves MG, Oliveira PF.
      The process that allows cells to control their pH and bicarbonate levels is essential for ionic and metabolic equilibrium. Carbonic anhydrases (CAs) catalyse the conversion of CO2 to HCO3 - and H+ and are thus essential for this process. Herein, we inhibited CAs with acetazolamide - ACT and SLC-0111 - to study their involvement in the metabolism, mitochondrial potential, mitochondrial biogenesis and lipid metabolism of human Sertoli cells (hSCs), obtained from biopsies from men with conserved spermatogenesis. We were able to identify three isoforms of CAs, one mitochondrial isoform (CA VB) and two cell membrane-bound isoforms (CA IX and CA XII) in hSCs. When assessing the expression of markers for mitochondrial biogenesis, we observed a decrease in HIF-1α, SIRT1, PGC1α and NRF-1 mRNAs after all CAs were inhibited, resulting in decreased mitochondrial DNA copy numbers. This was followed by an increased production of lactate and alanine in the same conditions. In addition, consumption of glucose was maintained after inhibition of all CAs in hSCs. These results indicate a reduced conversion of pyruvate to acetyl-coA, possibly due to decreased mitochondrial function, caused by CAs inhibition in hSCs. Inhibition of CAs also caused alterations in lipid metabolism, since we detected an increased expression of Hormone sensitive lipase (HSL) in hSCs. Our results suggest that CAs are essential for mitochondrial biogenesis, glucose and lipid metabolism in hSCs. This is the first report showing that CAs play an essential role in hSCs metabolic dynamics, being involved in mitochondrial biogenesis and controlling lactate production. This article is protected by copyright. All rights reserved.
    Keywords:  Acetazolamide; Bicarbonate; Sertoli cells; metabolism; mitochondria biogenesis
    DOI:  https://doi.org/10.1111/febs.14779
  43. Nature. 2019 Feb 06.
    Choi WS, Lee G, Song WH, Koh JT, Yang J, Kwak JS, Kim HE, Kim SK, Son YO, Nam H, Jin I, Park ZY, Kim J, Park IY, Hong JI, Kim HA, Chun CH, Ryu JH, Chun JS.
      Osteoarthritis-the most common form of age-related degenerative whole-joint disease1-is primarily characterized by cartilage destruction, as well as by synovial inflammation, osteophyte formation and subchondral bone remodelling2,3. However, the molecular mechanisms that underlie the pathogenesis of osteoarthritis are largely unknown. Although osteoarthritis is currently considered to be associated with metabolic disorders, direct evidence for this is lacking, and the role of cholesterol metabolism in the pathogenesis of osteoarthritis has not been fully investigated4-6. Various types of cholesterol hydroxylases contribute to cholesterol metabolism in extrahepatic tissues by converting cellular cholesterol to circulating oxysterols, which regulate diverse biological processes7,8. Here we show that the CH25H-CYP7B1-RORα axis of cholesterol metabolism in chondrocytes is a crucial catabolic regulator of the pathogenesis of osteoarthritis. Osteoarthritic chondrocytes had increased levels of cholesterol because of enhanced uptake, upregulation of cholesterol hydroxylases (CH25H and CYP7B1) and increased production of oxysterol metabolites. Adenoviral overexpression of CH25H or CYP7B1 in mouse joint tissues caused experimental osteoarthritis, whereas knockout or knockdown of these hydroxylases abrogated the pathogenesis of osteoarthritis. Moreover, retinoic acid-related orphan receptor alpha (RORα) was found to mediate the induction of osteoarthritis by alterations in cholesterol metabolism. These results indicate that osteoarthritis is a disease associated with metabolic disorders and suggest that targeting the CH25H-CYP7B1-RORα axis of cholesterol metabolism may provide a therapeutic avenue for treating osteoarthritis.
    DOI:  https://doi.org/10.1038/s41586-019-0920-1
  44. Autophagy. 2019 Feb 07.
    De Virgilio C, Hatakeyama R.
      The evolutionarily conserved target of rapamycin kinase complex 1 (TORC1) regulates cell growth in a homeostatic manner by tuning anabolic and catabolic processes in response to nutritional and hormonal cues. Interestingly, rather than being localized at the plasma membrane as perhaps expected for an integrator of extracellular signals, TORC1 mainly localizes at vacuolar (in yeast) and lysosomal (in more complex eukaryotes) membranes where it seems optimally placed to sense both the nutrient status within the cytoplasm and the vacuolar/lysosomal compartment. How TORC1 controls downstream targets that are distant from the vacuole/lysosome, is currently poorly understood. In this context, we recently identified and characterized 2 spatially and functionally distinct pools of TORC1 in the budding yeast Saccharomyces cerevisiae: one at the vacuole that promotes protein synthesis, and another at endosomes that inhibits protein degradation. Thus, our findings highlight the presence of spatially separated pools of TORC1 that are commissioned with functionally specific tasks within cells. In addition, they pinpoint the existence of signaling endosomes in yeast, which raises numerous new questions that are warranted to direct future research in this area.
    Keywords:  EGO complex; ESCRT; amino acid signaling; growth control; macroautophagy; microautophagy; protein homeostasis; target of rapamycin complex 1
    DOI:  https://doi.org/10.1080/15548627.2019.1580107
  45. Methods Mol Biol. 2019 ;1928 113-123
    Galenkamp KMO, Alas B, Commisso C.
      Macropinocytosis has emerged as an important nutrient supply pathway that sustains cell growth of cancer cells within the nutrient-poor tumor microenvironment. By internalizing extracellular fluid through this bulk endocytic pathway, albumin is supplied to the cancer cells, which, after degradation, serves as an amino acid source to meet the high nutrient demands of these highly proliferating cells. Here, we describe a streamlined protocol for visualization and quantitation of macropinosomes in adherent cancer cells grown in vitro. The determination of the "macropinocytic index" provides a tool for measuring the extent to which this internalization pathway is utilized within the cancer cells and allows for comparison between different cell lines and treatments. The protocol provided herein has been optimized for reproducibility and is readily adaptable to multiple conditions and settings.
    Keywords:  Cancer; Endocytosis; Fluorescence microscopy; Macropinocytic index; Macropinocytosis; Macropinosome; Membrane ruffles; Nutrient uptake; Quantification; Quantitation
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_8
  46. Int J Cancer. 2019 Feb 04.
    Alavi MV.
      The mitochondrial inner membrane proteins OMA1 and OPA1 belong to the BAX/BAK1-dependent apoptotic signaling pathway, which can be regulated by tumor protein p53 and the prohibitins PHB and PHB2 in the context of neoplastic disease. For the most part these proteins have been studied separate from each other. Here, I argue that the OMA1 mechanism of action represents the missing link between p53 and cytochrome c release. The mitochondrial fusion protein OPA1 is cleaved by OMA1 in a stress-dependent manner generating S-OPA1. Excessive S-OPA1 can facilitate outer membrane permeabilization upon BAX/BAK1 activation through its membrane shaping properties. p53 helps outer membrane permeabilization in a 2-step process. First, cytosolic p53 activates BAX/BAK1 at the mitochondrial surface. Then, in a second step, p53 binds to prohibitin thereby releasing the restraint on OMA1. This activates OMA1, which cleaves OPA1 and promotes cytochrome c release. Clearly, OMA1 and OPA1 are not root causes for cancer. Yet many cancer cells rely on this pathway for survival, which can explain why loss of p53 function promotes tumor growth and confers resistance to chemotherapies. This article is protected by copyright. All rights reserved.
    Keywords:  OMA1; OPA1; apoptosis; chemotherapy; mitochondria; outer membrane permeabilization; prohibitin; tumor protein p53
    DOI:  https://doi.org/10.1002/ijc.32177
  47. PLoS One. 2019 ;14(2): e0211515
    Verma K, Patel A.
      Pyruvate Kinase M2 (PKM2) mediates metabolic reshuffling and is ubiquitously upregulated in several cancer types. The non-metabolic function of PKM2 as key nuclear kinase and modulator of gene expression is instrumental in cancer progression and tumorigenesis. Here, we attempt to discern the non-canonical function of PKM2 as an epigenetic modulator and the underlying implication of this activity. Using 5'-FAM labelled reconstituted mononucleosome we have shown that PKM2 interacts with the complex through Histone H3 and possibly obstruct the access to DNA binding factors. Subsequently, the interaction negatively impacts the ATP dependent remodeling activity of Chromodomain Helicase DNA binding protein-7 (Chd7). Chd7 remodeling activity is required to ameliorate DNA damage and is crucial to genome stability. Our study shows that PKM2 blocks the Chd7 mediated sliding of nucleosome. It can be conjectured that stalling Chd7 may lead to impaired DNA damage and increased genomic instability. We propose a mechanism in which PKM2 negatively regulate nucleosome repositioning in chromatin and may exacerbate cancer by altering the nucleosome architecture. This research is imperative to our understanding of how altered cancer metabolism can potentially modulate the gene expression and sustain incessant proliferation by tweaking the chromatin topography.
    DOI:  https://doi.org/10.1371/journal.pone.0211515
  48. Methods Mol Biol. 2019 ;1928 479-489
    Quek LE, Turner N.
      Flux analysis is performed to infer intracellular metabolic activity using measured rates. By applying the highly curated human metabolic reconstruction Recon 2 as the reference model, the investigation of cancer cell metabolic fluxes can encompass the full metabolic potential of a human cell. But in its full form, Recon 2 is unsuitable for conventional metabolic flux analysis due to a large number of redundant elements. Here, we describe a procedure to reduce Recon 2 to an appropriate scale for cancer cell flux analysis such that calculated flux intervals are still informative, without compromising the opportunity to explore alternative pathways encoded in Recon 2 that may reveal novel metabolic features.
    Keywords:  COBRA Toolbox; Cancer metabolism; Constraint-based; Flux analysis; Genome-scale model; MATLAB; Model reduction
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_25
  49. Nat Chem Biol. 2019 Feb 04.
    Garner TP, Amgalan D, Reyna DE, Li S, Kitsis RN, Gavathiotis E.
      BAX is a critical effector of the mitochondrial cell death pathway in response to a diverse range of stimuli in physiological and disease contexts. Upon binding by BH3-only proteins, cytosolic BAX undergoes conformational activation and translocation, resulting in mitochondrial outer-membrane permeabilization. Efforts to rationally target BAX and develop inhibitors have been elusive, despite the clear therapeutic potential of inhibiting BAX-mediated cell death in a host of diseases. Here, we describe a class of small-molecule BAX inhibitors, termed BAIs, that bind directly to a previously unrecognized pocket and allosterically inhibit BAX activation. BAI binding around the hydrophobic helix α5 using hydrophobic and hydrogen bonding interactions stabilizes key areas of the hydrophobic core. BAIs inhibit conformational events in BAX activation that prevent BAX mitochondrial translocation and oligomerization. Our data highlight a novel paradigm for effective and selective pharmacological targeting of BAX to enable rational development of inhibitors of BAX-mediated cell death.
    DOI:  https://doi.org/10.1038/s41589-018-0223-0
  50. Methods Mol Biol. 2019 ;1928 1-27
    Crooks DR, Fan TW, Linehan WM.
      Stable isotope-resolved metabolomics (SIRM) methods are used increasingly by cancer researchers to probe metabolic pathways and identify vulnerabilities in cancer cells. Analytical and computational advances are being made constantly, but tissue culture and sample extraction procedures are often variable and not elaborated in the literature. This chapter discusses basic aspects of tissue culture practices as they relate to the use of stable isotope tracers and provides a detailed metabolic labeling and metabolite extraction procedure designed to maximize the amount of information that can be obtained from a single tracer experiment.
    Keywords:  Glucose; Glutamine; Metabolite extraction; Metabolomics; Stable isotopes; Tissue culture
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_1
  51. Proc Natl Acad Sci U S A. 2019 Feb 06. pii: 201812943. [Epub ahead of print]
    Pal R, Xiong Y, Sardiello M.
      Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that causes tumor formation in multiple organs. TSC is caused by inactivating mutations in the genes encoding TSC1/2, negative regulators of the mammalian target of rapamycin complex 1 (mTORC1). Diminished TSC function is associated with excess glycogen storage, but the causative mechanism is unknown. By studying human and mouse cells with defective or absent TSC2, we show that complete loss of TSC2 causes an increase in glycogen synthesis through mTORC1 hyperactivation and subsequent inactivation of glycogen synthase kinase 3β (GSK3β), a negative regulator of glycogen synthesis. Specific TSC2 pathogenic mutations, however, result in elevated glycogen levels with no changes in mTORC1 or GSK3β activities. We identify mTORC1-independent lysosomal depletion and impairment of autophagy as the driving causes underlying abnormal glycogen storage in TSC irrespective of the underlying mutation. The defective autophagic degradation of glycogen is associated with abnormal ubiquitination and degradation of essential proteins of the autophagy-lysosome pathway, such as LC3 and lysosomal associated membrane protein 1 and 2 (LAMP1/2) and is restored by the combined use of mTORC1 and Akt pharmacological inhibitors. In complementation to current models that place mTORC1 as the central therapeutic target for TSC pathogenesis, our findings identify mTORC1-independent pathways that are dysregulated in TSC and that should therefore be taken into account in the development of a therapeutic treatment.
    Keywords:  Akt; TSC; autophagy; glycogen; mTOR
    DOI:  https://doi.org/10.1073/pnas.1812943116
  52. Nat Commun. 2019 Feb 06. 10(1): 620
    Haikala HM, Anttila JM, Marques E, Raatikainen T, Ilander M, Hakanen H, Ala-Hongisto H, Savelius M, Balboa D, Von Eyss B, Eskelinen V, Munne P, Nieminen AI, Otonkoski T, Schüler J, Laajala TD, Aittokallio T, Sihto H, Mattson J, Heikkilä P, Leidenius M, Joensuu H, Mustjoki S, Kovanen P, Eilers M, Leverson JD, Klefström J.
      Elevated MYC expression sensitizes tumor cells to apoptosis but the therapeutic potential of this mechanism remains unclear. We find, in a model of MYC-driven breast cancer, that pharmacological activation of AMPK strongly synergizes with BCL-2/BCL-XL inhibitors to activate apoptosis. We demonstrate the translational potential of an AMPK and BCL-2/BCL-XL co-targeting strategy in ex vivo and in vivo models of MYC-high breast cancer. Metformin combined with navitoclax or venetoclax efficiently inhibited tumor growth, conferred survival benefits and induced tumor infiltration by immune cells. However, withdrawal of the drugs allowed tumor re-growth with presentation of PD-1+/CD8+ T cell infiltrates, suggesting immune escape. A two-step treatment regimen, beginning with neoadjuvant metformin+venetoclax to induce apoptosis and followed by adjuvant metformin+venetoclax+anti-PD-1 treatment to overcome immune escape, led to durable antitumor responses even after drug withdrawal. We demonstrate that pharmacological reactivation of MYC-dependent apoptosis is a powerful antitumor strategy involving both tumor cell depletion and immunosurveillance.
    DOI:  https://doi.org/10.1038/s41467-019-08541-2
  53. Cell. 2019 Feb 07. pii: S0092-8674(19)30039-X. [Epub ahead of print]176(4): 928-943.e22
    Schiebinger G, Shu J, Tabaka M, Cleary B, Subramanian V, Solomon A, Gould J, Liu S, Lin S, Berube P, Lee L, Chen J, Brumbaugh J, Rigollet P, Hochedlinger K, Jaenisch R, Regev A, Lander ES.
      Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.
    Keywords:  ancestors; descendants; development; iPSCs; optimal-transport; paracrine interactions; regulation; reprogramming; scRNA-seq; trajectories
    DOI:  https://doi.org/10.1016/j.cell.2019.01.006
  54. Biol Chem. 2018 Dec 01. pii: /j/bchm.just-accepted/hsz-2018-0477/hsz-2018-0477.xml. [Epub ahead of print]
    Chudoba C, Wardelmann K, Kleinridders A.
      The prevalence of obesity and its co-morbidities such as insulin resistance and type 2 diabetes are tightly linked to increased ingestion of palatable fat enriched food. Thus, it seems intuitive that the brain senses elevated amounts of fatty acids and affects adaptive metabolic response, which is connected to mitochondrial function and insulin signaling. This review will address the effect of dietary fatty acids on brain insulin and mitochondrial function with a special emphasis on the impact of different fatty acids on brain function and metabolism.
    Keywords:  brain; fatty acids; insulin signaling; ketone bodies; mitochondrial function; neuronal health
    DOI:  https://doi.org/10.1515/hsz-2018-0477
  55. Nat Cell Biol. 2019 Feb 04.
    Romani P, Brian I, Santinon G, Pocaterra A, Audano M, Pedretti S, Mathieu S, Forcato M, Bicciato S, Manneville JB, Mitro N, Dupont S.
      Extracellular matrix (ECM) mechanical cues have powerful effects on cell proliferation, differentiation and death. Here, starting from an unbiased metabolomics approach, we identify synthesis of neutral lipids as a general response to mechanical signals delivered by cell-matrix adhesions. Extracellular physical cues reverberate on the mechanical properties of the Golgi apparatus and regulate the Lipin-1 phosphatidate phosphatase. Conditions of reduced actomyosin contractility lead to inhibition of Lipin-1, accumulation of SCAP/SREBP to the Golgi apparatus and activation of SREBP transcription factors, in turn driving lipid synthesis and accumulation. This occurs independently of YAP/TAZ, mTOR and AMPK, and in parallel to feedback control by sterols. Regulation of SREBP can be observed in a stiffened diseased tissue, and contributes to the pro-survival activity of ROCK inhibitors in pluripotent stem cells. We thus identify a general mechanism centered on Lipin-1 and SREBP that links the physical cell microenvironment to a key metabolic pathway.
    DOI:  https://doi.org/10.1038/s41556-018-0270-5
  56. Front Microbiol. 2019 ;10 31
    Choi J, Antoniewicz MR.
      In the past two decades, 13C metabolic flux analysis (13C-MFA) has matured into a powerful and widely used scientific tool in metabolic engineering and systems biology. Traditionally, metabolic fluxes have been determined from measurements of isotopic labeling by means of mass spectrometry (MS) or nuclear magnetic resonance (NMR). In recent years, tandem MS has emerged as a new analytical technique that can provide additional information for high-resolution quantification of metabolic fluxes in complex biological systems. In this paper, we present recent advances in methods and algorithms for incorporating tandem MS measurements into existing 13C-MFA approaches that are based on the elementary metabolite units (EMU) framework. Specifically, efficient EMU-based algorithms are presented for simulating tandem MS data, tracing isotopic labeling in biochemical network models and for correcting tandem MS data for natural isotope abundances.
    Keywords:  elementary metabolite units; metabolic flux analysis; metabolism; stable isotope tracers; tandem mass spectrometry
    DOI:  https://doi.org/10.3389/fmicb.2019.00031
  57. J Inherit Metab Dis. 2018 Dec 27.
    Wanders RJA, Vaz FM, Ferdinandusse S, van Kuilenburg ABP, Kemp S, van Karnebeek CD, Waterham HR, Houtkooper RH.
      The laboratory diagnosis of inborn errors of metabolism has been revolutionized in recent years, thanks to the amazing developments in the field of DNA sequencing including whole exome and whole genome sequencing (WES and WGS). Interpretation of the results coming from WES and/or WGS analysis is definitely not trivial especially since the biological relevance of many of the variants identified by WES and/or WGS, have not been tested experimentally and prediction programs like POLYPHEN-2 and SIFT are far from perfect. Correct interpretation of WES and/or WGS results can only be achieved by performing functional studies at multiple levels (different metabolomics platforms, enzymology, in vitro and in vivo flux analysis), often requires studies in model organisms like zebra fish, Caenorhabditis elegans, Saccharomyces cerevisiae, mutant mice and others, and also requires the input of many different disciplines to make this Translational Metabolism approach effective.
    Keywords:  fatty acids; inborn errors of metabolism; metabolism; mitochondria; peroxisomes
    DOI:  https://doi.org/10.1002/jimd.12008
  58. Methods Mol Biol. 2019 ;1928 149-173
    von Muhlinen N.
      Autophagy, a dynamic pathway in which intracellular membrane structures sequester portions of the cytosol for degradation, plays multiple roles in physiological and pathological processes. Autophagy may have suppressive and promotive roles in the formation and progression of cancer. A growing number of methods to identify, quantify, and manipulate autophagy have been developed. Because most of these methods are semiquantitative and have significant limitations, it is important to emphasize that a combination of these assays is recommended for the analysis of autophagy. Here, I briefly discuss the autophagic process, its role in disease, and I summarize some of the best-known and most widely used methods to study autophagy in vitro in the context of cancer, including transmission electron microscopy (TEM), detection and quantification of the autophagy protein LC3 by western blot, and the use of GFP-LC3 to quantify puncta by fluorescence microscopy and tandem labeled RFP/mCherry-GFP-LC3 fluorescence microscopy to measure autophagic flux.
    Keywords:  Atg8; Autophagy; Autophagy flux; Cancer; Cancer therapy; GFP-LC3; LC3; Macroautophagy; Metabolism
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_10
  59. Methods Mol Biol. 2019 ;1928 175-204
    Islam SR, Manna SK.
      Due to their role in cellular structure, energetics, and signaling, characterization of changes in cellular and extracellular lipid composition is of key importance to understand cancer biology. In addition, several mass spectrometry-based profiling as well as imaging studies have indicated that lipid molecules may be useful to augment existing biochemical and histopathological methods for diagnosis, staging, and prognosis of cancer. Therefore, analysis of lipidomic changes associated with cancer cells and tumor tissues can be useful for both fundamental and translational studies. Here, we provide a high-throughput single-extraction-based method that can be used for simultaneous lipidomic and metabolomic analysis of cancer cells or healthy or tumor tissue samples. In this chapter, a modified Bligh-Dyer method is described for extraction of lipids followed by analysis of fatty acid composition by gas chromatography-mass spectrometry (GC-MS) or untargeted lipidomics using electrospray ionization mass spectrometry (ESIMS) coupled with reverse-phase (RP) ultraperformance liquid chromatography (UPLC) followed by multivariate data analysis to identify features of interest.
    Keywords:  Cancer; Fatty acid methyl ester; GC-MS; Lipidomics; RP-UPLC-ESIMS
    DOI:  https://doi.org/10.1007/978-1-4939-9027-6_11
  60. Dev Cell. 2019 Jan 30. pii: S1534-5807(18)31125-0. [Epub ahead of print]
    Pan Y, Chai X, Gao Q, Zhou L, Zhang S, Li L, Luan S.
      Calcium is a universal signal in all eukaryotes, but the mechanism for encoding calcium signatures remains largely unknown. Calcium oscillations control pollen tube growth and fertilization in flowering plants, serving as a model for dissecting the molecular machines that mediate calcium fluctuations. We report that pollen-tube-specific cyclic nucleotide-gated channels (CNGC18, CNGC8, and CNGC7) together with calmodulin 2 (CaM2) constitute a molecular switch that either opens or closes the calcium channel depending on cellular calcium levels. Under low calcium, calcium-free calmodulin 2 (Apo-CaM2) interacts with CNGC18-CNGC8 complex, leading to activation of the influx channel and consequently increasing cytosolic calcium levels. Calcium-bound CaM2 dissociates from CNGC18/8 heterotetramer, closing the channel and initiating a downturn of cellular calcium levels. We further reconstituted the calcium oscillator in HEK293 cells, supporting the model that Ca2+-CaM-dependent regulation of CNGC channel activity provides an auto-regulatory feedback mechanism for calcium oscillations during pollen tube growth.
    Keywords:  calcium oscillations; cyclic nucleotide-gated channels; ion channel subunit composition; pollen tube growth
    DOI:  https://doi.org/10.1016/j.devcel.2018.12.025
  61. Cell Death Differ. 2019 Feb 06.
    Nazio F, Bordi M, Cianfanelli V, Locatelli F, Cecconi F.
      Autophagy and mitophagy act in cancer as bimodal processes, whose differential functions strictly depend on cancer ontogenesis, progression, and type. For instance, they can act to promote cancer progression by helping cancer cells survive stress or, instead, when mutated or abnormal, to induce carcinogenesis by influencing cell signaling or promoting intracellular toxicity. For this reason, the study of autophagy in cancer is the main focus of many researchers and several clinical trials are already ongoing to manipulate autophagy and by this way determine the outcome of disease therapy. Since the establishment of the cancer stem cell (CSC) theory and the discovery of CSCs in individual cancer types, autophagy and mitophagy have been proposed as key mechanisms in their homeostasis, dismissal or spread, even though we still miss a comprehensive view of how and by which regulatory molecules these two processes drive cell fate. In this review, we will dive into the deep water of autophagy, mitophagy, and CSCs and offer novel viewpoints on possible therapeutic strategies, based on the modulation of these degradative systems.
    DOI:  https://doi.org/10.1038/s41418-019-0292-y
  62. Nature. 2019 Feb 06.
    Eaglesham JB, Pan Y, Kupper TS, Kranzusch PJ.
      Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.
    DOI:  https://doi.org/10.1038/s41586-019-0928-6
  63. JCI Insight. 2019 Feb 05. pii: 124989. [Epub ahead of print]
    Najjar YG, Menk AV, Sander C, Rao U, Karunamurthy A, Bhatia R, Zhai S, Kirkwood JM, Delgoffe GM.
      The tumor microenvironment presents physical, immunologic, and metabolic barriers to durable immunotherapy responses. We have recently described roles for both T cell metabolic insufficiency as well as tumor hypoxia as inhibitory mechanisms which prevent T cell activity in murine tumors, but whether intratumoral T cell activity or response to immunotherapy vary between patients as a function of distinct metabolic profiles in tumor cells remains unclear. Here we show that metabolic derangement can vary widely in both degree and type in patient-derived cell lines and in ex vivo analysis of patient samples, such that some cells demonstrate solely deregulated oxidative or glycolytic metabolism. Further, deregulated oxidative, but not glycolytic, metabolism was associated with increased generation of hypoxia upon implantation into immunodeficient animals. Generation of murine single cell melanoma cell lines that lacked either oxidative or glycolytic metabolism showed that elevated tumor oxygen consumption was associated with increased T cell exhaustion and decreased immune activity. Further, melanoma lines lacking oxidative metabolism were solely responsive to anti-PD1 therapy among those tested. Prospective analysis of patient samples immunotherapy revealed that oxidative, but not glycolytic, metabolism was associated with progression on PD-1 blockade. Our data highlight a role for oxygen as a crucial metabolite required for the tumor-infiltrating T cells to differentiate appropriately upon PD-1 blockade, and suggesting tumor oxidative metabolism may be a target to improve immunotherapeutic response.
    Keywords:  Cancer immunotherapy; Glucose metabolism; Immunology; T cells
    DOI:  https://doi.org/10.1172/jci.insight.124989
  64. Cell Metab. 2019 Jan 28. pii: S1550-4131(19)30002-6. [Epub ahead of print]
    Adebayo Michael AO, Ko S, Tao J, Moghe A, Yang H, Xu M, Russell JO, Pradhan-Sundd T, Liu S, Singh S, Poddar M, Monga JS, Liu P, Oertel M, Ranganathan S, Singhi A, Rebouissou S, Zucman-Rossi J, Ribback S, Calvisi D, Qvartskhava N, Görg B, Häussinger D, Chen X, Monga SP.
      Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-β-catenin signaling. Activating β-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between β-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were β-catenin-mutated liver tumors. Genetic disruption of β-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in β-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-β-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are β-catenin mutated and GS positive.
    Keywords:  Wnt; beta-catenin; glutamine synthetase; hepatocellular cancer; liver tumor; mTOR; metabolic zonation; personalized medicine; precision therapy; tumor metabolism
    DOI:  https://doi.org/10.1016/j.cmet.2019.01.002
  65. Nature. 2019 Feb;566(7743): 187-188
    Manning BD.
      
    Keywords:  Cardiovascular biology; Metabolism
    DOI:  https://doi.org/10.1038/d41586-019-00245-3
  66. Cell Death Differ. 2019 Feb 08.
    Germic N, Frangez Z, Yousefi S, Simon HU.
      Autophagy is an evolutionally conserved, highly regulated catabolic process that combines cellular functions required for the regulation of metabolic balance under conditions of stress with those needed for the degradation of damaged cell organelles via the lysosomal machinery. The importance of autophagy for cell homeostasis and survival has long been appreciated. Recent data suggest that autophagy is also involved in non-metabolic functions that impact the immune system. Here, we reflect in two review articles the recent literature pointing to an important role for autophagy in innate immune cells. In this article, we focus on neutrophils, eosinophils, mast cells, and natural killer cells. We mainly discuss the influence of autophagy on functional cellular responses and its importance for overall host defense. In the companion review, we present the role of autophagy in the functions performed by monocytes/macrophages and dendritic cells.
    DOI:  https://doi.org/10.1038/s41418-019-0295-8
  67. Elife. 2019 Feb 06. pii: e43128. [Epub ahead of print]8
    Guo H, Suzuki T, Rubinstein JL.
      ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit e shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.
    Keywords:  biochemistry; chemical biology; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.43128
  68. J Cell Physiol. 2019 Feb 04.
    Luo XL, Jiang JY, Huang Z, Chen LX.
      Platelets, developed from megakaryocytes, are characterized by anucleate and short-life span hemocyte in mammal vessel. Platelets are very important in the cardiovascular system. Studies indicate the occurrence of autophagy platelets and megakaryocytes. Moreover, abnormal autophagy decreases the number of platelets and suppresses platelet aggregation. In addition, mitophagy, as a kind of selective autophagy, could inhibit platelet aggregation under oxidative stress or hypoxic, whereas promote platelet aggregation after reperfusion. Finally, autophagy regulates hemorrhagic and thrombosis diseases by influencing the number and function of platelets. In this paper, the role of autophagy in platelets and megakaryocytes, as well as coupled with the promotive or inhibitory role of hemorrhagic and thrombosis diseases are elucidated. Therefore, autophagy may be a potentially therapeutic target in modulating the platelet-related diseases.
    Keywords:  autophagy; megakaryocytes; mitophagy; platelets
    DOI:  https://doi.org/10.1002/jcp.28243
  69. J Biol Chem. 2019 Feb 07. pii: jbc.RA119.007494. [Epub ahead of print]
    Dierschke SK, Miller WP, Favate JS, Shah P, Imamura Kawasawa Y, Salzberg AC, Kimball SR, Jefferson LS, Dennis MD.
      Diabetes promotes the post-translational modification of proteins by O-linked addition of N-acetylglucosamine (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation eIF4E-binding protein 1 (4E-BP1) is O-GlcNAcylated and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e., mRNAs undergoing translation), as <1% of mRNAs exhibited changes in abundance. Remarkably, ~19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), was dependent on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in wild-type cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in both cells in culture and retina. The retina of diabetic wild-type mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.
    Keywords:  O-linked N-acetylglucosamine (O-GlcNAc); Ribo-Seq; diabetes; eukaryotic translation initiation; eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1); oxidative stress; post-translational modification (PTM); retina; ribosome foot printing
    DOI:  https://doi.org/10.1074/jbc.RA119.007494
  70. J Inherit Metab Dis. 2019 Feb 06.
    Soria LR, Nitzahn M, De Angelis A, Khoja S, Attanasio S, Annunziata P, Palmer DJ, Ng P, Lipshutz GS, Brunetti-Pierri N.
      The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-AFP), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia. This article is protected by copyright. All rights reserved.
    Keywords:  carbamoyl phosphate synthetase 1 deficiency; glutamine synthetase; helper-dependent adenoviral vectors; hyperammonemia; urea cycle disorders
    DOI:  https://doi.org/10.1002/jimd.12070
  71. J Clin Invest. 2019 Feb 05. pii: 98288. [Epub ahead of print]
    Wang F, Gatica D, Ying ZX, Peterson LF, Kim PK, Bernard D, Saiya-Cork K, Wang S, Kaminski MS, Chang AE, Phillips T, Klionsky DJ, Malek SN.
      The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to MTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the v-ATPase subunit ATP6V1B2/Vma2 activate autophagic flux and maintain MTOR/Tor in an active state. Engineered lymphoma cell lines and primary follicular lymphoma B cells (FL B cells) carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutants. These data support mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a new possible therapeutically targetable pathway.
    Keywords:  Autophagy; Cell Biology; Lymphomas; Molecular pathology; Oncology
    DOI:  https://doi.org/10.1172/JCI98288