bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2020‒03‒22
fifty-five papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit

  1. Science. 2020 03 20. 367(6484): 1366-1371
    Nagashima S, Tábara LC, Tilokani L, Paupe V, Anand H, Pogson JH, Zunino R, McBride HM, Prudent J.
      Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIβ [PI(4)KIIIβ], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.
  2. Redox Biol. 2020 Mar 07. pii: S2213-2317(19)31503-4. [Epub ahead of print]32 101472
    Mailloux RJ.
      The pathogenesis of many human diseases has been attributed to the over production of reactive oxygen species (ROS), particularly superoxide (O2●-) and hydrogen peroxide (H2O2), by-products of metabolism that are generated by the premature reaction of electrons with molecular oxygen (O2) before they reach complex IV of the respiratory chain. To date, there are 32 known ROS generators in mammalian cells, 16 of which reside inside mitochondria. Importantly, although these ROS are deleterious at high levels, controlled and temporary bursts in H2O2 production is beneficial to mammalian cells. Mammalian cells use sophisticated systems to take advantage of the second messaging properties of H2O2. This includes controlling its availability using antioxidant systems and negative feedback loops that inhibit the genesis of ROS at sites of production. At its core, ROS production depends on fuel metabolism. Therefore, desensitizing H2O2 signals would also require the temporary inhibition of fuel combustion and fluxes through metabolic pathways that promote ROS production. Additionally, this would also demand the diversion of fuels and nutrients into pathways that generate NADPH and other molecules required to maintain cellular redox buffering capacity. Therefore, fuel selection and metabolic flux plays an integral role in dictating the strength and duration of cellular redox signals. In the present review I provide an updated view on the function of protein S-glutathionylation, a ubiquitous redox sensitive modification involving the formation of a disulfide between the small molecular antioxidant glutathione and a cysteine residue, in the regulation of cellular metabolism on a global scale. To date, these concepts have mostly been reviewed at the level of mitochondrial bioenergetics in the contexts of health and disease. Careful examination of the literature revealed that glutathionylation is a temporary inhibitor of most metabolic pathways including glycolysis, the Krebs cycle, oxidative phosphorylation, amino acid metabolism, and fatty acid combustion, resulting in the diversion of fuels towards NADPH-producing pathways and the inhibition of ROS production. Armed with this information, I propose that protein S-glutathionylation reactions desensitize H2O2 signals emanating from catabolic pathways using a three-pronged regulatory mechanism; 1) inhibition of metabolic flux through pathways that promote ROS production, 2) diversion of metabolites towards pathways that support antioxidant defenses, and 3) direct inhibition of ROS-generating enzymes.
    Keywords:  ROS; glutathoinylation; metabolic regulation; redox signaling
  3. Cell Metab. 2020 Mar 16. pii: S1550-4131(20)30075-9. [Epub ahead of print]
    van Dam E, van Leeuwen LAG, Dos Santos E, James J, Best L, Lennicke C, Vincent AJ, Marinos G, Foley A, Buricova M, Mokochinski JB, Kramer HB, Lieb W, Laudes M, Franke A, Kaleta C, Cochemé HM.
      High-sugar diets cause thirst, obesity, and metabolic dysregulation, leading to diseases including type 2 diabetes and shortened lifespan. However, the impact of obesity and water imbalance on health and survival is complex and difficult to disentangle. Here, we show that high sugar induces dehydration in adult Drosophila, and water supplementation fully rescues their lifespan. Conversely, the metabolic defects are water-independent, showing uncoupling between sugar-induced obesity and insulin resistance with reduced survival in vivo. High-sugar diets promote accumulation of uric acid, an end-product of purine catabolism, and the formation of renal stones, a process aggravated by dehydration and physiological acidification. Importantly, regulating uric acid production impacts on lifespan in a water-dependent manner. Furthermore, metabolomics analysis in a human cohort reveals that dietary sugar intake strongly predicts circulating purine levels. Our model explains the pathophysiology of high-sugar diets independently of obesity and insulin resistance and highlights purine metabolism as a pro-longevity target.
    Keywords:  Drosophila; aging; diabetes; high-sugar diet; obesity; purine catabolism; uric acid; water imbalance
  4. Proc Natl Acad Sci U S A. 2020 Mar 17. pii: 201918216. [Epub ahead of print]
    Di Bartolomeo F, Malina C, Campbell K, Mormino M, Fuchs J, Vorontsov E, Gustafsson CM, Nielsen J.
      Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.
    Keywords:  Saccharomyces cerevisiae; absolute proteomics; diauxic shift; mitochondria
  5. Nat Commun. 2020 Mar 17. 11(1): 1320
    Kodama M, Oshikawa K, Shimizu H, Yoshioka S, Takahashi M, Izumi Y, Bamba T, Tateishi C, Tomonaga T, Matsumoto M, Nakayama KI.
      Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.
  6. Front Oncol. 2020 ;10 256
    Ippolito L, Giannoni E, Chiarugi P, Parri M.
      Mitochondria play multifaceted roles in malignant tumor progression. Beyond their bioenergetic role, mitochondria are essential for providing malignant cells a higher plasticity to face the harsh environmental conditions. Cell-autonomous metabolic deregulation of cancer cells, or metabolic adaptation to microenvironmental cues (lack of nutrients, stromal supply, hypoxia, etc.), represent the triggering event of mitochondria overexploitation to orchestrate nutrient sensing and upload, signaling, and redox circuits. As readout of their higher function, mitochondria produce high amounts of reactive oxygen species (ROS) that are functional for multiple signaling networks underlying tumor proliferation, survival, and metastatic process. To compensate for the higher rate of mitochondrial ROS production, cancer cells have evolved adaptive mechanisms to increase their antioxidant systems and to address ROS activating pathways useful for the tumor cell adaptation to environmental changes. As these properties are critical for cancer progression, mitochondrial ROS have recently become an attractive target for anti-cancer therapies. We discuss how understanding of mitochondrial function in the tumor-specific generation of ROS will impact on the development of novel redox-based targeted therapeutic strategies.
    Keywords:  ROS—reactive oxygen species; anti oxidant; anticancer activity; mitochondria; redox targeting; tumor microenviroment
  7. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30161-X. [Epub ahead of print]155 545-555
    Wolf DM, Segawa M, Shirihai OS, Liesa M.
      The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.
    Keywords:  Airyscan; Cristae; Live-cell imaging; Mitochondrial membrane potential; Super-resolution imaging
  8. Cell Metab. 2020 Mar 16. pii: S1550-4131(20)30114-5. [Epub ahead of print]
    Yang L, Garcia Canaveras JC, Chen Z, Wang L, Liang L, Jang C, Mayr JA, Zhang Z, Ghergurovich JM, Zhan L, Joshi S, Hu Z, McReynolds MR, Su X, White E, Morscher RJ, Rabinowitz JD.
      NADH provides electrons for aerobic ATP production. In cells deprived of oxygen or with impaired electron transport chain activity, NADH accumulation can be toxic. To minimize such toxicity, elevated NADH inhibits the classical NADH-producing pathways: glucose, glutamine, and fat oxidation. Here, through deuterium-tracing studies in cultured cells and mice, we show that folate-dependent serine catabolism also produces substantial NADH. Strikingly, when respiration is impaired, serine catabolism through methylene tetrahydrofolate dehydrogenase (MTHFD2) becomes a major NADH source. In cells whose respiration is slowed by hypoxia, metformin, or genetic lesions, mitochondrial serine catabolism inhibition partially normalizes NADH levels and facilitates cell growth. In mice with engineered mitochondrial complex I deficiency (NDUSF4-/-), serine's contribution to NADH is elevated, and progression of spasticity is modestly slowed by pharmacological blockade of serine degradation. Thus, when respiration is impaired, serine catabolism contributes to toxic NADH accumulation.
    Keywords:  MTHFD2; NAD; NADH; SHMT2; complex I inhibitor; hypoxia; methylene tetrahydrofolate dehydrogenase; mitochondrial disease; redox; respiration inhibition; serine catabolism; serine hydroxymethyltransferase
  9. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30140-2. [Epub ahead of print]155 491-518
    Hoppins S, Lackner LL, Lee JE, Mears JA.
      Mitochondria are required for cell survival and are best known for their role in energy production. These organelles also participate in many other biological processes that are critical for cellular function, and thus, play a central role in cellular life and death decisions. In a majority of cell types, mitochondria form highly dynamic, reticular networks. Maintaining the shape of these complex, ever-changing networks is critical for mitochondrial and cellular function, and requires the conserved activities of mitochondrial fission and fusion. Great advances in our knowledge about the molecular machines that mediate these dynamic activities have been made over the past 2 decades. These advances have been driven by the use of highly complementary in vitro and in vivo approaches that have proven extremely powerful for studying the complex membrane remodeling processes that drive fission and fusion of the organelle. In this chapter, we detail current methods used to examine the mechanisms and regulation of mitochondrial fission and fusion in vitro and in vivo.
    Keywords:  Mitochondrial division; Mitochondrial dynamics; Mitochondrial fission; Mitochondrial fusion
  10. Biochem Pharmacol. 2020 Mar 11. pii: S0006-2952(20)30135-0. [Epub ahead of print] 113907
    Oleson BJ, Corbett JA.
      Insulin, produced by pancreatic β-cells, is responsible for the control of whole-body glucose metabolism. Insulin is secreted by pancreatic β-cells in a tightly regulated process that is controlled by the serum level of glucose, glucose sensing and glucose oxidative metabolism. The regulation of intermediary metabolism in β-cells is unique as these cells oxidize glucose to CO2 on substrate supply while mitochondrial oxidative metabolism occurs on demand, for the production of intermediates or energy production, in most cell types. This review discusses recent findings that the regulation of intermediary metabolism by nitric oxide attenuates the DNA damage response (DDR) and DNA damage-dependent apoptosis in a β-cell selective manner. Specific focus is placed on the mechanisms by which iNOS derived nitric oxide (low micromolar levels) regulates DDR activation via the inhibition of intermediary metabolism. The physiological significance of the association of metabolism, nitric oxide and DDR signaling for cancer biology and diabetes is discussed.
  11. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30121-9. [Epub ahead of print]155 369-379
    Carraro M, Bernardi P.
      The mitochondrial permeability transition (PT) is an increase in the inner membrane permeability caused by the opening of a Ca2+-activated high-conductance channel, the so-called PT pore (PTP) or mitochondrial megachannel (MMC). Recent data indicate that F-ATP synthase contributes substantially to the generation of the PTP, yet this hypothesis is the matter of controversy. In this chapter, we will describe an approach to study the pore, i.e., the evaluation of mitochondrial swelling by means of a decrease in the absorbance at 540nm. This method should be useful to resolve apparent discrepancies in the literature and help solve emerging issues on the identity of mitochondrial pores.
    Keywords:  Absorbance; Mitochondria; Permeability transition; Swelling
  12. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30156-6. [Epub ahead of print]155 33-44
    Montesinos J, Area-Gomez E.
      Organelles within cells are interconnected by physical associations or contact sites. In the last decade, many reports have shown that these interactions are functional domains that maintain cellular homeostasis. One of the best studied interactions is between endoplasmic reticulum (ER) and mitochondria via mitochondria-associated membranes or MAMs. MAMs are lipid rafts in the ER in close apposition to mitochondria, where multiple enzymatic activities converge to coordinately regulate cellular functions such as: the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, cholesterol esterification, calcium signaling, mitochondrial shape and motility, autophagy and apoptosis. In this chapter, we describe and discuss some of the methods to isolate and assay this interesting cellular region.
    Keywords:  Endoplasmic reticulum; Lipid raft; MAM; Mitochondria; Mitochondria-associated ER membranes; Organelle contact sites
  13. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30160-8. [Epub ahead of print]155 157-180
    Agrawal RR, Tamucci KA, Pera M, Larrea D.
      Mitochondria are responsible for the generation of ATP by oxidative phosphorylation; however, these multifaceted organelles regulate many other key cellular functions as well, such as calcium homeostasis, apoptosis, and biosynthesis of steroid hormones, heme and phospholipids. In order to carry out these functions, mitochondria establish physical and functional connections with other organelles such as the plasma membrane, lipid droplets/vesicles, peroxisomes, endosomes, and the endoplasmic reticulum. Dysregulation of any of the aforementioned processes or inter-organelle contacts can lead to mitochondrial dysfunction and subsequent changes in oxygen consumption and ATP production. Seahorse technology has become a critical tool for quantification of mitochondrial oxygen consumption and can help differentiate primary mitochondrial disorders from disorders where alterations in mitochondrial metabolism are consequences of a prior, upstream insult. In this chapter, we describe the application of Seahorse technology for assaying mitochondrial respiration in whole cells, permeabilized cells and isolated mitochondria. We leave it to the researcher's discretion to determine which of these approaches will generate the most physiologically relevant data based on the model system and research question at hand.
    Keywords:  Mitochondria; Oxygen consumption; Respiration
  14. Autophagy. 2020 Mar 18. 1-13
    Li C, Zhang Y, Liu J, Kang R, Klionsky DJ, Tang D.
      Pancreatic cancer tends to be highly resistant to current therapy and remains one of the great challenges in biomedicine with very low 5-year survival rates. Here, we report that zalcitabine, an antiviral drug for human immunodeficiency virus infection, can suppress the growth of primary and immortalized human pancreatic cancer cells through the induction of ferroptosis, an iron-dependent form of regulated cell death. Mechanically, this effect relies on zalcitabine-induced mitochondrial DNA stress, which activates the STING1/TMEM173-mediated DNA sensing pathway, leading to macroautophagy/autophagy-dependent ferroptotic cell death via lipid peroxidation, but not a type I interferon response. Consequently, the genetic and pharmacological inactivation of the autophagy-dependent ferroptosis pathway diminishes the anticancer effects of zalcitabine in cell culture and animal models. Together, these findings not only provide a new approach for pancreatic cancer therapy but also increase our understanding of the interplay between autophagy and DNA damage response in shaping cell death.Abbreviations: ALOX: arachidonate lipoxygenase; ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATM: ATM serine/threonine kinase; ATG: autophagy-related; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; FANCD2: FA complementation group D2; GPX4: glutathione peroxidase 4; IFNA1/IFNα: interferon alpha 1; IFNB1/IFNβ: interferon beta 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MDA: malondialdehyde; mtDNA: mitochondrial DNA; NCOA4: nuclear receptor coactivator 4; PDAC: pancreatic ductal adenocarcinoma; POLG: DNA polymerase gamma, catalytic subunit; qRT-PCR: quantitative polymerase chain reaction; RCD: regulated cell death; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; TFAM: transcription factor A, mitochondrial.
    Keywords:  Antiviral drug; CGAS; DNA damage; POLG; STING1; TFAM; autophagy; ferroptosis; mitochondria; tumor therapy
  15. Dev Cell. 2020 Mar 12. pii: S1534-5807(20)30142-8. [Epub ahead of print]
    Chi F, Sharpley MS, Nagaraj R, Roy SS, Banerjee U.
      The mouse embryo undergoes compaction at the 8-cell stage, and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. Here, we report that this first cell fate specification event is controlled by glucose. Glucose does not fuel mitochondrial ATP generation, and glycolysis is dispensable for blastocyst formation. Furthermore, glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1. In addition, glucose-dependent nucleotide synthesis by the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of Tfap2c. YAP1, TEAD4, and TFAP2C interact to form a complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, and this process of developmental metabolism specifically controls TE cell fate.
    Keywords:  S1P signaling; Tfap2c; YAP1; developmental metabolism; glucose; hexosamine biosynthetic pathway; morula blastocyst; pentose phosphate pathway; preimplantation embryo; trophectoderm
  16. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30159-1. [Epub ahead of print]155 441-487
    Bacman SR, Gammage PA, Minczuk M, Moraes CT.
      Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.
    Keywords:  Gene therapy; Heteroplasmy; Mitochondrial diseases; mitoTALEN; mtDNA; mtZFN
  17. Nat Commun. 2020 Mar 20. 11(1): 1487
    Schöpf B, Weissensteiner H, Schäfer G, Fazzini F, Charoentong P, Naschberger A, Rupp B, Fendt L, Bukur V, Giese I, Sorn P, Sant'Anna-Silva AC, Iglesias-Gonzalez J, Sahin U, Kronenberg F, Gnaiger E, Klocker H.
      Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.
  18. Nat Commun. 2020 Mar 17. 11(1): 1416
    Mutvei AP, Nagiec MJ, Hamann JC, Kim SG, Vincent CT, Blenis J.
      The kinase mTOR complex 1 (mTORC1) promotes cellular growth and is frequently dysregulated in cancers. In response to nutrients, mTORC1 is activated on lysosomes by Rag and Rheb guanosine triphosphatases (GTPases) and drives biosynthetic processes. How limitations in nutrients suppress mTORC1 activity remains poorly understood. We find that when amino acids are limited, the Rap1-GTPases confine lysosomes to the perinuclear region and reduce lysosome abundance, which suppresses mTORC1 signaling. Rap1 activation, which is independent of known amino acid signaling factors, limits the lysosomal surface available for mTORC1 activation. Conversely, Rap1 depletion expands the lysosome population, which markedly increases association between mTORC1 and its lysosome-borne activators, leading to mTORC1 hyperactivity. Taken together, we establish Rap1 as a critical coordinator of the lysosomal system, and propose that aberrant changes in lysosomal surface availability can impact mTORC1 signaling output.
  19. J Clin Invest. 2020 Mar 17. pii: 135026. [Epub ahead of print]
    Jauhari A, Baranov SV, Suofu Y, Kim J, Singh T, Yablonska S, Li F, Wang X, Oberly P, Minnigh MB, Poloyac SM, Carlisle DL, Friedlander RM.
      Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis resulting in mitochondrial DNA (mtDNA) release, activation of cytosolic DNA mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential with higher mitochondrial DNA (mtDNA) release in brain and primary cerebro-cortical neurons of melatonin deficient aralkylamine N-acetyltransferase (AANAT) knockout mice. Cytosolic mtDNA activated the cGAS/STING/IRF3 pathway, stimulating inflammatory cytokine generation. We found that Huntington's disease mice increased mtDNA release, cGAS activation, and inflammation, all inhibited by exogenous melatonin. Thus, we demonstrated that cytosolic mtDNA activated the inflammatory response in aging and neurodegeneration, a process modulated by melatonin. Furthermore, our data suggest that AANAT knockout mice are a model of accelerated aging.
    Keywords:  Inflammation; Mitochondria; Neurodegeneration; Neuroscience
  20. J Biol Chem. 2020 Mar 16. pii: jbc.RA120.012793. [Epub ahead of print]
    Santos ARS, Gerhardt ECM, Parize E, Pedrosa FO, Steffens MBR, Chubatsu LS, Souza EM, Passaglia LMP, Sant'Anna FH, de Souza GA, Huergo LF, Forchhammer K.
      Nicotinamide adenine dinucleotide (NAD+) is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use L-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+.  This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.
    Keywords:  2-oxoglutarate (2-OG); NadE; PII protein; allosteric regulation; bacterial metabolism; metabolic regulation; nicotinamide adenine dinucleotide (NAD); nutrient sensing; protein-protein interaction
  21. Nat Commun. 2020 Mar 20. 11(1): 1508
    von Roemeling CA, Wang Y, Qie Y, Yuan H, Zhao H, Liu X, Yang Z, Yang M, Deng W, Bruno KA, Chan CK, Lee AS, Rosenfeld SS, Yun K, Johnson AJ, Mitchell DA, Jiang W, Kim BYS.
      Tumour cell phagocytosis by antigen presenting cells (APCs) is critical to the generation of antitumour immunity. However, cancer cells can evade phagocytosis by upregulating anti-phagocytosis molecule CD47. Here, we show that CD47 blockade alone is inefficient in stimulating glioma cell phagocytosis. However, combining CD47 blockade with temozolomide results in a significant pro-phagocytosis effect due to the latter's ability to induce endoplasmic reticulum stress response. Increased tumour cell phagocytosis subsequently enhances antigen cross-presentation and activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in APCs, resulting in more efficient T cell priming. This bridging of innate and adaptive responses inhibits glioma growth, but also activates immune checkpoint. Sequential administration of an anti-PD1 antibody overcomes this potential adaptive resistance. Together, these findings reveal a dynamic relationship between innate and adaptive immune regulation in tumours and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.
  22. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30131-1. [Epub ahead of print]155 337-368
    Greotti E, De Stefani D.
      Calcium (Ca2+) is a universal intracellular messenger capable of governing a plethora of different biological functions. Its versatility is guaranteed on the one hand by a cell type-specific Ca2+ signaling toolkit. On the other hand, the fine compartmentalization of changes in Ca2+ concentration ([Ca2+]) into specific subcellular domains adds a level of complexity, thus generating a variety of signals that can be differentially decoded into specific cellular events. In this context, mitochondrial Ca2+ dynamics plays a central role, by regulating both specific organelle functions (e.g., regulation of substrate oxidation, release of caspase cofactors) and global cellular events (e.g., shaping of cytoplasmic Ca2+ waves). Here we describe a general method for the detection of intramitochondrial [Ca2+] using bioluminescent and fluorescent genetically-encoded Ca2+ indicators (GECIs). We will discuss the characteristics of different GECIs, as well as their strengths, limitations and applications.
    Keywords:  Aequorin; Calcium; Cameleon; Genetically-encoded calcium indicators; Mitochondria
  23. Sci Rep. 2020 Mar 17. 10(1): 4905
    Cai Y, Rattray NJW, Zhang Q, Mironova V, Santos-Neto A, Hsu KS, Rattray Z, Cross JR, Zhang Y, Paty PB, Khan SA, Johnson CH.
      Women have a lower incidence of colorectal cancer (CRC) than men, however, they have a higher incidence of right-sided colon cancer (RCC). This is of concern as patients with RCC have the poorest clinical outcomes among all CRC patients. Aberrant metabolism is a known hallmark and therapeutic target for cancer. We propose that metabolic subphenotypes exist between CRCs due to intertumoral molecular and genomic variation, and differences in environmental milieu of the colon which vary between the sexes. Metabolomics analysis of patient colon tumors (n = 197) and normal tissues (n = 39) revealed sex-specific metabolic subphenotypes dependent on anatomic location. Tumors from women with RCC were nutrient-deplete, showing enhanced energy production to fuel asparagine synthesis and amino acid uptake. The clinical importance of our findings were further investigated in an independent data set from The Cancer Genomic Atlas, and demonstrated that high asparagine synthetase (ASNS) expression correlated with poorer survival for women. This is the first study to show a unique, nutrient-deplete metabolic subphenotype in women with RCC, with implications for tumor progression and outcomes in CRC patients.
  24. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30124-4. [Epub ahead of print]155 199-219
    Morciano G, Imamura H, Patergnani S, Pedriali G, Giorgi C, Pinton P.
      Adenosine 5'-triphosphate (ATP) is the central metabolite in the energy metabolism of cells and is hydrolyzed to ADP and inorganic phosphate to provide free energy in various cellular processes. ATP also functions as an intracellular signaling molecule. Thus, it is important to know the ATP concentration within cells to understand cellular activities. Here, we describe two methods to detect ATP concentrations in the cytoplasm and mitochondrial matrix using genetically encoded luminescent or fluorescent biosensors. These methods enable quantitative investigation of ATP concentration dynamics in living cells, single cells and cell populations.
    Keywords:  ATP; Bioluminescence; Biosensor; FRET; Fluorescence; Live cell imaging; Luciferase assay; Mitochondria
  25. Proc Natl Acad Sci U S A. 2020 Mar 19. pii: 201914786. [Epub ahead of print]
    Yi Y, Chen D, Ao J, Zhang W, Yi J, Ren X, Fei J, Li F, Niu M, Chen H, Luo Y, Luo Z, Xiao ZJ.
      AMP-activated protein kinase (AMPK) functions as an energy sensor and is pivotal in maintaining cellular metabolic homeostasis. Numerous studies have shown that down-regulation of AMPK kinase activity or protein stability not only lead to abnormality of metabolism but also contribute to tumor development. However, whether transcription regulation of AMPK plays a critical role in cancer metastasis remains unknown. In this study, we demonstrate that AMPKα1 expression is down-regulated in advanced human breast cancer and is associated with poor clinical outcomes. Transcription of AMPKα1 is inhibited on activation of PI3K and HER2 through ΔNp63α. Ablation of AMPKα1 expression or inhibition of AMPK kinase activity leads to disruption of E-cadherin-mediated cell-cell adhesion in vitro and increased tumor metastasis in vivo. Furthermore, restoration of AMPKα1 expression significantly rescues PI3K/HER2-induced disruption of cell-cell adhesion, cell invasion, and cancer metastasis. Together, these results demonstrate that the transcription control is another layer of AMPK regulation and suggest a critical role for AMPK in regulating cell-cell adhesion and cancer metastasis.
    Keywords:  AMPK; cancer metastasis; cell adhesion; oncogenic signaling; ΔNp63α
  26. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30138-4. [Epub ahead of print]155 295-319
    Liao PC, Franco-Iborra S, Yang Y, Pon LA.
      The redox state of mitochondria is determined by the levels of reducing and oxidizing species in the organelle, which reflects mitochondrial metabolic activity and overall fitness. Mitochondria are also the primary endogenous source of reactive oxygen species (ROS). This chapter describes methods to measure the mitochondrial superoxide levels and the redox state of the organelle in mammalian cells and yeast. We describe the use of dihydroethidium (DHE) and MitoSOX (a derivative of dihydroethidium bound to a lipophilic cation) to detect mitochondrial superoxide in yeast and mammalian cells, respectively. We also describe the use of genetically encoded fluorescent biosensors for quantitative analysis of mitochondrial NADPH levels (iNap) in mammalian cells and mitochondrial redox state (mito-roGFP) in yeast.
    Keywords:  Dihydroethidium (DHE); MitoSOX; NADPH; Oxidative stress; Reactive oxygen species (ROS); iNap3; roGFP
  27. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30155-4. [Epub ahead of print]155 415-439
    Bacman SR, Nissanka N, Moraes CT.
      The study of the mitochondrial DNA (mtDNA) has been hampered by the lack of methods to genetically manipulate the mitochondrial genome in living animal cells. This limitation has been partially alleviated by the ability to transfer mitochondria (and their mtDNAs) from one cell into another, as long as they are from the same species. This is done by isolating mtDNA-containing cytoplasts and fusing these to cells lacking mtDNA. This transmitochondrial cytoplasmic hybrid (cybrid) technology has helped the field understand the mechanism of several pathogenic mutations. In this chapter, we describe procedures to obtain transmitochondrial cybrids.
    Keywords:  Mitochondrial diseases; Rho zero cells; Transmitochondrial cybrids; mtDNA
  28. Front Oncol. 2020 ;10 239
    Nazemi M, Rainero E.
      The extracellular matrix (ECM) is a complex network of secreted proteins which provides support for tissues and organs. Additionally, the ECM controls a plethora of cell functions, including cell polarity, migration, proliferation, and oncogenic transformation. One of the hallmarks of cancer is altered cell metabolism, which is currently being exploited to develop anti-cancer therapies. Several pieces of evidence indicate that the tumor microenvironment and the ECM impinge on tumor cell metabolism. Therefore, it is essential to understand the contribution of the complex 3D microenvironment in controlling metabolic plasticity and responsiveness to therapies targeting cell metabolism. In this mini-review, we will describe how the tumor microenvironment and cancer-associated fibroblasts dictate cancer cell metabolism, resulting in increased tumor progression. Moreover, we will define the cross-talk between nutrient signaling and the trafficking of the ECM receptors of the integrin family. Finally, we will present recent data highlighting the contribution of nutrient scavenging from the microenvironment to support cancer cells growth under nutrient starvation conditions.
    Keywords:  cancer associated fibroblasts; cell metabolism; extracellular matrix; nutrient scavenging; nutrient signaling
  29. Front Cardiovasc Med. 2020 ;7 28
    Mohammed SA, Ambrosini S, Lüscher T, Paneni F, Costantino S.
      The molecular signatures of epigenetic regulation and chromatin architecture are emerging as pivotal regulators of mitochondrial function. Recent studies unveiled a complex intersection among environmental factors, epigenetic signals, and mitochondrial metabolism, ultimately leading to alterations of vascular phenotype and increased cardiovascular risk. Changing environmental conditions over the lifetime induce covalent and post-translational chemical modification of the chromatin template which sensitize the genome to establish new transcriptional programs and, hence, diverse functional states. On the other hand, metabolic alterations occurring in mitochondria affect the availability of substrates for chromatin-modifying enzymes, thus leading to maladaptive epigenetic signatures altering chromatin accessibility and gene transcription. Indeed, several components of the epigenetic machinery require intermediates of cellular metabolism (ATP, AcCoA, NADH, α-ketoglutarate) for enzymatic function. In the present review, we describe the emerging role of epigenetic modifications as fine tuners of gene transcription in mitochondrial dysfunction and vascular disease. Specifically, the following aspects are described in detail: (i) mitochondria and vascular function, (ii) mitochondrial ROS, (iii) epigenetic regulation of mitochondrial function; (iv) the role of mitochondrial metabolites as key effectors for chromatin-modifying enzymes; (v) epigenetic therapies. Understanding epigenetic routes may pave the way for new approaches to develop personalized therapies to prevent mitochondrial insufficiency and its complications.
    Keywords:  endothelial function; epigenetics; mitochondria; oxidative stress; vascular disease
  30. Science. 2020 Mar 19. pii: eaay2494. [Epub ahead of print]
    Zhou Z, Torres M, Sha H, Halbrook CJ, Van den Bergh F, Reinert RB, Yamada T, Wang S, Luo Y, Hunter AH, Wang C, Sanderson TH, Liu M, Taylor A, Sesaki H, Lyssiotis CA, Wu J, Kersten S, Beard DA, Qi L.
      The endoplasmic reticulum (ER) engages mitochondria at specialized ER domains known as mitochondria-associated membranes (MAMs). Here, we used three-dimensional high-resolution imaging to investigate the formation of pleomorphic "megamitochondria" with altered MAMs in brown adipocytes lacking the Sel1L-Hrd1 protein complex of ER-associated protein degradation (ERAD). Mice with ERAD deficiency in brown adipocytes were cold sensitive and exhibited mitochondrial dysfunction. ERAD deficiency affected ER-mitochondria contacts and mitochondrial dynamics, at least in part, by regulating the turnover of the MAM protein, sigma receptor 1 (SigmaR1). Thus, our study provides molecular insights into ER-mitochondrial crosstalk and expands our understanding of the physiological importance of Sel1L-Hrd1 ERAD.
  31. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30119-0. [Epub ahead of print]155 3-31
    Liao PC, Bergamini C, Fato R, Pon LA, Pallotti F.
      Isolated mitochondria are useful to study fundamental processes including mitochondrial respiration, metabolic activity, protein import, membrane fusion, protein complex assembly, as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other organelles. In addition, studies of the mitochondrial proteome, phosphoproteome, and lipidome are dependent on preparation of highly purified mitochondria (Boldogh, Vojtov, Karmon, & Pon, 1998; Cui, Conte, Fox, Zara, & Winge, 2014; Marc et al., 2002; Meeusen, McCaffery, & Nunnari, 2004; Reinders et al., 2007; Schneiter et al., 1999; Stuart & Koehler, 2007). Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles. However, methods to disrupt cells and tissue vary. Moreover, density gradient centrifugation or affinity purification of the organelle are used to further purify mitochondria or to separate different populations of the organelle. Here, we describe protocols to isolate mitochondria from different cells and tissues as well as approaches to assess the purity and integrity of isolated organelles.
    Keywords:  Affinity purification; Mitochondria; Subcellular fractionation; Yeast
  32. Hum Mol Genet. 2020 Mar 19. pii: ddaa043. [Epub ahead of print]
    Butler TJ, Estep KN, Sommers JA, Maul RW, Moore AZ, Bandinelli S, Cucca F, Tuke MA, Wood AR, Bharti SK, Bogenhagen DF, Yakubovskaya E, Garcia-Diaz M, Guilliam TA, Byrd AK, Raney KD, Doherty AJ, Ferrucci L, Schlessinger D, Ding J, Brosh RM.
      As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.
  33. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30139-6. [Epub ahead of print]155 383-400
    Naini A, Gilkerson R, Shanske S, Pang J.
      The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569bp double stranded DNA that encodes 37 genes, 24 of which (2 rRNAs and 22 tRNAs) are necessary for transcription and translation of 13 polypeptides that are all subunits of respiratory chain. Pathogenic mutations in mtDNA cause respiratory chain dysfunction, and are the underlying defect in an ever-increasing number of mtDNA-related encephalomyopathies with distinct phenotypes. In this chapter, we present an overview of mtDNA mutations and describe the molecular techniques currently employed in our laboratory to detect two types of mtDNA mutations: single-large-scale rearrangements and point mutations.
    Keywords:  Mitochondria; Mutations; Next generation sequencing; Real-time PCR; mtDNA
  34. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30136-0. [Epub ahead of print]155 45-79
    Priesnitz C, Pfanner N, Becker T.
      Mitochondria are deeply integrated into crucial functions of eukaryotic cells, including ATP production via oxidative phosphorylation, biosynthesis of iron-sulfur clusters, amino acids, lipids and heme, signaling pathways, and programmed cell death. The import of about 1000 different proteins that are produced as precursors on cytosolic ribosomes is essential for mitochondrial functions and biogenesis. The translocase of the outer mitochondrial membrane (TOM) forms the entry gate for the vast majority of mitochondrial proteins. Research of the last years has uncovered a complicated network of protein translocases and pathways that sort proteins into the mitochondrial subcompartments: outer and inner membranes, intermembrane space, and matrix. The in vitro import of a large number of different precursor proteins into mitochondria has been a pivotal experimental assay to identify these protein-sorting routes. This experimental set-up enables studies on the kinetics of protein transport into isolated mitochondria, on the processing of precursor proteins, and on their assembly into functional protein machineries. In vitro protein import assays are widely used and are indispensable for research on mitochondrial protein biogenesis.
    Keywords:  Blue native electrophoresis; Mitochondria; Protein assembly; Protein import; Protein sorting; TIM23 complex; TOM complex
  35. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30154-2. [Epub ahead of print]155 247-270
    Franco-Iborra S, Tanji K.
      We describe here reliable histochemical and immunohistochemical techniques to visualize mitochondria and respiratory chain dysfunction in tissue sections. These morphological methods have been widely used for years, and yet remain relevant to obtain insight into the pathogenesis of mitochondrial diseases. Today, mitochondrial medicine is changing rapidly and genetic information plays an increasing role in the diagnostic process, owing to advances in next-generation sequencing. However, tissue analysis and morphological categorization remain essential, especially when genetic abnormalities of unknown significance might complicate a diagnostic odyssey. Furthermore, tissue assessment is an essential step in laboratory investigation using animal or cell models, in order to assess the distribution, severity, and/or progression of the disease, and to evaluate the effects of possible treatments. Optimized and reproducible staining and imaging methodology are the key elements for accurate tissue assessment. When these methods are used properly and integrated with wisely chosen genetic and biochemical approaches, powerful information can be obtained about the structure and function of mitochondria in both animal model systems and human patients. While the described protocols refer to skeletal muscle and brain mitochondria, the methods described can be applied to any tissue type.
    Keywords:  Cytochrome c oxidase; Histochemistry; Immunofluorescence; Immunohistochemistry; Muscle; Succinate dehydrogenase
  36. PLoS Genet. 2020 Mar 19. 16(3): e1008638
    Haeussler S, Köhler F, Witting M, Premm MF, Rolland SG, Fischer C, Chauve L, Casanueva O, Conradt B.
      Compromising mitochondrial fusion or fission disrupts cellular homeostasis; however, the underlying mechanism(s) are not fully understood. The loss of C. elegans fzo-1MFN results in mitochondrial fragmentation, decreased mitochondrial membrane potential and the induction of the mitochondrial unfolded protein response (UPRmt). We performed a genome-wide RNAi screen for genes that when knocked-down suppress fzo-1MFN(lf)-induced UPRmt. Of the 299 genes identified, 143 encode negative regulators of autophagy, many of which have previously not been implicated in this cellular quality control mechanism. We present evidence that increased autophagic flux suppresses fzo-1MFN(lf)-induced UPRmt by increasing mitochondrial membrane potential rather than restoring mitochondrial morphology. Furthermore, we demonstrate that increased autophagic flux also suppresses UPRmt induction in response to a block in mitochondrial fission, but not in response to the loss of spg-7, which encodes a mitochondrial metalloprotease. Finally, we found that blocking mitochondrial fusion or fission leads to increased levels of certain types of triacylglycerols and that this is at least partially reverted by the induction of autophagy. We propose that the breakdown of these triacylglycerols through autophagy leads to elevated metabolic activity, thereby increasing mitochondrial membrane potential and restoring mitochondrial and cellular homeostasis.
  37. Mol Cell. 2020 Mar 09. pii: S1097-2765(20)30111-8. [Epub ahead of print]
    Hao Z, Wu T, Cui X, Zhu P, Tan C, Dou X, Hsu KW, Lin YT, Peng PH, Zhang LS, Gao Y, Hu L, Sun HL, Zhu A, Liu J, Wu KJ, He C.
      N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.
    Keywords:  METTL4; N(6)-methyldeoxyadenosine (6mA); TFAM; methyltransferase; mitochondrial DNA methylation; mitochondrial replication; mitochondrial transcription regulation
  38. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30151-7. [Epub ahead of print]155 83-120
    Bennett MJ, Sheng F, Saada A.
      This chapter focuses on the methods to measure unique metabolites, specific enzymes, and metabolic flux in fatty acid β-oxidation, and on biochemical assays of tricarboxylic acid (TCA) cycle enzymes and the pyruvate dehydrogenase complex. These assays play an important role in the diagnosis of genetic diseases, newborn screening, and in cancer and metabolism research. The rationale, protocol, pros and cons, and alternative methods are discussed. Nevertheless, each laboratory should adapt the preferred method optimizing sample preparation and assay conditions for linearity and a low signal-to-noise ratio. The reader is also referred to the additional literature citing methods and clinical descriptions of the various diseases.
    Keywords:  Acylcarnitine; Acylglycines; Carnitine; Fatty acid oxidation; Krebs cycle; Mitochondria; Organic acids; Pyruvate dehydrogenase; TCA cycle
  39. Int J Biol Sci. 2020 ;16(7): 1194-1206
    Lin W, Zhou Q, Wang CQ, Zhu L, Bi C, Zhang S, Wang X, Jin H.
      Metabolic reprogramming is a hallmark of cancer. Mammalian genome is characterized by pervasive transcription, generating abundant non-coding RNAs (ncRNAs). Long non-coding RNAs (lncRNAs) are freshly discovered functional ncRNAs exerting extensive regulatory impact through diverse mechanisms. Emerging studies have revealed widespread roles of lncRNAs in the regulation of various cellular activities, including metabolic pathways. In this review, we summarize the latest advances regarding the regulatory roles of lncRNAs in cancer metabolism, particularly their roles in mitochondrial function, glucose, glutamine, and lipid metabolism. Moreover, we discuss the clinical application and challenges of targeting lncRNAs in cancer metabolism. Understanding the complex and special behavior of lncRNAs will allow a better depiction of cancer metabolic networks and permit the development of lncRNA-based clinical therapies by targeting cancer metabolism.
    Keywords:  Cancer; Long non-coding RNA; Metabolism
  40. Shock. 2020 Mar 17.
    Gu J, Zhang T, Guo J, Chen K, Li H, Wang J.
      Hepatic ischemia/reperfusion (I/R) injury is a major concern in liver surgery settings. Mitochondria are critical targets or the origin of tissue injury, particularly I/R injury. Mitophagy, a selective form of autophagy, is a fundamental process that removes damaged or unwanted mitochondria for mitochondrial quality control, but its role in hepatic I/R remains unclear. In the present study, we investigated the role of mitophagy in hepatic I/R by focusing on PTEN-induced putative kinase 1 (PINK1). Livers from 10-week-old mice and primary hepatocytes were subjected to in vivo hepatic I/R and in vitro hypoxia-reoxygenation (H/R), respectively. Analyses of oxidative stress, immunoblotting and ATP generation showed that hepatic I/R leads to mitochondrial damage. Dysfunctional mitochondria promoted reactive oxygen species (ROS) production and apoptosis. Hepatic I/R led to decreases in the mitochondrial proteins COX4 and TOM20 and mitochondrial DNA (mtDNA) and increases in the autophagy related indicators LC3 and P62, which indicates that hepatic I/R promotes mitophagy. We found that I/R also leads to endoplasmic reticulum (ER) stress, which has frequent signal communication with mitochondria through the mitochondria-associated membranes (MAMs). We showed that the mitophagy-related proteins Parkin, Beclin, optineurin (OPTN) were enhanced in hepatic I/R. No significant change in PINK1 but it translocated to MAMs region to initiate mitophagy. The silencing PINK1 by shRNA in cultured primary hepatocytes reduced the level of H/R-induced mitophagy, leading to the accumulation of dysfunctional mitochondria during H/R, increased production of ROS, mitochondria-induced apoptosis, and eventually hepatocyte death. Taken together, these findings indicate that PINK1-mediated mitophagy plays a key role in mitochondrial quality control and liver cell survival during I/R.
  41. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30122-0. [Epub ahead of print]155 401-414
    Tigano M, Phillips AF, Sfeir A.
      DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.
    Keywords:  DNA combing; DNA fibers; Mitochondrial DNA; Thymidine analogs; mtDNA; mtDNA replication; mtDNA replication speed
  42. Cancer Discov. 2020 Mar 18. pii: CD-19-1330. [Epub ahead of print]
    Liu Z, Yoshimi A, Wang J, Cho H, Chun-Wei Lee S, Ki M, Bitner L, Chu T, Shah H, Liu B, Mato AR, Ruvolo P, Fabbri G, Pasqualucci L, Abdel-Wahab O, Rabadan R.
      Although mutations in the RNA splicing factor SF3B1 are frequent in multiple cancers, their functional effects and therapeutic dependencies are poorly understood. Here we characterize 98 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations, identifying hundreds of cryptic 3' splice sites common and specific to different cancer types. Regulatory network analysis revealed that the most common SF3B1 mutation activates MYC via effects conserved across human and mouse cells. SF3B1 mutations promote decay of transcripts encoding the PP2A phosphatase subunit PPP2R5A, increasing c-MYC S62 and BCL2 S70 phosphorylation which, in turn, promote MYC protein stability and impair apoptosis, respectively. Genetic PPP2R5A restoration or pharmacologic PP2A activation impaired SF3B1-mutant tumorigenesis elucidating a therapeutic approach to aberrant splicing by mutant SF3B1.
  43. Proc Natl Acad Sci U S A. 2020 Mar 19. pii: 201915582. [Epub ahead of print]
    Zhu Q, Cheng X, Cheng Y, Chen J, Xu H, Gao Y, Duan X, Ji J, Li X, Yi W.
      Methionine metabolism is critical for the maintenance of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) pluripotency. However, little is known about the regulation of the methionine cycle to sustain ESC pluripotency. Here, we show that adenosylhomocysteinase (AHCY), an important enzyme in the methionine cycle, is critical for the maintenance and differentiation of mouse embryonic stem cells (mESCs). We show that mESCs exhibit high levels of methionine metabolism, whereas decreasing methionine metabolism via depletion of AHCY promotes mESCs to differentiate into the three germ layers. AHCY is posttranslationally modified with an O-linked β-N-acetylglucosamine sugar (O-GlcNAcylation), which is rapidly removed upon differentiation. O-GlcNAcylation of threonine 136 on AHCY increases its activity and is important for the maintenance of trimethylation of histone H3 lysine 4 (H3K4me3) to sustain mESC pluripotency. Blocking glycosylation of AHCY decreases the ratio of S-adenosylmethionine versus S-adenosylhomocysteine (SAM/SAH), reduces the level of H3K4me3, and poises mESC for differentiation. In addition, blocking glycosylation of AHCY reduces somatic cell reprogramming. Thus, our findings reveal a critical role of AHCY and a mechanistic understanding of O-glycosylation in regulating ESC pluripotency and differentiation.
    Keywords:  O-GlcNAcylation; metabolism; stem cell
  44. Proc Natl Acad Sci U S A. 2020 Mar 16. pii: 201910854. [Epub ahead of print]
    King C, Sengupta P, Seo AY, Lippincott-Schwartz J.
      The endoplasmic reticulum (ER) is the site of synthesis of secretory and membrane proteins and contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner. How the ER spatially segregates its numerous and diverse functions, including positioning nanoscopic contact sites with other organelles, is unclear. We demonstrate that hypotonic swelling of cells converts the ER and other membrane-bound organelles into micrometer-scale large intracellular vesicles (LICVs) that retain luminal protein content and maintain contact sites with each other through localized organelle tethers. Upon cooling, ER-derived LICVs phase-partition into microscopic domains having different lipid-ordering characteristics, which is reversible upon warming. Ordered ER lipid domains mark contact sites with ER and mitochondria, lipid droplets, endosomes, or plasma membrane, whereas disordered ER lipid domains mark contact sites with lysosomes or peroxisomes. Tethering proteins concentrate at ER-organelle contact sites, allowing time-dependent behavior of lipids and proteins to be studied at these sites. These findings demonstrate that LICVs provide a useful model system for studying the phase behavior and interactive properties of organelles in intact cells.
    Keywords:  contact sites; endoplasmic reticulum; organelle tethers; phase partitioning
  45. Nature. 2020 Mar;579(7799): 415-420
    Zhang Z, Zhang Y, Xia S, Kong Q, Li S, Liu X, Junqueira C, Meza-Sosa KF, Mok TMY, Ansara J, Sengupta S, Yao Y, Wu H, Lieberman J.
      Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis)1. Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss2-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells3-5. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer2,6, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8+ T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity.
  46. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30157-8. [Epub ahead of print]155 181-197
    Acín-Pérez R, Hernansanz-Agustín P, Enríquez JA.
      This review focuses on three independent and complementary approaches to obtain information on the combined function of respiratory complexes when present in different structural situations, either as individual complexes or when superassembled with other complexes. We review the utility of in-gel activity after blue native electrophoresis, integrated oxygen consumption of supercomplexes containing complex IV, and spectrophotometric activity measurements.
    Keywords:  Blue native gel electrophoresis; Electron transport chain; Mitochondria respiration; Oxidative phosphorylation; Supercomplexes
  47. Theranostics. 2020 ;10(6): 2696-2713
    Fabbri L, Dufies M, Lacas-Gervais S, Gardie B, Gad-Lapiteau S, Parola J, Nottet N, Meyenberg Cunha de Padua M, Contenti J, Borchiellini D, Ferrero JM, Leclercq NR, Ambrosetti D, Mograbi B, Richard S, Viotti J, Chamorey E, Sadaghianloo N, Rouleau M, Craigen WJ, Mari B, Clavel S, Pagès G, Pouysségur J, Bost F, Mazure NM.
      Rationale: Renal cell carcinoma (RCC) accounts for about 2% of all adult cancers, and clear cell RCC (ccRCC) is the most common RCC histologic subtype. A hallmark of ccRCC is the loss of the primary cilium, a cellular antenna that senses a wide variety of signals. Loss of this key organelle in ccRCC is associated with the loss of the von Hippel-Lindau protein (VHL). However, not all mechanisms of ciliopathy have been clearly elucidated. Methods: By using RCC4 renal cancer cells and patient samples, we examined the regulation of ciliogenesis via the presence or absence of the hypoxic form of the voltage-dependent anion channel (VDAC1-ΔC) and its impact on tumor aggressiveness. Three independent cohorts were analyzed. Cohort A was from PREDIR and included 12 patients with hereditary pVHL mutations and 22 sporadic patients presenting tumors with wild-type pVHL or mutated pVHL; Cohort B included tissue samples from 43 patients with non-metastatic ccRCC who had undergone surgery; and Cohort C was composed of 375 non-metastatic ccRCC tumor samples from The Cancer Genome Atlas (TCGA) and was used for validation. The presence of VDAC1-ΔC and legumain was determined by immunoblot. Transcriptional regulation of IFT20/GLI1 expression was evaluated by qPCR. Ciliogenesis was detected using both mouse anti-acetylated α-tubulin and rabbit polyclonal ARL13B antibodies for immunofluorescence. Results: Our study defines, for the first time, a group of ccRCC patients in which the hypoxia-cleaved form of VDAC1 (VDAC1-ΔC) induces resorption of the primary cilium in a Hypoxia-Inducible Factor-1 (HIF-1)-dependent manner. An additional novel group, in which the primary cilium is re-expressed or maintained, lacked VDAC1-ΔC yet maintained glycolysis, a signature of epithelial-mesenchymal transition (EMT) and more aggressive tumor progression, but was independent to VHL. Moreover, these patients were less sensitive to sunitinib, the first-line treatment for ccRCC, but were potentially suitable for immunotherapy, as indicated by the immunophenoscore and the presence of PDL1 expression. Conclusion: This study provides a new way to classify ccRCC patients and proposes potential therapeutic targets linked to metabolism and immunotherapy.
    Keywords:  Ciliopathy; HIFs; VDAC1.; Voltage-Dependent Anion Channel 1; clear cell Renal Cell Carcinoma; immunotherapy; poor prognosis; primary cilium
  48. J Intern Med. 2020 Mar 18.
    Wei W, Chinnery PF.
      The first draft human mitochondrial DNA (mtDNA) sequence was published in 1981, paving the way for two decades of discovery linking mtDNA variation with human disease. Severe pathogenic mutations cause sporadic and inherited rare disorders that often involve the nervous system. However, some mutations cause mild organ-specific phenotypes that have a reduced clinical penetrance, and polymorphic variation of mtDNA is associated with an altered risk of developing several late-onset common human diseases including Parkinson's disease. mtDNA mutations also accumulate during human life and are enriched in affected organs in a number of age-related diseases. Thus, mtDNA contributes to a wide range of human pathologies. For many decades, it has generally been accepted that mtDNA is inherited exclusively down the maternal line in humans. Although recent evidence has challenged this dogma, whole-genome sequencing has identified nuclear-encoded mitochondrial sequences (NUMTs) that can give the false impression of paternally inherited mtDNA. This provides a more likely explanation for recent reports of 'bi-parental inheritance', where the paternal alleles are actually transmitted through the nuclear genome. The presence of both mutated and wild-type variant alleles within the same individual (heteroplasmy) and rapid shifts in allele frequency can lead to offspring with variable severity of disease. In addition, there is emerging evidence that selection can act for and against specific mtDNA variants within the developing germ line, and possibly within developing tissues. Thus, understanding how mtDNA is inherited has far-reaching implications across medicine. There is emerging evidence that this highly dynamic system is amenable to therapeutic manipulation, raising the possibility that we can harness new understanding to prevent and treat rare and common human diseases where mtDNA mutations play a key role.
    Keywords:  human mitochondrial DNA; mitochondrial DNA mutation; mitochondrial bottleneck; mitochondrial disorders; mitochondrial inheritance
  49. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30120-7. [Epub ahead of print]155 221-245
    Esteras N, Adjobo-Hermans MJW, Abramov AY, Koopman WJH.
      Assessment of the mitochondrial membrane potential (Δψ) in living cells, although not trivial, can be used to estimate mitochondrial bioenergetic state. For this purpose, fluorescent lipophilic cations are broadly applied. These cations enter cells and accumulate primarily in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of the cations tetramethylrhodamine methyl ester (TMRM) and rhodamine 123 (Rhod123) for semi-quantitative Δψ analysis in living mammalian cells. Two different strategies are presented: (1) steady-state measurements that are suited to compare Δψ between different conditions (i.e., for comparing disease states or treatments) and (2) dynamic measurements allowing temporal monitoring of Δψ changes (i.e., to assess the effect of acute perturbations). We discuss the rationale for the use of TMRM and Rhod123 in their non-quenching/redistribution and quenching mode, how these modes are associated with different fluorescence responses, and how data can be interpreted. Practically, three experimental protocols are provided describing the use of TMRM and/or Rhod123 to assess Δψ in primary human skin fibroblasts (PHSFs) and neuron/astrocyte co-cultures by live-cell fluorescence microscopy.
    Keywords:  Fluorescence; Microscopy; Quenching; Rhodamine 123; TMRM
  50. J Proteome Res. 2020 Mar 16.
    Guo X, Park JE, Gallart-Palau X, Sze SK.
      Aging can have profound effects on the mammalian brain leading to neurodegeneration and cognitive impairment. The brain has exceptionally high-energy requirements and is particularly susceptible to damage within its bioenergetic pathways. Here we asked how the bioenergetic proteome of the murine brain changed with age and how this might affect brain function. Using label free LC-MS/MS proteomics for the discovery phase and quantitative multiple reaction monitoring LC-MRM-MS/MS for the validation phase we found dysregulated expression of multiple components of the tricarboxylic acid cycle, which is key for mitochondrial energy production, including SULA2, IDH1, IDH2, SDHB, PDHB, MDH1, FH1 and NDUFS3, in old murine brains. We also saw that the oxidoreductases, thioredoxin (Trx) and glutaredoxin (Grx) were significantly downregulated in the old mouse brain, and showed through MS that this correlated with the accumulation of trioxidation in the key metabolic enzyme MDH1 at Cys137. 3D modeling of MDH1 predicted that the damaged sites were located at the protein active zone, and enzymatic kinetic analysis confirmed that MDH1 function was significantly reduced in the old mouse brain. These findings identify the tricarboxylic acid cycle as a key target of degenerative protein modifications with deleterious effects on the aging brain's bioenergetic function.
  51. Proc Natl Acad Sci U S A. 2020 Mar 18. pii: 201921694. [Epub ahead of print]
    Nozaki Y, Petersen MC, Zhang D, Vatner DF, Perry RJ, Abulizi A, Haedersdal S, Zhang XM, Butrico GM, Samuel VT, Mason GF, Cline GW, Petersen KF, Rothman DL, Shulman GI.
      Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.
    Keywords:  glucokinase; hepatic glycogen synthesis; hepatic insulin resistance; in vivo; metabolic control analysis
  52. Trends Endocrinol Metab. 2020 Apr;pii: S1043-2760(20)30032-1. [Epub ahead of print]31(4): 269-271
    Martín-Rodríguez S, de Pablos-Velasco P, Calbet JAL.
      Metformin has antidiabetic, anticancer, and prolongevity effects, but seems to interfere with aerobic training mitochondrial adaptations. The primary mechanism of action has been suggested to be the inhibition of mitochondrial complex I. Recent papers (Wang et al. and Cameron et al.), however, provide evidence to deny the hypothesis of a direct action of metformin on complex I.
    Keywords:  exercise; metformin; mitochondrial complex I
  53. Sci Immunol. 2020 Mar 20. pii: eaaz2738. [Epub ahead of print]5(45):
    Nicolai CJ, Wolf N, Chang IC, Kirn G, Marcus A, Ndubaku CO, McWhirter SM, Raulet DH.
      Several immunotherapy approaches that mobilize CD8+ T cell responses stimulate tumor rejection, and some, such as checkpoint blockade, have been approved for several cancer indications and show impressive increases in patient survival. However, tumors may evade CD8+ T cell recognition via loss of MHC molecules or because they contain few or no neoantigens. Therefore, approaches are needed to combat CD8+ T cell-resistant cancers. STING-activating cyclic dinucleotides (CDNs) are a new class of immune-stimulating agents that elicit impressive CD8+ T cell-mediated tumor rejection in preclinical tumor models and are now being tested in clinical trials. Here, we demonstrate powerful CDN-induced, natural killer (NK) cell-mediated tumor rejection in numerous tumor models, independent of CD8+ T cells. CDNs enhanced NK cell activation, cytotoxicity, and antitumor effects in part by inducing type I interferon (IFN). IFN acted in part directly on NK cells in vivo and in part indirectly via the induction of IL-15 and IL-15 receptors, which were important for CDN-induced NK activation and tumor control. After in vivo administration of CDNs, dendritic cells (DCs) up-regulated IL-15Rα in an IFN-dependent manner. Mice lacking the type I IFN receptor specifically on DCs had reduced NK cell activation and tumor control. Therapeutics that activate NK cells, such as CDNs, checkpoint inhibitors, NK cell engagers, and cytokines, may represent next-generation approaches to cancer immunotherapy.
  54. Nat Commun. 2020 Mar 20. 11(1): 1507
    Liu J, Liu ZX, Wu QN, Lu YX, Wong CW, Miao L, Wang Y, Wang Z, Jin Y, He MM, Ren C, Wang DS, Chen DL, Pu HY, Feng L, Li B, Xie D, Zeng MS, Huang P, Lin A, Lin D, Xu RH, Ju HQ.
      Tumor cells often reprogram their metabolism for rapid proliferation. The roles of long noncoding RNAs (lncRNAs) in metabolism remodeling and the underlying mechanisms remain elusive. Through screening, we found that the lncRNA Actin Gamma 1 Pseudogene (AGPG) is required for increased glycolysis activity and cell proliferation in esophageal squamous cell carcinoma (ESCC). Mechanistically, AGPG binds to and stabilizes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). By preventing APC/C-mediated ubiquitination, AGPG protects PFKFB3 from proteasomal degradation, leading to the accumulation of PFKFB3 in cancer cells, which subsequently activates glycolytic flux and promotes cell cycle progression. AGPG is also a transcriptional target of p53; loss or mutation of TP53 triggers the marked upregulation of AGPG. Notably, inhibiting AGPG dramatically impaired tumor growth in patient-derived xenograft (PDX) models. Clinically, AGPG is highly expressed in many cancers, and high AGPG expression levels are correlated with poor prognosis, suggesting that AGPG is a potential biomarker and cancer therapeutic target.