bims-camemi Biomed news
on Mitochondrial metabolism in cancer
Issue of 2019‒03‒10
sixty-three papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit

  1. Nature. 2019 Mar 06.
    Lee J, Yesilkanal AE, Wynne JP, Frankenberger C, Liu J, Yan J, Elbaz M, Rabe DC, Rustandy FD, Tiwari P, Grossman EA, Hart PC, Kang C, Sanderson SM, Andrade J, Nomura DK, Bonini MG, Locasale JW, Rosner MR.
      Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.
  2. Metabolomics. 2019 Feb 28. 15(3): 32
    Hertig D, Felser A, Diserens G, Kurth S, Vermathen P, Nuoffer JM.
      INTRODUCTION: A decline in mitochondrial function represents a key factor of a large number of inborn errors of metabolism, which lead to an extremely heterogeneous group of disorders.OBJECTIVES: To gain insight into the biochemical consequences of mitochondrial dysfunction, we performed a metabolic profiling study in human skin fibroblasts using galactose stress medium, which forces cells to rely on mitochondrial metabolism.
    METHODS: Fibroblasts from controls, complex I and pyruvate dehydrogenase (PDH) deficient patients were grown under glucose or galactose culture condition. We investigated extracellular flux using Seahorse XF24 cell analyzer and assessed metabolome fingerprints using NMR spectroscopy.
    RESULTS: Incubation of fibroblasts in galactose leads to an increase in oxygen consumption and decrease in extracellular acidification rate, confirming adaptation to a more aerobic metabolism. NMR allowed rapid profiling of 41 intracellular metabolites and revealed clear separation of mitochondrial defects from controls under galactose using partial least squares discriminant analysis. We found changes in classical markers of mitochondrial metabolic dysfunction, as well as unexpected markers of amino acid and choline metabolism. PDH deficient cell lines showed distinct upregulation of glutaminolytic metabolism and accumulation of branched-chain amino acids, while complex I deficient cell lines were characterized by increased levels in choline metabolites under galactose.
    CONCLUSION: Our results show the relevance of selective culture methods in discriminating normal from metabolic deficient cells. The study indicates that untargeted fingerprinting NMR profiles provide physiological insight on metabolic adaptations and can be used to distinguish cellular metabolic adaptations in PDH and complex I deficient fibroblasts.
    Keywords:  Complex I; Galactose; Mitochondrial dysfunction; NMR; Pyruvate dehydrogenase
  3. Cell Mol Life Sci. 2019 Mar 06.
    Byrne JJ, Soh MS, Chandhok G, Vijayaraghavan T, Teoh JS, Crawford S, Cobham AE, Yapa NMB, Mirth CK, Neumann B.
      Mitochondria are essential components of eukaryotic cells, carrying out critical physiological processes that include energy production and calcium buffering. Consequently, mitochondrial dysfunction is associated with a range of human diseases. Fundamental to their function is the ability to transition through fission and fusion states, which is regulated by several GTPases. Here, we have developed new methods for the non-subjective quantification of mitochondrial morphology in muscle and neuronal cells of Caenorhabditis elegans. Using these techniques, we uncover surprising tissue-specific differences in mitochondrial morphology when fusion or fission proteins are absent. From ultrastructural analysis, we reveal a novel role for the fusion protein FZO-1/mitofusin 2 in regulating the structure of the inner mitochondrial membrane. Moreover, we have determined the influence of the individual mitochondrial fission (DRP-1/DRP1) and fusion (FZO-1/mitofusin 1,2; EAT-3/OPA1) proteins on animal behaviour and lifespan. We show that loss of these mitochondrial fusion or fission regulators induced age-dependent and progressive deficits in animal movement, as well as in muscle and neuronal function. Our results reveal that disruption of fusion induces more profound defects than lack of fission on animal behaviour and tissue function, and imply that while fusion is required throughout life, fission is more important later in life likely to combat ageing-associated stressors. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology, with no correlation found between morphological changes and behavioural defects. Surprisingly, we find that disruption of either mitochondrial fission or fusion significantly reduces median lifespan, but maximal lifespan is unchanged, demonstrating that mitochondrial dynamics play an important role in limiting variance in longevity across isogenic populations. Overall, our study provides important new insights into the central role of mitochondrial dynamics in maintaining organismal health.
    Keywords:  Caenorhabditis elegans; DRP-1; DRP1; EAT-3; FZO-1; Mitochondria; Mitochondrial dynamics; Mitofusin 1; Mitofusin 2; OPA1; Transmission electron microscopy
  4. Redox Biol. 2019 Feb 23. pii: S2213-2317(19)30109-0. [Epub ahead of print]22 101152
    Nanadikar MS, Vergel Leon AM, Borowik S, Hillemann A, Zieseniss A, Belousov VV, Bogeski I, Rehling P, Dudek J, Katschinski DM.
      Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (EGSH) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct EGSH of the mitochondrial matrix (-278.9 ± 0.4 mV) in isolated cardiomyocytes is observed. When analyzing the EGSH in isolated mitochondria from the transgenic hearts, however, the EGSH in the mitochondrial matrix is significantly oxidized (-247.7 ± 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% O2) conditions that mimics mitochondrial pO2 levels in cellulo. The reduced EGSH is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.
    Keywords:  Glutathione redox potential; Grx1-roGFP; Hypoxia; Mitochondrial matrix
  5. Nature. 2019 Mar 06.
    Shi Y, Lim SK, Liang Q, Iyer SV, Wang HY, Wang Z, Xie X, Sun D, Chen YJ, Tabar V, Gutin P, Williams N, De Brabander JK, Parada LF.
      Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.
  6. Dis Model Mech. 2019 Mar 04. pii: dmm.037226. [Epub ahead of print]
    Martorano L, Peron M, Laquatra C, Lidron E, Facchinello N, Meneghetti G, Tiso N, Rasola A, Ghezzi D, Argenton F.
      Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance, and characterized by both a strong reduction of mtDNA content and severe mitochondrial defects in affected tissues. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been associated with hepatocerebral forms of MDS. Zebrafish mpv17 null mutant lacks the guanine-based reflective skin cells named iridophores and represents a promising model to clarify the role of Mpv17. In our work, we have characterized the mitochondrial phenotype of mpv17 -/- larvae and found early and severe ultrastructural alterations in liver mitochondria as well as a significant impairment of the respiratory chain leading to activation of the mitochondrial quality control. Our results provide evidences for zebrafish Mpv17 being essential for maintaining mitochondrial structure and functionality while its effect on mtDNA copy number seems to be subordinate. Considering that a role in nucleotides availability had already been postulated for MPV17, that embryos blocked in pyrimidine synthesis do phenocopy mpv17 -/- KO and that mpv17 -/- KO have an impaired Dihydroorotate dehydrogenase activity, we provided mpv17 mutants with the pyrimidine precursor orotic acid (OA). The treatment with OA, an easily available food supplement, significantly increased both iridophores number and mtDNA content of mpv17 -/- mutants, thus linking the loss of Mpv17 to pyrimidine de novo synthesis and opening a new simple therapeutic approach for MPV17-related MDS.
    Keywords:  Dehydroorotate dehydrogenase; Iridophores; Mitochondrial DNA depletion syndrome; Pyrimidine; Vitamin b13
  7. Biochim Biophys Acta Mol Basis Dis. 2019 Apr 01. pii: S0925-4439(18)30318-1. [Epub ahead of print]1865(4): 810-821
    Dudek J, Hartmann M, Rehling P.
      Mitochondria play an essential role in the energy metabolism of the heart. Many of the essential functions are associated with mitochondrial membranes and oxidative phosphorylation driven by the respiratory chain. Mitochondrial membranes are unique in the cell as they contain the phospholipid cardiolipin. The important role of cardiolipin in cardiovascular health is highlighted by several cardiac diseases, in which cardiolipin plays a fundamental role. Barth syndrome, Sengers syndrome, and Dilated cardiomyopathy with ataxia (DCMA) are genetic disorders, which affect cardiolipin biosynthesis. Other cardiovascular diseases including ischemia/reperfusion injury and heart failure are also associated with changes in the cardiolipin pool. Here, we summarize molecular functions of cardiolipin in mitochondrial biogenesis and morphology. We highlight the role of cardiolipin for the respiratory chain, metabolite carriers, and mitochondrial metabolism and describe links to apoptosis and mitochondria specific autophagy (mitophagy) with possible implications in cardiac disease.
    Keywords:  Barth syndrome; Cardiolipin; Mitochondria; Respiratory chain; Sengers syndrome
  8. Cancer Discov. 2019 Mar 05. pii: CD-18-1212. [Epub ahead of print]
    Auciello FR, Bulusu V, Oon C, Tait-Mulder J, Berry M, Bhattacharyya S, Tumanov S, Allen-Petersen BL, Link J, Kendsersky ND, Vringer E, Schug M, Novo D, Hwang RF, Evans RM, Nixon C, Dorrell C, Morton JP, Norman JC, Sears RC, Kamphorst JJ, Sherman MH.
      Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features trans-differentiation of tissue-resident pancreatic stellate cells (PSCs) into activated cancer-associated fibroblasts (CAFs), a process induced by PDAC cells but of unclear significance for PDAC progression. Here we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound healing mediators to stimulate their own growth and migration.
  9. Cell Metab. 2019 Feb 16. pii: S1550-4131(19)30064-6. [Epub ahead of print]
    Thaker SK, Chapa T, Garcia G, Gong D, Schmid EW, Arumugaswami V, Sun R, Christofk HR.
      Zika virus is a pathogen that poses serious consequences, including congenital microcephaly. Although many viruses reprogram host cell metabolism, whether Zika virus alters cellular metabolism and the functional consequences of Zika-induced metabolic changes remain unknown. Here, we show that Zika virus infection differentially reprograms glucose metabolism in human versus C6/36 mosquito cells by increasing glucose use in the tricarboxylic acid cycle in human cells versus increasing glucose use in the pentose phosphate pathway in mosquito cells. Infection of human cells selectively depletes nucleotide triphosphate levels, leading to elevated AMP/ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and caspase-mediated cell death. AMPK is also phosphorylated in Zika virus-infected mouse brain. Inhibiting AMPK in human cells decreases Zika virus-mediated cell death, whereas activating AMPK in mosquito cells promotes Zika virus-mediated cell death. These findings suggest that the differential metabolic reprogramming during Zika virus infection of human versus mosquito cells determines whether cell death occurs.
    Keywords:  AMPK; Zika virus; apoptosis; virus metabolism
  10. Nat Med. 2019 Mar 04.
    Bryant KL, Stalnecker CA, Zeitouni D, Klomp JE, Peng S, Tikunov AP, Gunda V, Pierobon M, Waters AM, George SD, Tomar G, Papke B, Hobbs GA, Yan L, Hayes TK, Diehl JN, Goode GD, Chaika NV, Wang Y, Zhang GF, Witkiewicz AK, Knudsen ES, Petricoin EF, Singh PK, Macdonald JM, Tran NL, Lyssiotis CA, Ying H, Kimmelman AC, Cox AD, Der CJ.
      Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth, but the role of KRAS in supporting autophagy has not been established. We show that, to our surprise, suppression of KRAS increased autophagic flux, as did pharmacological inhibition of its effector ERK MAPK. Furthermore, we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. We speculated that ERK inhibition might thus enhance PDAC dependence on autophagy, in part by impairing other KRAS- or ERK-driven metabolic processes. Accordingly, we found that the autophagy inhibitor chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven PDAC. We conclude that combinations of pharmacologic inhibitors that concurrently block both ERK MAPK and autophagic processes that are upregulated in response to ERK inhibition may be effective treatments for PDAC.
  11. Sci Rep. 2019 Mar 08. 9(1): 3941
    Castellano P, Prevedel L, Valdebenito S, Eugenin EA.
      Currently, a major barrier to curing HIV infection is the generation of tissue-associated, non-replicating, long-lasting viral reservoirs that are refractory to therapy and can be reactivated upon anti-retroviral therapy interruption. One of these reservoirs are latently HIV-infected macrophages. Here, we show that HIV infection of macrophages results in survival of a small population of infected cells that are metabolically altered and characterized by mitochondrial fusion, lipid accumulation, and reduced mitochondrial ATP production. No changes in glycolysis were detected. Metabolic analysis indicated an essential role of succinate and other TCA metabolites in the tricarboxylic acid (TCA) cycle in mediating lipid accumulation and oxidative phosphorylation (OXPHOS) in the mitochondria. Furthermore, we show that while uninfected and HIV infected macrophages use fatty acids and glucose as primary sources of energy, surviving HIV infected macrophages also use glutamine/glutamate as a major energy source, and blocking these new sources of energy resulted in the killing of latent HIV infected macrophages. Together, our data provide a new understanding of the formation, properties, and potential novel ways to eliminate macrophage viral reservoirs.
  12. Nature. 2019 Mar 06.
    Li L, Mao Y, Zhao L, Li L, Wu J, Zhao M, Du W, Yu L, Jiang P.
      Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.
  13. Mol Metab. 2019 Feb 06. pii: S2212-8778(18)31112-8. [Epub ahead of print]
    Orang AV, Petersen J, McKinnon RA, Michael MZ.
      BACKGROUND: Cancer cells possess a common metabolic phenotype, rewiring their metabolic pathways from mitochondrial oxidative phosphorylation to aerobic glycolysis and anabolic circuits, to support the energetic and biosynthetic requirements of continuous proliferation and migration. While, over the past decade, molecular and cellular studies have clearly highlighted the association of oncogenes and tumor suppressors with cancer-associated glycolysis, more recent attention has focused on the role of microRNAs (miRNAs) in mediating this metabolic shift. Accumulating studies have connected aberrant expression of miRNAs with direct and indirect regulation of aerobic glycolysis and associated pathways.SCOPE OF REVIEW: This review discusses the underlying mechanisms of metabolic reprogramming in cancer cells and provides arguments that the earlier paradigm of cancer glycolysis needs to be updated to a broader concept, which involves interconnecting biological pathways that include miRNA-mediated regulation of metabolism. For these reasons and in light of recent knowledge, we illustrate the relationships between metabolic pathways in cancer cells. We further summarize our current understanding of the interplay between miRNAs and these metabolic pathways. This review aims to highlight important metabolism-associated molecular components in the hunt for selective preventive and therapeutic treatments.
    MAJOR CONCLUSIONS: Metabolism in cancer cells is influenced by driver mutations but is also regulated by posttranscriptional gene silencing. Understanding the nuanced regulation of gene expression in these cells and distinguishing rapid cellular responses from chronic adaptive mechanisms provides a basis for rational drug design and novel therapeutic strategies.
    Keywords:  Aerobic glycolysis; Cancer; Metabolism; Warburg effect; microRNA
  14. Sci Rep. 2019 Mar 04. 9(1): 3402
    Najac C, Radoul M, Le Page LM, Batsios G, Subramani E, Viswanath P, Gillespie AM, Ronen SM.
      Dysregulation in NAD+/NADH levels is associated with increased cell division and elevated levels of reactive oxygen species in rapidly proliferating cancer cells. Conversion of the ketone body acetoacetate (AcAc) to β-hydroxybutyrate (β-HB) by the mitochondrial enzyme β-hydroxybutyrate dehydrogenase (BDH) depends upon NADH availability. The β-HB-to-AcAc ratio is therefore expected to reflect mitochondrial redox. Previous studies reported the potential of hyperpolarized 13C-AcAc to monitor mitochondrial redox in cells, perfused organs and in vivo. However, the ability of hyperpolarized 13C-AcAc to cross the blood brain barrier (BBB) and its potential to monitor brain metabolism remained unknown. Our goal was to assess the value of hyperpolarized [1,3-13C2]AcAc in healthy and tumor-bearing mice in vivo. Following hyperpolarized [1,3-13C2]AcAc injection, production of [1,3-13C2]β-HB was detected in normal and tumor-bearing mice. Significantly higher levels of [1-13C]AcAc and lower [1-13C]β-HB-to-[1-13C]AcAc ratios were observed in tumor-bearing mice. These results were consistent with decreased BDH activity in tumors and associated with increased total cellular NAD+/NADH. Our study confirmed that AcAc crosses the BBB and can be used for monitoring metabolism in the brain. It highlights the potential of AcAc for future clinical translation and its potential utility for monitoring metabolic changes associated with glioma, and other neurological disorders.
  15. JCI Insight. 2019 Mar 07. pii: 123130. [Epub ahead of print]4(5):
    Osataphan S, Macchi C, Singhal G, Chimene-Weiss J, Sales V, Kozuka C, Dreyfuss JM, Pan H, Tangcharoenpaisan Y, Morningstar J, Gerszten R, Patti ME.
      Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia. Given that SGLT2 inhibitors (SGLT2i) reduce mortality and cardiovascular risk in type 2 diabetes, improved understanding of molecular mechanisms mediating these metabolic effects is required. Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. Utilizing an integrated transcriptomic-metabolomics approach, we demonstrate that CANA modulates key nutrient-sensing pathways, with activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR), independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Given that these phenotypes mirror the effects of FGF21 to promote lipid oxidation, ketogenesis, and reduction in adiposity, we hypothesized that FGF21 is required for CANA action. Using FGF21-null mice, we demonstrate that FGF21 is not required for SGLT2i-mediated induction of lipid oxidation and ketogenesis but is required for reduction in fat mass and activation of lipolysis. Taken together, these data demonstrate that SGLT2 inhibition triggers a fasting-like transcriptional and metabolic paradigm but requires FGF21 for reduction in adiposity.
    Keywords:  Fatty acid oxidation; Hepatology; Intermediary metabolism; Metabolism; Obesity
  16. Int J Cancer. 2019 Mar 08.
    Luo X, Liao C, Quan J, Cheng C, Zhao X, Bode AM, Cao Y.
      Deregulation of cellular metabolism is well established in cancer. The mitochondria are dynamic organelles and act as the center stage for energy metabolism. Central to mitochondrial regulatory network is peroxisome proliferator- activated receptorγcoactivator 1a (PGC-1α), which serves as a master regulator of mitochondrial proliferation and metabolism. The activity and stability of PGC-1α are subject to dynamic and versatile posttranslational modifications including phosphorylation, ubiquitination, methylation and acetylation in response to metabolic stress and other environmental signals. In this review, we describe the structure of PGC-1α. Then, we discuss recent advances in the posttranslational regulatory machinery of PGC-1α, which affects its transcriptional activity, stability and organelle localization. Furthermore, we address the important roles of PGC-1α in tumorigenesis and malignancy. Finally, we also mention the clinical therapeutic potentials of PGC-1α modulators. A better understanding of the elegant function of PGC-1α in cancer progression could provide novel insights into therapeutic interventions through the targeting of PGC-1α signaling. This article is protected by copyright. All rights reserved.
    Keywords:  PGC-1α; cancer metabolism; post-translational modifications
  17. J Pharmacol Exp Ther. 2019 Mar 07. pii: jpet.118.254300. [Epub ahead of print]
    Mohsin AA, Chen Q, Quan N, Rousselle T, Maceyka MW, Samidurai A, Thompson J, Hu Y, Li J, Lesnefsky EJ.
      Transient, reversible blockade of complex I during early reperfusion after ischemia limits cardiac injury. We studied the cardioprotection of high dose of metformin in cultured cells and mouse hearts via the novel mechanism of acute downregulation of complex I. The effect of high dose of metformin on complex I activity was studied in isolated heart mitochondria and cultured H9c2 cells. Protection with metformin was evaluated in H9c2 cells at reoxygenation and at early reperfusion in isolated perfused mouse hearts and in vivo regional ischemia-reperfusion. Acute, high dose metformin treatment inhibited complex I in ischemia-damaged mitochondria and in H9c2 cells following hypoxia. Accompanying the complex I modulation, high dose metformin at reoxygenation decreased death in H9c2 cells. Acute treatment with high dose metformin at the end of ischemia reduced infarct size following ischemia-reperfusion in vitro and in vivo, including in the AMP kinase dead mouse. Metformin treatment during early reperfusion improved mitochondrial calcium retention capacity, indicating decreased permeability transition pore (MPTP) opening. Acute, high dose metformin therapy decreased cardiac injury through inhibition of complex I accompanied by attenuation of MPTP opening. Moreover, in contrast to chronic metformin treatment, protection by acute, high dose metformin is independent of AMPK activation. Thus, a single, high dose metformin treatment at reperfusion reduces cardiac injury via modulation of complex I.
    Keywords:  cardiac ischemia; cell death; cell injury; drug development; free radicals; ischemia / reperfusion injury; mitochondria; pharmacodynamics; reactive oxygen species (ROS)
  18. Oncotarget. 2019 Feb 05. 10(11): 1217-1223
    Barnoud T, Parris JLD, Murphy ME.
      Mutations in the TP53 tumor suppressor gene remain a hallmark of human cancer. In addition to mutation of TP53, single nucleotide polymorphisms (SNPs) in this gene can have a profound impact on p53 function, and can affect cancer risk as well as other p53 functions. Wild type (WT) p53 contains a proline at amino acid 47, but approximately 1% of African-Americans express a p53 allele with a serine at amino acid 47 (Pro47Ser, hereafter S47). In a mouse model for this variant, mice expressing S47 are predisposed to spontaneous cancers. The S47 variant also is associated with increased pre-menopausal breast cancer risk in African American women. We recently reported that S47 tumor cells are resistant to the majority of cytotoxic chemotherapeutic agents, but show increased sensitivity to a subset of anti-cancer agents, compared to tumors with WT p53. In this work, we report on another potentially promising therapeutic vulnerability of S47 tumors. We find that S47 tumors show decreased mitochondrial metabolism, along with increased dependency on glycolysis. S47 tumor cells also show increased sensitivity to the glycolytic poison 2-deoxy-glucose. We propose that the altered metabolism in S47 tumor cells may be yet another potentially-actionable therapeutic vulnerability to exploit in cancer-prone individuals with this genotype.
    Keywords:  Pro47Ser; RRAD; metabolism; mitochondria; p53
  19. Cell Signal. 2019 Mar 01. pii: S0898-6568(19)30045-2. [Epub ahead of print]
    Li Y, Ngo A, Hoffmann P, Ferrante A, Hii CS.
      Endothelial cell injury and death precede atherosclerosis development. Thus, it is important to understand the mechanisms that lead to these early changes in endothelial cells. Although members of the MAP kinase/ERK kinase (MEK) kinase 3 (MEKK3)-MEK5-ERK5 module play an essential role in underpinning endothelial cell survival, how they execute these actions remain poorly understood. Furthermore, there is poor understanding of death-inducing pathways in endothelial cells and it is also unclear whether there are direct interactions between the kinase module and death-inducing pathways. Using immunoprecipitation and liquid chromatography-electrospray ionisation tandem mass spectrometry approaches, we show in human umbilical vein endothelial cells that the MEKK3-MEK5-ERK5 ternary complex contains glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme that can trigger the death of certain cell-types. GAPDH binds directly to MEKK3. Interestingly, serum depletion, a trigger of endothelial cell death, results in a rapid loss of cytosolic MEKK3 and MEKK3-GAPDH interaction. MEKK3 rapidly reappears in the cytosol upon serum replenishment, accompanied by the restoration of MEKK3-GAPDH interaction. During serum starvation or exposure to cytotoxic concentrations of H2O2, GAPDH accumulates in the nucleus. Inhibition of the nuclear accumulation of GAPDH with R-(-)-deprenyl hydrochloride attenuates the degree of cell death. Serum replenishment of serum-starved cells reduces the level of nuclear GAPDH and prevents cell death. Cell-free assays show phosphorylation of GAPDH on four residues by MEKK3. These data not only strongly implicate nuclear GAPDH in causing endothelial cell death but also reveal a potential mechanism for MEKK3 to regulate GAPDH function and hence promote endothelial cell survival.
    Keywords:  Endothelial cell survival; Glyceraldehyde-3-phosphate dehydrogenase; MAP kinase/ERK kinase kinase 3; Mitogen activated protein kinases; Protein-protein interaction
  20. FASEB J. 2019 Mar 08. fj201801591R
    Blázquez-Bermejo C, Carreño-Gago L, Molina-Granada D, Aguirre J, Ramón J, Torres-Torronteras J, Cabrera-Pérez R, Martín MÁ, Domínguez-González C, de la Cruz X, Lombès A, García-Arumí E, Martí R, Cámara Y.
      Polymerase γ catalytic subunit ( POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.
    Keywords:  deoxynucleosides; mitochondria; mitochondrial DNA replication; polymerase γ; therapy
  21. Aging Cell. 2019 Mar 05. e12941
    Song C, Zhang J, Qi S, Liu Z, Zhang X, Zheng Y, Andersen JP, Zhang W, Strong R, Martinez PA, Musi N, Nie J, Shi Y.
      Cardiolipin (CL) is a mitochondrial signature phospholipid that is required for membrane structure, respiration, dynamics, and mitophagy. Oxidative damage of CL by reactive oxygen species is implicated in the pathogenesis of Parkinson's disease (PD), but the underlying cause remains elusive. This work investigated the role of ALCAT1, an acyltransferase that catalyzes pathological remodeling of CL in various aging-related diseases, in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP). We show that MPTP treatment caused oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain. In contrast, ablation of the ALCAT1 gene or pharmacological inhibition of ALCAT1 prevented MPTP-induced neurotoxicity, apoptosis, and motor deficits. ALCAT1 deficiency also mitigated mitochondrial dysfunction by modulating DRP1 translocation to the mitochondria. Moreover, pharmacological inhibition of ALCAT1 significantly improved mitophagy by promoting the recruitment of Parkin to dysfunctional mitochondria. Finally, ALCAT1 expression was upregulated by MPTP and by α-synucleinopathy, a key hallmark of PD, whereas ALCAT1 deficiency prevented α-synuclein oligomerization and S-129 phosphorylation, implicating a key role of ALCAT1 in the etiology of mouse models of PD. Together, these findings identify ALCAT1 as a novel drug target for the treatment of PD.
    Keywords:  MPTP; cardiolipin; mitochondrial dysfunction; mitophagy; α-synuclein
  22. Proc Natl Acad Sci U S A. 2019 Mar 08. pii: 201817259. [Epub ahead of print]
    Deng P, Uma Naresh N, Du Y, Lamech LT, Yu J, Zhu LJ, Pukkila-Worley R, Haynes CM.
      Mitochondria generate most cellular energy and are targeted by multiple pathogens during infection. In turn, metazoans employ surveillance mechanisms such as the mitochondrial unfolded protein response (UPRmt) to detect and respond to mitochondrial dysfunction as an indicator of infection. The UPRmt is an adaptive transcriptional program regulated by the transcription factor ATFS-1, which induces genes that promote mitochondrial recovery and innate immunity. The bacterial pathogen Pseudomonas aeruginosa produces toxins that disrupt oxidative phosphorylation (OXPHOS), resulting in UPRmt activation. Here, we demonstrate that Pseudomonas aeruginosa exploits an intrinsic negative regulatory mechanism mediated by the Caenorhabditis elegans bZIP protein ZIP-3 to repress UPRmt activation. Strikingly, worms lacking zip-3 were impervious to Pseudomonas aeruginosa-mediated UPRmt repression and resistant to infection. Pathogen-secreted phenazines perturbed mitochondrial function and were the primary cause of UPRmt activation, consistent with these molecules being electron shuttles and virulence determinants. Surprisingly, Pseudomonas aeruginosa unable to produce phenazines and thus elicit UPRmt activation were hypertoxic in zip-3-deletion worms. These data emphasize the significance of virulence-mediated UPRmt repression and the potency of the UPRmt as an antibacterial response.
    Keywords:  ATFS-1; UPRmt; ZIP-3; immunity; mitochondrial UPR
  23. Cell Death Dis. 2019 Mar 04. 10(3): 222
    Zhang S, Sheng H, Zhang X, Qi Q, Chan CB, Li L, Shan C, Ye K.
      Phosphoinositide 3-kinase enhancer-activating Akt (PIKE-A), which associates with and potentiates Akt activity, is a pro-oncogenic factor that play vital role in cancer cell survival and growth. However, PIKE-A physiological functions under energy/nutrient deficiency are poorly understood. The AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that is a principal regulator of energy homeostasis and has a critical role in metabolic disorders and cancers. In this present study, we show that cellular energy stress induces PIKE-A phosphorylation mediated by AMPK activation, thereby preventing its carcinogenic action. Moreover, AMPK directly phosphorylates PIKE-A Ser-351 and Ser-377, which become accessible for the interaction with 14-3-3β, and in turn stimulates nuclear translocation of PIKE-A. Nuclear PIKE-A associates with CDK4 and then disrupts CDK4-cyclinD1 complex and inhibits the Rb pathway, resulting in cancer cell cycle arrest. Our data uncover a molecular mechanism and functional significance of PIKE-A phosphorylation response to cellular energy status mediated by AMPK.
  24. Mol Cell. 2019 Mar 07. pii: S1097-2765(19)30139-X. [Epub ahead of print]73(5): 861-862
    Edwards R, Tokatlidis K.
      Porin is crucial for metabolite flux in mitochondria. In this issue of Molecular Cell, Sakaue et al. (2019) and Ellenrieder et al. (2019) describe an unexpected role for Porin in mitochondrial protein import by regulating the oligomeric state of the major protein import gate, the TOM complex, and the inner membrane insertion of metabolite carriers.
  25. Front Microbiol. 2019 ;10 219
    Neumann-Schaal M, Jahn D, Schmidt-Hohagen K.
      Strains of Clostridioides difficile cause detrimental diarrheas with thousands of deaths worldwide. The infection process by the Gram-positive, strictly anaerobic gut bacterium is directly related to its unique metabolism, using multiple Stickland-type amino acid fermentation reactions coupled to Rnf complex-mediated sodium/proton gradient formation for ATP generation. Major pathways utilize phenylalanine, leucine, glycine and proline with the formation of 3-phenylproprionate, isocaproate, butyrate, 5-methylcaproate, valerate and 5-aminovalerate. In parallel a versatile sugar catabolism including pyruvate formate-lyase as a central enzyme and an incomplete tricarboxylic acid cycle to prevent unnecessary NADH formation completes the picture. However, a complex gene regulatory network that carefully mediates the continuous adaptation of this metabolism to changing environmental conditions is only partially elucidated. It involves the pleiotropic regulators CodY and SigH, the known carbon metabolism regulator CcpA, the proline regulator PrdR, the iron regulator Fur, the small regulatory RNA CsrA and potentially the NADH-responsive regulator Rex. Here, we describe the current knowledge of the metabolic principles of energy generation by C. difficile and the underlying gene regulatory scenarios.
    Keywords:  Clostridioides (Clostridium) difficile; Stickland reactions; TCA cycle; Wood-Ljungdahl pathway; fermentation; metabolism
  26. Cardiovasc Res. 2019 Mar 07. pii: cvz061. [Epub ahead of print]
    Nikolaou PE, Boengler K, Efentakis P, Vougogianopoulou K, Zoga A, Gaboriaud-Kolar N, Myrianthopoulos V, Alexakos P, Kostomitsopoulos N, Rerras I, Tsantil-Kakoulidou A, Skaltsounis AL, Papapetropoulos A, Iliodromitis EK, Schulz R, Andreadou I.
      AIMS: Glycogen synthase kinase 3 beta (GSK3β) link with the mitochondrial Permeability Transition Pore (mPTP) in cardioprotection is debated. We investigated the role of GSK3β in Ischemia (I)/Reperfusion (R) injury using pharmacological tools.METHODS AND RESULTS: Infarct size (IS) using the GSK3β inhibitor BIO and several novel analogues (MLS2776-MLS2779) was determined in anesthetized rabbits and mice. In myocardial tissue GSK3β inhibition and the specificity of the compounds was tested. The mechanism of protection focused on autophagy-related proteins. GSK3β localization was determined in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from Langendorff-perfused murine hearts (30'I/10'R or normoxic conditions). Calcium Retention Capacity (CRC) was determined in mitochondria after administration of the inhibitors in mice and in vitro. The effects of the inhibitors on mitochondrial respiration, reactive oxygen species (ROS) formation, ATP-production or hydrolysis were measured in SSM at baseline. Cyclosporine A (CsA) was co-administered with the inhibitors to address putative additive cardioprotective effects. Rabbits and mice treated with MLS compounds had smaller IS compared to control. In rabbits, MLS2776 and MLS2778 possessed greater infarct-sparing effects than BIO. GSK3β inhibition was confirmed at the 10th min and 2 hours of reperfusion while upregulation of autophagy-related proteins was evident at late reperfusion. The mitochondrial amount of GSK3β was similar in normoxic SSM and IFM and was not altered by I/R. The inhibitors did not affect CRC or respiration, ROS and ATP-production/hydrolysis at baseline. The co-administration of CsA ensured that cardioprotection was CypD-independent.
    CONCLUSIONS: Pharmacological inhibition of GSK3β attenuates IS beyond mPTP inhibition.
    Keywords:  glycogen synthase kinase 3 beta (GSK3β); infarct size; mitochondrial permeability transition pore; novel selective analogues of BIO
  27. Methods Mol Biol. 2019 ;1943 313-322
    Hall A, Moghimi SM.
      A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.
    Keywords:  ATP; Mitochondrial respiratory states; Oxidative phosphorylation; Oxygen consumption; Polyethylenimine
  28. EMBO J. 2019 Mar 06. pii: e99748. [Epub ahead of print]
    Yu R, Jin SB, Lendahl U, Nistér M, Zhao J.
      Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin-related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1-mediated mitochondrial fragmentation occurs in the absence of Drp1 and Dyn2, suggesting that they are dispensable for hFis1 function. hFis1 instead binds to Mfn1, Mfn2, and OPA1 and inhibits their GTPase activity, thus blocking the fusion machinery. Consistent with this, disruption of the fusion machinery in Drp1-/- cells phenocopies the fragmentation phenotype induced by hFis1 overexpression. In sum, our data suggest a novel role for hFis1 as an inhibitor of the fusion machinery, revealing an important functional evolutionary divergence between yeast and mammalian Fis1 proteins.
    Keywords:  Drp1; OPA1; hFis1; mitochondrial dynamics; mitofusins
  29. Cancer Sci. 2019 Mar 07.
    Hayashi Y, Yokota A, Harada H, Huang G.
      Since the first identification of hypoxic cells in sections of carcinomas in the 1950s, hypoxia has been known as a central hallmark of cancer cells and their microenvironment. Indeed, hypoxia benefits cancer cells in their growth, survival, and metastasis. The historical discovery of hypoxia inducible factor 1 alpha (HIF1A) in the early 1990s had a great influence on the field as many phenomena in hypoxia could be explained by HIF1A. However, all regions or types of tumors are not necessarily hypoxic. Thus, it is difficult to explain whole cancer pathobiology by hypoxia especially in the early stage of cancer. On the other hand, upregulation of glucose metabolism in cancer cells has been well known. Oxygen-independent glycolysis is activated in cancer cells even in the normoxia condition, which is known as the Warburg effect. Accumulating evidences and recent advances of the cancer metabolism research suggest that hypoxia-independent mechanisms for HIF signaling activation is a hallmark for cancer. There are various mechanisms which generate pseudo-hypoxic conditions even in the normoxia. Given the importance of HIF1A for cancer pathobiology, the pseudohypoxia concept could shed light on the longstanding mystery of the Warburg effect and accelerate better understanding of the diverse phenomena seen in a variety of cancers. This article is protected by copyright. All rights reserved.
    Keywords:  HIF1A; Hypoxia; Oncometabolites; Pseudohypoxia; Warburg effect
  30. Neurochem Res. 2019 Mar 07.
    Jordan F, Nemeria N, Gerfen G.
      According to recent findings, the human 2-oxoglutarate dehydrogenase complex (hOGDHc) could be an important source of the reactive oxygen species in the mitochondria and could contribute to mitochondrial abnormalities associated with multiple neurodegenerative diseases, including Alzheimer's disease, Huntington disease, and Parkinson's disease. The human 2-oxoadipate dehydrogenase (hE1a) is a novel protein, which is encoded by the DHTKD1 gene. Both missence and nonsense mutations were identified in the DHTKD1 that lead to alpha-aminoadipic and alpha-oxoadipic aciduria, a metabolic disorder with a wide variety of the neurological abnormalities, and Charcot-Marie-Tooth disease type 2Q, an inherited neurological disorder affecting the peripheral nervous system. Recently, the rare pathogenic mutations in DHTKD1 and an increased H2O2 production were linked to the genetic ethiology of Eosinophilic Esophagitis (EoE), a chronic allergic inflammatory esophageal disorder. In view of the importance of hOGDHc in the tricarboxylic acid cycle (TCA cycle) and hE1a on the L-lysine, L-hydroxylysine and L-tryptophan degradation pathway in mitochondria, and to enhance our current understanding of the mechanism of superoxide/H2O2 generation by hOGDHc, and by human 2-oxoadipate dehydrogenase complex (hOADHc), this review focuses on several novel and unanticipated recent findings in vitro that emerged from the Jordan group's research. Most significantly, the hE1o and hE1a now join the hE3 as being able to generate the superoxide/H2O2 in mitochondria.
    Keywords:  2-Oxoglutarate and 2-oxoadipate dehydrogenase complexes; Hydrogen peroxide; Oxidative stress; Thiamin diphosphate-enamine radical
  31. Rev Neurosci. 2019 Mar 06. pii: /j/revneuro.ahead-of-print/revneuro-2018-0095/revneuro-2018-0095.xml. [Epub ahead of print]
    Di Rita A, Strappazzon F.
      During aging, the process of mitophagy, a system that allows the removal of dysfunctional mitochondria through lysosomal degradation, starts to malfunction. Because of this defect, damaged mitochondria are not removed correctly, and their decomposing components accumulate inside the cells. Dysfunctional mitochondria that are not removed by mitophagy produce high amounts of reactive oxygen species (ROS) and, thus, cause oxidative stress. Oxidative stress, in turn, is very harmful for the cells, neuronal cells, in particular. Consequently, the process of mitophagy plays a crucial role in mitochondria-related disease. Mitochondrial dysfunctions and oxidative stress are well-established factors contributing to Parkinson's disease (PD), one of the most common neurodegenerative disorders. In this review, we report various known antioxidants for PD treatments and describe the stimulation of mitophagy process as a novel and exciting method for reducing oxidative stress in PD patients. We describe the different mechanisms responsible for mitochondria removal through the mitophagy process. In addition, we review the functional connection between mitophagy induction and reduction of oxidative stress in several in vitro models of PD and also agents (drugs and natural compounds) already known to be antioxidants and to be able to activate mitophagy. Finally, we propose that there is an urgent need to test the use of mitophagy-inducing antioxidants in order to fight PD.
    Keywords:  Parkinsonism; mitochondrial selective degradation; neurodegeneration; oxidative stress
  32. Nat Commun. 2019 Mar 04. 10(1): 1037
    Toomer KH, Lui JB, Altman NH, Ban Y, Chen X, Malek TR.
      IL-2R signaling is essential for regulatory T cell (Treg) function. However, the precise contribution of IL-2 during Treg thymic development, peripheral homeostasis and lineage stability remains unclear. Here we show that IL-2R signaling is required by thymic Tregs at an early step for expansion and survival, and a later step for functional maturation. Using inducible, conditional deletion of CD25 in peripheral Tregs, we also find that IL-2R signaling is indispensable for Treg homeostasis, whereas Treg lineage stability is largely IL-2-independent. CD25 knockout peripheral Tregs have increased apoptosis, oxidative stress, signs of mitochondrial dysfunction, and reduced transcription of key enzymes of lipid and cholesterol biosynthetic pathways. A divergent IL-2R transcriptional signature is noted for thymic Tregs versus peripheral Tregs. These data indicate that IL-2R signaling in the thymus and the periphery leads to distinctive effects on Treg function, while peripheral Treg survival depends on a non-conventional mechanism of metabolic regulation.
  33. Metabolomics. 2018 Oct 01. 14(10): 136
    Dhami N, Trivedi DK, Goodacre R, Mainwaring D, Humphreys DP.
      INTRODUCTION: Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.OBJECTIVES: The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.
    METHODS: To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.
    RESULTS: Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.
    CONCLUSION: Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.
    Keywords:  CHO cells; Glucose metabolism; Oxidative stress; TCA cycle; m-Aconitase
  34. Int Neurourol J. 2019 Feb;23(Suppl 1): S5-10
    Tang J, Oliveros A, Jang MH.
      Synapses are sites of high energy demand which are dependent on high levels of mitochondrial derived adenosine triphosphate. Mitochondria within synaptic structures are key for maintenance of functional neurotransmission and this critical biological process is modulated by energy metabolism, mitochondrial distribution, mitochondrial trafficking, and cellular synaptic calcium flux. Synapse loss is presumed to be an early yet progressive pathological event in Alzheimer disease (AD), resulting in impaired cognitive function and memory loss which is particularly prevalent at later stages of disease. Supporting evidence from AD patients and animal models suggests that pathological mitochondrial dynamics indeed occurs early and is highly associated with synaptic lesions and degeneration in AD neurons. This review comprehensively highlights recent findings that describe how synaptic mitochondria pathology involves dysfunctional trafficking of this organelle, to maladaptive epigenetic contributions affecting mitochondrial function in AD. We further discuss how these negative, dynamic alterations impact synaptic function associated with AD. Finally, this review explores how antioxidant therapeutic approaches targeting mitochondria in AD can further clinical research and basic science investigations to advance our in-depth understanding of the pathogenesis of AD.
    Keywords:  Alzheimer disease; Cognitive dysfunction; Mitochondria; Synaptic plasticity
  35. J Biomed Sci. 2019 Mar 08. 26(1): 24
    Chi HC, Tsai CY, Tsai MM, Yeh CT, Lin KH.
      The liver is controlled by several metabolic hormones, including thyroid hormone, and characteristically displays high lysosomal activity as well as metabolic stress-triggered autophagy, which is stringently regulated by the levels of hormones and metabolites. Hepatic autophagy provides energy through catabolism of glucose, amino acids and free fatty acids for starved cells, facilitating the generation of new macromolecules and maintenance of the quantity and quality of cellular organelles, such as mitochondria. Dysregulation of autophagy and defective mitochondrial homeostasis contribute to hepatocyte injury and liver-related diseases, such as non-alcoholic fatty liver disease (NAFLD) and liver cancer.Thyroid hormones (TH) mediate several critical physiological processes including organ development, cell differentiation, metabolism and cell growth and maintenance. Accumulating evidence has revealed dysregulation of cellular TH activity as the underlying cause of several liver-related diseases, including alcoholic or non-alcoholic fatty liver disease and liver cancer. Data from epidemiologic, animal and clinical studies collectively support preventive functions of THs in liver-related diseases, highlighting the therapeutic potential of TH analogs. Elucidation of the molecular mechanisms and downstream targets of TH should thus facilitate the development of therapeutic strategies for a number of major public health issues.Here, we have reviewed recent studies focusing on the involvement of THs in hepatic homeostasis through induction of autophagy and their implications in liver-related diseases. Additionally, the potential underlying molecular pathways and therapeutic applications of THs in NAFLD and HCC are discussed.
    Keywords:  Autophagy; Thyroid hormone; Thyroid hormone receptor; hepatocellular carcinoma; non-alcoholic fatty liver disease
  36. Mol Cell. 2019 Feb 26. pii: S1097-2765(19)30057-7. [Epub ahead of print]
    Zhu XG, Nicholson Puthenveedu S, Shen Y, La K, Ozlu C, Wang T, Klompstra D, Gultekin Y, Chi J, Fidelin J, Peng T, Molina H, Hang HC, Min W, Birsoy K.
      Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.
    Keywords:  CHP1; CRISPR; GPAT4; cellular metabolism; fatty acids; genetic screens; glycerolipid synthesis; lipid metabolism; lipidomics; triacylglycerol accumulation
  37. Front Cardiovasc Med. 2019 ;6 10
    Chen Q, Thompson J, Hu Y, Das A, Lesnefsky EJ.
      Endoplasmic reticulum (ER) stress contributes to cardiovascular disease including heart failure. Interactions between the ER and mitochondria during ER stress can impair the mitochondrial respiratory chain and increase cell injury. p53 is a tumor suppressor protein that regulates apoptosis. p53 contributes to the regulation of mitochondrial and ER interactions, especially during the progression of ER stress. The knockout (KO) of p53 leads to decreased injury in hearts following ischemia-reperfusion. We asked if KO of p53 can protect mitochondria during the induction of ER stress and decrease cell injury. Floxed p53 mice were crossed with mice carrying an α-myosin heavy chain cre to generate cardiac specific p53 KO mice. Thapsigargin (THAP) was used to induce ER stress in wild type (WT) and p53 KO mice. Mice were euthanized after 48 h THAP treatment. Cardiac mitochondria were isolated for functional measurement. TUNEL staining was used to assess myocyte death. In WT mice, THAP treatment decreased the rate of oxidative phosphorylation using pyruvate + malate as complex I substrates compared to vehicle-treated control. Complex I activity was also decreased in the THAP-treated WT mice. The rate of oxidative phosphorylation and complex I activity were not altered in THAP-treated p53 KO mice. The content of pyruvate dehydrogenase (PDH) α1 subunit was decreased in THAP-treated WT mice but not in p53 KO mice. ER stress led to a release of cytochrome c and apoptosis inducing factor from mitochondria into cytosol in WT but not in KO mice. Knockout of p53 also preserved mitochondrial bcl-2 content in THAP-treated mice. In WT mice, THAP treatment markedly increased cell death compared to vehicle treated hearts. In contrast, cell injury was decreased in THAP-treated p53 KO mice compared to corresponding wild type. Thus, KO of p53 decreased cell injury by protecting mitochondria during the ER stress.
    Keywords:  apoptosis; complex I; mitochondria; pyruvate dehydrogenase; thapsigargin
  38. Science. 2019 Mar 08. 363(6431): 1088-1092
    Hoxhaj G, Ben-Sahra I, Lockwood SE, Timson RC, Byles V, Henning GT, Gao P, Selfors LM, Asara JM, Manning BD.
      Nicotinamide adenine dinucleotide phosphate (NADP+) is essential for producing NADPH, the primary cofactor for reductive metabolism. We find that growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP+ and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP+ from NAD+ (the oxidized form of NADH), on three serine residues (Ser44, Ser46, and Ser48) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP+ production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP+ production through relief of an autoinhibitory function inherent to its amino terminus.
  39. Int Neurourol J. 2019 Feb;23(Suppl 1): S22-31
    Yoo SZ, No MH, Heo JW, Park DH, Kang JH, Kim JH, Seo DY, Han J, Jung SJ, Kwak HB.
      PURPOSE: This study aimed to investigate the effects of single-bout exercise on mitochondrial function, dynamics (fusion, fission), and mitophagy in cardiac and skeletal muscles.METHODS: Fischer 344 rats (4 months old) were randomly divided into the control (CON) or acute exercise (EX) group (n=10 each). The rats performed a single bout of treadmill exercise for 60 minutes. Mitochondrial function (e.g., O2 respiration, H2O2 emission, Ca2+ retention capacity), mitochondrial fusion (e.g., Mfn1, Mfn2, Opa1), mitochondrial fission (e.g., Drp1, Fis1), and mitophagy (e.g., Parkin, Pink1, LC3II, Bnip3) were measured in permeabilized cardiac (e.g., left ventricle) and skeletal (e.g., soleus, white gastrocnemius) muscles.
    RESULTS: Mitochondrial O2 respiration and Ca2+ retention capacity were significantly increased in all tissues of the EX group compared with the CON group. Mitochondrial H2O2 emissions showed tissue-specific results; the emissions showed no significant differences in the left ventricle or soleus (type I fibers) but was significantly increased in the white gastrocnemius (type II fibers) after acute exercise. Mitochondrial fusion and fission were not altered in any tissues of the EX group. Mitophagy showed tissue-specific differences: It was not changed in the left ventricle or white gastrocnemius, whereas Parkin and LC3II were significantly elevated in the soleus muscle.
    CONCLUSION: A single bout of aerobic exercise may improve mitochondrial function (e.g., O2 respiration and Ca2+ retention capacity) in the heart and skeletal muscles without changes in mitochondrial dynamics or mitophagy.
    Keywords:  Acute exercise; Dynamics; Heart; Mitochondrial function; Skeletal muscle
  40. Cell Mol Life Sci. 2019 Mar 04.
    Andersen JV, Skotte NH, Aldana BI, Nørremølle A, Waagepetersen HS.
      Huntington's disease (HD) is a hereditary and fatal disease causing profound neurodegeneration. Deficits in cerebral energy and neurotransmitter metabolism have been suggested to play a central role in the neuronal dysfunction and death associated with HD. The branched-chain amino acids (BCAAs), leucine, isoleucine and valine, are important for cerebral nitrogen homeostasis, neurotransmitter recycling and can be utilized as energy substrates in the tricarboxylic acid (TCA) cycle. Reduced levels of BCAAs in HD have been validated by several reports. However, it is still unknown how cerebral BCAA metabolism is regulated in HD. Here we investigate the metabolism of leucine and isoleucine in the R6/2 mouse model of HD. Acutely isolated cerebral cortical and striatal slices of control and R6/2 mice were incubated in media containing 15N- or 13C-labeled leucine or isoleucine and slice extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine isotopic enrichment of derived metabolites. Elevated BCAA transamination was found from incubations with [15N]leucine and [15N]isoleucine, in both cerebral cortical and striatal slices of R6/2 mice compared to controls. Metabolism of [U-13C]leucine and [U-13C]isoleucine, entering oxidative metabolism as acetyl CoA, was maintained in R6/2 mice. However, metabolism of [U-13C]isoleucine, entering the TCA cycle as succinyl CoA, was elevated in both cerebral cortical and striatal slices of R6/2 mice, suggesting enhanced metabolic flux via this anaplerotic pathway. To support the metabolic studies, expression of enzymes in the BCAA metabolic pathway was assessed from a proteomic resource. Several enzymes related to BCAA metabolism were found to exhibit augmented expression in the R6/2 brain, particularly related to isoleucine metabolism, suggesting an increase in the BCAA metabolic machinery. Our results show that the capacity for cerebral BCAA metabolism, predominantly of isoleucine, is amplified in the R6/2 brain and indicates that perturbations in cerebral BCAA homeostasis could have functional consequences for HD pathology.
    Keywords:  Anaplerosis; BCAA; Brain energy metabolism; Isoleucine; Leucine; Neurodegeneration
  41. Cell. 2019 Mar 07. pii: S0092-8674(19)30055-8. [Epub ahead of print]176(6): 1325-1339.e22
    Ludwig LS, Lareau CA, Ulirsch JC, Christian E, Muus C, Li LH, Pelka K, Ge W, Oren Y, Brack A, Law T, Rodman C, Chen JH, Boland GM, Hacohen N, Rozenblatt-Rosen O, Aryee MJ, Buenrostro JD, Regev A, Sankaran VG.
      Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.
    Keywords:  chronic myeloid leukemia; colon cancer; hematopoiesis; lineage tracing; mitochondrial DNA; mtDNA; sequencing; single cell genomics; somatic mutations
  42. Nat Commun. 2019 Mar 06. 10(1): 1072
    Kidoya H, Muramatsu F, Shimamura T, Jia W, Satoh T, Hayashi Y, Naito H, Kunisaki Y, Arai F, Seki M, Suzuki Y, Osawa T, Akira S, Takakura N.
      The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.
  43. Meat Sci. 2019 Feb 28. pii: S0309-1740(18)30706-X. [Epub ahead of print]152 121-126
    Krischek C, Popp J, Sharifi AR.
      Muscle-to-meat-transition is influenced by alterations of the energy metabolism. Porcine Musculus triceps brachii (MT) consisted of more fast-twitch-glycolytic muscle fibers and samples, collected 0, 10 and 20 min after slaughter (p.m.), showed higher mitochondrial respiratory activities and ATP concentrations than Musculus longissimus thoracis (LT) samples. Enzyme activities in MT were higher at 0 min (glycogen phosphorylase (GP)), 10 min (GP, citrate synthase (CS)) and at 20 min p.m. (CS). However, LT results were higher at 0 min (lactate dehydrogenase (LDH)), 10 min (phosphofructokinase (PFK), LDH) and at 20 min p.m. (PFK, F0F1-ATPase (F0F1)). Between 0 min and 10 min p.m. CS activities decreased in LT and MT samples, PFK increased in LT and GP in MT samples. Between 10 min and 20 min p.m. PFK and LDH decreased in LT and GP in MT samples, whereas F0F1 increased in LT and CS in MT samples. The data indicate that muscles with different mitochondria contents show clearly different energy metabolism characteristics.
    Keywords:  Metabolic enzymes; Mitochondrial respiration; Musculus longissimus thoracis; Musculus triceps brachii; Pig; Postmortem metabolism
  44. Cell Metab. 2019 Feb 21. pii: S1550-4131(19)30065-8. [Epub ahead of print]
    Halbrook CJ, Pontious C, Kovalenko I, Lapienyte L, Dreyer S, Lee HJ, Thurston G, Zhang Y, Lazarus J, Sajjakulnukit P, Hong HS, Kremer DM, Nelson BS, Kemp S, Zhang L, Chang D, Biankin A, Shi J, Frankel TL, Crawford HC, Morton JP, Pasca di Magliano M, Lyssiotis CA.
      Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant infiltration of tumor-associated macrophages (TAMs). TAMs have been reported to drive resistance to gemcitabine, a frontline chemotherapy in PDA, though the mechanism of this resistance remains unclear. Profiling metabolite exchange, we demonstrate that macrophages programmed by PDA cells release a spectrum of pyrimidine species. These include deoxycytidine, which inhibits gemcitabine through molecular competition at the level of drug uptake and metabolism. Accordingly, genetic or pharmacological depletion of TAMs in murine models of PDA sensitizes these tumors to gemcitabine. Consistent with this, patients with low macrophage burden demonstrate superior response to gemcitabine treatment. Together, these findings provide insights into the role of macrophages in pancreatic cancer therapy and have potential to inform the design of future treatments. Additionally, we report that pyrimidine release is a general function of alternatively activated macrophage cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells.
    Keywords:  deoxycytidine; gemcitabine resistance; immunometabolism; macrophage; metabolic crosstalk; metabolomics; pancreatic cancer; pancreatic ductal adenocarcinoma; tumor microenvironment; tumor-associated macrophage
  45. Metabolomics. 2018 Nov 03. 14(11): 150
    Stander Z, Luies L, Mienie LJ, Keane KM, Howatson G, Clifford T, Stevenson EJ, Loots DT.
      INTRODUCTION: Endurance races have been associated with a substantial amount of adverse effects which could lead to chronic disease and long-term performance impairment. However, little is known about the holistic metabolic changes occurring within the serum metabolome of athletes after the completion of a marathon.OBJECTIVES: Considering this, the aim of this study was to better characterize the acute metabolic changes induced by a marathon.
    METHODS: Using an untargeted two dimensional gas chromatography time-of-flight mass spectrometry metabolomics approach, pre- and post-marathon serum samples of 31 athletes were analyzed and compared to identify those metabolites varying the most after the marathon perturbation.
    RESULTS: Principle component analysis of the comparative groups indicated natural differentiation due to variation in the total metabolite profiles. Elevated concentrations of carbohydrates, fatty acids, tricarboxylic acid cycle intermediates, ketones and reduced concentrations of amino acids indicated a metabolic shift between various fuel substrate systems. Additionally, elevated odd-chain fatty acids and α-hydroxy acids indicated the utilization of α-oxidation and autophagy as alternative energy-producing mechanisms. Adaptations in gut microbe-associated markers were also observed and correlated with the metabolic flexibility of the athlete.
    CONCLUSION: From these results it is evident that a marathon places immense strain on the energy-producing pathways of the athlete, leading to extensive protein degradation, oxidative stress, mammalian target of rapamycin complex 1 inhibition and autophagy. A better understanding of this metabolic shift could provide new insights for optimizing athletic performance, developing more efficient nutrition regimens and identify strategies to improve recovery.
    Keywords:  Fuel substrates; Marathon; Metabolite markers; Metabolomics; Serum
  46. Autophagy. 2019 Mar 08. 1-3
    Sharif T, Martell E, Dai C, Singh SK, Gujar S.
      Cancer stem-like cells (CSLCs) reside as a small population within tumors, which mostly contain a larger population of differentiated cells. With their unique self-renewing abilities, CSLCs remain refractory to various therapeutic interventions, which otherwise kill differentiated cancer cells, and thus are a major culprit behind cancer treatment failures and cancer relapse. Recently, the process of macroautophagy/autophagy has emerged as a potential therapeutic target for eliminating CSLCs, as autophagic homeostasis has been discovered to play an important role in the growth of cancer and normal stem cells, and is required for the maintenance of the non-differentiated state of CSLCs. Our current work now shows that the so-called 'tumor suppressor' TP73/p73 plays an unconventional role in CSLC biology, and positively regulates the growth and stemness of CSLCs through the modulation of autophagy. Our data show that TP73/p73 deficiency, promotes autophagy in CSLCs by activating the autophagy machinery involving AMPK-TSC-MTOR signaling. Mechanistically, TP73/p73 deficiency-induced autophagy occurs as a result of reduced ATP levels resulting from the metabolic perturbations within the proline regulatory axis. Collectively, these findings unveil novel therapeutically-relevant implications for autophagy in the TP73/p73-dependent regulation of stemness within CSLCs.
    Keywords:  Autophagy; TP73/p73; brain tumor-initiating cells; cancer stem cells; glutamine; metabolism; proline-regulatory axis; tumor suppressors
  47. Mol Biol Cell. 2019 Mar 06. mbcE19010044
    Farmer T, O'Neill KL, Naslavsky N, Luo X, Caplan S.
      The anti-apoptotic Bcl-2 family protein Bcl-xL plays a critical role in cell survival by protecting the integrity of the mitochondrial outer membrane (MOM). The mechanism through which Bcl-xL is recruited to the MOM has not been fully discerned. The retromer is a conserved endosomal scaffold complex involved in membrane trafficking.  Here we identify VPS35 and VPS26, two core components of the retromer, as novel regulators of Bcl-xL. We observed interactions and co-localization between Bcl-xL, VPS35, VPS26 and MICAL-L1, a protein involved in recycling endosome biogenesis that also interacts with the retromer. We also found that upon VPS35 depletion, levels of non-mitochondrial Bcl-xL were increased.  In addition, retromer-depleted cells displayed more rapid Bax activation and apoptosis. These results suggest that the retromer regulates apoptosis by facilitating Bcl-xL's transport to the MOM. Importantly, our studies suggest a previously uncharacterized relationship between the machineries of cell death/survival and endosomal trafficking. Movie S1 Movie S1.
  48. Nat Commun. 2019 Mar 05. 10(1): 1044
    Brock CK, Wallin ST, Ruiz OE, Samms KM, Mandal A, Sumner EA, Eisenhoffer GT.
      Epithelial tissues require the removal and replacement of damaged cells to sustain a functional barrier. Dying cells provide instructive cues that can influence surrounding cells to proliferate, but how these signals are transmitted to their healthy neighbors to control cellular behaviors during tissue homeostasis remains poorly understood. Here we show that dying stem cells facilitate communication with adjacent stem cells by caspase-dependent production of Wnt8a-containing apoptotic bodies to drive cellular turnover in living epithelia. Basal stem cells engulf apoptotic bodies, activate Wnt signaling, and are stimulated to divide to maintain tissue-wide cell numbers. Inhibition of either cell death or Wnt signaling eliminated the apoptosis-induced cell division, while overexpression of Wnt8a signaling combined with induced cell death led to an expansion of the stem cell population. We conclude that ingestion of apoptotic bodies represents a regulatory mechanism linking death and division to maintain overall stem cell numbers and epithelial tissue homeostasis.
  49. Nat Med. 2019 Mar 04.
    Kinsey CG, Camolotto SA, Boespflug AM, Gullien KP, Foth M, Truong A, Schuman SS, Shea JE, Seipp MT, Yap JT, Burrell LD, Lum DH, Whisenant JR, Gilcrease GW, Cavalieri CC, Rehbein KM, Cutler SL, Affolter KE, Welm AL, Welm BE, Scaife CL, Snyder EL, McMahon M.
      Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.
  50. Dev Cell. 2019 Feb 27. pii: S1534-5807(19)30055-3. [Epub ahead of print]
    Kumar S, Gu Y, Abudu YP, Bruun JA, Jain A, Farzam F, Mudd M, Anonsen JH, Rusten TE, Kasof G, Ktistakis N, Lidke KA, Johansen T, Deretic V.
      Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13+FIP200+ mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17pS202 transfers from the Golgi, where its steady-state pools localize, to the ATG13+FIP200+ mPAS. Stx17pS202 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17S202A was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17pS202 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.
    Keywords:  TBK1; ULK1; autophagy; pre-autophagosomal structure
  51. Biochim Biophys Acta Rev Cancer. 2019 Feb 28. pii: S0304-419X(18)30195-1. [Epub ahead of print]
    Amin S, Yang P, Li Z.
      Cancer cells upregulate pyruvate kinase M2 isoform (PKM2) for their imperiously high energetic and biosynthetic demands to achieve enhanced proliferation, a common fingerprint of all type of cancers. PKM2 was primitively discovered as a cytosolic glycolytic enzyme, now has been extensively reported in non-canonical localizations such as extracellular secretion, mitochondrial localization, nuclear translocation, and exclusively pertained to novel biological functions in recent decades. Hence, such moonlighting functions of PKM2 have opened a new itinerary for cancer researchers. This review highlights the up-to-date moonlighting functions of PKM2 at various subcellular localizations and draws attention to the molecular mechanism of PKM2 translocation from cytosol into the nucleus where it plays crucial roles in cancer progression. Moreover, non-canonical PKM2 in tumor cells could have an important role in resistance acquisition processes against various chemotherapeutic drugs. We finally accentuate on the future perspectives of improving the anticancer therapeutic strategies by targeting PKM2 and associated signaling pathways.
    Keywords:  Cancer progression; Moonlighting functions; Non-canonical localization; PKM2 nuclear translocation; Pyruvate kinase M2
  52. Nat Commun. 2019 Mar 04. 10(1): 1034
    Ma B, Cheng H, Mu C, Geng G, Zhao T, Luo Q, Ma K, Chang R, Liu Q, Gao R, Nie J, Xie J, Han J, Chen L, Ma G, Zhu Y, Chen Q.
      The interactions between tumor cells with their microenvironments, including hypoxia, acidosis and immune cells, lead to the tumor heterogeneity which promotes tumor progression. Here, we show that SIAH2-NRF1 axis remodels tumor microenvironment through regulating tumor mitochondrial function, tumor-associated macrophages (TAMs) polarization and cell death for tumor maintenance and progression. Mechanistically, low mitochondrial gene expression in breast cancers is associated with a poor clinical outcome. The hypoxia-activated E3 ligase SIAH2 spatially downregulates nuclear-encoded mitochondrial gene expression including pyruvate dehydrogenase beta via degrading NRF1 (Nuclear Respiratory Factor 1) through ubiquitination on lysine 230, resulting in enhanced Warburg effect, metabolic reprogramming and pro-tumor immune response. Dampening NRF1 degradation under hypoxia not only impairs the polarization of TAMs, but also promotes tumor cells to become more susceptible to apoptosis in a FADD-dependent fashion, resulting in secondary necrosis due to the impairment of efferocytosis. These data represent that inhibition of NRF1 degradation is a potential therapeutic strategy against cancer.
  53. Cell Metab. 2019 Feb 26. pii: S1550-4131(19)30066-X. [Epub ahead of print]
    Chiche J, Reverso-Meinietti J, Mouchotte A, Rubio-Patiño C, Mhaidly R, Villa E, Bossowski JP, Proics E, Grima-Reyes M, Paquet A, Fragaki K, Marchetti S, Briere J, Ambrosetti D, Michiels JF, Molina TJ, Copie-Bergman C, Lehmann-Che J, Peyrottes I, Peyrade F, de Kerviler E, Taillan B, Garnier G, Verhoeyen E, Paquis-Flucklinger V, Shintu L, Delwail V, Delpech-Debiais C, Delarue R, Bosly A, Petrella T, Brisou G, Nadel B, Barbry P, Mounier N, Thieblemont C, Ricci JE.
      Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.
    Keywords:  DLBCL; GAPDH; L-asparaginase; OxPhos; R-CHOP; glycolysis; mTOR; predictive marker
  54. Cancer Cell. 2019 Feb 11. pii: S1535-6108(19)30052-2. [Epub ahead of print]
    Reina-Campos M, Linares JF, Duran A, Cordes T, L'Hermitte A, Badur MG, Bhangoo MS, Thorson PK, Richards A, Rooslid T, Garcia-Olmo DC, Nam-Cha SY, Salinas-Sanchez AS, Eng K, Beltran H, Scott DA, Metallo CM, Moscat J, Diaz-Meco MT.
      Increasingly effective therapies targeting the androgen receptor have paradoxically promoted the incidence of neuroendocrine prostate cancer (NEPC), the most lethal subtype of castration-resistant prostate cancer (PCa), for which there is no effective therapy. Here we report that protein kinase C (PKC)λ/ι is downregulated in de novo and during therapy-induced NEPC, which results in the upregulation of serine biosynthesis through an mTORC1/ATF4-driven pathway. This metabolic reprogramming supports cell proliferation and increases intracellular S-adenosyl methionine (SAM) levels to feed epigenetic changes that favor the development of NEPC characteristics. Altogether, we have uncovered a metabolic vulnerability triggered by PKCλ/ι deficiency in NEPC, which offers potentially actionable targets to prevent therapy resistance in PCa.
    Keywords:  ATF4; PKClambda; aPKC; cancer metabolism; epigenetics; lineage plasticity; mTOR; neuroendocrine; prostate cancer; serine metabolism
  55. Nat Commun. 2019 Mar 05. 10(1): 1060
    Wittemans LBL, Lotta LA, Oliver-Williams C, Stewart ID, Surendran P, Karthikeyan S, Day FR, Koulman A, Imamura F, Zeng L, Erdmann J, Schunkert H, Khaw KT, Griffin JL, Forouhi NG, Scott RA, Wood AM, Burgess S, Howson JMM, Danesh J, Wareham NJ, Butterworth AS, Langenberg C.
      Circulating levels of glycine have previously been associated with lower incidence of coronary heart disease (CHD) and type 2 diabetes (T2D) but it remains uncertain if glycine plays an aetiological role. We present a meta-analysis of genome-wide association studies for glycine in 80,003 participants and investigate the causality and potential mechanisms of the association between glycine and cardio-metabolic diseases using genetic approaches. We identify 27 genetic loci, of which 22 have not previously been reported for glycine. We show that glycine is genetically associated with lower CHD risk and find that this may be partly driven by blood pressure. Evidence for a genetic association of glycine with T2D is weaker, but we find a strong inverse genetic effect of hyperinsulinaemia on glycine. Our findings strengthen evidence for a protective effect of glycine on CHD and show that the glycine-T2D association may be driven by a glycine-lowering effect of insulin resistance.
  56. Metabolomics. 2019 Jan 28. 15(2): 18
    Batushansky A, Matsuzaki S, Newhardt MF, West MS, Griffin TM, Humphries KM.
      INTRODUCTION: As an insulin sensitive tissue, the heart decreases glucose usage during fasting. This response is mediated, in part, by decreasing phosphofructokinase-2 (PFK-2) activity and levels of its product fructose-2,6-bisphosphate. However, the importance of fructose-2,6-bisphosphate in the fasting response on other metabolic pathways has not been evaluated.OBJECTIVES: The goal of this study is to determine how sustaining cardiac fructose-2,6-bisphosphate levels during fasting affects the metabolomic profile.
    METHODS: Control and transgenic mice expressing a constitutively active form of PFK-2 (GlycoHi) were subjected to either 12-h fasting or regular feeding. Animals (n = 4 per group) were used for whole-heart extraction, followed by gas chromatography-mass spectrometry metabolic profiling and multivariate data analysis.
    RESULTS: Principal component analysis displayed differences between Control and GlycoHi groups under both fasting and fed conditions while a clear response to fasting was observed only for Control animals. However, pathway analysis revealed that these smaller changes in the GlycoHi group were significantly associated with branched-chain amino acid (BCAA) metabolism (~ 40% increase in all BCAAs). Correlation network analysis demonstrated clear differences in response to fasting between Control and GlycoHi groups amongst most parameters. Notably, fasting caused an increase in network density in the Control group from 0.12 to 0.14 while the GlycoHi group responded oppositely (0.17-0.15).
    CONCLUSIONS: Elevated cardiac PFK-2 activity during fasting selectively increases BCAAs levels and decreases global changes in metabolism.
    Keywords:  Cardiac metabolism; Correlation network; GC–MS metabolomics; Heart pathologies
  57. PLoS Genet. 2019 Mar 07. 15(3): e1007633
    Liu YJ, Janssens GE, McIntyre RL, Molenaars M, Kamble R, Gao AW, Jongejan A, Weeghel MV, MacInnes AW, Houtkooper RH.
      The deregulation of metabolism is a hallmark of aging. As such, changes in the expression of metabolic genes and the profiles of amino acid levels are features associated with aging animals. We previously reported that the levels of most amino acids decline with age in Caenorhabditis elegans (C. elegans). Glycine, in contrast, substantially accumulates in aging C. elegans. In this study we show that this is coupled to a decrease in gene expression of enzymes important for glycine catabolism. We further show that supplementation of glycine significantly prolongs C. elegans lifespan, and early adulthood is important for its salutary effects. Moreover, supplementation of glycine ameliorates specific transcriptional changes that are associated with aging. Glycine feeds into the methionine cycle. We find that mutations in components of this cycle, methionine synthase (metr-1) and S-adenosylmethionine synthetase (sams-1), completely abrogate glycine-induced lifespan extension. Strikingly, the beneficial effects of glycine supplementation are conserved when we supplement with serine, which also feeds into the methionine cycle. RNA-sequencing reveals a similar transcriptional landscape in serine- and glycine-supplemented worms both demarked by widespread gene repression. Taken together, these data uncover a novel role of glycine in the deceleration of aging through its function in the methionine cycle.
  58. Nat Commun. 2019 Mar 04. 10(1): 1038
    Pelosse M, Cottet-Rousselle C, Bidan CM, Dupont A, Gupta K, Berger I, Schlattner U.
      AMP-activated protein kinase AMPK senses and regulates cellular energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineered AMPK fused to fluorescent proteins, the sensor allows direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). AMPfret faithfully and dynamically reports the binding of AMP and ADP to AMPK γ-CBS sites, competed by Mg2+-free ATP. FRET signals correlate with activation of AMPK by allosteric mechanisms and protection from dephosphorylation, attributed here to specific CBS sites, but does not require activation loop phosphorylation. Moreover, AMPfret detects binding of pharmacological compounds to the AMPK α/β-ADaM site enabling activator screening. Cellular assays demonstrate that AMPfret is applicable in vivo for spatiotemporal analysis of energy state and allosteric AMPK activation.
  59. Proc Natl Acad Sci U S A. 2019 Mar 08. pii: 201816317. [Epub ahead of print]
    Xia C, Fu Z, Battaile KP, Kim JP.
      Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.6-Å resolution. The biological unit of the protein is α2β2 The overall structure of the heterotetramer is the same as that observed by cryo-EM methods. The two β-subunits make a tightly bound homodimer at the center, and two α-subunits are bound to each side of the β2 dimer, creating an arc, which binds on its concave side to the mitochondrial innermembrane. The catalytic residues in all three active sites are arranged similarly to those of the corresponding, soluble monofunctional enzymes. A structure-based, substrate channeling pathway from the ECH active site to the HAD and KT sites is proposed. The passage from the ECH site to the HAD site is similar to those found in the two bacterial TFPs. However, the passage from the HAD site to the KT site is unique in that the acyl-CoA intermediate can be transferred between the two sites by passing along the mitochondrial inner membrane using the hydrophobic nature of the acyl chain. The 3'-AMP-PPi moiety is guided by the positively charged residues located along the "ceiling" of the channel, suggesting that membrane integrity is an essential part of the channel and is required for the activity of the enzyme.
    Keywords:  fatty acid oxidation; metabolon; mitochondrial trifunctional protein; substrate channeling
  60. Cell Metab. 2019 Mar 05. pii: S1550-4131(19)30074-9. [Epub ahead of print]29(3): 505
    Mott R, Fabbiano S, Levinson R, Emambokus N.
  61. Proc Biol Sci. 2019 Mar 13. 286(1898): 20190098
    Sambamoorthy G, Sinha H, Raman K.
      Microorganisms are ubiquitous and adapt to various dynamic environments to sustain growth. These adaptations accumulate, generating new traits forming the basis of evolution. Organisms adapt at various levels, such as gene regulation, signalling, protein-protein interactions and metabolism. Of these, metabolism forms the integral core of an organism for maintaining the growth and function of a cell. Therefore, studying adaptations in metabolic networks is crucial to understand the emergence of novel metabolic capabilities. Metabolic networks, composed of enzyme-catalysed reactions, exhibit certain repeating paradigms or design principles that arise out of different selection pressures. In this review, we discuss the design principles that are known to exist in metabolic networks, such as functional redundancy, modularity, flux coupling and exaptations. We elaborate on the studies that have helped gain insights highlighting the interplay of these design principles and adaptation. Further, we discuss how evolution plays a role in exploiting such paradigms to enhance the robustness of organisms. Looking forward, we predict that with the availability of ever-increasing numbers of bacterial, archaeal and eukaryotic genomic sequences, novel design principles will be identified, expanding our understanding of these paradigms shaped by varied evolutionary processes.
    Keywords:  adaptation; design principles; metabolic networks; robustness; systems biology
  62. Nature. 2019 Mar 06.
    Lu E, Wolfreys FD, Muppidi JR, Xu Y, Cyster JG.
      Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a Gα13-coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues1,2. P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma1,3-6, and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-L-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues.
  63. Cell Stem Cell. 2019 Mar 07. pii: S1934-5909(19)30062-1. [Epub ahead of print]24(3): 405-418.e7
    Vannini N, Campos V, Girotra M, Trachsel V, Rojas-Sutterlin S, Tratwal J, Ragusa S, Stefanidis E, Ryu D, Rainer PY, Nikitin G, Giger S, Li TY, Semilietof A, Oggier A, Yersin Y, Tauzin L, Pirinen E, Cheng WC, Ratajczak J, Canto C, Ehrbar M, Sizzano F, Petrova TV, Vanhecke D, Zhang L, Romero P, Nahimana A, Cherix S, Duchosal MA, Ho PC, Deplancke B, Coukos G, Auwerx J, Lutolf MP, Naveiras O.
      It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD+-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD+-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.
    Keywords:  HSC; UPRmt; aplastic anemia; asymmetric stem cell division; autophagy; bone marrow aplasia; bone marrow failure; bone marrow transplantation; chemotherapy; hematopoietic stem cell; hematopoietic stem cell transplantation; human CD34+ progenitors; immune thrombocytopenia; long-term hematopoietic stem cell; mitochondria; mitochondrial clearance; mitochondrial recycling; mitonuclear protein imbalance; mitophagy; myelodysplasia; myelodysplastic syndrome; radiotherapy; unfolded protein response mitochondria