bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2020‒06‒14
forty-four papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit


  1. Mol Cell. 2020 Jun 02. pii: S1097-2765(20)30319-1. [Epub ahead of print]
    Bose S, Allen AE, Locasale JW.
      Malignant cells remodel their metabolism to meet the demands of uncontrolled cell proliferation. These demands lead to differential requirements in energy, biosynthetic precursors, and signaling intermediates. Both genetic programs arising from oncogenic events and transcriptional programs and epigenomic events are important in providing the necessary metabolic network activity. Accumulating evidence has established that environmental factors play a major role in shaping cancer cell metabolism. For metabolism, diet and nutrition are the major environmental aspects and have emerged as key components in determining cancer cell metabolism. In this review, we discuss these emerging concepts in cancer metabolism and how diet and nutrition influence cancer cell metabolism.
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.018
  2. Cell Metab. 2020 Jun 02. pii: S1550-4131(20)30257-6. [Epub ahead of print]
    Ibrahim A, Yucel N, Kim B, Arany Z.
      Most organs use fatty acids (FAs) as a key nutrient, but little is known of how blood-borne FAs traverse the endothelium to reach underlying tissues. We conducted a small-molecule screen and identified niclosamide as a suppressor of endothelial FA uptake and transport. Structure/activity relationship studies demonstrated that niclosamide acts through mitochondrial uncoupling. Inhibitors of oxidative phosphorylation and the ATP/ADP translocase also suppressed FA uptake, pointing principally to ATP production. Decreasing total cellular ATP by blocking glycolysis did not decrease uptake, indicating that specifically mitochondrial ATP is required. Endothelial FA uptake is promoted by fatty acid transport protein 4 (FATP4) via its ATP-dependent acyl-CoA synthetase activity. Confocal microscopy revealed that FATP4 resides in the endoplasmic reticulum (ER), and that endothelial ER is intimately juxtaposed with mitochondria. Together, these data indicate that mitochondrial ATP production, but not total ATP levels, drives endothelial FA uptake and transport via acyl-CoA formation in mitochondrial/ER microdomains.
    Keywords:  ATP; FATP4; endothelial; fatty acid; mitochondria; niclosamide; vectorial acylation
    DOI:  https://doi.org/10.1016/j.cmet.2020.05.018
  3. Nat Commun. 2020 Jun 09. 11(1): 2894
    Yap YW, Rusu PM, Chan AY, Fam BC, Jungmann A, Solon-Biet SM, Barlow CK, Creek DJ, Huang C, Schittenhelm RB, Morgan B, Schmoll D, Kiens B, Piper MDW, Heikenwälder M, Simpson SJ, Bröer S, Andrikopoulos S, Müller OJ, Rose AJ.
      Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.
    DOI:  https://doi.org/10.1038/s41467-020-16568-z
  4. Exp Biol Med (Maywood). 2020 Jun 09. 1535370220932856
    Lee WH, Bhute VJ, Higuchi H, Ikeda S, Palecek SP, Ikeda A.
      IMPACT STATEMENT: Mitochondria are dynamic organelles undergoing fission and fusion. Proper regulation of this process is important for healthy aging process, as aberrant mitochondrial dynamics are associated with several age-related diseases/pathologies. However, it is not well understood how imbalanced mitochondrial dynamics may lead to those diseases and pathologies. Here, we aimed to determine metabolic alterations in tissues and cells from mouse models with over-fused (fusion > fission) and over-fragmented (fusion < fission) mitochondria that display age-related disease pathologies. Our results indicated tissue-dependent sensitivity to these mitochondrial changes, and metabolic pathways likely affected by aberrant mitochondrial dynamics. This study provides new insights into how dysregulated mitochondrial dynamics could lead to functional abnormalities of tissues and cells.
    Keywords:  Mitochondrial dynamics; Tmem135; heart; metabolomics; mouse models; retinal pigment epithelium
    DOI:  https://doi.org/10.1177/1535370220932856
  5. Cell Death Dis. 2020 Jun 09. 11(6): 444
    Sreedhar A, Aguilera-Aguirre L, Singh KK.
      The skin is a high turnover organ, and its constant renewal depends on the rapid proliferation of its progenitor cells. The energy requirement for these metabolically active cells is met by mitochondrial respiration, an ATP generating process driven by a series of protein complexes collectively known as the electron transport chain (ETC) that is located on the inner membrane of the mitochondria. However, reactive oxygen species (ROS) like superoxide, singlet oxygen, peroxides are inevitably produced during respiration and disrupt macromolecular and cellular structures if not quenched by the antioxidant system. The oxidative damage caused by mitochondrial ROS production has been established as the molecular basis of multiple pathophysiological conditions, including aging and cancer. Not surprisingly, the mitochondria are the primary organelle affected during chronological and UV-induced skin aging, the phenotypic manifestations of which are the direct consequence of mitochondrial dysfunction. Also, deletions and other aberrations in the mitochondrial DNA (mtDNA) are frequent in photo-aged skin and skin cancer lesions. Recent studies have revealed a more innate role of the mitochondria in maintaining skin homeostasis and pigmentation, which are affected when the essential mitochondrial functions are impaired. Some common and rare skin disorders have a mitochondrial involvement and include dermal manifestations of primary mitochondrial diseases as well as congenital skin diseases caused by damaged mitochondria. With studies increasingly supporting the close association between mitochondria and skin health, its therapeutic targeting in the skin-either via an ATP production boost or free radical scavenging-has gained attention from clinicians and aestheticians alike. Numerous bioactive compounds have been identified that improve mitochondrial functions and have proved effective against aged and diseased skin. In this review, we discuss the essential role of mitochondria in regulating normal and abnormal skin physiology and the possibility of targeting this organelle in various skin disorders.
    DOI:  https://doi.org/10.1038/s41419-020-2649-z
  6. Dev Cell. 2020 Jun 04. pii: S1534-5807(20)30403-2. [Epub ahead of print]
    Wang TS, Coppens I, Saorin A, Brady NR, Hamacher-Brady A.
      Mitochondrial outer membrane permeabilization (MOMP) is a core event in apoptosis signaling. However, the underlying mechanism of BAX and BAK pore formation remains incompletely understood. We demonstrate that mitochondria are globally and dynamically targeted by endolysosomes (ELs) during MOMP. In response to pro-apoptotic BH3-only protein signaling and pharmacological MOMP induction, ELs increasingly form transient contacts with mitochondria. Subsequently, ELs rapidly accumulate within the entire mitochondrial compartment. This switch-like accumulation period temporally coincides with mitochondrial BAX clustering and cytochrome c release. Remarkably, interactions of ELs with mitochondria control BAX recruitment and pore formation. Knockdown of Rab5A, Rab5C, or USP15 interferes with EL targeting of mitochondria and functionally uncouples BAX clustering from cytochrome c release, while knockdown of the Rab5 exchange factor Rabex-5 impairs both BAX clustering and cytochrome c release. Together, these data reveal that EL-mitochondrial inter-organelle communication is an integral regulatory component of functional MOMP execution during cellular apoptosis signaling.
    Keywords:  Apoptosis; BCL-2-associated X protein (BAX); Rab5; Rabex-5/RabGEF1; USP15; endolysosomes; endosomes; mitochondria; mitochondrial outer membrane permeabilization (MOMP); regulated cell death
    DOI:  https://doi.org/10.1016/j.devcel.2020.05.014
  7. Nat Commun. 2020 Jun 10. 11(1): 2932
    Khawaja A, Itoh Y, Remes C, Spåhr H, Yukhnovets O, Höfig H, Amunts A, Rorbach J.
      Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.
    DOI:  https://doi.org/10.1038/s41467-020-16503-2
  8. Front Oncol. 2020 ;10 660
    Klepinin A, Zhang S, Klepinina L, Rebane-Klemm E, Terzic A, Kaambre T, Dzeja P.
      A hallmark of cancer cells is the ability to rewire their bioenergetics and metabolic signaling circuits to fuel their uncontrolled proliferation and metastasis. Adenylate kinase (AK) is the critical enzyme in the metabolic monitoring of cellular adenine nucleotide homeostasis. It also directs AK→ AMP→ AMPK signaling controlling cell cycle and proliferation, and ATP energy transfer from mitochondria to distribute energy among cellular processes. The significance of AK isoform network in the regulation of a variety of cellular processes, which include cell differentiation and motility, is rapidly growing. Adenylate kinase 2 (AK2) isoform, localized in intermembrane and intra-cristae space, is vital for mitochondria nucleotide exchange and ATP export. AK2 deficiency disrupts cell energetics, causes severe human diseases, and is embryonically lethal in mice, signifying the importance of catalyzed phosphotransfer in cellular energetics. Suppression of AK phosphotransfer and AMP generation in cancer cells and consequently signaling through AMPK could be an important factor in the initiation of cancerous transformation, unleashing uncontrolled cell cycle and growth. Evidence also builds up that shift in AK isoforms is used later by cancer cells for rewiring energy metabolism to support their high proliferation activity and tumor progression. As cell motility is an energy-consuming process, positioning of AK isoforms to increased energy consumption sites could be an essential factor to incline cancer cells to metastases. In this review, we summarize recent advances in studies of the significance of AK isoforms involved in cancer cell metabolism, metabolic signaling, metastatic potential, and a therapeutic target.
    Keywords:  adenylate kinase; cancer; energy metabolism; mitochondria; phosphotransfer
    DOI:  https://doi.org/10.3389/fonc.2020.00660
  9. Front Oncol. 2020 ;10 792
    Sun NY, Yang MH.
      Metabolic reprogramming and epithelial-mesenchymal plasticity are both hallmarks of the adaptation of cancer cells for tumor growth and progression. For metabolic changes, cancer cells alter metabolism by utilizing glucose, lipids, and amino acids to meet the requirement of rapid proliferation and to endure stressful environments. Dynamic changes between the epithelial and mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are critical steps for cancer invasion and metastatic colonization. Compared to the extensively studied metabolic reprogramming in tumorigenesis, the metabolic changes in metastasis are relatively unclear. Here, we review metabolic reprogramming, epithelial-mesenchymal plasticity, and their mutual influences on tumor cells. We also review the developing treatments for targeting cancer metabolism and the impact of metabolic targeting on EMT. In summary, understanding the metabolic adaption and phenotypic plasticity will be mandatory for developing new strategies to target metastatic and refractory cancers that are intractable to current treatments.
    Keywords:  aerobic glycolysis; cancer metabolism; drug resistance; epithelial-mesenchymal plasticity; metastasis
    DOI:  https://doi.org/10.3389/fonc.2020.00792
  10. JCI Insight. 2020 Jun 09. pii: 129070. [Epub ahead of print]
    Soulet F, Bodineau C, Hooks KB, Descarpentrie J, Alves ID, Dubreuil M, Mouchard A, Eugenie M, Hoepffner JL, Lopez JJ, Rosado JA, Soubeyran I, Tomé M, Durán RV, Nikolski M, Villoutreix BO, Evrard S, Siegfried G, Khatib AM.
      Apelin is a well-established mediator of survival and mitogenic signalling through apelin receptor (Aplnr) and have been implicated in various cancers, however little is known regarding Elabela (ELA/APELA) signalling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here we identify a function of mTORC1 signalling as an essential mediator of ELA that represses kidney tumour cells growth, migration and survival. Moreover, sunitinib and ELA show synergistic effect in repressing tumour growth and angiogenesis in mice. The use of site directed mutagenesis and pharmacologic experiments provide evidence that the alteration of the cleavage site of proELA by Furin induced improved ELA anti-tumorigenic activity. Finally, cohort of tumours and public data sets revealed that ELA is only repressed in the main human kidney cancer subtypes namely clear cell, papillary, and chromophobe renal cell carcinoma. While Aplnr is expressed by various kidney cells, ELA is generally expressed by epithelial cells. Collectively, these results show the tumour-suppressive role of mTORC1 signalling mediated by ELA and establish the potential use of ELA or derivatives in kidney cancers treatment.
    Keywords:  Calcium; Nephrology
    DOI:  https://doi.org/10.1172/jci.insight.129070
  11. Cells. 2020 Jun 05. pii: E1407. [Epub ahead of print]9(6):
    Chvanov M, Voronina S, Zhang X, Telnova S, Chard R, Ouyang Y, Armstrong J, Tanton H, Awais M, Latawiec D, Sutton R, Criddle DN, Tepikin AV.
      Acute pancreatitis is a frequent disease that lacks specific drug treatment. Unravelling the molecular mechanisms of acute pancreatitis is essential for the development of new therapeutics. Several inducers of acute pancreatitis trigger sustained Ca2+ increases in the cytosol and mitochondria of pancreatic acinar cells. The mitochondrial calcium uniporter (MCU) mediates mitochondrial Ca2+ uptake that regulates bioenergetics and plays an important role in cell survival, damage and death. Aberrant Ca2+ signaling and mitochondrial damage in pancreatic acinar cells have been implicated in the initiation of acute pancreatitis. The primary aim of this study was to assess the involvement of the MCU in experimental acute pancreatitis. We found that pancreatic acinar cells from MCU-/- mice display dramatically reduced mitochondrial Ca2+ uptake. This is consistent with the drastic changes of stimulus-metabolism coupling, manifested by the reduction of mitochondrial NADH/FAD+ responses to cholecystokinin and in the decrease of cholecystokinin-stimulated oxygen consumption. However, in three experimental models of acute pancreatitis (induced by caerulein, taurolithocholic acid 3-sulfate or palmitoleic acid plus ethanol), MCU knockout failed to reduce the biochemical and histological changes characterizing the severity of local and systemic damage. A possible explanation of this surprising finding is the redundancy of damaging mechanisms activated by the inducers of acute pancreatitis.
    Keywords:  Ca2+ signaling; acute pancreatitis; mitochondrial calcium uniporter; pancreatic acinar cells
    DOI:  https://doi.org/10.3390/cells9061407
  12. Cell Stem Cell. 2020 Jun 10. pii: S1934-5909(20)30206-X. [Epub ahead of print]
    Zhu H, Blum RH, Bernareggi D, Ask EH, Wu Z, Hoel HJ, Meng Z, Wu C, Guan KL, Malmberg KJ, Kaufman DS.
      Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.
    Keywords:  CISH; IL-15; JAK-STAT; acute myelogenous leukemia; cell therapy; iPSCs; immunotherapy; mTOR; metabolic reprograming; natural killer cells
    DOI:  https://doi.org/10.1016/j.stem.2020.05.008
  13. Cancer Lett. 2020 Jun 05. pii: S0304-3835(20)30296-2. [Epub ahead of print]
    Bacigalupa ZA, Rathmell WK.
      The relationship between kidney cancer, specifically clear cell renal cell carcinoma (ccRCC), and the hypoxia signaling program has been extensively characterized. Its underlying role as the primary driver of the disease has led to the development of the most effective targeted therapies to date. Cellular responses to hypoxia or mutations affecting the von Hippel-Lindau (VHL) tumor suppressor gene stabilize the hypoxia inducible factor (HIF) transcription factors which then orchestrate elaborate downstream signaling events resulting in adaptations to key biological processes, such as reprogramming metabolism. The direct link of hypoxia signaling to glucose uptake and glycolysis has long been appreciated; however, the HIF family of proteins directly regulate many downstream targets, including other transcription factors with their own extensive networks. In this review, we will summarize our current understanding of how hypoxia signaling regulates other metabolic pathways and how this contributes to the development and progression of clear cell renal cell carcinomas.
    Keywords:  HIF; Metabolism; Mitochondria; Pseudohypoxia; ccRCC
    DOI:  https://doi.org/10.1016/j.canlet.2020.05.034
  14. Front Endocrinol (Lausanne). 2020 ;11 319
    Martínez J, Marmisolle I, Tarallo D, Quijano C.
      Secretion is an energy consuming process that plays a relevant role in cell communication and adaptation to the environment. Among others, endocrine cells producing hormones, immune cells producing cytokines or antibodies, neurons releasing neurotransmitters at synapsis, and more recently acknowledged, senescent cells synthesizing and secreting multiple cytokines, growth factors and proteases, require energy to successfully accomplish the different stages of the secretion process. Calcium ions (Ca2+) act as second messengers regulating secretion in many of these cases. In this setting, mitochondria appear as key players providing ATP by oxidative phosphorylation, buffering Ca2+ concentrations and acting as structural platforms. These tasks also require the concerted actions of the mitochondrial dynamics machinery. These proteins mediate mitochondrial fusion and fission, and are also required for transport and tethering of mitochondria to cellular organelles where the different steps of the secretion process take place. Herein we present a brief overview of mitochondrial energy metabolism, mitochondrial dynamics, and the different steps of the secretion processes, along with evidence of the interaction between these pathways. We also analyze the role of mitochondria in secretion by different cell types in physiological and pathological settings.
    Keywords:  ATP; bioenergetics; calcium; dynamics; endoplasmic reticulum; exocytosis; mitochondria; secretion
    DOI:  https://doi.org/10.3389/fendo.2020.00319
  15. Front Physiol. 2020 ;11 515
    Quiles JM, Gustafsson ÅB.
      Mitochondrial dysfunction is a hallmark of cardiac pathophysiology. Defects in mitochondrial performance disrupt contractile function, overwhelm myocytes with reactive oxygen species (ROS), and transform these cellular powerhouses into pro-death organelles. Thus, quality control (QC) pathways aimed at identifying and removing damaged mitochondrial proteins, components, or entire mitochondria are crucial processes in post-mitotic cells such as cardiac myocytes. Almost all of the mitochondrial proteins are encoded by the nuclear genome and the trafficking of these nuclear-encoded proteins necessitates significant cross-talk with the cytosolic protein QC machinery to ensure that only functional proteins are delivered to the mitochondria. Within the organelle, mitochondria contain their own protein QC system consisting of chaperones and proteases. This system represents another level of QC to promote mitochondrial protein folding and prevent aggregation. If this system is overwhelmed, a conserved transcriptional response known as the mitochondrial unfolded protein response is activated to increase the expression of proteins involved in restoring mitochondrial proteostasis. If the mitochondrion is beyond repair, the entire organelle must be removed before it becomes cytotoxic and causes cellular damage. Recent evidence has also uncovered mitochondria as participants in cytosolic protein QC where misfolded cytosolic proteins can be imported and degraded inside mitochondria. However, this process also places increased pressure on mitochondrial QC pathways to ensure that the imported proteins do not cause mitochondrial dysfunction. This review is focused on discussing the pathways involved in regulating mitochondrial QC and their relationship to cellular proteostasis and mitochondrial health in the heart.
    Keywords:  Parkin; UPR; UPS; import; mitochondria; mitophagy; proteasome; proteotoxicity
    DOI:  https://doi.org/10.3389/fphys.2020.00515
  16. Sci Transl Med. 2020 Jun 10. pii: eaay2163. [Epub ahead of print]12(547):
    Cissé MY, Pyrdziak S, Firmin N, Gayte L, Heuillet M, Bellvert F, Fuentes M, Delpech H, Riscal R, Arena G, Chibon F, Le Gellec S, Maran-Gonzalez A, Chateau MC, Theillet C, Carrere S, Portais JC, Le Cam L, Linares LK.
      Well-differentiated and dedifferentiated liposarcomas (LPSs) are characterized by a systematic amplification of the MDM2 oncogene, which encodes a key negative regulator of the p53 pathway. The molecular mechanisms underlying MDM2 overexpression while sparing wild-type p53 in LPS remain poorly understood. Here, we show that the p53-independent metabolic functions of chromatin-bound MDM2 are exacerbated in LPS and mediate an addiction to serine metabolism that sustains nucleotide synthesis and tumor growth. Treatment of LPS cells with Nutlin-3A, a pharmacological inhibitor of the MDM2-p53 interaction, stabilized p53 but unexpectedly enhanced MDM2-mediated control of serine metabolism by increasing its recruitment to chromatin, likely explaining the poor clinical efficacy of this class of MDM2 inhibitors. In contrast, genetic or pharmacological inhibition of chromatin-bound MDM2 by SP141, a distinct MDM2 inhibitor triggering its degradation, or interfering with de novo serine synthesis, impaired LPS growth both in vitro and in clinically relevant patient-derived xenograft models. Our data indicate that targeting MDM2 functions in serine metabolism represents a potential therapeutic strategy for LPS.
    DOI:  https://doi.org/10.1126/scitranslmed.aay2163
  17. J Exp Med. 2020 Sep 07. pii: e20200388. [Epub ahead of print]217(9):
    Recouvreux MV, Moldenhauer MR, Galenkamp KMO, Jung M, James B, Zhang Y, Lowy A, Bagchi A, Commisso C.
      Tumor cells rely on glutamine to fulfill their metabolic demands and sustain proliferation. The elevated consumption of glutamine can lead to intratumoral nutrient depletion, causing metabolic stress that has the potential to impact tumor progression. Here, we show that nutrient stress caused by glutamine deprivation leads to the induction of epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, we demonstrate that glutamine deficiency regulates EMT through the up-regulation of the EMT master regulator Slug, a process that is dependent on both MEK/ERK signaling and ATF4. We find that Slug is required in PDAC cells for glutamine deprivation-induced EMT, cell motility, and nutrient stress survival. Importantly, we decipher that Slug is associated with nutrient stress in PDAC tumors and is required for metastasis. These results delineate a novel role for Slug in the nutrient stress response and provide insight into how nutrient depletion might influence PDAC progression.
    DOI:  https://doi.org/10.1084/jem.20200388
  18. Autophagy. 2020 Jun 09. 1-19
    Ponsford AH, Ryan TA, Raimondi A, Cocucci E, Wycislo SA, Fröhlich F, Swan LE, Stagi M.
      Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines.ABBREVIATIONS: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A1; BLOC-1: biogenesis of lysosome-related organelles complex 1; BSA: bovine serum albumin; Cos7: African green monkey kidney fibroblast-like cell line; CQ: chloroquine; CTSB: cathepsin B; CYCS: cytochrome c, somatic; DAPI: 4',6-diamidino -2- phenylindole; DIC: differential interference contrast; DIV: days in vitro; DMEM: Dulbecco's modified Eagle's medium;‎ E8: embryonic day 8; EEA1: early endosome antigen 1; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GTP: guanosine triphosphate; HEK293T: human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen; HeLa: Henrietta Lacks-derived cell; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: horseradish peroxidase; IGF2R/ciM6PR: insulin like growth factor 2 receptor; LAMP1/2: lysosomal associated membrane protein 1/2; LMAN2/VIP36: lectin, mannose binding 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PCR: polymerase chain reaction; PDL: poly-d-lysine; PGK1p: promotor from human phosphoglycerate kinase 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPT1/CLN1: palmitoyl-protein thioesterase 1; RPS6KB1/p70: ribosomal protein S6 kinase B1; STAT3: signal transducer and activator of transcription 3; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGN: trans-Golgi network; TGOLN2/TGN46: trans-Golgi network protein 2; TIRF: total internal reflection fluorescence; TMEM106B: transmembrane protein 106B; TOR: target of rapamycin; TRPM2: transient receptor potential cation channel subfamily M member 2; V-ATPase: vacuolar-type proton-translocating ATPase; VPS35: VPS35 retromer complex component.
    Keywords:  Chloroquine; MTOR protein; V-type ATPase; fluorescence microscopy; lysosomes; pH
    DOI:  https://doi.org/10.1080/15548627.2020.1771858
  19. J Biol Chem. 2020 Jun 09. pii: jbc.REV120.011438. [Epub ahead of print]
    Klein MA, Denu JM.
      Sirtuin 6 (SIRT6) is a nuclear NAD+-dependent deacetylase of histone H3 that regulates genome stability and gene expression. However, non-histone substrates and additional catalytic activities of SIRT6, including long-chain deacylation and mono-ADP-ribosylation of other proteins have also been reported, but many of these non-canonical roles remain enigmatic. Genetic studies have revealed critical homeostatic cellular functions of SIRT6, underscoring the need to better understand which catalytic functions and molecular pathways are driving SIRT6-associated phenotypes. At the physiological level, SIRT6 activity promotes increased longevity by regulating metabolism andDNA repair. Recent work has identified natural products and synthetic small-molecules capable of activating the inefficient in vitrodeacetylase activity of SIRT6. Here, we discuss the cellular functions of SIRT6 with a focus on attributing its catalytic activity to its proposed biological functions.  We cover the molecular architecture and catalytic mechanisms that distinguish SIRT6 from other NAD+-dependent deacylases. We propose that combining specific SIRT6 amino-acid substitutions identified in enzymology studies, and activity-selective compounds could help delineate SIRT6 functions in specific biological contexts and resolve the apparently conflicting roles of SIRT6 in processes such as tumor development. We further highlight the recent development of small-molecule modulators that provide additional biologicalinsight into SIRT6 functions and offer therapeutic approaches to manage metabolic and age-associated diseases.
    Keywords:  activator; aging; cancer; cell metabolism; chromatin; gene expression; histone deacetylase (HDAC); longevity; metabolic disorder; sirt6; sirtuin; small molecule
    DOI:  https://doi.org/10.1074/jbc.REV120.011438
  20. Cell Biol Toxicol. 2020 Jun 08.
    Wang W, Liu X, Wang X.
      Mitochondrial DNA (mtDNA) plays an important role in regulating mitochondrial homeostasis, transcription, cell metabolism, and drug sensitivity. Patterns and regulations of mtDNA methylation vary among cell types, species, functions, and diseases. High-resolution mtDNA methylation maps of human and animal mitochondrial genomes addressed that the light (L)-strand non-CpG methylation of mtDNA varied among species, developing stages, and ages. Of DNA methyltransferases, DNMT3A was a critical enzyme in the dynamic regulation of mtDNA regional methylation patterns and strand bias. Altered mtDNA methylations may regulate dynamic occurrences of pathogenic mtDNA mutations. The number and sites of control regions, involved enzymes, and regulators during mtDNA methylation may vary among cell types and diseases. Specific regulatory and functional networks associated with mtDNA methylation mainly include mtDNA, regulatory factors, methyltransferases, nucleotides, mt-rRNAs, and other epigenetic modifications. Those carry out precise functions and regulations of mtDNA methylation-associated network, interactions with genome DNA and other signal pathways, and decisive roles in patient phenomes. A breakthrough in mtDNA methylation-associated networks will be a crucial milestone in the journey of understanding mitochondrial function at a higher level and discovering new mitochondria-based biomarkers and therapeutic targets.
    Keywords:  Epigenetics; Methylation; Mitochondria; Mitochondrial DNA; Networks
    DOI:  https://doi.org/10.1007/s10565-020-09539-z
  21. Trends Analyt Chem. 2019 Nov;pii: 115322. [Epub ahead of print]120
    Lane AN, Higashi RM, Fan TW.
      There is considerable interest in defining metabolic reprogramming in human diseases, which is recognized as a hallmark of human cancer. Although radiotracers have a long history in specific metabolic studies, stable isotope-enriched precursors coupled with modern high resolution mass spectrometry and NMR spectroscopy have enabled systematic mapping of metabolic networks and fluxes in cells, tissues and living organisms including humans. These analytical platforms are high in information content, are complementary and cross-validating in terms of compound identification, quantification, and isotope labeling pattern analysis of a large number of metabolites simultaneously. Furthermore, new developments in chemoselective derivatization and in vivo spectroscopy enable tracking of labile/low abundance metabolites and metabolic kinetics in real-time. Here we review developments in Stable Isotope Resolved Metabolomics (SIRM) and recent applications in cancer metabolism using a wide variety of stable isotope tracers that probe both broad and specific aspects of cancer metabolism required for proliferation and survival.
    Keywords:  NMR; SIRM; cancer metabolism; mass spectrometry; model systems
    DOI:  https://doi.org/10.1016/j.trac.2018.11.020
  22. Elife. 2020 Jun 12. pii: e53247. [Epub ahead of print]9
    Tiwari SK, Toshniwal AG, Mandal S, Mandal L.
      Cell-intrinsic and extrinsic signals regulate the state and fate of stem and progenitor cells. Recent advances in metabolomics illustrate that various metabolic pathways are also important in regulating stem cell fate. However, our understanding of the metabolic control of the state and fate of progenitor cells is in its infancy. Using Drosophila hematopoietic organ: lymph gland, we demonstrate that Fatty Acid Oxidation (FAO) is essential for the differentiation of blood cell progenitors. In the absence of FAO, the progenitors are unable to differentiate and exhibit altered histone acetylation. Interestingly, acetate supplementation rescues both histone acetylation and the differentiation defects. We further show that the CPT1/whd (withered), the rate-limiting enzyme of FAO, is transcriptionally regulated by Jun-Kinase (JNK), which has been previously implicated in progenitor differentiation. Our study thus reveals how the cellular signaling machinery integrates with the metabolic cue to facilitate the differentiation program.
    Keywords:  D. melanogaster; developmental biology
    DOI:  https://doi.org/10.7554/eLife.53247
  23. Antioxid Redox Signal. 2020 Jun 10.
    Plecitá-Hlavatá L, Engstová H, Holendova B, Tauber J, Spacek T, Petrásková L, Kren V, Špačková J, Gotvaldová K, Jezek J, Dlasková A, Smolková K, Jezek P.
      AIMS: Glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria.RESULTS: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H<sub>2</sub>O<sub>2</sub> (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH<sub>F</sub> indicated its ~20% decrease. Matrix NAD<sup>+</sup><sub>F</sub> increased upon GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD<sup>+</sup><sub>F</sub>. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH<sub>F</sub> (iNAP1 fluorescence monitoring) at the expense of matrix NADH<sub>F</sub>, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: all changes vanished upon these manipulations. <sup>13</sup>C-incorporation from <sup>13</sup>C-1-glutamine into <sup>13</sup>C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH<sub>F</sub>, (iNAP3 monitored) decreased. With decreasing glucose, the S3QEL suppressor caused a higher Complex I I<sub>F</sub> site contribution, but a lower superoxide fraction ascribed to the Complex III site III<sub>Qo</sub>. Thus the diminished matrix NADH<sub>F</sub>/NAD<sup>+</sup><sub>F</sub> decreased Complex I flavin site I<sub>F</sub> superoxide formation upon GSIS.
    INNOVATION: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β-cells upon GSIS, due to the decreasing matrix NADH<sub>F</sub>/NAD<sup>+</sup><sub>F</sub> (NADPH<sub>F</sub>/NADP<sup>+</sup><sub>F</sub>) at increasing cytosolic NADPH<sub>F</sub> levels. The developed innovative methods enable real-time NADH/NAD<sup>+</sup> and NADPH/NADP<sup>+</sup> monitoring in any distinct cell compartment.
    CONCLUSION: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production upon GSIS, but does not prevent oxidative stress in pancreatic β-cells.
    DOI:  https://doi.org/10.1089/ars.2019.7800
  24. Methods Mol Biol. 2020 ;2159 129-140
    Samanas NB, Hoppins S.
      Dynamin-related proteins on both the mitochondrial outer and inner membranes mediate membrane fusion. Mitochondrial fusion is regulated in many different physiological contexts including cell cycle progression, differentiation pathways, stress responses, and cell death. Mitochondrial fusion is opposed by mitochondrial division and requires movement of mitochondria on microtubules. We developed a cell-free reconstituted mitochondrial fusion assay to circumvent the complexity of the pathways impinging on the activity of the mitochondrial fusion machinery in vivo. This allows for quantification of mitochondrial fusion in defined conditions and in the absence of other processes such as mitochondrial division or transport. The impact of proteins or small molecules on mitochondria fusion can also be assessed. Here we describe the cell-free mitochondrial fusion assay using mitochondria isolated from mouse embryonic fibroblasts.
    Keywords:  Cell-free; Fusion; Isolated mitochondria; Microscopy; Mitochondrial dynamics; Mitofusin; Opa1
    DOI:  https://doi.org/10.1007/978-1-0716-0676-6_10
  25. Nat Immunol. 2020 Jun 08.
    Zhou QD, Chi X, Lee MS, Hsieh WY, Mkrtchyan JJ, Feng AC, He C, York AG, Bui VL, Kronenberger EB, Ferrari A, Xiao X, Daly AE, Tarling EJ, Damoiseaux R, Scumpia PO, Smale ST, Williams KJ, Tontonoz P, Bensinger SJ.
      Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.
    DOI:  https://doi.org/10.1038/s41590-020-0695-4
  26. Nat Cell Biol. 2020 Jun 08.
    Martínez-Zamudio RI, Roux PF, de Freitas JANLF, Robinson L, Doré G, Sun B, Belenki D, Milanovic M, Herbig U, Schmitt CA, Gil J, Bischof O.
      Senescent cells affect many physiological and pathophysiological processes. While select genetic and epigenetic elements for senescence induction have been identified, the dynamics, epigenetic mechanisms and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding the deliberate therapeutic targeting of senescence for health benefits. Here, we examined the possibility that the epigenetic state of enhancers determines senescent cell fate. We explored this by generating time-resolved transcriptomes and epigenome profiles during oncogenic RAS-induced senescence and validating central findings in different cell biology and disease models of senescence. Through integrative analysis and functional validation, we reveal links between enhancer chromatin, transcription factor recruitment and senescence competence. We demonstrate that activator protein 1 (AP-1) 'pioneers' the senescence enhancer landscape and defines the organizational principles of the transcription factor network that drives the transcriptional programme of senescent cells. Together, our findings enabled us to manipulate the senescence phenotype with potential therapeutic implications.
    DOI:  https://doi.org/10.1038/s41556-020-0529-5
  27. Trends Biochem Sci. 2020 Jul;pii: S0968-0004(20)30085-2. [Epub ahead of print]45(7): 578-592
    Song S, Lam EW, Tchkonia T, Kirkland JL, Sun Y.
      Aging is a major risk factor for numerous human pathologies, including cardiovascular, metabolic, musculoskeletal, and neurodegenerative conditions and various malignancies. While our understanding of aging is far from complete, recent advances suggest that targeting fundamental aging processes can delay, prevent, or alleviate age-related disorders. Cellular senescence is physiologically beneficial in several contexts, but it has causal roles in multiple chronic diseases. New studies have illustrated the promising feasibility and safety to selectively ablate senescent cells from tissues, a therapeutic modality that holds potential for treating multiple chronic pathologies and extending human healthspan. Here, we review molecular links between cellular senescence and age-associated complications and highlight novel therapeutic avenues that may be exploited to target senescent cells in future geriatric medicine.
    Keywords:  SASP; cellular senescence; clinical trials; geroscience; senolytics
    DOI:  https://doi.org/10.1016/j.tibs.2020.03.008
  28. Redox Biol. 2020 May 26. pii: S2213-2317(20)30358-X. [Epub ahead of print]36 101568
    Brown JA, Sammy MJ, Ballinger SW.
      The incidence of common, metabolic diseases (e.g. obesity, cardiovascular disease, diabetes) with complex genetic etiology has been steadily increasing nationally and globally. While identification of a genetic model that explains susceptibility and risk for these diseases has been pursued over several decades, no clear paradigm has yet been found to disentangle the genetic basis of polygenic/complex disease development. Since the evolution of the eukaryotic cell involved a symbiotic interaction between the antecedents of the mitochondrion and nucleus (which itself is a genetic hybrid), we suggest that this history provides a rational basis for investigating whether genetic interaction and co-evolution of these genomes still exists. We propose that both mitochondrial and Mendelian, or "mito-Mendelian" genetics play a significant role in cell function, and thus disease risk. This paradigm contemplates the natural variation and co-evolution of both mitochondrial and nuclear DNA backgrounds on multiple mitochondrial functions that are discussed herein, including energy production, cell signaling and immune response, which collectively can influence disease development. At the nexus of these processes is the economy of mitochondrial metabolism, programmed by both mitochondrial and nuclear genomes.
    DOI:  https://doi.org/10.1016/j.redox.2020.101568
  29. Nat Commun. 2020 Jun 10. 11(1): 2926
    Verma K, Saxena K, Donaka R, Chaphalkar A, Rai MK, Shukla A, Zaidi Z, Dandage R, Shanmugam D, Chakraborty K.
      Metabolic changes alter the cellular milieu; can this also change intracellular protein folding? Since proteostasis can modulate mutational buffering, if change in metabolism has the ability to change protein folding, arguably, it should also alter mutational buffering. Here we find that altered cellular metabolic states in E. coli buffer distinct mutations on model proteins. Buffered-mutants have folding problems in vivo and are differently chaperoned in different metabolic states. Notably, this assistance is dependent upon the metabolites and not on the increase in canonical chaperone machineries. Being able to reconstitute the folding assistance afforded by metabolites in vitro, we propose that changes in metabolite concentrations have the potential to alter protein folding capacity. Collectively, we unravel that the metabolite pools are bona fide members of proteostasis and aid in mutational buffering. Given the plasticity in cellular metabolism, we posit that metabolic alterations may play an important role in cellular proteostasis.
    DOI:  https://doi.org/10.1038/s41467-020-16804-6
  30. Methods Mol Biol. 2020 ;2159 115-127
    Ban T, Ishihara N.
      Mitochondria are highly dynamic organelles, which move and fuse to regulate their shape, size, and fundamental function. The dynamin-related GTPases play a critical role in mitochondrial membrane fusion. In vitro reconstitution of membrane fusion using recombinant proteins and model membranes is quite useful in elucidating the molecular mechanisms underlying membrane fusion and to identify the essential elements involved in fusion. However, only a few reconstituting approaches have been reported for mitochondrial fusion machinery due to the difficulty of preparing active recombinant mitochondrial fusion GTPases. Recently, we succeeded in preparing a sufficient amount of recombinant OPA1 involved in mitochondrial inner membrane fusion using a BmNPV bacmid-silkworm expression system. In this chapter, we describe the method for the expression and purification of a membrane-anchored form of OPA1 and liposome-based in vitro reconstitution of membrane fusion.
    Keywords:  Baculovirus expression system; Fluorescence resonance energy transfer- (FRET-) based lipid mixing assay; GTPase protein; In vitro reconstitution; Membrane fusion; Mitochondria; Optic atrophy 1(OPA1); Proteoliposome; Silkworm
    DOI:  https://doi.org/10.1007/978-1-0716-0676-6_9
  31. Front Genet. 2020 ;11 510
    Cronan JE.
      Three human mitochondrial diseases that directly affect lipoic acid metabolism result from heterozygous missense and nonsense mutations in the LIAS, LIPT1, and LIPT2 genes. However, the functions of the proteins encoded by these genes in lipoic acid metabolism remained uncertain due to a lack of biochemical analysis at the enzyme level. An exception was the LIPT1 protein for which a perplexing property had been reported, a ligase lacking the ability to activate its substrate. This led to several models, some contradictory, to accommodate the role of LIPT1 protein activity in explaining the phenotypes of the afflicted neonatal patients. Recent evidence indicates that this LIPT1 protein activity is a misleading evolutionary artifact and that the physiological role of LIPT1 is in transfer of lipoic acid moieties from one protein to another. This and other new biochemical data now define a straightforward pathway that fully explains each of the human disorders specific to the assembly of lipoic acid on its cognate enzyme proteins.
    Keywords:  LIAS; LIPT1; LIPT2; glycine cleavage system; lipoate assembly; lipoic acid; pyruvate dehydrogenase; α-ketoglutarate dehydrogenase
    DOI:  https://doi.org/10.3389/fgene.2020.00510
  32. Cancer Discov. 2020 Jun 12.
      Metabolites produced in cancer cells interfered with resolution of DNA double-strand breaks.
    DOI:  https://doi.org/10.1158/2159-8290.CD-RW2020-089
  33. Cancer Discov. 2020 Jun 08. pii: CD-19-1262. [Epub ahead of print]
    Liu H, Paddock MN, Wang H, Murphy CJ, Geck RC, Navarro AJ, Wulf GM, Elemento O, Haucke V, Cantley LC, Toker A.
      Inactivation of the tumor suppressor lipid phosphatase INPP4B is common in triple negative breast cancer (TNBC). We generated a genetically-engineered TNBC mouse model deficient in INPP4B. We found a dose-dependent increase in tumor incidence in INPP4B homozygous and heterozygous knockout mice compared to wild-type, supporting a role for INPP4B as a tumor suppressor in TNBC. Tumors derived from INPP4B knockout mice are enriched for AKT and MEK gene signatures. Consequently, mice with INPP4B deficiency are more sensitive to PI3K or MEK inhibitors, compared to wild-type mice. Mechanistically, we found that INPP4B deficiency increases PI(3,4)P2 levels in endocytic vesicles but not at the plasma membrane. Moreover, INPP4B loss delays degradation of EGFR and MET, while promoting recycling of RTKs, thus enhancing the duration and amplitude of signaling output upon growth factor stimulation. Therefore, INPP4B inactivation in TNBC promotes tumorigenesis by modulating RTK recycling and signaling duration.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-1262
  34. Commun Biol. 2020 Jun 12. 3(1): 305
    Ahl PJ, Hopkins RA, Xiang WW, Au B, Kaliaperumal N, Fairhurst AM, Connolly JE.
      A complex interaction of anabolic and catabolic metabolism underpins the ability of leukocytes to mount an immune response. Their capacity to respond to changing environments by metabolic reprogramming is crucial to effector function. However, current methods lack the ability to interrogate this network of metabolic pathways at single-cell level within a heterogeneous population. We present Met-Flow, a flow cytometry-based method capturing the metabolic state of immune cells by targeting key proteins and rate-limiting enzymes across multiple pathways. We demonstrate the ability to simultaneously measure divergent metabolic profiles and dynamic remodeling in human peripheral blood mononuclear cells. Using Met-Flow, we discovered that glucose restriction and metabolic remodeling drive the expansion of an inflammatory central memory T cell subset. This method captures the complex metabolic state of any cell as it relates to phenotype and function, leading to a greater understanding of the role of metabolic heterogeneity in immune responses.
    DOI:  https://doi.org/10.1038/s42003-020-1027-9
  35. Redox Biol. 2020 May 30. pii: S2213-2317(20)30634-0. [Epub ahead of print]36 101599
    Seo BJ, Choi J, La H, Habib O, Choi Y, Hong K, Do JT.
      Mitochondria, the major organelles that produce energy for cell survival and function, dynamically change their morphology via fusion and fission, a process called mitochondrial dynamics. The details of the underlying mechanism of mitochondrial dynamics have not yet been elucidated. Here, we aimed to investigate the function of mitochondrial fission genes in embryonic stem cells (ESCs). To this end, we generated homozygous knockout ESC lines, namely, Fis1-/-, Mff-/-, and Dnm1l-/- ESCs, using the CRISPR-Cas9 system. Interestingly, the Fis1-/-, Mff-/-, and Dnm1l-/- ESCs showed normal morphology, self-renewal, and the ability to differentiate into all three germ layers in vitro. However, transmission electron microscopy showed a significant increase in the cytoplasm to nucleus ratio and mitochondrial elongation in Dnm1l-/- ESCs, which was due to incomplete fission. To assess the change in metabolic energy, we analyzed oxidative phosphorylation (OXPHOS), glycolysis, and the intracellular ATP concentration. The ESC knockout lines showed an increase in OXPHOS, decrease in glycolysis, and an increase in intracellular ATP concentration, which was related to mitochondrial elongation. In particular, the Dnm1l knockout most significantly affected mitochondrial morphology, energy metabolism, and ATP production in ESCs. Furthermore, RNA sequencing and gene ontology analysis showed that the differentially expressed genes in Mff-/- ESCs were distinct from those in Dnm1l-/- or Fis1-/- ESCs. In total, five metabolism-related genes, namely, Aass, Cdo1, Cyp2b23, Nt5e, and Pck2, were expressed in all three knockout ESC lines, and three of them were associated with regulation of ATP generation.
    Keywords:  Cellular metabolism; Dynamin 1 like (Dnm1l); Embryonic stem cells (ESCs); Knockout; Mitochondrial fission 1 protein (Fis1); Mitochondrial fission factor (Mff)
    DOI:  https://doi.org/10.1016/j.redox.2020.101599
  36. PLoS Biol. 2020 Jun 10. 18(6): e3000741
    Doleželová E, Kunzová M, Dejung M, Levin M, Panicucci B, Regnault C, Janzen CJ, Barrett MP, Butter F, Zíková A.
      Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.
    DOI:  https://doi.org/10.1371/journal.pbio.3000741
  37. Sci Signal. 2020 Jun 09. pii: eaaz2597. [Epub ahead of print]13(635):
    Lovisa S, Fletcher-Sananikone E, Sugimoto H, Hensel J, Lahiri S, Hertig A, Taduri G, Lawson E, Dewar R, Revuelta I, Kato N, Wu CJ, Bassett RL, Putluri N, Zeisberg M, Zeisberg EM, LeBleu VS, Kalluri R.
      Endothelial-to-mesenchymal transition (EndMT) is a cellular transdifferentiation program in which endothelial cells partially lose their endothelial identity and acquire mesenchymal-like features. Renal capillary endothelial cells can undergo EndMT in association with persistent damage of the renal parenchyma. The functional consequence(s) of EndMT in kidney fibrosis remains unexplored. Here, we studied the effect of Twist or Snail deficiency in endothelial cells on EndMT in kidney fibrosis. Conditional deletion of Twist1 (which encodes Twist) or Snai1 (which encodes Snail) in VE-cadherin+ or Tie1+ endothelial cells inhibited the emergence of EndMT and improved kidney fibrosis in two different kidney injury/fibrosis mouse models. Suppression of EndMT limited peritubular vascular leakage, reduced tissue hypoxia, and preserved tubular epithelial health and function. Hypoxia, which was exacerbated by EndMT, resulted in increased Myc abundance in tubular epithelial cells, enhanced glycolysis, and suppression of fatty acid oxidation. Pharmacological suppression or epithelial-specific genetic ablation of Myc in tubular epithelial cells ameliorated fibrosis and restored renal parenchymal function and metabolic homeostasis. Together, these findings demonstrate a functional role for EndMT in the response to kidney capillary endothelial injury and highlight the contribution of endothelial-epithelial cross-talk in the development of kidney fibrosis with a potential for therapeutic intervention.
    DOI:  https://doi.org/10.1126/scisignal.aaz2597
  38. Cell Metab. 2020 May 30. pii: S1550-4131(20)30242-4. [Epub ahead of print]
    Hsieh WY, Zhou QD, York AG, Williams KJ, Scumpia PO, Kronenberger EB, Hoi XP, Su B, Chi X, Bui VL, Khialeeva E, Kaplan A, Son YM, Divakaruni AS, Sun J, Smale ST, Flavell RA, Bensinger SJ.
      Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems-level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary "shotgun" and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. "Shotgun" lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity.
    Keywords:  MyD88; acetylated-LDL; host defense; inflammation; interferon; lipidomics; macrophages; stable isotope tracer analysis; stearoyl-CoA desaturase; toll-like receptors
    DOI:  https://doi.org/10.1016/j.cmet.2020.05.003
  39. Front Oncol. 2020 ;10 851
    Neagu M.
      Tumor microenvironment is a network of complex cellular and molecular systems where cells will gain specific phenotypes and specific functions that would drive tumorigenesis. In skin cancers, tumor microenvironment is characterized by tumor infiltrating immune cells that sustain immune suppression, mainly lymphocytes. Melanoma cellular heterogeneity can be described on genetic, proteomic, transcriptomic and metabolomic levels. Melanoma cells display a metabolic reprogramming triggered by both genetic alterations and adaptation to a microenvironment that lacks nutrients and oxygen supply. Tumor cells present clear metabolic adaptations and identifying deregulated glycolysis pathway could offer new therapy targets. Moreover, the immune cells (T lymphocytes, macrophages, NK cells, neutrophils and so on) that infiltrate melanoma tumors have metabolic particularities that, upon interaction within tumor microenvironment, would favor tumorigenesis. Analyzing both tumor cell metabolism and the metabolic outline of immune cells can offer innovative insights in new therapy targets and cancer therapeutical approaches. In addition to already approved immune- and targeted therapy in melanoma, approaching metabolic check-points could improve therapy efficacy and hinder resistance to therapy.
    Keywords:  immune cells; melanoma; metabolism; therapy; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2020.00851
  40. Curr Diab Rep. 2020 Jun 10. 20(8): 30
    Romeo GR, Jain M.
      PURPOSE OF REVIEW: This article reviews evidence linking cardiometabolic conditions with changes in purine metabolites, including increased serum uric acid (sUA), and discusses intervention studies that investigated the therapeutic relevance of these associations.RECENT FINDINGS: Metabolic and epidemiological findings support a correlation between sUA and circulating levels of other purines with insulin resistance (IR) and risk factors for cardiovascular disease (CVD). In addition, increased activity of xanthine oxidoreductase (XOR), the rate-limiting enzyme for UA production, has been detected in tissues targeted by obesity. Yet, inhibition of XOR in pre-clinical and clinical studies generally failed to support a causal role for excess sUA in IR and CVD. The lack of efficacy of XOR inhibitors strongly suggests that UA is a marker of, rather than a direct contributory factor for, cardiometabolic diseases. Validation of the function of other purines will require a paradigm shift, from a "UA-centric" view to a more granular assessment of the entire purine network and its interaction with other pathways.
    Keywords:  Allopurinol; Cardiovascular disease; Insulin resistance; Non-alcoholic fatty liver disease; Xanthine oxidoreductase
    DOI:  https://doi.org/10.1007/s11892-020-01313-z
  41. Nat Commun. 2020 Jun 10. 11(1): 2936
    Reich S, Nguyen CDL, Has C, Steltgens S, Soni H, Coman C, Freyberg M, Bichler A, Seifert N, Conrad D, Knobbe-Thomsen CB, Tews B, Toedt G, Ahrends R, Medenbach J.
      Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.
    DOI:  https://doi.org/10.1038/s41467-020-16747-y
  42. Proc Natl Acad Sci U S A. 2020 Jun 08. pii: 201916851. [Epub ahead of print]
    Wanrooij PH, Tran P, Thompson LJ, Carvalho G, Sharma S, Kreisel K, Navarrete C, Feldberg AL, Watt DL, Nilsson AK, Engqvist MKM, Clausen AR, Chabes A.
      Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1 -/- mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.
    Keywords:  SAMHD1; dNTP pool; mitochondrial DNA; mtDNA; ribonucleotide incorporation
    DOI:  https://doi.org/10.1073/pnas.1916851117
  43. J Theor Biol. 2020 Jun 06. pii: S0022-5193(20)30215-0. [Epub ahead of print] 110360
    Guerrero-Morín JG, Santillán M.
      Crosstalk between the circadian clock clockwork and cellular metabolic regulatory networks is crucial to ensure an adequate response of an organism to the day/night cycle. mTOR (mammalian/mechanistic target of rapamycin) is a master growth regulator and sensor of nutrient status, which is part of the mTOR complex 1 (mTORC1). While the circadian clock confers rhythmicity to the mTOR protein by regulating its degradation rate, mTORC1 activity diminishes period and augments amplitude of circadian oscillations at the cellular level by a currently unknown mechanism. Here, we develop a mathematical deterministic DAE (differential-algebraic equation) model, to explore the possible interactions that allow mTORC1 to display such regulation of the core circadian clock. Our results suggest that mTORC1 is capable of regulating amplitude by exerting translational control on core the clock protein BMAL1, and that period-tuning is achieved by controlling post-translational localization of BMAL1. Since, in our model, mTORC1 control of BMAL1 localization greatly diminishes the ability of the clock to oscillate, and regulation of BMAL1 translation reduces this effect, our results also suggest that both levels of regulation must be present to ensure the robustness of oscillations. Together, the above results emphasize the importance of the influence of mTORC1 on the circadian rhythms.
    DOI:  https://doi.org/10.1016/j.jtbi.2020.110360
  44. Trends Genet. 2020 Jun 09. pii: S0168-9525(20)30124-4. [Epub ahead of print]
    Song Y, Shvartsman SY.
      New studies of metabolic reactions and networks in embryos are making important additions to regulatory models of development, so far dominated by genes and signals. Metabolic control of development is not a new idea and can be traced back to Joseph Needham's 'Chemical Embryology', published in the 1930s. Even though Needham's ideas fell by the wayside with the advent of genetic studies of embryogenesis, they demonstrated that embryos provide convenient models for addressing fundamental questions in biochemistry and are now experiencing a comeback, enabled by the powerful merger of detailed mechanistic studies and systems-level techniques. Here we review recent results from studies that quantified the energy budget of embryogenesis in Drosophila and started to untangle the intricate connections between core anabolic processes and developmental transitions. Dynamic coordination of metabolic, genetic, and signaling networks appears to be essential for seamless progression of development.
    Keywords:  dNTP metabolism; embryonic cell cycle; embryonic energy budget; midblastula transition; negative feedback regulation; ribonucleotide reductase
    DOI:  https://doi.org/10.1016/j.tig.2020.05.007