bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒09‒12
37 papers selected by
Kelsey Fisher-Wellman
East Carolina University
  1. Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition.
  2. Pyruvate-Driven Oxidative Phosphorylation is Downregulated in Sepsis-Induced Cardiomyopathy: A Study Of Mitochondrial Proteome.
  3. MCU-complex-mediated mitochondrial calcium signaling is impaired in Barth syndrome.
  4. T cells critically depend on pyruvate oxidation.
  5. A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation.
  6. Targeting Mitochondrial Oxidative Phosphorylation in Glioblastoma Therapy.
  7. Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression.
  8. Mitochondrial dysfunction induces radioresistance in colorectal cancer by activating [Ca2+]m-PDP1-PDH-histone acetylation retrograde signaling.
  9. The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids.
  10. The Combined Treatment With the FLT3-Inhibitor AC220 and the Complex I Inhibitor IACS-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells.
  11. Functional basis of electron transport within photosynthetic complex I.
  12. DHX30 Coordinates Cytoplasmic Translation and Mitochondrial Function Contributing to Cancer Cell Survival.
  13. Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia.
  14. ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division.
  15. The Mitochondrial Ca2+ Uniporter is a Central Regulator of Interorganellar Ca2+ Transfer and NFAT Activation.
  16. Discovery of bactericides as an acute mitochondrial membrane damage inducer.
  17. The OMM-severed and IMM-ubiquitinated mitochondria are intermediates of mitochondrial proteotoxicity-induced autophagy in PRKN/parkin-deficient cells.
  18. Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells.
  19. Overcoming the compensatory elevation of NRF2 renders hepatocellular carcinoma cells more vulnerable to disulfiram/copper-induced ferroptosis.
  20. A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.
  21. A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.
  22. Dynamic Modeling of Mitochondrial Membrane Potential Upon Exposure to Mitochondrial Inhibitors.
  23. PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability.
  24. Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion.
  25. Low lysophosphatidylcholine induces skeletal muscle myopathy that is aggravated by high-fat diet feeding.
  26. Elucidating the molecular mechanisms associated with TARS2-related mitochondrial disease.
  27. High-throughput Single-cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.
  28. Autophagic flux in cancer cells at the invasive front in the tumor-stroma border.
  29. Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.
  30. Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium.
  31. Revisiting energy expenditure: how to correct mouse metabolic rate for body mass.
  32. Diurnal regulation of oxidative phosphorylation restricts hepatocyte proliferation and inflammation.
  33. Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL.
  34. β-hydroxybutyrate accumulates in the rat heart during low-flow ischaemia with implications for functional recovery.
  35. Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.
  36. A novel integrated approach to estimate the mitochondrial content of neuronal cells and brain tissues.
  37. Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH.
  1. Blood Adv. 2021 Sep 10. pii: bloodadvances.2020003661. [Epub ahead of print]
    Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition.
    Kaori Saito, Qi Zhang, Haeun Yang, Kotoko Yamatani, Tomohiko Ai, Vivian Ruvolo, Natalia Baran, Tianyu Cai, Helen Ma, Rodrigo Jacamo, Vinitha Mary Kuruvilla, Junichi Imoto, Sonoko Kinjo, Kazuho Ikeo, Kaori Moriya, Koya Suzuki, Takashi Miida, Yong-Mi Kim, Christopher P Vellano, Michael Andreeff, Joseph R Marszalek, Yoko Tabe, Marina Y Konopleva.
      Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact coculture conditions, and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased OxPhos inhibition-triggered mitochondrial transfer from MSCs to AML cells. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.
    DOI:  https://doi.org/10.1182/bloodadvances.2020003661
  2. Shock. 2021 Sep 09.
    Pyruvate-Driven Oxidative Phosphorylation is Downregulated in Sepsis-Induced Cardiomyopathy: A Study Of Mitochondrial Proteome.
    Briana K Shimada, Liron Boyman, Weiliang Huang, Jing Zhu, Yang Yang, Fengqian Chen, Maureen A Kane, Nagendra Yadava, Lin Zou, W Jonathan Lederer, Brian M Polster, Wei Chao.
      BACKGROUND: Sepsis-induced cardiomyopathy (SIC) is a major contributing factor for morbidity and mortality in sepsis. Accumulative evidence has suggested that cardiac mitochondrial oxidative phosphorylation is attenuated in sepsis, but the underlying molecular mechanisms remain incompletely understood.METHODS: Adult male mice of 9-12 weeks old were subjected to sham or cecal ligation and puncture procedure. Echocardiography in vivo and Langendorff-perfused hearts were used to assess cardiac function 24 hours after the procedures. Unbiased proteomics analysis was performed to profile mitochondrial proteins in the hearts of both sham and SIC mice. Seahorse respirator technology was used to evaluate oxygen consumption in purified mitochondria.
    RESULTS: Of the 665 mitochondrial proteins identified in the proteomics assay, 35 were altered in septic mice. The mitochondrial remodeling involved various energy metabolism pathways including subunits of the electron transport chain, fatty acid catabolism, and carbohydrate oxidative metabolism. We also identified a significant increase of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) and inhibition of PDH activity in septic hearts. Furthermore, compared to sham mice, mitochondrial oxygen consumption of septic mice was significantly reduced when pyruvate was provided as a substrate. However, it was unchanged when PDH was bypassed by directly supplying the Complex I substrate NADH, or by using the Complex II substrate succinate, or using Complex IV substrate, or by providing the beta-oxidation substrate palmitoylcarnitine, neither of which require PDH for mitochondrial oxygen consumption.
    CONCLUSIONS: These data demonstrate a broad mitochondrial protein remodeling, PDH inactivation and impaired pyruvate-fueled oxidative phosphorylation during SIC, and provide a molecular framework for further exploration.
    DOI:  https://doi.org/10.1097/SHK.0000000000001858
  3. Hum Mol Genet. 2021 Sep 07. pii: ddab254. [Epub ahead of print]
    MCU-complex-mediated mitochondrial calcium signaling is impaired in Barth syndrome.
    Sagnika Ghosh, Mohammad Zulkifli, Alaumy Joshi, Manigandan Venkatesan, Allen Cristel, Neelanjan Vishnu, Muniswamy Madesh, Vishal M Gohil.
      Calcium signaling via mitochondrial calcium uniporter (MCU) complex coordinates mitochondrial bioenergetics with cellular energy demands. Emerging studies show that the stability and activity of the pore-forming subunit of the complex, MCU, is dependent on the mitochondrial phospholipid, cardiolipin (CL), but how this impacts calcium-dependent mitochondrial bioenergetics in CL-deficiency disorder like Barth syndrome (BTHS) is not known. Here we utilized multiple models of BTHS including yeast, mouse muscle cell line, as well as BTHS patient cells and cardiac tissue to show that CL is required for the abundance and stability of the MCU-complex regulatory subunit MICU1. Interestingly, the reduction in MICU1 abundance in BTHS mitochondria is independent of MCU. Unlike MCU and MICU1/MICU2, other subunit and associated factor of the uniporter complex, EMRE and MCUR1, respectively, are not affected in BTHS models. Consistent with the decrease in MICU1 levels, we show that the kinetics of MICU1-dependent mitochondrial calcium uptake is perturbed and acute stimulation of mitochondrial calcium signaling in BTHS myoblasts fails to activate pyruvate dehydrogenase, which in turn impairs the generation of reducing equivalents and blunts mitochondrial bioenergetics. Taken together, our findings suggest that defects in mitochondrial calcium signaling could contribute to cardiac and skeletal muscle pathologies observed in BTHS patients.
    DOI:  https://doi.org/10.1093/hmg/ddab254
  4. Cell Metab. 2021 Sep 07. pii: S1550-4131(21)00373-9. [Epub ahead of print]33(9): 1719-1720
    T cells critically depend on pyruvate oxidation.
    Christopher Schrecker, Andrew M Intlekofer.
      Supporting the notion that cell lineage is a key determinant of cancer cell metabolism, Jun et al. (2021) identify a selective requirement for pyruvate dehydrogenase (PDH) activity in T cells and T cell leukemia, but not hematopoietic stem cells (HSCs) or myeloid leukemia, in this issue of Cell Metabolism.
    DOI:  https://doi.org/10.1016/j.cmet.2021.08.010
  5. Nat Metab. 2021 Sep 09.
    A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation.
    Li Li, Devon M Conradson, Vinita Bharat, Min Joo Kim, Chung-Han Hsieh, Paras S Minhas, Amanda M Papakyrikos, Aarooran Sivakumaran Durairaj, Anthony Ludlam, Katrin I Andreasson, Linda Partridge, Michael A Cianfrocco, Xinnan Wang.
      Mitochondria are the main site for generating reactive oxygen species, which are key players in diverse biological processes. However, the molecular pathways of redox signal transduction from the matrix to the cytosol are poorly defined. Here we report an inside-out redox signal of mitochondria. Cysteine oxidation of MIC60, an inner mitochondrial membrane protein, triggers the formation of disulfide bonds and the physical association of MIC60 with Miro, an outer mitochondrial membrane protein. The oxidative structural change of this membrane-crossing complex ultimately elicits cellular responses that delay mitophagy, impair cellular respiration and cause oxidative stress. Blocking the MIC60-Miro interaction or reducing either protein, genetically or pharmacologically, extends lifespan and health-span of healthy fruit flies, and benefits multiple models of Parkinson's disease and Friedreich's ataxia. Our discovery provides a molecular basis for common treatment strategies against oxidative stress.
    DOI:  https://doi.org/10.1038/s42255-021-00443-2
  6. Neuromolecular Med. 2021 Sep 06.
    Targeting Mitochondrial Oxidative Phosphorylation in Glioblastoma Therapy.
    Zhihao Wu, Winson S Ho, Rongze Lu.
      As a multi-functional cellular organelle, mitochondrial metabolic reprogramming is well recognized as a hallmark of cancer. The center of mitochondrial metabolism is oxidative phosphorylation (OXPHOS), in which cells use enzymes to oxidize nutrients, thereby converting the chemical energy to the biological energy currency ATPs. OXPHOS also creates the mitochondrial membrane potential and serve as the driving force of other mitochondrial metabolic pathways and experiences significant reshape in the different stages of tumor progression. In this minireview, we reviewed the major mitochondrial pathways that are connected to OXPHOS and are affected in cancer cells. In addition, we summarized the function of novel bio-active molecules targeting mitochondrial metabolic processes such as OXPHOS, mitochondrial membrane potential and mitochondrial dynamics. These molecules exhibit intriguing preclinical and clinical results and have been proven to be promising antitumor candidates in recent studies.
    Keywords:  Glioblastoma; Mitochondrial dysfunction; OXPHOS inhibitors
    DOI:  https://doi.org/10.1007/s12017-021-08678-8
  7. Cancers (Basel). 2021 Sep 04. pii: 4457. [Epub ahead of print]13(17):
    Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression.
    Jesusa Capera, Mireia Pérez-Verdaguer, María Navarro-Pérez, Antonio Felipe.
      The voltage-gated potassium channel Kv1.3 is a potential therapeutic target for obesity and diabetes. The genetic ablation and pharmacological inhibition of Kv1.3 lead to a lean phenotype in rodents. The mechanism of regulation of body weight and energy homeostasis involves Kv1.3 expression in different organs, including white and brown adipose tissues. Here, we show that Kv1.3 promotes the proliferation of preadipocytes through the control of mitochondrial dynamics. Kv1.3 is expressed in mitochondria exhibiting high affinity for the perinuclear population. The mitochondrial network is highly dynamic during the cell cycle, showing continuous fusion-fission events. The formation of a hyperfused mitochondrial network at the G1/S phase of the cell cycle is dependent on Kv1.3 expression. Our results demonstrate that Kv1.3 promotes preadipocyte proliferation and differentiation by controlling mitochondrial membrane potential and mitochondrial dynamics at the G1 phase of the cell cycle.
    Keywords:  adipocytes; fusion/fission; mitochondria; potassium channels; proliferation
    DOI:  https://doi.org/10.3390/cancers13174457
  8. Cell Death Dis. 2021 Sep 06. 12(9): 837
    Mitochondrial dysfunction induces radioresistance in colorectal cancer by activating [Ca2+]m-PDP1-PDH-histone acetylation retrograde signaling.
    Yingying Shi, You Wang, Huangang Jiang, Xuehua Sun, Hui Xu, Xue Wei, Yan Wei, Guohui Xiao, Zhiyin Song, Fuxiang Zhou.
      Mitochondrial retrograde signaling (mito-RTG) triggered by mitochondrial dysfunction plays a potential role in regulating tumor metabolic reprogramming and cellular sensitivity to radiation. Our previous studies showed phos-pyruvate dehydrogenase (p-PDH) and PDK1, which involved in aerobic glycolysis, were positively correlated with radioresistance, but how they initiate and work in the mito-RTG pathway is still unknown. Our further genomics analysis revealed that complex I components were widely downregulated in mitochondrial dysfunction model. In the present study, high expression of p-PDH was found in the complex I deficient cells and induced radioresistance. Mechanistically, complex I defects led to a decreased PDH both in cytoplasm and nucleus through [Ca2+]m-PDP1-PDH axis, and decreased PDH in nucleus promote DNA damage repair (DDR) response via reducing histone acetylation. Meanwhile, NDUFS1 (an important component of the complex I) overexpression could enhance the complex I activity, reverse glycolysis and resensitize cancer cells to radiation in vivo and in vitro. Furthermore, low NDUFS1 and PDH expression were validated to be correlated with poor tumor regression grading (TRG) in local advanced colorectal cancer (CRC) patients underwent neoadjuvant radiotherapy. Here, we propose that the [Ca2+]m-PDP1-PDH-histone acetylation retrograde signaling activated by mitochondrial complex I defects contribute to cancer cell radioresistance, which provides new insight in the understanding of the mito-RTG. For the first time, we reveal that NDUFS1 could be served as a promising predictor of radiosensitivity and modification of complex I function may improve clinical benefits of radiotherapy in CRC.
    DOI:  https://doi.org/10.1038/s41419-021-03984-2
  9. J Biol Chem. 2021 Sep 01. pii: S0021-9258(21)00957-1. [Epub ahead of print] 101155
    The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids.
    Jan Frankovsky, Barbora Keresztesová, Jana Bellová, Nina Kunová, Nikola Čanigová, Katerina Hanakova, Jacob A Bauer, Gabriela Ondrovičová, Veronika Lukáčová, Barbara Siváková, Zbynek Zdrahal, Vladimír Pevala, Katarína Procházková, Jozef Nosek, Peter Baráth, Eva Kutejova, Lubomir Tomaska.
      Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-coenzyme A, is primarily generated in the tricarboxylic acid (TCA) cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here we performed a proteomic mass spectrometry analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA (mtDNA) and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mt-nucleoids responsible for compacting mtDNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in concentration of TCA intermediates may affect mitochondrial functions.
    Keywords:  DNA-protein interaction; lysine succinylation; mitochondria; mitochondrial DNA; mitochondrial nucleoid; post-translational modification (PTM); proteomics; succinylome; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2021.101155
  10. Front Oncol. 2021 ;11 686765
    The Combined Treatment With the FLT3-Inhibitor AC220 and the Complex I Inhibitor IACS-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells.
    Xiyuan Lu, Lina Han, Jonathan Busquets, Meghan Collins, Alessia Lodi, Joseph R Marszalek, Marina Konopleva, Stefano Tiziani.
      Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high mortality rate and relapse risk. Although progress on the genetic and molecular understanding of this disease has been made, the standard of care has changed minimally for the past 40 years and the five-year survival rate remains poor, warranting new treatment strategies. Here, we applied a two-step screening platform consisting of a primary cell viability screening and a secondary metabolomics-based phenotypic screening to find synergistic drug combinations to treat AML. A novel synergy between the oxidative phosphorylation inhibitor IACS-010759 and the FMS-like tyrosine kinase 3 (FLT3) inhibitor AC220 (quizartinib) was discovered in AML and then validated by ATP bioluminescence and apoptosis assays. In-depth stable isotope tracer metabolic flux analysis revealed that IACS-010759 and AC220 synergistically reduced glucose and glutamine enrichment in glycolysis and the TCA cycle, leading to impaired energy production and de novo nucleotide biosynthesis. In summary, we identified a novel drug combination, AC220 and IACS-010759, which synergistically inhibits cell growth in AML cells due to a major disruption of cell metabolism, regardless of FLT3 mutation status.
    Keywords:  FLT3-inhibitor; acute myeloid leukemia; complex I inhibitor; high-throughput screening; metabolomics
    DOI:  https://doi.org/10.3389/fonc.2021.686765
  11. Nat Commun. 2021 Sep 10. 12(1): 5387
    Functional basis of electron transport within photosynthetic complex I.
    Katherine H Richardson, John J Wright, Mantas Šimėnas, Jacqueline Thiemann, Ana M Esteves, Gemma McGuire, William K Myers, John J L Morton, Michael Hippler, Marc M Nowaczyk, Guy T Hanke, Maxie M Roessler.
      Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme - the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.
    DOI:  https://doi.org/10.1038/s41467-021-25527-1
  12. Cancers (Basel). 2021 Aug 31. pii: 4412. [Epub ahead of print]13(17):
    DHX30 Coordinates Cytoplasmic Translation and Mitochondrial Function Contributing to Cancer Cell Survival.
    Bartolomeo Bosco, Annalisa Rossi, Dario Rizzotto, Meriem Hadjer Hamadou, Alessandra Bisio, Sebastiano Giorgetta, Alicia Perzolli, Francesco Bonollo, Angeline Gaucherot, Frédéric Catez, Jean-Jacques Diaz, Erik Dassi, Alberto Inga.
      DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here, we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, the depletion of both DHX30 isoforms leads to higher global translation but slower proliferation and lower mitochondrial energy metabolism. Isoform-specific silencing supports a role for cytoplasmic DHX30 in modulating global translation. The impact on translation and proliferation was confirmed in U2OS and MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified fourteen mitoribosome transcripts we propose as direct DHX30 targets that can be used to explore the prognostic value of this mechanism in cancer. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.
    Keywords:  DHX30; RNA binding proteins; mitoribosome; polysomal profiling; ribosome biogenesis; translation efficiency
    DOI:  https://doi.org/10.3390/cancers13174412
  13. Free Radic Biol Med. 2021 Sep 05. pii: S0891-5849(21)00715-2. [Epub ahead of print]
    Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia.
    Werner J Geldenhuys, Debbie Piktel, Javohn C Moore, Stephanie L Rellick, Ethan Meadows, Mark V Pinti, John M Hollander, Amanda G Ammer, Karen H Martin, Laura F Gibson.
      B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.
    Keywords:  Chemoresistance; Glitazones; Mitochondrial dysfunction; cisd2
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.09.003
  14. Nat Commun. 2021 Sep 09. 12(1): 5354
    ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division.
    Maxime Boutry, Peter K Kim.
      Mitochondrial division is not an autonomous event but involves multiple organelles, including the endoplasmic reticulum (ER) and lysosomes. Whereas the ER drives the constriction of mitochondrial membranes, the role of lysosomes in mitochondrial division is not known. Here, using super-resolution live-cell imaging, we investigate the recruitment of lysosomes to the site of mitochondrial division. We find that the ER recruits lysosomes to the site of division through the interaction of VAMP-associated proteins (VAPs) with the lysosomal lipid transfer protein ORP1L to induce a three-way contact between the ER, lysosome, and the mitochondrion. We also show that ORP1L might transport phosphatidylinositol-4-phosphate (PI(4)P) from lysosomes to mitochondria, as inhibiting its transfer or depleting PI(4)P at the mitochondrial division site impairs fission, demonstrating a direct role for PI(4)P in the division process. Our findings support a model where the ER recruits lysosomes to act in concert at the fission site for the efficient division of mitochondria.
    DOI:  https://doi.org/10.1038/s41467-021-25621-4
  15. J Biol Chem. 2021 Sep 06. pii: S0021-9258(21)00976-5. [Epub ahead of print] 101174
    The Mitochondrial Ca2+ Uniporter is a Central Regulator of Interorganellar Ca2+ Transfer and NFAT Activation.
    Ryan E Yoast, Scott M Emrich, Xuexin Zhang, Ping Xin, Vikas Arige, Trayambak Pathak, J Cory Benson, Martin T Johnson, Ahmed Emam Abdelnaby, Natalia Lakomski, Nadine Hempel, Jung Min Han, Geneviève Dupont, David I Yule, James Sneyd, Mohamed Trebak.
      Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked inter-organellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, sub-cellular Ca2+ imaging and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum (ER) Ca2+ refilling, nuclear translocation of nuclear factor for activated T-cells (NFAT) transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate inter-organellar Ca2+ transfer and NFAT nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping ER and cytosolic Ca2+ signals that regulate cellular transcription and function.
    Keywords:  CRAC channels; MCU; NFAT; SOCE; calcium oscillations; calcium signaling; mitochondria
    DOI:  https://doi.org/10.1016/j.jbc.2021.101174
  16. Mol Biol Cell. 2021 Sep 08. mbcE21040191
    Discovery of bactericides as an acute mitochondrial membrane damage inducer.
    Ryan Houston, Yusuke Sekine, Mads B Larsen, Kei Murakami, Steven J Mullett, Stacy G Wendell, Derek P Narendra, Bill B Chen, Shiori Sekine.
      Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, Chlorhexidine and Alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found Alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that Alexidine severely perturbated the cristae structure. Notably, Alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of Alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.
    DOI:  https://doi.org/10.1091/mbc.E21-04-0191
  17. Autophagy. 2021 Sep 05. 1-3
    The OMM-severed and IMM-ubiquitinated mitochondria are intermediates of mitochondrial proteotoxicity-induced autophagy in PRKN/parkin-deficient cells.
    Yumiko Oshima, Nicolas Verhoeven, Etienne Cartier, Mariusz Karbowski.
      Among other mechanisms, mitochondrial membrane dynamics including mitochondrial fission and fusion, and the activity of the ubiquitin (Ub)-proteasome system (UPS) both are critical for maintaining mitochondrial function. To advance our knowledge of the role of mitochondrial fission, the UPS, and how they coordinatively affect mitochondrial response to proteotoxicity, we analyzed mitochondrial ubiquitination and mitochondria-specific autophagy (mitophagy) in E3 Ub ligase PRKN/parkin-expressing and -deficient cells. Through imaging, biochemical, and genetic analyses, we found that in a model of acute reduction of mitochondrial translation fidelity (MTF) some population of mitochondria within a single cell are enriched, while some showed reduced levels of CYCS (cytochrome c, somatic) and CPOX (coproporphyrinogen oxidase) proteins, both located in the intermembrane space (IMS); henceforth called "mosaic distribution". Formation of mosaic mitochondria requires mitochondrial fission and active mitochondrial translation. In cell lines deficient in PRKN activity, this process is followed by severing the outer mitochondrial membrane (OMM) and ubiquitination of the inner mitochondrial membrane (IMM) proteins (including TRAP1 and CPOX), recruitment of autophagy receptors, and formation of mito-autophagosomes. In contrast, in PRKN-expressing cells, mitochondria with high CYCS and CPOX levels are preferentially targeted by PRKN, leading to OMM ubiquitination and canonical PRKN-PINK1-mediated autophagy.
    Keywords:  DRP1; Parkin; mitochondria; mitochondrial translation; mitophagy; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2021.1964887
  18. Int J Mol Sci. 2021 Aug 27. pii: 9283. [Epub ahead of print]22(17):
    Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells.
    Seyedeh Tayebeh Ahmadpour, Valérie Desquiret-Dumas, Ulku Yikilmaz, Julie Dartier, Isabelle Domingo, Celine Wetterwald, Charlotte Orre, Naïg Gueguen, Lucie Brisson, Karine Mahéo, Jean-François Dumas.
      Since its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMPO.
    Keywords:  breast cancer; doxorubicin; mitochondria; mitophagy; reactive oxygen species
    DOI:  https://doi.org/10.3390/ijms22179283
  19. Redox Biol. 2021 Aug 31. pii: S2213-2317(21)00281-0. [Epub ahead of print]46 102122
    Overcoming the compensatory elevation of NRF2 renders hepatocellular carcinoma cells more vulnerable to disulfiram/copper-induced ferroptosis.
    Xueying Ren, Yanchun Li, Yi Zhou, Wanye Hu, Chen Yang, Qiangan Jing, Chaoting Zhou, Xu Wang, Jiayu Hu, Luyang Wang, Jing Yang, Hairui Wang, Haifeng Xu, Huanjuan Li, Xiangmin Tong, Ying Wang, Jing Du.
      Hepatocellular carcinoma (HCC) is one of the paramount causes of cancer-related death worldwide. Despite recent advances have been made in clinical treatments of HCC, the general prognosis of patients remains poor. Therefore, it is imperative to develop a less toxic and more effective therapeutic strategy. Currently, series of cellular, molecular, and pharmacological experimental approaches were utilized to address the unrecognized characteristics of disulfiram (DSF), pursuing the goal of repurposing DSF for cancer therapy. We found that DSF/Cu selectively exerted an efficient cytotoxic effect on HCC cell lines, and potently inhibited migration, invasion, and angiogenesis of HCC cells. Importantly, we confirmed that DSF/Cu could intensively impair mitochondrial homeostasis, increase free iron pool, enhance lipid peroxidation, and eventually result in ferroptotic cell death. Of note, a compensatory elevation of NRF2 accompanies the process of ferroptosis, and contributes to the resistance to DSF/Cu. Mechanically, we found that DSF/Cu dramatically activated the phosphorylation of p62, which facilitates competitive binding of Keap1, thus prolonging the half-life of NRF2. Notably, inhibition of NRF2 expression via RNA interference or pharmacological inhibitors significantly facilitated the accumulation of lipid peroxidation, and rendered HCC cells more sensitive to DSF/Cu induced ferroptosis. Conversely, fostering NRF2 expression was capable of ameliorating the cell death activated by DSF/Cu. Additionally, DSF/Cu could strengthen the cytotoxicity of sorafenib, and arrest tumor growth both in vitro and in vivo, by simultaneously inhibiting the signal pathway of NRF2 and MAPK kinase. In summary, these results provide experimental evidence that inhibition of the compensatory NRF2 elevation strengthens HCC cells more vulnerable to DSF/Cu induced ferroptosis, which facilitates the synergistic cytotoxicity of DSF/Cu and sorafenib.
    Keywords:  Disulfiram; Ferroptosis; Hepatocellular carcinoma; Oxidative stress; Sorafenib
    DOI:  https://doi.org/10.1016/j.redox.2021.102122
  20. mSphere. 2021 Sep 08. e0061421
    A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.
    Selma L van Esveld, Lisette Meerstein-Kessel, Cas Boshoven, Jochem F Baaij, Konstantin Barylyuk, Jordy P M Coolen, Joeri van Strien, Ronald A J Duim, Bas E Dutilh, Daniel R Garza, Marijn Letterie, Nicholas I Proellochs, Michelle N de Ridder, Prashanna Balaji Venkatasubramanian, Laura E de Vries, Ross F Waller, Taco W A Kooij, Martijn A Huynen.
      Plasmodium species have a single mitochondrion that is essential for their survival and has been successfully targeted by antimalarial drugs. Most mitochondrial proteins are imported into this organelle, and our picture of the Plasmodium mitochondrial proteome remains incomplete. Many data sources contain information about mitochondrial localization, including proteome and gene expression profiles, orthology to mitochondrial proteins from other species, coevolutionary relationships, and amino acid sequences, each with different coverage and reliability. To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight data sets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 445 proteins with a sensitivity of 87% and a specificity of 97%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. Mitochondrial localization of seven Plasmodium berghei orthologs was confirmed by epitope labeling and colocalization with a mitochondrial marker protein. One of these belongs to a newly identified apicomplexan mitochondrial protein family that in P. falciparum has four members. With the experimentally validated mitochondrial proteins and the complete ranked P. falciparum proteome, which we have named PlasmoMitoCarta, we present a resource to study unique proteins of Plasmodium mitochondria. IMPORTANCE The unique biology and medical relevance of the mitochondrion of the malaria parasite Plasmodium falciparum have made it the subject of many studies. However, we actually do not have a comprehensive assessment of which proteins reside in this organelle. Many omics data are available that are predictive of mitochondrial localization, such as proteomics data and expression data. Individual data sets are, however, rarely complete and can provide conflicting evidence. We integrated a wide variety of available omics data in a manner that exploits the relative strengths of the data sets. Our analysis gave a predictive score for the mitochondrial localization to each nuclear encoded P. falciparum protein and identified 445 likely mitochondrial proteins. We experimentally validated the mitochondrial localization of seven of the new mitochondrial proteins, confirming the quality of the complete list. These include proteins that have not been observed mitochondria before, adding unique mitochondrial functions to P. falciparum.
    Keywords:  Bayesian data integration; Plasmodium; mitochondria
    DOI:  https://doi.org/10.1128/mSphere.00614-21
  21. Biochem Biophys Res Commun. 2021 Aug 31. pii: S0006-291X(21)01270-5. [Epub ahead of print]576 93-99
    A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.
    Nobuko Koshikawa, Yuki Kida, Nanami Yasui, Yoshinao Shinozaki, Kohei Tsuji, Takayoshi Watanabe, Jason Lin, Seigi Yamamoto, Keizo Takenaga, Hiroki Nagase.
      Somatic mutations in mitochondrial DNA may provide a new avenue for cancer therapy due to their associations to a number of cancers and a tendency of homoplasmicity. In consideration of mitochondrial features and its relatively small genome size, a nucleotide-based targeting approach is a considerably more promising option. To explore the efficacy of short linear N-methylpyrrole-N-methylimidazole polyamide (PI polyamide), we synthesized a five-ring short PI polyamide that provided sequence-specific homing for the A3243G mitochondrial mutation upon conjugation with triphenylphosphonium cation (TPP). This PI polyamide-TPP was able to induce cytotoxicity in HeLamtA3243G cybrid cells, while preserving preferential binding for oligonucleotides containing the A3243G motif from melting temperature assays. The PI polyamide-TPP also localized in the mitochondria in HeLamtA3243G cells and induced mitochondrial reactive oxygen species production, mitophagy and apoptosis in a mutation-specific fashion compared to the wild-type HeLamtHeLa cybrids; normal human dermal fibroblasts were also relatively unaffected to suggest discriminating selectivity for the mutant mitochondria, offering a novel outlook for cancer therapy via mitochondrial homing of short linear PIP-TPPs.
    Keywords:  Apoptosis; Mitochondrial DNA mutation; Mitophagy; Pyrrole-imidazole polyamide; Triphenylphosphonium; mtROS
    DOI:  https://doi.org/10.1016/j.bbrc.2021.08.088
  22. Front Pharmacol. 2021 ;12 679407
    Dynamic Modeling of Mitochondrial Membrane Potential Upon Exposure to Mitochondrial Inhibitors.
    Huan Yang, Wanda van der Stel, Randy Lee, Caroline Bauch, Sam Bevan, Paul Walker, Bob van de Water, Erik H J Danen, Joost B Beltman.
      Mitochondria are the main bioenergetic organelles of cells. Exposure to chemicals targeting mitochondria therefore generally results in the development of toxicity. The cellular response to perturbations in cellular energy production is a balance between adaptation, by reorganisation and organelle biogenesis, and sacrifice, in the form of cell death. In homeostatic conditions, aerobic mitochondrial energy production requires the maintenance of a mitochondrial membrane potential (MMP). Chemicals can perturb this MMP, and the extent of this perturbation depends both on the pharmacokinetics of the chemicals and on downstream MMP dynamics. Here we obtain a quantitative understanding of mitochondrial adaptation upon exposure to various mitochondrial respiration inhibitors by applying mathematical modeling to partially published high-content imaging time-lapse confocal imaging data, focusing on MMP dynamics in HepG2 cells over a period of 24 h. The MMP was perturbed using a set of 24 compounds, either acting as uncoupler or as mitochondrial complex inhibitor targeting complex I, II, III or V. To characterize the effect of chemical exposure on MMP dynamics, we adapted an existing differential equation model and fitted this model to the observed MMP dynamics. Complex III inhibitor data were better described by the model than complex I data. Incorporation of pharmacokinetic decay into the model was required to obtain a proper fit for the uncoupler FCCP. Furthermore, oligomycin (complex V inhibitor) model fits were improved by either combining pharmacokinetic (PK) decay and ion leakage or a concentration-dependent decay. Subsequent mass spectrometry measurements showed that FCCP had a significant decay in its PK profile as predicted by the model. Moreover, the measured oligomycin PK profile exhibited only a limited decay at high concentration, whereas at low concentrations the compound remained below the detection limit within cells. This is consistent with the hypothesis that oligomycin exhibits a concentration-dependent decay, yet awaits further experimental verification with more sensitive detection methods. Overall, we show that there is a complex interplay between PK and MMP dynamics within mitochondria and that data-driven modeling is a powerful combination to unravel such complexity.
    Keywords:  dynamic modeling; high-throughput microscopy imaging; mitochondrial membrane potential; mitochondrial respiration; parameter identifiability; uncertainty quantification
    DOI:  https://doi.org/10.3389/fphar.2021.679407
  23. EMBO J. 2021 Sep 06. e108065
    PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability.
    Fabao Liu, Min Ma, Ang Gao, Fengfei Ma, Gui Ma, Peng Liu, Chenxi Jia, Yidan Wang, Kristine Donahue, Shengjie Zhang, Irene M Ong, Sunduz Keles, Lingjun Li, Wei Xu.
      The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.
    Keywords:  PKM2; SCAP degradation; TMEM33; total cholesterol levels; tumor growth
    DOI:  https://doi.org/10.15252/embj.2021108065
  24. Nat Commun. 2021 09 06. 12(1): 5277
    Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion.
    Jana Škerlová, Jens Berndtsson, Hendrik Nolte, Martin Ott, Pål Stenmark.
      The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.
    DOI:  https://doi.org/10.1038/s41467-021-25570-y
  25. FASEB J. 2021 Oct;35(10): e21867
    Low lysophosphatidylcholine induces skeletal muscle myopathy that is aggravated by high-fat diet feeding.
    Patrick J Ferrara, Anthony R P Verkerke, J Alan Maschek, Justin L Shahtout, Piyarat Siripoksup, Hiroaki Eshima, Jordan M Johnson, Jonathan J Petrocelli, Ziad S Mahmassani, Thomas D Green, Joseph M McClung, James E Cox, Micah J Drummond, Katsuhiko Funai.
      Obesity alters skeletal muscle lipidome and promotes myopathy, but it is unknown whether aberrant muscle lipidome contributes to the reduction in skeletal muscle contractile force-generating capacity. Comprehensive lipidomic analyses of mouse skeletal muscle revealed a very strong positive correlation between the abundance of lysophosphatidylcholine (lyso-PC), a class of lipids that is known to be downregulated with obesity, with maximal tetanic force production. The level of lyso-PC is regulated primarily by lyso-PC acyltransferase 3 (LPCAT3), which acylates lyso-PC to form phosphatidylcholine. Tamoxifen-inducible skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) was sufficient to reduce muscle lyso-PC content in both standard chow diet- and high-fat diet (HFD)-fed conditions. Strikingly, the assessment of skeletal muscle force-generating capacity ex vivo revealed that muscles from LPCAT3-MKI mice were weaker regardless of diet. Defects in force production were more apparent in HFD-fed condition, where tetanic force production was 40% lower in muscles from LPCAT3-MKI compared to that of control mice. These observations were partly explained by reductions in the cross-sectional area in type IIa and IIx fibers, and signs of muscle edema in the absence of fibrosis. Future studies will pursue the mechanism by which LPCAT3 may alter protein turnover to promote myopathy.
    Keywords:  diabetes; lysophospholipid; myopathy; skeletal muscle
    DOI:  https://doi.org/10.1096/fj.202101104R
  26. Hum Mol Genet. 2021 Sep 11. pii: ddab257. [Epub ahead of print]
    Elucidating the molecular mechanisms associated with TARS2-related mitochondrial disease.
    Wen-Qiang Zheng, Signe Vandal Pedersen, Kyle Thompson, Emanuele Bellacchio, Courtney E French, Benjamin Munro, Toni S Pearson, Julie Vogt, Daria Diodato, Tue Diemer, Anja Ernst, Rita Horvath, Manali Chitre, Jakob Ek, Flemming Wibrand, Dorothy K Grange, Lucy Raymond, Xiao-Long Zhou, Robert W Taylor, Elsebet Ostergaard.
      TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. Additionally, we document seven novel TARS2 variants-one nonsense variant and six missense variants-that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modelling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.
    DOI:  https://doi.org/10.1093/hmg/ddab257
  27. Angew Chem Int Ed Engl. 2021 Sep 10.
    High-throughput Single-cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.
    Qinlei Liu, Wenjie Ge, Tongtong Wang, Jiayi Lan, Sandra Martínez-Jarquín, Christian Wolfrum, Markus Stoffel, Renato Zenobi.
      Even populations of clonal cells are heterogeneous, which requires high-throughput analysis methods with single-cell sensitivity. Here, we propose a rapid, label-free single-cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK-293T, PANC-1, CFPAC-1, H6c7, HeLa and iBAs) were discriminated successfully. We found that abnormal lipid metabolism occurs in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor or a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.
    Keywords:  ATP citrate lyase; abnormal lipid metabolism; active capillary dielectric barrier discharge ionization source; pancreatic ductal adenocarcinoma target; single-cell mass spectrometry
    DOI:  https://doi.org/10.1002/anie.202107223
  28. Aging (Albany NY). 2021 Aug 17. 13(16): 20229-20245
    Autophagic flux in cancer cells at the invasive front in the tumor-stroma border.
    Fei Yan, Xuexi Zhang, Rongying Tan, Mingchen Li, Zengtuan Xiao, Hao Wang, Zhenfa Zhang, Zhenyi Ma, Zhe Liu.
      Cancer cells at the invasive front directly interact with stromal tissue that provides a microenvironment with mechanical, nutrient, and oxygen supply characteristics distinct from those of intratumoral tissues. It has long been known that cancer cells at the invasive front and cancer cells inside the tumor body exhibit highly differentiated functions and behaviors. However, it is unknown whether cancer cells at different locations exhibit a variety of autophagic flux, an important catabolic process to maintain cellular homeostasis in response to environmental changes. Here, using transmission electron microscopy (TEM), we found that invading cancer cells at the invasive front, which show mesenchymal transcriptomic traits, exhibit higher autophagic flux than cancer cells inside the tumor body in human primary non-small cell lung cancer (NSCLC) tissues. This autophagic feature was further confirmed by a live cell autophagic flux monitoring system combined with a 3D organotypic invasion coculture system. Additionally, the increased autophagic flux endows cancer cells with invasive behavior and positively correlates with the advanced tumor stages and the reduced survival period of lung cancer patients. These findings expand the understanding of autophagic dynamics during cancer invasion.
    Keywords:  autophagy; invasion; invasive front; lung cancer; tumor-stroma border
    DOI:  https://doi.org/10.18632/aging.203406
  29. Nature. 2021 Sep 08.
    Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.
    Roarke A Kamber, Yoko Nishiga, Bhek Morton, Allison M Banuelos, Amira A Barkal, Felipe Vences-Catalán, Mingxin Gu, Daniel Fernandez, Jose A Seoane, David Yao, Katherine Liu, Sijie Lin, Kaitlyn Spees, Christina Curtis, Livnat Jerby-Arnon, Irving L Weissman, Julien Sage, Michael C Bassik.
      Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.
    DOI:  https://doi.org/10.1038/s41586-021-03879-4
  30. Invest Ophthalmol Vis Sci. 2021 Sep 02. 62(12): 4
    Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium.
    Moonjung Choi, Joseph A Bonanno.
      Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown.Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking.
    Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11-transfected fibroblasts.
    Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.
    DOI:  https://doi.org/10.1167/iovs.62.12.4
  31. Nat Metab. 2021 Sep 06.
    Revisiting energy expenditure: how to correct mouse metabolic rate for body mass.
    Timo D Müller, Martin Klingenspor, Matthias H Tschöp.
      
    DOI:  https://doi.org/10.1038/s42255-021-00451-2
  32. Cell Rep. 2021 Sep 07. pii: S2211-1247(21)01103-7. [Epub ahead of print]36(10): 109659
    Diurnal regulation of oxidative phosphorylation restricts hepatocyte proliferation and inflammation.
    Xiaojie Bai, Yilie Liao, Fangfang Sun, Xia Xiao, Suneng Fu.
      The principles guiding the diurnal organization of biological pathways remain to be fully elucidated. Here, we perturb the hepatic transcriptome through nutrient regulators (high-fat diet and mTOR signaling components) to identify enduring properties of pathway organization. Temporal separation and counter-regulation between pathways of energy metabolism and inflammation/proliferation emerge as persistent transcriptome features across animal models, and network analysis identifies the G0s2 and Rgs16 genes as potential mediators at the metabolism-inflammation interface. Mechanistically, G0s2 and Rgs16 are sequentially induced during the light phase, promoting amino acid oxidation and suppressing overall mitochondrial respiration. In their absence, sphingolipids and diacylglycerides accumulate, accompanied by hepatic inflammation and hepatocyte proliferation. Notably, the expression of G0s2 and Rgs16 is further induced in obese mouse livers, and silencing of their expression accentuates hepatic fibrosis. Therefore, diurnal regulation of energy metabolism alleviates inflammatory and proliferative stresses under physiological and pathological conditions.
    Keywords:  G0S2; RGS16; circadian rhythm; diacylglyceride; fibrosis; hepatic steatosis; inflammation; liver regeneration; oxidative phosphorylation; sphingolipid
    DOI:  https://doi.org/10.1016/j.celrep.2021.109659
  33. Proc Natl Acad Sci U S A. 2021 Sep 14. pii: e2025834118. [Epub ahead of print]118(37):
    Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL.
    Victor Vasquez-Montes, Mykola V Rodnin, Alexander Kyrychenko, Alexey S Ladokhin.
      Regulation of apoptosis is tightly linked with the targeting of numerous Bcl-2 proteins to the mitochondrial outer membrane (MOM), where their activation or inhibition dictates cell death or survival. According to the traditional view of apoptotic regulation, BH3-effector proteins are indispensable for the cytosol-to-MOM targeting and activation of proapoptotic and antiapoptotic members of the Bcl-2 protein family. This view is challenged by recent studies showing that these processes can occur in cells lacking BH3 effectors by as yet to be determined mechanism(s). Here, we exploit a model membrane system that recapitulates key features of MOM to demonstrate that the proapoptotic Bcl-2 protein BAX and antiapoptotic Bcl-xL have an inherent ability to interact with membranes in the absence of BH3 effectors, but only in the presence of cellular concentrations of Mg2+/Ca2+ Under these conditions, BAX and Bcl-xL are selectively targeted to membranes, refolded, and activated in the presence of anionic lipids especially the mitochondrial-specific lipid cardiolipin. These results provide a mechanistic explanation for the mitochondrial targeting and activation of Bcl-2 proteins in cells lacking BH3 effectors. At cytosolic Mg2+ levels, the BH3-independent activation of BAX could provide localized amplification of apoptotic signaling at regions enriched in cardiolipin (e.g., contact sites between MOM and mitochondrial inner membrane). Increases in MOM cardiolipin, as well as cytosolic [Ca2+] during apoptosis could further contribute to its MOM targeting and activity. Meanwhile, the BH3-independent targeting and activation of Bcl-xL to the MOM is expected to counter the action of proapoptotic BAX, thereby preventing premature commitment to apoptosis.
    Keywords:  apoptosis; divalent cations; membrane protein folding; mitochondria permeabilization; protein–lipid interactions
    DOI:  https://doi.org/10.1073/pnas.2025834118
  34. Elife. 2021 09 07. pii: e71270. [Epub ahead of print]10
    β-hydroxybutyrate accumulates in the rat heart during low-flow ischaemia with implications for functional recovery.
    Ross T Lindsay, Sophie Dieckmann, Dominika Krzyzanska, Dominic Manetta-Jones, James A West, Cecilia Castro, Julian L Griffin, Andrew J Murray.
      Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic β-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of β-hydroxy-β-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.
    Keywords:  Heart; Ischaemia; Langendorff; biochemistry; cardiomyocyte; cell biology; chemical biology; rat
    DOI:  https://doi.org/10.7554/eLife.71270
  35. Proc Natl Acad Sci U S A. 2021 Sep 14. pii: e2025932118. [Epub ahead of print]118(37):
    Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.
    Joshua C Drake, Rebecca J Wilson, Rhianna C Laker, Yuntian Guan, Hannah R Spaulding, Anna S Nichenko, Wenqing Shen, Huayu Shang, Maya V Dorn, Kian Huang, Mei Zhang, Aloka B Bandara, Matthew H Brisendine, Jennifer A Kashatus, Poonam R Sharma, Alexander Young, Jitendra Gautam, Ruofan Cao, Horst Wallrabe, Paul A Chang, Michael Wong, Eric M Desjardins, Simon A Hawley, George J Christ, David F Kashatus, Clint L Miller, Matthew J Wolf, Ammasi Periasamy, Gregory R Steinberg, D Grahame Hardie, Zhen Yan.
      Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.
    Keywords:  AMPK; exercise; mitochondria; mitophagy; skeletal muscle
    DOI:  https://doi.org/10.1073/pnas.2025932118
  36. J Neurosci Methods. 2021 Sep 02. pii: S0165-0270(21)00286-7. [Epub ahead of print] 109351
    A novel integrated approach to estimate the mitochondrial content of neuronal cells and brain tissues.
    Sara Spadini, Gabriella Racchetti, Alice Adiletta, Jacopo Lamanna, Andrea Stefano Moro, Mattia Ferro, Vincenzo Zimarino, Antonio Malgaroli.
      BACKGROUND: Mitochondria and their dynamics fuel most cellular processes both in physiological and pathological conditions. In the central nervous system, mitochondria sustain synaptic transmission and plasticity via multiple mechanisms which include their redistribution and/or expansion to higher energy demanding sites, sustaining activity changes and promoting morphological circuit adaptations.NEW METHOD: To be able to evaluate changes in mitochondrial number and protein phenotype, we propose a novel methodological approach where the simultaneous analysis of both mitochondrial DNA and protein content is performed on each individual microsample, avoiding non-homogeneous loss of material.
    RESULTS: We validated this method on neuronal-like cells and tissue samples and obtained estimates for the mitochondrial/genomic DNA ratio as well as for the abundance of protein counterparts. When the mitochondrial content per cell was evaluated in different brain areas, our results matched the known regional variation in aerobic-anaerobic metabolism. When long-term potentiation (LTP) was induced on hippocampal neurons, we detected increases in the abundance of mitochondria that correlated with the degree of synaptic enhancement.
    CONCLUSIONS: Our approach can be effectively used to study the mitochondrial content andits changes in different brain cells and tissues.
    Keywords:  Energy metabolism; LTP; Mitochondria; Neuronal metabolism; Synaptic plasticity; Synaptic transmission
    DOI:  https://doi.org/10.1016/j.jneumeth.2021.109351
  37. Anal Chem. 2021 Sep 10.
    Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH.
    Liam T Wilson, William J Tipping, Corinna Wetherill, Zoë Henley, Karen Faulds, Duncan Graham, Simon P Mackay, Nicholas C O Tomkinson.
      Mitochondrial pH (pHmito) is intimately related to mitochondrial function, and aberrant values for pHmito are linked to several disease states. We report the design, synthesis, and application of mitokyne 1-the first small molecule pHmito sensor for stimulated Raman scattering (SRS) microscopy. This ratiometric probe can determine subtle changes in pHmito in response to external stimuli and the inhibition of both the electron transport chain and ATP synthase with small molecule inhibitors. In addition, 1 was also used to monitor mitochondrial dynamics in a time-resolved manner with subcellular spatial resolution during mitophagy providing a powerful tool for dissecting the molecular and cell biology of this critical organelle.
    DOI:  https://doi.org/10.1021/acs.analchem.1c03075
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