bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒06‒28
forty-eight papers selected by
Kelsey Fisher-Wellman
East Carolina University


  1. Int J Mol Sci. 2020 Jun 20. pii: E4405. [Epub ahead of print]21(12):
    Nakhle J, Rodriguez AM, Vignais ML.
      Mitochondria are essential cellular components that ensure physiological metabolic functions. They provide energy in the form of adenosine triphosphate (ATP) through the electron transport chain (ETC). They also constitute a metabolic hub in which metabolites are used and processed, notably through the tricarboxylic acid (TCA) cycle. These newly generated metabolites have the capacity to feed other cellular metabolic pathways; modify cellular functions; and, ultimately, generate specific phenotypes. Mitochondria also provide intracellular signaling cues through reactive oxygen species (ROS) production. As expected with such a central cellular role, mitochondrial dysfunctions have been linked to many different diseases. The origins of some of these diseases could be pinpointed to specific mutations in both mitochondrial- and nuclear-encoded genes. In addition to their impressive intracellular tasks, mitochondria also provide intercellular signaling as they can be exchanged between cells, with resulting effects ranging from repair of damaged cells to strengthened progression and chemo-resistance of cancer cells. Several therapeutic options can now be envisioned to rescue mitochondria-defective cells. They include gene therapy for both mitochondrial and nuclear defective genes. Transferring exogenous mitochondria to target cells is also a whole new area of investigation. Finally, supplementing targeted metabolites, possibly through microbiota transplantation, appears as another therapeutic approach full of promises.
    Keywords:  cancer; electron transport chain (ETC); metabolism; metabolites; microbiota; mitochondria; mitochondria exchange; mitochondrial DNA (mtDNA); therapy; tricarboxylic acid (TCA) cycle
    DOI:  https://doi.org/10.3390/ijms21124405
  2. Arch Biochem Biophys. 2020 Jun 17. pii: S0003-9861(20)30424-0. [Epub ahead of print] 108415
    Wang R, Liu Y, Mi X, Chen Q, Jiang P, Hou J, Lin Y, Li S, Ji B, Fang Y.
      Regorafenib, a multiple kinase inhibitor, is recently approved for treatment of patients with advanced hepatocellular carcinoma (HCC). Previous studies demonstrated that regorafenib was a mitochondrial toxicant, which associated with the impairment of mitochondria. Sirt3 is involved in the regulation of mitochondrial function in cancers. This study aimed to investigate the mechanism of Sirt3 involved in the mitochondrial dysfunction which associated with regorafenib treatment in liver cancer cells. We found regorafenib inhibited Sirt3 and p-ERK expression in HCC cells in a dose-dependent manner. Bioinformatics analysis showed that Sirt3 expression was down-regulated in liver cancer tissues and its low expression was correlated with worse overall survival (OS) in liver cancer patients. After transfected with Sirt3 overexpression plasmid, we found that Sirt3 sensitized liver cancer cells to regorafenib and resulted in much more apoptosis with a significant increase of ROS level. However, exogenous antioxidant could not weaken the apoptosis. Mitochondrial membrane potential assay indicated that Sirt3 overexpression accelerated the mitochondrial depolarization process induced by regorafenib and aggravated mitochondrial injury. Cellular oxygen consumption assay showed that mitochondrial dysfunction was caused by the damage of the electron transport chain. The results demonstrated that Sirt3 overexpression promoted the increase of ROS and apoptosis induced by regorafenib through the acceleration of mitochondrial dysfunction by impairing function of the electron transport chain in liver cancer cells. Our studies verified the functional role of Sirt3 in regorafenib treatment and suggested that regorafenib accompanied with Sirt3 activator as a novel treatment strategy for HCC.
    Keywords:  Liver cancer; Mitochondria; Regorafenib; Sirt3
    DOI:  https://doi.org/10.1016/j.abb.2020.108415
  3. FEBS J. 2020 Jun 20.
    Hurtado E, Núñez-Álvarez Y, Muñoz M, Gutiérrez-Caballero C, Casas J, Pendás AM, Peinado MA, Suelves M.
      Skeletal muscle is the largest tissue in mammalian organisms and is a key determinant of basal metabolic rate and whole-body energy metabolism. Histone deacetylase 11 (HDAC11) is the only member of the class IV subfamily of HDACs and it is highly expressed in skeletal muscle, but its role in skeletal muscle physiology has never been investigated. Here, we describe for the first time the consequences of HDAC11 genetic deficiency in skeletal muscle, which results in the improvement of muscle function enhancing fatigue resistance and muscle strength. Loss of HDAC11 had no obvious impact on skeletal muscle structure but increased the number of oxidative myofibers by promoting a glycolytic-to-oxidative muscle fiber switch. Unexpectedly, HDAC11 was localized in muscle mitochondria and its deficiency enhanced mitochondrial content. In particular, we showed that HDAC11 depletion increased mitochondrial fatty acid β-oxidation through activating the AMPK-ACC pathway and reducing acylcarnitine levels in vivo, thus providing a mechanistic explanation for the improved muscle strength and fatigue resistance. Overall, our data reveal a unique role of HDAC11 in the maintenance of muscle fiber-type balance and the mitochondrial lipid oxidation. These findings shed light on the mechanisms governing muscle metabolism and may have implications for chronic muscle metabolic diseases management.
    Keywords:  HDAC11; acylcarnitines; fatigue resistance; fatty acid oxidation; fiber type; mitochondria; oxidative metabolism; skeletal muscle
    DOI:  https://doi.org/10.1111/febs.15456
  4. Cell Metab. 2020 Jun 16. pii: S1550-4131(20)30304-1. [Epub ahead of print]
    McElroy GS, Reczek CR, Reyfman PA, Mithal DS, Horbinski CM, Chandel NS.
      Mitochondrial complex I regenerates NAD+ and proton pumps for TCA cycle function and ATP production, respectively. Mitochondrial complex I dysfunction has been implicated in many brain pathologies including Leigh syndrome and Parkinson's disease. We sought to determine whether NAD+ regeneration or proton pumping, i.e., bioenergetics, is the dominant function of mitochondrial complex I in protection from brain pathology. We generated a mouse that conditionally expresses the yeast NADH dehydrogenase (NDI1), a single enzyme that can replace the NAD+ regeneration capability of the 45-subunit mammalian mitochondrial complex I without proton pumping. NDI1 expression was sufficient to dramatically prolong lifespan without significantly improving motor function in a mouse model of Leigh syndrome driven by the loss of NDUFS4, a subunit of mitochondrial complex I. Therefore, mitochondrial complex I activity in the brain supports organismal survival through its NAD+ regeneration capacity, while optimal motor control requires the bioenergetic function of mitochondrial complex I.
    Keywords:  Leigh syndrome; NAD; ataxia; metabolism; microglia; mitochondria; mitochondrial complex I; mitochondrial disease; neurodegeneration; neurometabolism
    DOI:  https://doi.org/10.1016/j.cmet.2020.06.003
  5. FASEB J. 2020 Jun 22.
    Ruiz CF, Montal ED, Haley JA, Bott AJ, Haley JD.
      Cancer cells require extensive metabolic reprograming in order to provide the bioenergetics and macromolecular precursors needed to sustain a malignant phenotype. Mutant KRAS is a driver oncogene that is well-known for its ability to regulate the ERK and PI3K signaling pathways. However, it is now appreciated that KRAS can promote the tumor growth via upregulation of anabolic metabolism. We recently reported that oncogenic KRAS promotes a gene expression program of de novo lipogenesis in non-small cell lung cancer (NSCLC). To define the mechanism(s) responsible, we focused on the lipogenic transcription factor SREBP1. We observed that KRAS increases SREBP1 expression and genetic knockdown of SREBP1 significantly inhibited the cell proliferation of mutant KRAS-expressing cells. Unexpectedly, lipogenesis was not significantly altered in cells subject to SREBP1 knockdown. Carbon tracing metabolic studies showed a significant decrease in oxidative phosphorylation and RNA-seq data revealed a significant decrease in mitochondrial encoded subunits of the electron transport chain (ETC). Taken together, these data support a novel role, distinct from lipogenesis, of SREBP1 on mitochondrial function in mutant KRAS NSCLC.
    Keywords:  cancer metabolism; de novo lipogenesis; electron transport chain; oxidative phosphorylation
    DOI:  https://doi.org/10.1096/fj.202000052R
  6. Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30796-8. [Epub ahead of print]31(12): 107815
    Utley A, Chavel C, Lightman S, Holling GA, Cooper J, Peng P, Liu W, Barwick BG, Gavile CM, Maguire O, Murray-Dupuis M, Rozanski C, Jordan MS, Kambayashi T, Olejniczak SH, Boise LH, Lee KP.
      Durable humoral immunity against epidemic infectious disease requires the survival of long-lived plasma cells (LLPCs). LLPC longevity is dependent on metabolic programs distinct from short-lived plasma cells (SLPCs); however, the mechanistic basis for this difference is unclear. We have previously shown that CD28, the prototypic T cell costimulatory receptor, is expressed on both LLPCs and SLPCs but is essential only for LLPC survival. Here we show that CD28 transduces pro-survival signaling specifically in LLPCs through differential SLP76 expression. CD28 signaling in LLPCs increased glucose uptake, mitochondrial mass/respiration, and reactive oxygen species (ROS) production. Unexpectedly, CD28-mediated regulation of mitochondrial respiration, NF-κB activation, and survival was ROS dependent. IRF4, a target of NF-κB, was upregulated by CD28 activation in LLPCs and decreased IRF4 levels correlated with decreased glucose uptake, mitochondrial mass, ROS, and CD28-mediated survival. Altogether, these data demonstrate that CD28 signaling induces a ROS-dependent metabolic program required for LLPC survival.
    Keywords:  CD28; IRF4; ROS; SLP76; metabolism; mitochondria; plasma cell
    DOI:  https://doi.org/10.1016/j.celrep.2020.107815
  7. FASEB J. 2020 Jun 27.
    Shao S, Zhao L, An G, Zhang L, Jing X, Luo M, Li W, Meng D, Ning Q, Zhao X, Lei J.
      The tumor microenvironment (TME) is a crucial factor in cancer progression. In breast cancer, cancer-associated fibroblasts (CAFs) and the derived stromal components have been recognized as comprising the majority of the pathological structure of the TME. In this study, we show that metformin (Met), a diabetes drug, transforms CAFs in the TME. Met disrupts tumor-stromal cross talk by preventing breast cancer cell transforming growth factor-β (TGF-β) signaling and the production of stromal-derived factor-1 (SDF-1) and interleukin-8 (IL-8) by CAFs. The suppression of bidirectional signaling between tumor cells and CAFs by Met is attributed to increased phospho-AMP kinase (p-AMPK) levels. By upregulating p-AMPK in CAFs, Met induces prolyl hydroxylases (PHDs), leading to the degradation of hypoxia-inducible factor-1α (HIF-1α) in CAFs. Moreover, interruption of HIF-1α-driven SDF-1 signaling in CAFs by Met leads to decreased breast cancer cell invasion. These findings suggest that Met may be used to target tumor-promoting signaling between CAFs and breast cancer cells in the TME.
    Keywords:  cancer-associated fibroblasts; hypoxia-inducible factor-1α; metformin; phospho-AMPK; tumor microenvironment
    DOI:  https://doi.org/10.1096/fj.202000951RR
  8. Front Cell Dev Biol. 2020 ;8 397
    Reina S, Pittalà MGG, Guarino F, Messina A, De Pinto V, Foti S, Saletti R.
      Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.
    Keywords:  Orbitrap Fusion Tribrid; ROS; cysteine overoxidation; outer mitochondrial membrane; posttranslational modification; voltage-dependent anion selective channel isoforms
    DOI:  https://doi.org/10.3389/fcell.2020.00397
  9. J Exp Clin Cancer Res. 2020 Jun 23. 39(1): 119
    Cao Y, Wang J, Tian H, Fu GH.
      BACKGROUND: Gastric cancer (GC) is a common form of malignant cancer in worldwide which has a poor prognosis. Despite recent improvements in the treatment of GC, the prognosis is not yet satisfactory for GC patients. CYT997, a novel microtubule-targeting agent, recently has been identified to be a promising anticancer candidate for the treatment of cancers; however, the effects of CYT997 in GC remain largely unknown.METHODS: Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry. The mitochondrial ROS were detected by confocal microscope and flow cytometry. Gastric cancer patient-derived xenograft (PDX) model was used to evaluate its antitumor activity of CYT997 in vivo.
    RESULTS: CYT997 inhibited gastric cancer cell proliferation and induced cell apoptosis and triggered autophagy. CYT997 induced apoptosis through triggering intracellular mitochondrial ROS generation in GC cells. ROS scavengers N-acetylcysteine (NAC) and Mitoquinone (MitoQ) distinctly weakened CYT997-induced cell cycle G2/M arrest and apoptosis in GC cells. Pretreatment with autophagy inhibitor 3-MA promoted the effect of CYT997 on cells apoptosis. Mechanistically, CYT997 performed its function through regulation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in GC cells. In addition, CYT997 inhibited growth of gastric cancer patient-derived xenograft (PDX) tumors.
    CONCLUSIONS: CYT997 induces autophagy and apoptosis in gastric cancer by triggering mitochondrial ROS accumulation to silence JAK2/STAT3 pathway. CYT997 might be a potential antitumor drug candidate to treat GC.
    Keywords:  Apoptosis; CYT997; Gastric cancer; JAK2/STAT3; ROS
    DOI:  https://doi.org/10.1186/s13046-020-01621-y
  10. Cancers (Basel). 2020 Jun 21. pii: E1643. [Epub ahead of print]12(6):
    Anania S, Peiffer R, Rademaker G, Hego A, Thiry M, Deldicque L, Francaux M, Maloujahmoum N, Agirman F, Bellahcène A, Castronovo V, Peulen O.
      Pancreas ductal adenocarcinoma is one of the deadliest cancers where surgery remains the main survival factor. Mitochondria were described to be involved in tumor aggressiveness in several cancer types including pancreas cancer. We have previously reported that myoferlin controls mitochondrial structure and function, and demonstrated that myoferlin depletion disturbs the mitochondrial dynamics culminating in a mitochondrial fission. In order to unravel the mechanism underlying this observation, we explored the myoferlin localization in pancreatic cancer cells and showed a colocalization with the mitochondrial dynamic machinery element: mitofusin. This colocalization was confirmed in several pancreas cancer cell lines and in normal cell lines as well. Moreover, in pancreas cancer cell lines, it appeared that myoferlin interacted with mitofusin. These discoveries open-up new research avenues aiming at modulating mitofusin function in pancreas cancer.
    Keywords:  mitochondria; mitofusin; myoferlin; pancreas cancer
    DOI:  https://doi.org/10.3390/cancers12061643
  11. Cells. 2020 Jun 23. pii: E1529. [Epub ahead of print]9(6):
    Fiorillo M, Tóth F, Brindisi M, Sotgia F, Lisanti MP.
      Deferiprone (DFP), also known as Ferriprox, is an FDA-approved, orally active, iron chelator that is currently used clinically for the treatment of iron-overload, especially in thalassaemia major. As iron is a critical factor in Fe-S cluster assembly that is absolutely required for the metabolic function of mitochondria, we hypothesized that DFP treatment could be used to selectively target mitochondria in cancer stem cells (CSCs). For this purpose, we used two ER(+) human breast cancer cell lines, namely MCF7 and T47D cells, as model systems. More specifically, a 3D tumorsphere assay was employed as a functional readout of CSC activity which measures anchorage-independent growth under low attachment conditions. Here, we show that DFP dose dependently inhibited the propagation of CSCs, with an IC-50 of ~100 nM for MCF7 and an IC-50 of ~0.5 to 1 μM for T47D cells, making DFP one the most potent FDA-approved drugs that we and others have thus far identified for targeting CSCs. Mechanistically, we show that high concentrations of DFP metabolically targeted both mitochondrial oxygen consumption (OCR) and glycolysis (extracellular acidification rates (ECAR)) in MCF7 and T47D cell monolayers. Most importantly, we demonstrate that DFP also induced a generalized increase in reactive oxygen species (ROS) and mitochondrial superoxide production, and its effects reverted in the presence of N-acetyl-cysteine (NAC). Therefore, we propose that DFP is a new candidate therapeutic for drug repurposing and for Phase II clinical trials aimed at eradicating CSCs.
    Keywords:  CSCs; FDA-approved drugs; ROS; deferiprone; iron chelators; mitochondrial metabolism
    DOI:  https://doi.org/10.3390/cells9061529
  12. Cell Metab. 2020 Jun 22. pii: S1550-4131(20)30307-7. [Epub ahead of print]
    Gnad T, Navarro G, Lahesmaa M, Reverte-Salisa L, Copperi F, Cordomi A, Naumann J, Hochhäuser A, Haufs-Brusberg S, Wenzel D, Suhr F, Jespersen NZ, Scheele C, Tsvilovskyy V, Brinkmann C, Rittweger J, Dani C, Kranz M, Deuther-Conrad W, Eltzschig HK, Niemi T, Taittonen M, Brust P, Nuutila P, Pardo L, Fleischmann BK, Blüher M, Franco R, Bloch W, Virtanen KA, Pfeifer A.
      The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.
    Keywords:  GPCR; adenosine; adenosine receptor A2B; aging; brown adipose tissue; browning; energy metabolism; muscle; obesity; sarcopenia
    DOI:  https://doi.org/10.1016/j.cmet.2020.06.006
  13. Cells. 2020 Jun 20. pii: E1505. [Epub ahead of print]9(6):
    Battaglia AM, Chirillo R, Aversa I, Sacco A, Costanzo F, Biamonte F.
      Ferroptosis is a new type of oxidative regulated cell death (RCD) driven by iron-dependent lipid peroxidation. As major sites of iron utilization and master regulators of oxidative metabolism, mitochondria are the main source of reactive oxygen species (ROS) and, thus, play a role in this type of RCD. Ferroptosis is, indeed, associated with severe damage in mitochondrial morphology, bioenergetics, and metabolism. Furthermore, dysregulation of mitochondrial metabolism is considered a biochemical feature of neurodegenerative diseases linked to ferroptosis. Whether mitochondrial dysfunction can, per se, initiate ferroptosis and whether mitochondrial function in ferroptosis is context-dependent are still under debate. Cancer cells accumulate high levels of iron and ROS to promote their metabolic activity and growth. Of note, cancer cell metabolic rewiring is often associated with acquired sensitivity to ferroptosis. This strongly suggests that ferroptosis may act as an adaptive response to metabolic imbalance and, thus, may constitute a new promising way to eradicate malignant cells. Here, we review the current literature on the role of mitochondria in ferroptosis, and we discuss opportunities to potentially use mitochondria-mediated ferroptosis as a new strategy for cancer therapy.
    Keywords:  ROS; cancer; cell death; ferroptosis; iron; mitochondria
    DOI:  https://doi.org/10.3390/cells9061505
  14. Aging (Albany NY). 2020 Jun 23. 12
    Yen K, Mehta HH, Kim SJ, Lue Y, Hoang J, Guerrero N, Port J, Bi Q, Navarrete G, Brandhorst S, Lewis KN, Wan J, Swerdloff R, Mattison JA, Buffenstein R, Breton CV, Wang C, Longo V, Atzmon G, Wallace D, Barzilai N, Cohen P.
      Humanin is a member of a new family of peptides that are encoded by short open reading frames within the mitochondrial genome. It is conserved in animals and is both neuroprotective and cytoprotective. Here we report that in C. elegans the overexpression of humanin is sufficient to increase lifespan, dependent on daf-16/Foxo. Humanin transgenic mice have many phenotypes that overlap with the worm phenotypes and, similar to exogenous humanin treatment, have increased protection against toxic insults. Treating middle-aged mice twice weekly with the potent humanin analogue HNG, humanin improves metabolic healthspan parameters and reduces inflammatory markers. In multiple species, humanin levels generally decline with age, but here we show that levels are surprisingly stable in the naked mole-rat, a model of negligible senescence. Furthermore, in children of centenarians, who are more likely to become centenarians themselves, circulating humanin levels are much greater than age-matched control subjects. Further linking humanin to healthspan, we observe that humanin levels are decreased in human diseases such as Alzheimer's disease and MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes). Together, these studies are the first to demonstrate that humanin is linked to improved healthspan and increased lifespan.
    Keywords:  aging; humanin; mitochondria; peptides
    DOI:  https://doi.org/10.18632/aging.103534
  15. PLoS One. 2020 ;15(6): e0234606
    Pérez-Rial S, Barreiro E, Fernández-Aceñero MJ, Fernández-Valle ME, González-Mangado N, Peces-Barba G.
      Skeletal muscle dysfunction is a common complication and an important prognostic factor in patients with chronic obstructive pulmonary disease (COPD). It is associated with intrinsic muscular abnormalities of the lower extremities, but it is not known whether there is an easy way to predict its presence. Using a mouse model of chronic cigarette smoke exposure, we tested the hypothesis that magnetic resonance spectroscopy allows us to detect muscle bioenergetic deficit in early stages of lung disease. We employed this technique to evaluate the synthesis rate of adenosine triphosphate (ATP) and characterize concomitant mitochondrial dynamics patterns in the gastrocnemius muscle of emphysematous mice. The fibers type composition and citrate synthase (CtS) and cytochrome c oxidase subunit IV (COX4) enzymatic activities were evaluated. We found that the rate of ATP synthesis was reduced in the distal skeletal muscle of mice exposed to cigarette smoke. Emphysematous mice showed a significant reduction in body weight gain, in the cross-sectional area of the total fiber and in the COX4 to CtS activity ratio, due to a significant increase in CtS activity of the gastrocnemius muscle. Taken together, these data support the hypothesis that in the early stage of lung disease, we can detect a decrease in ATP synthesis in skeletal muscle, partly caused by high oxidative mitochondrial enzyme activity. These findings may be relevant to predict the presence of skeletal bioenergetic deficit in the early stage of lung disease besides placing the mitochondria as a potential therapeutic target for the treatment of COPD comorbidities.
    DOI:  https://doi.org/10.1371/journal.pone.0234606
  16. Crit Rev Biochem Mol Biol. 2020 Jun 24. 1-13
    Nesci S, Pagliarani A, Algieri C, Trombetti F.
      Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic FO domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The FO complexity in mitochondria is apparently related to additional enzyme functions that lack in bacterial and thylakoid complexes. Indeed, the F-type ATP synthase has the main bioenergetic role to synthesize ATP by exploiting the electrochemical gradient built by respiratory complexes. The FO membrane domain, essential in the enzyme machinery, also participates in the bioenergetic cost of synthesizing ATP and in the formation of the cristae, thus contributing to mitochondrial morphology. The recent enzyme involvement in a high-conductance channel, which forms in the inner mitochondrial membrane and promotes the mitochondrial permeability transition, highlights a new F-type ATP synthase role. Point mutations which cause amino acid substitutions in FO subunits produce mitochondrial dysfunctions and lead to severe pathologies. The FO variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.
    Keywords:  F1FO-ATPase; FO domain; membrane; mitochondria; molecular mechanism; structure
    DOI:  https://doi.org/10.1080/10409238.2020.1784084
  17. Cancer Discov. 2020 Jun 22. pii: CD-19-1228. [Epub ahead of print]
    Ngo B, Kim E, Osorio-Vasquez V, Doll S, Bustraan S, Liang RJ, Luengo A, Davidson SM, Ali A, Ferraro GB, Fischer GM, Eskandari R, Kang DS, Ni J, Plasger A, Rajasekhar VK, Kastenhuber ER, Bacha S, Sriram RK, Stein BD, Bakhoum SF, Snuderl M, Cotzia P, Healey JH, Mainolfi N, Suri V, Friedman A, Manfredi M, Sabatini DM, Jones DR, Yu M, Zhao JJ, Jain RK, Keshari KR, Davies MA, Vander Heiden MG, Hernando E, Mann M, Cantley LC, Pacold ME.
      A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacological inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggests that PHGDH inhibitors may be useful in the treatment of brain metastasis.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-1228
  18. Cells. 2020 Jun 22. pii: E1520. [Epub ahead of print]9(6):
    Shah SI, Ullah G.
      Mitochondrial Ca2+ ([Ca2+]M) uptake through its Ca2+ uniporter (MCU) is central to many cell functions such as bioenergetics, spatiotemporal organization of Ca2+ signals, and apoptosis. MCU activity is regulated by several intrinsic proteins including MICU1, MICU2, and EMRE. While significant details about the role of MICU1, MICU2, and EMRE in MCU function have emerged recently, a key challenge for the future experiments is to investigate how these regulatory proteins modulate mitochondrial Ca2+ influx through MCU in intact cells under pathophysiological conditions. This is further complicated by the fact that several variables affecting MCU function change dynamically as cell functions. To overcome this void, we develop a data-driven model that closely replicates the behavior of MCU under a wide range of cytosolic Ca2+ ([Ca2+]C), [Ca2+]M, and mitochondrial membrane potential values in WT, MICU1 knockout (KO), and MICU2 KO cells at the single mitochondrion and whole-cell levels. The model is extended to investigate how MICU1 or MICU2 KO affect mitochondrial function. Moreover, we show how Ca2+ buffering proteins, the separation between mitochondrion and Ca2+-releasing stores, and the duration of opening of Ca2+-releasing channels affect mitochondrial function under different conditions. Finally, we demonstrate an easy extension of the model to single channel function of MCU.
    Keywords:  Ca2+ overload; EMRE; MICU1; MICU2; mitochondrial Ca2+ uniporter; mitochondrial Ca2+ uptake
    DOI:  https://doi.org/10.3390/cells9061520
  19. Redox Biol. 2020 Jun 10. pii: S2213-2317(20)30807-7. [Epub ahead of print]36 101602
    Love DT, Guo C, Nikelshparg EI, Brazhe NA, Sosnovtseva O, Hawkins CL.
      A host of chronic inflammatory diseases are accelerated by the formation of the powerful oxidant hypochlorous acid (HOCl) by myeloperoxidase (MPO). In the presence of thiocyanate (SCN-), the production of HOCl by MPO is decreased in favour of the formation of a milder oxidant, hypothiocyanous acid (HOSCN). The role of HOSCN in disease has not been fully elucidated, though there is increasing interest in using SCN- therapeutically in different disease settings. Unlike HOCl, HOSCN can be detoxified by thioredoxin reductase, and reacts selectively with thiols to result in reversible modifications, which could potentially reduce the extent of MPO-induced damage during chronic inflammation. In this study, we show that exposure of macrophages, a key inflammatory cell type, to HOSCN results in the reversible modification of multiple mitochondrial proteins, leading to increased mitochondrial membrane permeability, decreased oxidative phosphorylation and reduced formation of ATP. The increased permeability and reduction in ATP could be reversed by pre-treatment of the macrophages with cyclosporine A, implicating a role for the mitochondrial permeability transition pore. HOSCN also drives cells to utilise fatty acids as an energetic substrate after the inhibition of oxidative phosphorylation. Raman imaging studies highlighted the ability of HOSCN to perturb the electron transport chain of mitochondria and redistribute these organelles within the cell. Taken together, these data provide new insight into the pathways by which HOSCN can induce cytotoxicity and cellular damage, which may have relevance for the development of inflammatory disease, and therapeutic strategies to reduce HOCl-induced damage by supplementation with SCN-.
    Keywords:  Atherosclerosis; Hypothiocyanous acid; Inflammation; Mitochondria; Myeloperoxidase; Raman spectroscopy; Thiocyanate
    DOI:  https://doi.org/10.1016/j.redox.2020.101602
  20. Int J Mol Sci. 2020 Jun 19. pii: E4378. [Epub ahead of print]21(12):
    Tsao YC, Chang YJ, Wang CH, Chen L.
      Isoplumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone), a naturally occurring quinone from Lawsonia inermis and Plumbago europaea, has been reported to have anti-inflammatory and antimicrobial activity. Inflammation has long been implicated in cancer progression. In this study, we examined the anticancer effect of chemically synthesized isoplumbagin. Our results revealed that isoplumbagin treatment suppressed cell viability and invasion of highly invasive oral squamous cell carcinoma (OSCC) OC3-IV2 cells, glioblastoma U87 cells, non-small cell lung carcinoma H1299 cells, prostate cancer PC3 cells, and cervical cancer HeLa cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Boyden chamber assays. In vivo studies demonstrate the inhibitory effect of 2 mg/kg isoplumbagin on the growth of orthotopic xenograft tumors derived from OSCC cells. Mechanistically, isoplumbagin exerts its cytotoxic effect through acting as a substrate of reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] dehydrogenase quinone 1 (NQO1) to generate hydroquinone, which reverses mitochondrial fission phenotype, reduces mitochondrial complex IV activity, and thus compromises mitochondrial function. Collectively, this work reveals an anticancer activity of isoplumbagin mainly through modulating mitochondrial dynamics and function.
    Keywords:  NQO1; cancer; isoplumbagin; metastasis; quinone
    DOI:  https://doi.org/10.3390/ijms21124378
  21. Nat Commun. 2020 Jun 26. 11(1): 3243
    Guo Y, Ye Q, Deng P, Cao Y, He D, Zhou Z, Wang C, Zaytseva YY, Schwartz CE, Lee EY, Mark Evers B, Morris AJ, Liu S, She QB.
      Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC), but the underlying mechanism is incompletely characterized. Here, we report that spermine synthase (SMS), a polyamine biosynthetic enzyme, is overexpressed in CRC. Targeted disruption of SMS in CRC cells results in spermidine accumulation, which inhibits FOXO3a acetylation and allows subsequent translocation to the nucleus to transcriptionally induce expression of the proapoptotic protein Bim. However, this induction is blunted by MYC-driven expression of miR-19a and miR-19b that repress Bim production. Pharmacological or genetic inhibition of MYC activity in SMS-depleted CRC cells dramatically induces Bim expression and apoptosis and causes tumor regression, but these effects are profoundly attenuated by silencing Bim. These findings uncover a key survival signal in CRC through convergent repression of Bim expression by distinct SMS- and MYC-mediated signaling pathways. Thus, combined inhibition of SMS and MYC signaling may be an effective therapy for CRC.
    DOI:  https://doi.org/10.1038/s41467-020-17067-x
  22. J Immunother Cancer. 2020 Jun;pii: e000289. [Epub ahead of print]8(1):
    Chen D, Barsoumian HB, Fischer G, Yang L, Verma V, Younes AI, Hu Y, Masropour F, Klein K, Vellano C, Marszalek J, Davies M, Cortez MA, Welsh J.
      BACKGROUND: Despite outstanding responses to anti-PD-1 agents in a subset of non-small cell lung cancer (NSCLC) patients, approximately 80% of patients fail to have prolonged favorable response. Recent studies show that tumor cell oxidative metabolism is a barrier to PD-1 immunotherapy and radiotherapy could overcome PD-1 resistance, so it is urgent to determine if combination treatment with radiotherapy and a novel oxidative phosphorylation (OXPHOS) inhibitor (IACS-010759) is an effective strategy against PD-1 resistance in NSCLC.METHODS: The antitumor effect of this combinational treatment was evaluated in vitro and in vivo. For in vivo experiments, we treated 129Sv/Ev mice with anti-PD1-sensitive and anti-PD1-resistant 344SQ NSCLC adenocarcinoma xenografts with oral IACS-010759 combined with radiotherapy (XRT). In vitro experiments included PCR, seahorse bioenergetic profiling, flow cytometry phenotyping, and clonogenic survival assay.
    RESULTS: In the current study, we found that our PD-1-resistant model utilized OXPHOS to a significantly greater extent than the PD-1-sensitive model and XRT increased OXPHOS in vitro and in vivo. Thus, we explored the effect of the novel OXPHOS inhibitor IACS-010759 on PD-1-resistant NSCLC in an effort to overcome XRT-induced immunosuppression and maximize response to PD-1. Additionally, combined XRT and IACS-010759 promoted antitumor effects in the PD-1-resistant model, but not in the sensitive model. After elucidation of the most optimal dose/fractionation scheme of XRT with IACS-010759, the combinatorial therapy with this regimen did not increase the abscopal antitumor effect, although IACS-010549 did not decrease CD45+, CD4+, and CD8+ immune cells. Finally, triple therapy with IACS-010759, XRT, and anti-PD-1 promoted abscopal responses and prolonged survival time.
    CONCLUSION: OXPHOS inhibition as part of a combinatorial regimen with XRT is a promising strategy to address PD-1-resistant NSCLC, and this combination is being tested clinically.
    Keywords:  immunology; radiotherapy; tumor
    DOI:  https://doi.org/10.1136/jitc-2019-000289
  23. Biochemistry (Mosc). 2020 Jun;85(6): 651-659
    Sokolov SS, Severin FF.
      Up to now numerous studies in the field of gerontology have been published. Nevertheless, a well-known food restriction remains the most reliable and efficient way of lifespan extension. Physical activity is also a well-documented anti-aging intervention being especially efficient in slowing down the age-associated decline of skeletal muscle mass. In this review we focus on the molecular mechanisms of the effect of physical exercise on muscle tissues. We also discuss the possibilities of pharmacological extension of this effect to the rest of the tissues. During the exercise, the level of ATP decreases triggering activation of AMP-dependent protein kinase (AMPK). This kinase stimulates antioxidant potential of the cells and their mitochondrial respiratory capacity. The exercise also induces mild oxidative stress, which, in turn, mediates the stimulation via hormetic response. Furthermore, during the exercise cells generate activators of mammalian target of rapamycin (mTOR). The intracellular ATP level increases during the rest periods between exercises thus promoting mTOR activation. Therefore, regular exercise intermittently activates anti-oxidant defenses and mitochondrial biogenesis (via AMPK and the hormetic response) of the muscle tissue, as well as its proliferative potential (via mTOR), which, in turn, impedes the age-dependent muscle atrophy. Thus, the intermittent treatment with activators of (i) AMPK combined with the inducers of hormetic response and of (ii) mTOR might partly mimic the effects of physical exercise. Importantly, pharmacological activation of AMPK takes place in the absence of ATP level decrease. The use of uncouplers of respiration and oxidative phosphorylation at the phase of AMPK activation could also prevent negative consequences of the cellular hyper-energization. It is believed that the decline of both antioxidant and proliferative potentials of the cells causes the age-dependent decline of multiple tissues, rather than only the muscular one. We argue that the approach above is applicable for the majority of tissues in an organism.
    DOI:  https://doi.org/10.1134/S0006297920060024
  24. Molecules. 2020 Jun 21. pii: E2857. [Epub ahead of print]25(12):
    Boguszewska K, Szewczuk M, Kaźmierczak-Barańska J, Karwowski BT.
      Mitochondria emerged from bacterial ancestors during endosymbiosis and are crucial for cellular processes such as energy production and homeostasis, stress responses, cell survival, and more. They are the site of aerobic respiration and adenosine triphosphate (ATP) production in eukaryotes. However, oxidative phosphorylation (OXPHOS) is also the source of reactive oxygen species (ROS), which are both important and dangerous for the cell. Human mitochondria contain mitochondrial DNA (mtDNA), and its integrity may be endangered by the action of ROS. Fortunately, human mitochondria have repair mechanisms that allow protecting mtDNA and repairing lesions that may contribute to the occurrence of mutations. Mutagenesis of the mitochondrial genome may manifest in the form of pathological states such as mitochondrial, neurodegenerative, and/or cardiovascular diseases, premature aging, and cancer. The review describes the mitochondrial structure, genome, and the main mitochondrial repair mechanism (base excision repair (BER)) of oxidative lesions in the context of common features between human mitochondria and bacteria. The authors present a holistic view of the similarities of mitochondria and bacteria to show that bacteria may be an interesting experimental model for studying mitochondrial diseases, especially those where the mechanism of DNA repair is impaired.
    Keywords:  BER; DNA repair; ROS; mitochondria; mtDNA
    DOI:  https://doi.org/10.3390/molecules25122857
  25. Proc Natl Acad Sci U S A. 2020 Jun 25. pii: 202004223. [Epub ahead of print]
    Greenway R, Barts N, Henpita C, Brown AP, Arias Rodriguez L, Rodríguez Peña CM, Arndt S, Lau GY, Murphy MP, Wu L, Lin D, Tobler M, Kelley JL, Shaw JH.
      Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)-a toxicant that impairs mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and-in some instances-codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.
    Keywords:  adaptive evolution; comparative physiology; ecological genomics; hydrogen sulfide; phylogenetic comparative analysis
    DOI:  https://doi.org/10.1073/pnas.2004223117
  26. Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30787-7. [Epub ahead of print]31(12): 107806
    Triki M, Rinaldi G, Planque M, Broekaert D, Winkelkotte AM, Maier CR, Janaki Raman S, Vandekeere A, Van Elsen J, Orth MF, Grünewald TGP, Schulze A, Fendt SM.
      Cancer cells display an increased plasticity in their lipid metabolism, which includes the conversion of palmitate to sapienate via the enzyme fatty acid desaturase 2 (FADS2). We find that FADS2 expression correlates with mammalian target of rapamycin (mTOR) signaling and sterol regulatory element-binding protein 1 (SREBP-1) activity across multiple cancer types and is prognostic in some cancer types. Accordingly, activating mTOR signaling by deleting tuberous sclerosis complex 2 (Tsc2) or overexpression of SREBP-1/2 is sufficient to increase FADS2 mRNA expression and sapienate metabolism in mouse embryonic fibroblasts (MEFs) and U87 glioblastoma cells, respectively. Conversely, inhibiting mTOR signaling decreases FADS2 expression and sapienate biosynthesis in MEFs with Tsc2 deletion, HUH7 hepatocellular carcinoma cells, and orthotopic HUH7 liver xenografts. In conclusion, we show that mTOR signaling and SREBP activity are sufficient to activate sapienate metabolism by increasing FADS2 expression. Consequently, targeting mTOR signaling can reduce sapienate metabolism in vivo.
    Keywords:  FADS2; SCD1; SREBP; cancer; fatty acid metabolism; glioblastoma; hepatocellular carcinoma; mTOR; palmitate; palmitoleate; sapienate
    DOI:  https://doi.org/10.1016/j.celrep.2020.107806
  27. Trends Mol Med. 2020 Jul;pii: S1471-4914(20)30062-9. [Epub ahead of print]26(7): 698-709
    Jackson CB, Turnbull DM, Minczuk M, Gammage PA.
      Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease.
    Keywords:  gene therapy; heteroplasmy; mitoTALEN; mitochondrial disease; mtDNA; mtZFN
    DOI:  https://doi.org/10.1016/j.molmed.2020.02.006
  28. Biochem Soc Trans. 2020 Jun 23. pii: BST20190033. [Epub ahead of print]
    Griffiths HBS, Williams C, King SJ, Allison SJ.
      Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH are essential coupled redox metabolites that primarily promote cellular oxidative (catabolic) metabolic reactions. This enables energy generation through glycolysis and mitochondrial respiration to support cell growth and survival. In addition, many key enzymes that regulate diverse cell functions ranging from gene expression to proteostasis require NAD+ as a co-substrate for their catalytic activity. This includes the NAD+-dependent sirtuin family of protein deacetylases and the PARP family of DNA repair enzymes. Whilst their vital activity consumes NAD+ which is cleaved to nicotinamide, several pathways exist for re-generating NAD+ and sustaining NAD+ homeostasis. However, there is growing evidence of perturbed NAD+ homeostasis and NAD+-regulated processes contributing to multiple disease states. NAD+ levels decline in the human brain and other organs with age and this is associated with neurodegeneration and other age-related diseases. Dietary supplementation with NAD+ precursors is being investigated to counteract this. Paradoxically, many cancers have increased dependency on NAD+. Clinical efforts to exploit this have so far shown limited success. Emerging new opportunities to exploit dysregulation of NAD+ metabolism in cancers are critically discussed. An update is also provided on other key NAD+ research including perturbation of the NAD+ salvage enzyme NAMPT in the context of the tumour microenvironment (TME), methodology to study subcellular NAD+ dynamics in real-time and the regulation of differentiation by competing NAD+ pools.
    Keywords:  NAMPT; PARP; cancer therapeutics; lactate dehydrogenase A; nicotinamide adenine dinucleotide; sirtuins
    DOI:  https://doi.org/10.1042/BST20190033
  29. Redox Biol. 2020 Jun 01. pii: S2213-2317(20)30563-2. [Epub ahead of print]36 101595
    Borah K, Rickman OJ, Voutsina N, Ampong I, Gao D, Baple EL, Dias IH, Crosby AH, Griffiths HR.
      Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples.
    Keywords:  Blood; Brain oxysterol; Cholesterol; Dihydroxycholesterol; Liquid chromatography-mass spectrometry; Metabolism; Mitochondria; Monocytes; Neuroblastoma; Oxysterol; Peripheral blood mononuclear cell; Subcellular; Whole cell
    DOI:  https://doi.org/10.1016/j.redox.2020.101595
  30. Biochemistry (Mosc). 2020 Mar;85(3): 257-263
    Levitskii SA, Baleva MV, Chicherin IV, Krasheninnikov IA, Kamenski PA.
      Mitochondria are obligate organelles of most eukaryotic cells that perform many different functions important for cellular homeostasis. The main role of mitochondria is supplying cells with energy in a form of ATP, which is synthesized in a chain of oxidative phosphorylation reactions on the organelle inner membrane. It is commonly believed now that mitochondria have the endosymbiotic origin. In the course of evolution, they have lost most of their genetic material as a result of genome reduction and gene transfer to the nucleus. The majority of mitochondrial proteins are synthesized in the cytosol and then imported to the mitochondria. However, almost all known mitochondria still contain genomes that are maintained and expressed. The processes of protein biosynthesis in the mitochondria - mitochondrial translation - substantially differs from the analogous processes in bacteria and the cytosol of eukaryotic cells. Mitochondrial translation is characterized by a high degree of specialization and specific regulatory mechanisms. In this review, we analyze available information on the common principles of mitochondrial translation with emphasis on the molecular mechanisms of translation initiation in the mitochondria of yeast and mammalian cells.
    DOI:  https://doi.org/10.1134/S0006297920030013
  31. Mol Ther. 2020 Jun 12. pii: S1525-0016(20)30300-2. [Epub ahead of print]
    Luna-Sanchez M, Benincá C, Cerutti R, Brea-Calvo G, Yeates A, Scorrano L, Zeviani M, Viscomi C.
      Moderate overexpression of Opa1, the master regulator of mitochondrial cristae morphology, significantly improved mitochondrial damage induced by drugs, surgical denervation, or oxidative phosphorylation (OXPHOS) defects due to specific impairment of a single mitochondrial respiratory chain complex. Here, we investigated the effectiveness of this approach in the Mpv17-/- mouse, characterized by profound, multisystem mitochondrial DNA (mtDNA) depletion. After the crossing with Opa1tg mice, we found a surprising anticipation of the severe, progressive focal segmental glomerulosclerosis, previously described in Mpv17-/- animals as a late-onset clinical feature (after 12-18 months of life). In contrast, Mpv17-/- animals from this new "mixed" strain died at 8-9 weeks after birth because of severe kidney failure However, Mpv17-/-::Opa1tg mice lived much longer than Mpv17-/- littermates and developed the kidney dysfunction much later. mtDNA content and OXPHOS activities were significantly higher in Mpv17-/-::Opa1tg than in Mpv17-/- kidneys and similar to those for wild-type (WT) littermates. Mitochondrial network and cristae ultrastructure were largely preserved in Mpv17-/-::Opa1tg versus Mpv17-/- kidney and isolated podocytes. Mechanistically, the protective effect of Opa1 overexpression in this model was mediated by a block in apoptosis due to the stabilization of the mitochondrial cristae. These results demonstrate that strategies aiming at increasing Opa1 expression or activity can be effective against mtDNA depletion syndromes.
    Keywords:  Mpv17; Opa1; apoptosis; focal segmental glomerulosclerosis; mitochondrial DNA depletion; mitochondrial cristae; oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.ymthe.2020.06.010
  32. Metabolism. 2020 Jun 23. pii: S0026-0495(20)30166-9. [Epub ahead of print] 154302
    Noh KH, Kang HM, Yoo W, Min Y, Kim D, Kim M, Wang S, Lim JH, Jung CR.
      BACKGROUND: Intracellular lipid accumulation is associated with various diseases, particularly cancer. Mitochondrial dysfunction is considered as a cause of lipid accumulation; however, the related underlying mechanism remains unclear. Findings We found that Von Hippel-Lindau (VHL)-deficiency led to lipid accumulation and mitochondrial dysfunction in renal cell carcinoma cells. Moreover, VHL downregulated ATP-citrate lyase (ACLY), a key enzyme in de novo lipid synthesis, at the transcriptional level, which inhibited intracellular lipid accumulation in human renal carcinoma tissues. We identified PPARγ as the transcription factor regulating ACLY expression by binding to the cis-regulatory site PPRE on its promoter. VHL directly interacted with and promoted ubiquitination of PPARγ, leading to its degradation both in vitro and in vivo, resulting in the downregulation of ACLY. Furthermore, adenovirus-mediated VHL overexpression substantially ameliorated hepatic steatosis induced by a high-fat diet in db/db mice. Importantly, low VHL expression was associated with high ACLY expression and poor prognosis in human liver carcinoma in a dataset in The Cancer Genome Atlas.CONCLUSIONS: VHL plays role in cellular lipid metabolism via regulating mitochondria and targeting PPARγ, a transcription factor for ACLY independent of hypoxia-inducible factor 1α. A novel VHL-PPARγ-ACLY axis and its implication in fatty liver disease and cancer were uncovered.
    Keywords:  ACLY; Lipid metabolism; PPAR-gamma; Ubiquitination; Von Hippel-Lindau
    DOI:  https://doi.org/10.1016/j.metabol.2020.154302
  33. Int J Mol Sci. 2020 Jun 19. pii: E4374. [Epub ahead of print]21(12):
    Menduti G, Vitaliti A, Capo CR, Lettieri-Barbato D, Aquilano K, Malaspina P, Rossi L.
      Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.
    Keywords:  4-HNE; ALDH5A1; GABA; SSA; SSADH; U87 cells; mitochondria; paraquat
    DOI:  https://doi.org/10.3390/ijms21124374
  34. PLoS One. 2020 ;15(6): e0234653
    Pereira GC, Lee L, Rawlings N, Ouwendijk J, Parker JE, Andrienko TN, Henley JM, Halestrap AP.
      We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].
    DOI:  https://doi.org/10.1371/journal.pone.0234653
  35. Bioorg Chem. 2020 Jun 15. pii: S0045-2068(20)30057-2. [Epub ahead of print]101 103901
    Kim D, Kim SY, Kim D, Yoon NG, Yun J, Hong KB, Lee C, Lee JH, Kang BH, Kang S.
      TNF Receptor Associated Protein 1 (TRAP1) is a mitochondrial paralog of Hsp90 related to the promotion of tumorigenesis in various cancers via maintaining mitochondrial integrity, reducing the production of reactive oxygen species, and reprogramming cellular metabolism. Consequently, Hsp90 and TRAP1 have been targeted to develop cancer therapeutics. Herein, we report a series of pyrazolo[3,4-d]pyrimidine derivatives that are mitochondria-permeable TRAP1 inhibitors. Structure-based drug design guided the optimization of potency, leading to the identification of compounds 47 and 48 as potent TRAP1 and Hsp90 inhibitors with good metabolic and plasma stability as well as acceptable CYP and hERG inhibition. X-ray co-crystallization studies confirmed both 47 and 48 interact with the ATP binding pocket in the TRAP1 protein. Compounds 47 and 48 demonstrated excellent anticancer efficiency in various cancer cells, with limited toxicity over normal hepatocyte and prostate cells. Mouse PC3 xenograft studies showed 47 and 48 significantly reduced tumor growth.
    Keywords:  Anticancer; Drug; Hsp90; Mitochondria; Selectivity; TRAP1
    DOI:  https://doi.org/10.1016/j.bioorg.2020.103901
  36. EMBO J. 2020 Jun 22. e105714
    McArthur K, Ryan MT.
      The many functions of mitochondria-the powerhouses of our cells-are intimately linked with their ultrastructure and network morphology. In this issue, Stephan et al (2020) apply a tour de force of microscopic techniques to examine the contributions of specific mitochondrial proteins to crista architecture.
    DOI:  https://doi.org/10.15252/embj.2020105714
  37. Angew Chem Int Ed Engl. 2020 Jun 22.
    Yang G, Liu Z, Zhang R, Tian X, Chen J, Han G, Liu B, Han X, Fu Y, Hu Z, Zhang Z.
      Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties, however, are that molecular probes suitable for super-resolution nanoscopy are rather rare, and more adversely the use of multiple probes complicates their cellular uptakes, biodistributions, and spectral distinctions. Here, we report a quinolinium derivative probe that is selectively enriched in mitochondria, and switches on in three different fluorescence modes in response to hydrogen peroxide (H2O2), protein, and nucleic acid, enabling the visualization of mitochondrial nucleoprotein dynamics. Stimulated emission depletion (STED) nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H2O2 level leads to disassociation of nucleic acid-protein complexes.
    Keywords:  Reactive Oxygen Species; imaging probe; nucleic acids; proteins; super-resolution
    DOI:  https://doi.org/10.1002/anie.202005959
  38. EMBO J. 2020 Jun 22. e104105
    Stephan T, Brüser C, Deckers M, Steyer AM, Balzarotti F, Barbot M, Behr TS, Heim G, Hübner W, Ilgen P, Lange F, Pacheu-Grau D, Pape JK, Stoldt S, Huser T, Hell SW, Möbius W, Rehling P, Riedel D, Jakobs S.
      Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F1 Fo -ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super-resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re-expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre-existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60-subcomplex is sufficient for CJ formation, whereas the Mic10-subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F1 Fo -ATP synthase, fine-tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.
    Keywords:   MINFLUX ; cristae biogenesis; electron microscopy; nanoscopy; super-resolution microscopy
    DOI:  https://doi.org/10.15252/embj.2019104105
  39. Front Cell Dev Biol. 2020 ;8 413
    Wang Y, Liu HH, Cao YT, Zhang LL, Huang F, Yi C.
      Mitochondria are key cellular organelles and play vital roles in energy metabolism, apoptosis regulation and cellular homeostasis. Mitochondrial dynamics refers to the varying balance between mitochondrial fission and mitochondrial fusion that plays an important part in maintaining mitochondrial homeostasis and quality. Mitochondrial malfunction is involved in aging, metabolic disease, neurodegenerative disorders, and cancers. Mitophagy, a selective autophagy of mitochondria, can efficiently degrade, remove and recycle the malfunctioning or damaged mitochondria, and is crucial for quality control. In past decades, numerous studies have identified a series of factors that regulate mitophagy and are also involved in carcinogenesis, cancer cell migration and death. Therefore, it has become critically important to analyze signal pathways that regulate mitophagy to identify potential therapeutic targets. Here, we review recent progresses in mitochondrial dynamics, the mechanisms of mitophagy regulation, and the implications for understanding carcinogenesis, metastasis, treatment, and drug resistance.
    Keywords:  carcinogenesis; mitochondria; mitochondrial dynamics; mitophagy; therapy
    DOI:  https://doi.org/10.3389/fcell.2020.00413
  40. Elife. 2020 Jun 23. pii: e53618. [Epub ahead of print]9
    Contat C, Ancey PB, Zangger N, Sabatino S, Pascual J, Escrig S, Jensen L, Goepfert C, Lanz B, Lepore M, Gruetter R, Rossier A, Berezowska S, Neppl C, Zlobec I, Clerc-Rosset S, Knott GW, Rathmell JC, Abel ED, Meibom A, Meylan E.
      Glucose utilization increases in tumors, a metabolic process that is observed clinically by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). However, is increased glucose uptake important for tumor cells, and which transporters are implicated in vivo? In a genetically-engineered mouse model of lung adenocarcinoma, we show that the deletion of only one highly expressed glucose transporter, Glut1 or Glut3, in cancer cells does not impair tumor growth, whereas their combined loss diminishes tumor development. 18F-FDG-PET analyses of tumors demonstrate that Glut1 and Glut3 loss decreases glucose uptake, which is mainly dependent on Glut1. Using 13C-glucose tracing with correlated nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy, we also report the presence of lamellar body-like organelles in tumor cells accumulating glucose-derived biomass, depending partially on Glut1. Our results demonstrate the requirement for two glucose transporters in lung adenocarcinoma, the dual blockade of which could reach therapeutic responses not achieved by individual targeting.
    Keywords:  NanoSIMS; cancer biology; genetically engineered mouse model of cancer; glucose transporters; human; lamellar bodies; lung adenocarcinoma; mouse
    DOI:  https://doi.org/10.7554/eLife.53618
  41. Nat Commun. 2020 Jun 26. 11(1): 3244
    Kleinpenning F, Steigenberger B, Wu W, Heck AJR.
      Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.
    DOI:  https://doi.org/10.1038/s41467-020-17010-0
  42. iScience. 2020 Jun 12. pii: S2589-0042(20)30449-1. [Epub ahead of print]23(7): 101263
    Dubois C, Kondratskyi A, Bidaux G, Noyer L, Vancauwenberghe E, Farfariello V, Toillon RA, Roudbaraki M, Tierny D, Bonnal JL, Prevarskaya N, Vanden Abeele F.
      Mitochondria are important cell death checkpoints, and mitochondrial Ca2+ overload is considered as a potent apoptotic intrinsic pathway inducer. Here, we report that this Ca2+ apoptosis link is largely ineffective in inducing cell-death just by itself and required a concomitant inhibition of autophagy to counteract its pro-survival action. In such condition, an acute mitochondrial stress revealed by a DRP1-mediated mitochondrial dynamic remodeling is observed concomitantly with mitochondrial depolarization, release of cytochrome c, and efficient apoptosis induction. We also uncover that mitochondrial Ca2+ status modulates the function of autophagy as a sensitizer for chemotherapies. This priming mediated by mitochondrial Ca2+ overload and inhibition of autophagy sensitizes many cancer cells types to different chemotherapies with independent mechanisms of action. Collectively, our results redefine an important cell signaling pathway, uncovering new combined therapies for the treatment of diseases associated with mitochondrial Ca2+ homeostasis disorders such as cancer.
    Keywords:  Biological Sciences; Cancer; Cell Biology
    DOI:  https://doi.org/10.1016/j.isci.2020.101263
  43. Biochemistry (Mosc). 2020 May;85(5): 636-641
    Chernyak BV.
      "Mitochondrial transplantation" refers to a procedure for introducing isolated mitochondria into a damaged area of a heart or other organ. A considerable amount of data has been accumulated on the therapeutic effects of "mitochondrial transplantation" in animals with ischemic heart damage. In 2017, the first attempts were made to apply this procedure in a clinic. The authors of the method suggest that exogenous mitochondria penetrate into cardiomyocytes, retaining functional activity, and compensate for impaired energy output of endogenous mitochondria. This hypothesis contradicts the well-known fact of loss of mitochondrial functions in the presence of high concentrations of Ca2+, which are characteristic of the extracellular medium. This review critically considers the possible mechanisms of the therapeutic effect of "mitochondrial transplantation".
    DOI:  https://doi.org/10.1134/S0006297920050132
  44. J Cell Sci. 2020 Jun 23. pii: jcs.240374. [Epub ahead of print]
    Lee RG, Gao J, Siira SJ, Shearwood AM, Ermer JA, Hofferek V, Mathews JC, Zheng M, Reid GE, Rackham O, Filipovska A.
      The mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the cardiolipin biosynthesis gene Crls1 to investigate the effects of cardiolipin loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by reduced uncoordinated mitochondrial translation rates and impaired respiratory supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of cardiolipin resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that cardiolipin is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.
    Keywords:  Mitochondrial membranes; Mitochondrial ribosomes; Protein synthesis
    DOI:  https://doi.org/10.1242/jcs.240374
  45. Emerg Top Life Sci. 2020 Jun 23. pii: ETLS20190196. [Epub ahead of print]
    Craven L, Murphy JL, Turnbull DM.
      In 2015, the UK became the first country to approve the use of mitochondrial donation. This novel in vitro fertilisation treatment was developed to prevent transmission of mitochondrial DNA (mtDNA) disease and ultimately give more reproductive choice to women at risk of having severely affected offspring. The policy change was a major advance that surmounted many scientific, legislative and clinical challenges. Further challenges have since been addressed and there is now an NHS clinical service available to families with pathogenic mtDNA mutations that provides reproductive advice and options, and a research study to look at the outcome at 18 months of children born after mitochondrial donation.
    Keywords:  mitochondrial DNA disease; mitochondrial donation; mtDNA
    DOI:  https://doi.org/10.1042/ETLS20190196
  46. Aging (Albany NY). 2020 Jun 21. 12
    Chen C, Wei M, Wang C, Sun D, Liu P, Zhong X, Yu W.
      In this study, we investigated the mechanistic role and prognostic significance of the long coding RNA (lncRNA) KCNQ1OT1 in colorectal cancer (CRC). KCNQ1OT1 levels were significantly higher in CRC tissues than adjacent normal colorectal tissues (n=79). High KCNQ1OT1 expression correlated with poorer prognosis in CRC patients. KCNQ1OT1-silenced CRC cells showed reduced proliferation, colony formation, extracellular acidification, and lactate and glucose secretion. This suggests KCNQ1OT1 promotes CRC cell proliferation by increasing aerobic glycolysis. RNA pull-down assays with biotinylated KCNQ1OT1 followed by mass spectrometry analysis showed that KCNQ1OT1 directly binds to hexokinase 2 (HK2). This was confirmed by RNA immunoprecipitation assays using anti-hexokinase 2 antibody. HK2 protein levels were reduced in KCNQ1OT1 knockdown CRC cells, but were restored by treatment with the proteasomal inhibitor MG132. KCNQ1OT1 knockdown CRC cells also showed higher ubiquitinated-HK2 levels, suggesting KCNQ1OT1 enhances aerobic glycolysis by stabilizing HK2. HK2 overexpression in KCNQ1OT1 knockdown CRC cells restored proliferation and aerobic glycolysis. KCNQ1OT1 levels correlated positively with HK2 expression and prognosis in CRC patients. These findings show that KCNQ1OT1 promotes colorectal carcinogenesis by increasing aerobic glycolysis through HK2.
    Keywords:  HK2; aerobic glycolysis; colorectal cancer; lncRNA-KCNQ1OT1
    DOI:  https://doi.org/10.18632/aging.103334
  47. Cancer Cell. 2020 Jun 09. pii: S1535-6108(20)30268-3. [Epub ahead of print]
    Kudo Y, Sugimoto M, Arias E, Kasashima H, Cordes T, Linares JF, Duran A, Nakanishi Y, Nakanishi N, L'Hermitte A, Campos A, Senni N, Rooslid T, Roberts LR, Cuervo AM, Metallo CM, Karin M, Diaz-Meco MT, Moscat J.
      Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during reactive oxygen species (ROS) generation, and how ROS promote tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) λ/ι loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC through cell-autonomous and non-autonomous mechanisms. Although PKCλ/ι promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKCλ/ι levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.
    Keywords:  NRF2; PKCζ; PKCι; PKCλ; atypical PKC; autophagy; hepatocellular carcinoma; metabolic reprogramming; oxidative phosphorylation; reactive oxygen species
    DOI:  https://doi.org/10.1016/j.ccell.2020.05.018
  48. Metabolomics. 2020 Jun 23. 16(7): 78
    van der Walt G, Louw R.
      INTRODUCTION: Mitochondria represent an important milieu for studying the pathogenesis of several major diseases. The need for organelle-level metabolic resolution exists, as mitochondrial/cytosolic metabolites are often diluted beyond detection limits in complex samples. Compartment-specific studies are still hindered by the lack of efficient, cost-effective fractioning methods-applicable to laboratories of all financial/analytical standing.OBJECTIVES: We established a novel mitochondrial/cytosolic purification pipeline for complimentary GC-TOF-MS and 1H-NMR metabolomics using robust, commercially available fractionation strategies.
    METHODS: Magnetic based mitochondria isolation kits (MACS) were adapted for this purpose, accompanied by cytosolic filtering. Yield was assessed through the percentage recovery of citrate synthase (CS; a mitochondrial marker), purity by immunoblotting against compartment-specific proteins and integrity interrogated through the respiratory coupling ratio (RCR). The effects of the kit-based buffers on MS/NMR analyses of pure metabolite standards were evaluated. Finally, biological applicability to mammalian disease models was shown using Ndufs4 mouse brain tissue.
    RESULTS: With minor modifications, MACS produced around 60% more mitochondria compared to a differential centrifugation method. Less than 15% of lysosomal LAMP-2 protein was found in the MACS isolates, confirming relative purity-while RCR's above 6 indicate sufficient mitochondrial integrity. The filtering approach effectively depleted mitochondria from the cytosolic fraction, as indicated by negligible Hsp60 and CS levels. Our GC-MS pilot yielded 60-70 features per fraction, while NMR analyses could quantify 6-10 of the most abundant compounds in each fraction.
    CONCLUSION: This study provides a simple and flexible solution for mitochondrial and cytosolic metabolomics in animal model tissues, towards large-scale application of such methodologies in disease research.
    Keywords:  Compartment-specific metabolomics; Cytosol; Gas chromatography time-of-flight mass spectrometry (GC-TOF–MS); MACS; Mitochondria; Proton nuclear magnetic resonance (1H-NMR)
    DOI:  https://doi.org/10.1007/s11306-020-01697-9