bims-mimbat Biomed News
on Mitochondrial metabolism in brown adipose tissue
Issue of 2023–03–05
nineteen papers selected by
José Carlos de Lima-Júnior, Washington University
  1. Cold acclimation and pioglitazone combined increase thermogenic capacity of BAT and WAT but this does not translate into higher energy expenditure.
  2. Ssu72 phosphatase is essential for thermogenic adaptation by regulating cytosolic translation.
  3. Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice.
  4. IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes.
  5. Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress.
  6. Proton-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans plasma membranes catalyzes FMN-independent reverse electron transfer to hexaammineruthenium (III).
  7. Novel UCP1 knockout models broaden our understanding of mammalian non-shivering thermogenesis.
  8. Fibroblastic reticular cells in lymph node potentiate white adipose tissue beiging through neuro-immune crosstalk in male mice.
  9. Armeniaspirol analogues disrupt the electrical potential (ΔΨ) of the proton motive force.
  10. On the cutting edge: perspectives in bioenergetics.
  11. Ubiquinone deficiency drives reverse electron transport to disrupt hepatic metabolic homeostasis in obesity.
  12. Energy Coupling and Stoichiometry of Zn 2+ /H + Antiport by the Cation Diffusion Facilitator YiiP.
  13. Thermal modulation of mitochondrial function is affected by environmental nickel in rainbow trout (Oncorhynchus mykiss).
  14. Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions.
  15. Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.
  16. FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery.
  17. Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome.
  18. Conserved allosteric inhibition mechanism in SLC1 transporters.
  19. Protocol for mitochondrial isolation and sub-cellular localization assay for mitochondrial proteins.
  1. Am J Physiol Endocrinol Metab. 2023 Mar 01.
    Cold acclimation and pioglitazone combined increase thermogenic capacity of BAT and WAT but this does not translate into higher energy expenditure.
    Luís Felipe Galvão Valdívia, Érique Castro, Rosangela Aparecida Dos Santos Eichler, Mayara Franzoi Moreno, Érica de Sousa, Giovanna Freitas Rodrigues Jardim, Albert Souza Peixoto, Maria Nathália Moraes, Ana Maria de Lauro Castrucci, Jan Nedergard, Natasa Petrovic, William T Festuccia, Patrícia Reckziegel.
      Cold acclimation and pharmacological peroxisome proliferator-activated receptor γ (PPARγ) activation have each earlier been shown to recruit brown adipose tissue (BAT) and beige adipocytes thermogenic machinery, enhancing uncoupling protein 1 (UCP1)-mediated thermogenic capacity. We here investigated whether cold acclimation and PPARγ agonism combined have additive effects in inducing brown and beige adipocytes UCP1 content and whether this translates into a higher thermogenic capacity and energy expenditure. C57BL/6J mice treated or not with pioglitazone (30 mg/kg/day) were maintained at 21°C or exposed to cold (7°C) for 15 days and evaluated for thermogenic capacity, energy expenditure and interscapular BAT (iBAT) and inguinal white adipose tissue (iWAT) mass, morphology, UCP1 content and gene expression, glucose uptake and oxygen consumption. Cold acclimation and PPARγ agonism combined synergistically increased iBAT and iWAT total UCP1 content and mRNA levels of the thermogenesis-related proteins PGC1a, CIDEA, FABP4, GYK, PPARa, LPL, GLUTs (GLUT1 in iBAT and GLUT4 in iWAT), and ATG when compared to cold and pioglitazone individually. This translated into a stronger increase in body temperature in response to the β3-adrenergic agonist CL316,243 and iBAT and iWAT respiration induced by succinate and pyruvate in comparison to that seen in either cold-acclimated or pioglitazone-treated mice. However, basal energy expenditure, BAT glucose uptake and glucose tolerance were not increased above that seen in cold-acclimated untreated mice. In conclusion, cold acclimation and PPARγ agonism combined induced a robust increase in brown and beige adipocytes UCP1 content and thermogenic capacity, much higher than each treatment individually. However, our findings enforce the concept that increases in total UCP1 do not innately lead to higher energy expenditure.
    Keywords:  Adipogenesis; UCP1; browning; pioglitazone; thermogenesis
    DOI:  https://doi.org/10.1152/ajpendo.00217.2022
  2. Nat Commun. 2023 Feb 25. 14(1): 1097
    Ssu72 phosphatase is essential for thermogenic adaptation by regulating cytosolic translation.
    Eun-Ji Park, Hyun-Soo Kim, Do-Hyoung Lee, Su-Min Kim, Joon-Sup Yoon, Ji-Min Lee, Se Jin Im, Ho Lee, Min-Woo Lee, Chang-Woo Lee.
      Brown adipose tissue (BAT) plays a pivotal role in maintaining body temperature and energy homeostasis. BAT dysfunction is associated with impaired metabolic health. Here, we show that Ssu72 phosphatase is essential for mRNA translation of genes required for thermogenesis in BAT. Ssu72 is found to be highly expressed in BAT among adipose tissue depots, and the expression level of Ssu72 is increased upon acute cold exposure. Mice lacking adipocyte Ssu72 exhibit cold intolerance during acute cold exposure. Mechanistically, Ssu72 deficiency alters cytosolic mRNA translation program through hyperphosphorylation of eIF2α and reduces translation of mitochondrial oxidative phosphorylation (OXPHOS) subunits, resulting in mitochondrial dysfunction and defective thermogenesis in BAT. In addition, metabolic dysfunction in Ssu72-deficient BAT returns to almost normal after restoring Ssu72 expression. In summary, our findings demonstrate that cold-responsive Ssu72 phosphatase is involved in cytosolic translation of key thermogenic effectors via dephosphorylation of eIF2α in brown adipocytes, providing insights into metabolic benefits of Ssu72.
    DOI:  https://doi.org/10.1038/s41467-023-36836-y
  3. Nat Commun. 2023 Mar 01. 14(1): 1172
    Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice.
    Blanca Jiménez-Gómez, Patricia Ortega-Sáenz, Lin Gao, Patricia González-Rodríguez, Paula García-Flores, Navdeep Chandel, José López-Barneo.
      The hypoxic ventilatory response (HVR) is a life-saving reflex, triggered by the activation of chemoreceptor glomus cells in the carotid body (CB) connected with the brainstem respiratory center. The molecular mechanisms underlying glomus cell acute oxygen (O2) sensing are unclear. Genetic disruption of mitochondrial complex I (MCI) selectively abolishes the HVR and glomus cell responsiveness to hypoxia. However, it is unknown what functions of MCI (metabolic, proton transport, or signaling) are essential for O2 sensing. Here we show that transgenic mitochondrial expression of NDI1, a single-molecule yeast NADH/quinone oxidoreductase that does not directly contribute to proton pumping, fully recovers the HVR and glomus cell sensitivity to hypoxia in MCI-deficient mice. Therefore, maintenance of mitochondrial NADH dehydrogenase activity and the electron transport chain are absolutely necessary for O2-dependent regulation of breathing. NDI1 expression also rescues other systemic defects caused by MCI deficiency. These data explain the role of MCI in acute O2 sensing by arterial chemoreceptors and demonstrate the optimal recovery of complex organismal functions by gene therapy.
    DOI:  https://doi.org/10.1038/s41467-023-36894-2
  4. Front Physiol. 2023 ;14 1106662
    IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes.
    Pablo Sánchez-Aguilera, Camila López-Crisosto, Ignacio Norambuena-Soto, Christian Penannen, Jumo Zhu, Nils Bomer, Matijn F Hoes, Peter Van Der Meer, Mario Chiong, B Daan Westenbrink, Sergio Lavandero.
      A physiological increase in cardiac workload results in adaptive cardiac remodeling, characterized by increased oxidative metabolism and improvements in cardiac performance. Insulin-like growth factor-1 (IGF-1) has been identified as a critical regulator of physiological cardiac growth, but its precise role in cardiometabolic adaptations to physiological stress remains unresolved. Mitochondrial calcium (Ca2+) handling has been proposed to be required for sustaining key mitochondrial dehydrogenase activity and energy production during increased workload conditions, thus ensuring the adaptive cardiac response. We hypothesized that IGF-1 enhances mitochondrial energy production through a Ca2+-dependent mechanism to ensure adaptive cardiomyocyte growth. We found that stimulation with IGF-1 resulted in increased mitochondrial Ca2+ uptake in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes, estimated by fluorescence microscopy and indirectly by a reduction in the pyruvate dehydrogenase phosphorylation. We showed that IGF-1 modulated the expression of mitochondrial Ca2+ uniporter (MCU) complex subunits and increased the mitochondrial membrane potential; consistent with higher MCU-mediated Ca2+ transport. Finally, we showed that IGF-1 improved mitochondrial respiration through a mechanism dependent on MCU-mediated Ca2+ transport. In conclusion, IGF-1-induced mitochondrial Ca2+ uptake is required to boost oxidative metabolism during cardiomyocyte adaptive growth.
    Keywords:  MCU complex; human embryonic stem cell derived-cardiomyocytes (hES-CMs); insulin-like growth factor 1 (IGF-1); mitochondrial calcium handling; neonatal rat ventricular myocytes (NRVMs); physiological cardiac hypertrophy
    DOI:  https://doi.org/10.3389/fphys.2023.1106662
  5. Cell Rep. 2023 Feb 27. pii: S2211-1247(23)00166-3. [Epub ahead of print]42(3): 112155
    Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress.
    Travis R Madaris, Manigandan Venkatesan, Soumya Maity, Miriam C Stein, Neelanjan Vishnu, Mridula K Venkateswaran, James G Davis, Karthik Ramachandran, Sukanthathulse Uthayabalan, Cristel Allen, Ayodeji Osidele, Kristen Stanley, Nicholas P Bigham, Terry M Bakewell, Melanie Narkunan, Amy Le, Varsha Karanam, Kang Li, Aum Mhapankar, Luke Norton, Jean Ross, M Imran Aslam, W Brian Reeves, Brij B Singh, Jeffrey Caplan, Justin J Wilson, Peter B Stathopulos, Joseph A Baur, Muniswamy Madesh.
      The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-μM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, β-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2-/- mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome.
    Keywords:  CP: Metabolism; HCC; HIF1; MCU; Mrs2; NAFLD; Western diet; adipose expansion; adipose tissue; calcium channel; cardiometabolic disease; diabetes; energy imbalance; hepatocytes; liver; magnesium channel; metabolic disease; metabolic syndrome; mitochondrial dysfunction; obesity; whole-body metabolism
    DOI:  https://doi.org/10.1016/j.celrep.2023.112155
  6. Biochim Biophys Acta Bioenerg. 2023 Feb 24. pii: S0005-2728(23)00009-9. [Epub ahead of print]1864(2): 148963
    Proton-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans plasma membranes catalyzes FMN-independent reverse electron transfer to hexaammineruthenium (III).
    Grigory V Gladyshev, Tatyana V Zharova, Alexandra V Kareyeva, Vera G Grivennikova.
      NADH-OH, the specific inhibitor of NADH-binding site of the mammalian complex I, is shown to completely block FMN-dependent reactions of P. denitrificans enzyme in plasma membrane vesicles: NADH oxidation (in a competitive manner with Ki of 1 nM) as well as reduction of pyridine nucleotides, ferricyanide and oxygen in the reverse electron transfer. In contrast to these activities, the reverse electron transfer to hexaammineruthenium (III) catalyzed by plasma membrane vesicles is insensitive to NADH-OH. To explain these results, we hypothesize the existence of a non-FMN redox group of P. denitrificans complex I that is capable of reducing hexaammineruthenium (III), which is corroborated by the complex kinetics of NADH: hexaammineruthenium (III)-reductase activity, catalyzed by this enzyme. A new assay procedure for measuring succinate-driven reverse electron transfer catalyzed by P. denitrificans complex I to hexaammineruthenium (III) is proposed.
    Keywords:  Bioenergetics; FMN; Hexaammineruthenium (III); NADH-OH; NADH:ubiquinone oxidoreductase (complex I); Reverse electron transfer
    DOI:  https://doi.org/10.1016/j.bbabio.2023.148963
  7. Acta Physiol (Oxf). 2023 Mar 03. e13956
    Novel UCP1 knockout models broaden our understanding of mammalian non-shivering thermogenesis.
    Martin Jastroch.
      
    DOI:  https://doi.org/10.1111/apha.13956
  8. Nat Commun. 2023 Mar 03. 14(1): 1213
    Fibroblastic reticular cells in lymph node potentiate white adipose tissue beiging through neuro-immune crosstalk in male mice.
    Lai Yee Cheong, Baile Wang, Qin Wang, Leigang Jin, Kelvin H M Kwok, Xiaoping Wu, Lingling Shu, Huige Lin, Sookja Kim Chung, Kenneth K Y Cheng, Ruby L C Hoo, Aimin Xu.
      Lymph nodes (LNs) are always embedded in the metabolically-active white adipose tissue (WAT), whereas their functional relationship remains obscure. Here, we identify fibroblastic reticular cells (FRCs) in inguinal LNs (iLNs) as a major source of IL-33 in mediating cold-induced beiging and thermogenesis of subcutaneous WAT (scWAT). Depletion of iLNs in male mice results in defective cold-induced beiging of scWAT. Mechanistically, cold-enhanced sympathetic outflow to iLNs activates β1- and β2-adrenergic receptor (AR) signaling in FRCs to facilitate IL-33 release into iLN-surrounding scWAT, where IL-33 activates type 2 immune response to potentiate biogenesis of beige adipocytes. Cold-induced beiging of scWAT is abrogated by selective ablation of IL-33 or β1- and β2-AR in FRCs, or sympathetic denervation of iLNs, whereas replenishment of IL-33 reverses the impaired cold-induced beiging in iLN-deficient mice. Taken together, our study uncovers an unexpected role of FRCs in iLNs in mediating neuro-immune interaction to maintain energy homeostasis.
    DOI:  https://doi.org/10.1038/s41467-023-36737-0
  9. Bioorg Med Chem Lett. 2023 Feb 27. pii: S0960-894X(23)00088-4. [Epub ahead of print] 129210
    Armeniaspirol analogues disrupt the electrical potential (ΔΨ) of the proton motive force.
    Michael G Darnowski, Taylor D Lanosky, André R Paquette, Christopher N Boddy.
      The armeniaspirol family of natural products antibiotics have been shown to inhibit the ATP-dependent proteases ClpXP and ClpYQ and disrupt membrane potential through shuttling of protons across the membrane. Herein we investigate their ability to disrupt the proton motive force (PMF). We show, using a voltage sensitive, that armeniaspiols disrupt the electrical membrane potential (ΔΨ) component of the PMF and not the transmembrane proton gradient (ΔpH). Using checkerboard assays, we confirm this by showing antagonism, with kanamycin, an antibiotic that required ΔΨ for penetration. By evaluating the antibiotic activity and disruption of the PMF by fourteen armeniaspirol analogs, we find that disruption of the PMF is necessary but not sufficient for antibiotic activity. Analogs that are potent disruptors of the PMF without possessing the ability to inhibit ClpXP and ClpYQ are not potent antibiotics. Thus we propose that the armeniaspirols utilize a dual mechanism of action where they disrupt PMF and inhibit the ATP-dependent proteases ClpXP and ClpYQ. This type of dual mechanism has been observed in other natural product-based antibiotics, most notably chelocardin.
    DOI:  https://doi.org/10.1016/j.bmcl.2023.129210
  10. Nat Rev Endocrinol. 2023 Mar 03.
    On the cutting edge: perspectives in bioenergetics.
    Melia Granath-Panelo, Anna Krook, Jared Rutter, Shingo Kajimura.
      
    DOI:  https://doi.org/10.1038/s41574-023-00820-9
  11. bioRxiv. 2023 Feb 22. pii: 2023.02.21.528863. [Epub ahead of print]
    Ubiquinone deficiency drives reverse electron transport to disrupt hepatic metabolic homeostasis in obesity.
    Renata L S Goncalves, Zeqiu Branden Wang, Karen E Inouye, Grace Yankun Lee, Xiaorong Fu, Jani Saksi, Clement Rosique, Gunes Parlakgul, Ana Paula Arruda, Sheng Tony Hui, Mar Coll Loperena, Shawn C Burgess, Isabel Graupera, Gökhan S Hotamisligil.
      Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generation in vivo remain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH 2 /Q ratio, driving excessive mROS production via reverse electron transport (RET) from site I Q in complex I. Using multiple complementary genetic and pharmacological models in vivo we demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH 2 /Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.
    DOI:  https://doi.org/10.1101/2023.02.21.528863
  12. bioRxiv. 2023 Feb 23. pii: 2023.02.23.529644. [Epub ahead of print]
    Energy Coupling and Stoichiometry of Zn 2+ /H + Antiport by the Cation Diffusion Facilitator YiiP.
    Adel Hussein, Shujie Fan, Maria Lopez-Redondo, Ian Kenney, Xihui Zhang, Oliver Beckstein, David L Stokes.
      YiiP is a prokaryotic Zn 2+ /H + antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn 2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites and the interplay between Zn 2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn 2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn 2+ to 2-3 H + depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn 2+ .
    DOI:  https://doi.org/10.1101/2023.02.23.529644
  13. Aquat Toxicol. 2023 Feb 24. pii: S0166-445X(23)00058-9. [Epub ahead of print]257 106451
    Thermal modulation of mitochondrial function is affected by environmental nickel in rainbow trout (Oncorhynchus mykiss).
    Elyas Aissia, Pierre U Blier, Mariem Fadhlaoui, Patrice Couture.
      In this study, we investigated the combined effects of temperature and nickel (Ni) contamination on liver mitochondria electron transport system (ETS) enzymes, citrate synthase (CS), phospholipid fatty acid composition and lipid peroxidation in rainbow trout (Oncorhynchus mykiss). Juvenile trout were acclimated for two weeks to two different temperatures (5˚C and 15˚C) and exposed to nickel (Ni; 520 μg/L) for three weeks. Using ratios of ETS enzymes and CS activities, our data suggest that Ni and an elevated temperature acted synergistically to induce a higher capacity for reduction status of the ETS. The response of phospholipid fatty acid profiles to thermal variation was also altered under nickel exposure. In control conditions, the proportion of saturated fatty acids (SFA) was higher at 15˚C than at 5˚C, while the opposite was observed for monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA). However, in nickel contaminated fish, the proportion of SFA was higher at 5˚C than at 15˚C, while PUFA and MUFA followed the opposite direction. A higher PUFA ratio is associated with higher vulnerability to lipid peroxidation. Thiobarbituric Acid Reactive Substances (TBARS) content was higher when the PUFA were in higher proportions, except for Ni-exposed, warm-acclimated fish, in which we reported the lowest level of TBARS but the highest proportion of PUFA. We suspect that the interaction of nickel and temperature on lipid peroxidation is due to their synergistic effects on aerobic energy metabolism, as supported by the decrease in the activity of complex IV of the ETS enzyme activity in those fish, or on antioxidant enzymes and pathways. Overall, our study demonstrates that Ni exposure in heat-challenged fish can lead to the remodelling of the mitochondrial phenotype and potentially stimulate alternative antioxidant mechanisms.
    Keywords:  Electron transport system; Fatty acids; Fish, Mitochondria; Nickel; Temperature
    DOI:  https://doi.org/10.1016/j.aquatox.2023.106451
  14. Biochem J. 2022 Mar 02. pii: BCJ20220611. [Epub ahead of print]
    Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions.
    Edwin T Gibbs Ii, Chad A Lerner, Mark A Watson, Hoi-Shan Wong, Akos A Gerencser, Martin D Brand.
      Superoxide/hydrogen peroxide production by site IQ in complex I of the electron transport chain is conventionally assayed during reverse electron transport from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site IQ) have potent effects in cells and in vivo during presumed forward electron transport. Therefore, we tested whether site IQ generates S1QEL-sensitive superoxide/hydrogen peroxide during forward electron transport (site IQf), or alternatively, whether reverse electron transport and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site IQ can be equally great whether reverse electron transport or forward electron transport is running. We show that sites IQr and IQf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site IQr during forward electron transport is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site IQ. Finally, we show that superoxide/hydrogen peroxide production by site IQ in cells occurs during forward electron transport, and is S1QEL-sensitive.
    Keywords:  Reverse electron transport; S1QEL; complex I; electron transport chain; mitochondria; reactive oxygen species
    DOI:  https://doi.org/10.1042/BCJ20220611
  15. Nat Commun. 2023 Feb 27. 14(1): 1120
    Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.
    James S Davies, Michael J Currie, Rachel A North, Mariafrancesca Scalise, Joshua D Wright, Jack M Copping, Daniela M Remus, Ashutosh Gulati, Dustin R Morado, Sam A Jamieson, Michael C Newton-Vesty, Gayan S Abeysekera, Subramanian Ramaswamy, Rosmarie Friemann, Soichi Wakatsuki, Jane R Allison, Cesare Indiveri, David Drew, Peter D Mace, Renwick C J Dobson.
      In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.
    DOI:  https://doi.org/10.1038/s41467-023-36590-1
  16. Proc Natl Acad Sci U S A. 2023 Mar 07. 120(10): e2216722120
    FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery.
    Mohammad Zulkifli, Amy N Spelbring, Yuteng Zhang, Shivatheja Soma, Si Chen, Luxi Li, Trung Le, Vinit Shanbhag, Michael J Petris, Tai-Yen Chen, Martina Ralle, David P Barondeau, Vishal M Gohil.
      Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.
    Keywords:  FDX1; copper; cytochrome c oxidase; elesclomol; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2216722120
  17. Nat Commun. 2023 Mar 01. 14(1): 1167
    Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome.
    Luis O Romero, Rebeca Caires, A Kaitlyn Victor, Juanma Ramirez, Francisco J Sierra-Valdez, Patrick Walsh, Vincent Truong, Jungsoo Lee, Ugo Mayor, Lawrence T Reiter, Valeria Vásquez, Julio F Cordero-Morales.
      Angelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability and atypical behaviors. AS results from loss of expression of the E3 ubiquitin-protein ligase UBE3A from the maternal allele in neurons. Individuals with AS display impaired coordination, poor balance, and gait ataxia. PIEZO2 is a mechanosensitive ion channel essential for coordination and balance. Here, we report that PIEZO2 activity is reduced in Ube3a deficient male and female mouse sensory neurons, a human Merkel cell carcinoma cell line and female human iPSC-derived sensory neurons with UBE3A knock-down, and de-identified stem cell-derived neurons from individuals with AS. We find that loss of UBE3A decreases actin filaments and reduces PIEZO2 expression and function. A linoleic acid (LA)-enriched diet increases PIEZO2 activity, mechano-excitability, and improves gait in male AS mice. Finally, LA supplementation increases PIEZO2 function in stem cell-derived neurons from individuals with AS. We propose a mechanism whereby loss of UBE3A expression reduces PIEZO2 function and identified a fatty acid that enhances channel activity and ameliorates AS-associated mechano-sensory deficits.
    DOI:  https://doi.org/10.1038/s41467-023-36818-0
  18. Elife. 2023 Mar 01. pii: e83464. [Epub ahead of print]12
    Conserved allosteric inhibition mechanism in SLC1 transporters.
    Yang Dong, Jiali Wang, Rachel-Ann Garibsingh, Keino Hutchinson, Yueyue Shi, Gilad Eisenberg, Xiaozhen Yu, Avner Schlessinger, Christof Grewer.
      Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers . It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 mM), much closer to the EAAT1 value of 0.6 mM. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.
    Keywords:  human; molecular biophysics; rat; structural biology
    DOI:  https://doi.org/10.7554/eLife.83464
  19. STAR Protoc. 2023 Feb 03. pii: S2666-1667(23)00046-1. [Epub ahead of print]4(1): 102088
    Protocol for mitochondrial isolation and sub-cellular localization assay for mitochondrial proteins.
    Danyi Zhou, Sheng Zhong, Xinyu Han, Dandan Liu, Hezhi Fang, Ya Wang.
      Here, we provide a protocol to isolate mitochondria from cultured cells and extract differently located mitochondrial proteins. We detail steps to separate both integral and peripheral membrane proteins from soluble proteins using sonication. We describe the separation of integral membrane proteins from the peripheral membrane and soluble proteins using sodium carbonate extraction. Furthermore, we detail the use of proteinase K and Triton X-100 to distinguish outer membrane proteins from mitochondrial proteins.
    Keywords:  Cell Membrane; Cell culture; Cell separation/fractionation; Protein Biochemistry
    DOI:  https://doi.org/10.1016/j.xpro.2023.102088
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