- Email: [email protected]
Chloroplast tomography allows revisiting diatoms photosynthesis
e12
Abstracts
mitochondrial population, elevated intramitochondrial NADH and dephosphorylated PDH insensitive to dichloroacetate, all consistent with a high resting mitochondrial calcium content. We propose that this reflects dysregulation of the thresholding of mitochondrial calcium uptake leading to a futile cycle, whereby import of Ca2 + at resting [Ca2 +]c is balanced by export in exchange for Na+ by the Na+/Ca2 + exchanger (NCX). This is balanced by Na+/H+ proton exchange, undermining oxidative phosphorylation. Blockade of the NCX with CGP37157 caused a rapid increase in intramitochondrial calcium in patient but not control cells associated with a small but significant increase in ATP content. Thus, MICU1 plays a key role in thresholding mitochondrial calcium uptake, protecting cells from futile calcium cycling, and serving as a signal/noise discriminator. References 1. Logan CV, Szabadkai G, et al. Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling. Nat Genet. 46(2) (2014) 188–93.
Seahorse metabolic analyzers are redefining disease relevant, cell-based assays. Basic cellular functions such as proliferation, differentiation, activation and stress create different metabolic demands for cells in terms of energy, biosynthesis and redox potential. The unique metabolic properties and liabilities of agerelated diseases are leading to many fresh perspectives of how to intervene in chronic, progressive disorders. More recently, a growing body of evidence points to a primary role for energy metabolism in cancer, immune function, cell fate and functional competence. The ability to explore these metabolic programs and liabilities has been enhanced by new tools that enable researchers to rapidly map the metabolic phenotype of any cell, regardless of its energy and metabolic status. This is accomplished by integrating real-time measures of mitochondrial respiration and glycolysis into a single powerful test. This talk will show how metabolism is key to understanding cancer and immune cell function. This knowledge is being employed to target cancer and enhance immune-based therapies, respectively.
doi:10.1016/j.bbabio.2016.04.039
doi:10.1016/j.bbabio.2016.04.374
Coenzyme Q redox status controls the dynamic configuration of the mitochondrial electron transport chain by reverse electron transport Jose Antonio Enriquez Centro Nacional de Invesitgaciones Cardiovasculares, Madrid, Spain E-mail address: [email protected] (J.A. Enriquez) The mitochondrial electron transport chain (mETC), composed of four multi-subunit complexes (complex I [CI]–complex IV [CIV]) and two electron carriers (coenzyme Q [CoQ, or ubiquinone] and cytochrome c [cyt c]), generates a transmembrane proton gradient that drives ATP synthesis by complex V (CV; ATP synthase). Freely moving respiratory complexes and mobile carriers co-exist in the inner mitochondrial membrane with larger structures called respiratory supercomplexes (SCs). Most CI occurs in SCs with CIII, with or without CIV (CI + CIII + CIV, known as the respirasome, and CI + CIII). The dynamic organization into SCs creates two functional CIII populations, defined by association or non-association with CI and functional pools of CoQ are defined for carrying electrons from NADH or FAD electrons (Lapuente-Brun et al., 2013). A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of harmful reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. CoQ redox status thus acts as a metabolic sensor that fine-tunes mETC configuration to match the prevailing substrate profile. doi:10.1016/j.bbabio.2016.04.373
Probing the energy metabolism of cancer and immunotherapy David A. Ferrick Seahorse Bioscience, A part of Agilent Technologies, N. Billerica, MA, USA E-mail address: [email protected] (D.A. Ferrick)
Chloroplast tomography allows revisiting diatoms photosynthesis Serena Flori, Dimitris Petroutsos, Denis Falconet, Giovanni Finazzi Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, Centre National de la Recherche Scientifique (CNRS), Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Université Grenoble Alpes, Institut National Recherche Agronomique (INRA), Institut de Recherche en Sciences et Technologies pour le Vivant (iRTSV), CEA Grenoble, F38054 Grenoble cedex 9, France E-mail address: giovanni.fi[email protected] (G. Finazzi) Diatoms are key ecological players in contemporary oceans, which actively contribute to CO2 sequestration via their photosynthesis. Although this process is similar to that of plants, it occurs in a different chloroplast environment with no apparent differentiation between appressed stacks (the grana, rich in photosystem-PS- 2) and non-appressed regions (the stroma lamellae, rich in PS1). This lack of segregation could affect photosynthesis, by promoting physical contacts between the two photosystems, and reduce the quantum yield of this process via direct energy spillover from PS2 to PS1. In this study, we combined in vivo spectroscopic studies with biochemical analysis and chloroplast tomography to reevaluate the structural/functional features of diatom chloroplasts in the model species Phaeodactylum tricornutum. We found [1] that photosynthetic complexes are structurally segregated in defined chloroplast compartments, which are connected by linking structures to ensure efficient diffusion of soluble electron flow carriers. This segregation optimizes light partitioning between the two photosystems, while introducing a need for active mechanisms of photoprotection. Refined electron microscopy analysis also reveals the existence of direct connections between the chloroplast and mitochondria, which likely funnel exchanges of reducing equivalents and ATP, as required for optimum carbon assimilation in diatoms [2]. Overall, the structural features of diatom chloroplasts disclose the sophisticated organization of chloroplasts derived from a secondary endosymbiosis event are “poorly” organized, and illustrate alternative solutions to optimize photosynthesis. References 1. S Flori et al., manuscript in preparation. 2. B. Bailleul, N.,Berne, O. Murik, D. Petroutsos, J. Prihoda, A. Tanaka, V. Villanova, R. Bligny, S. Flori, D. Falconet, A. Krieger-Liszkay, S; Santabarbara, F. Rappaport, P. Joliot, L. Tirichine, P.G. Falkowski, P. Cardol,
Abstracts
C. Bowler, G. Finazzi Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms, Nature, 524 (2015) :366–369. doi:10.1016/j.bbabio.2016.04.375
Shutting down the pore: The search for small molecule inhibitors of the mitochondrial permeability transition Michael Forte Vollum Institute, Oregon Health & Science University, Portland, OR, USA E-mail address: [email protected] (M. Forte) The mitochondrial permeability transition pore (PTP) is now recognized as playing a key role in a wide variety of human diseases whose common pathology may be based in mitochondrial dysfunction. Recently, PTP assays have been adapted to high-throughput screening approaches to identify small molecules specifically inhibiting the PTP. Following extensive secondary chemistry, the most potent inhibitors of the PTP described to date have been developed. We will provide an overview of each of these screening efforts, use of resulting compounds in animal models of PTP-based diseases, and problems that will require further study. doi:10.1016/j.bbabio.2016.04.376
Mechanisms of rotary ATP synthases revealed by single-molecule rotation studies Wayne D. Frasch Arizona State University, Tempe, AZ 85287-4501, USA E-mail address: [email protected] (W.D. Frasch) The effects of pH and site-directed mutations on the ability of the Fo motor to rotate clockwise against the counterclockwise rotation of the F1-ATPase motor from Escherichia coli support a model in which the Fo motor adopts alternate conformations that facilitate rotation in the ATP synthase direction. High resolution measurements of angular velocity of the γ-subunit as a function of rotational position during power strokes of F1-ATPases from the F-type ATP synthases of mesophilic E. coli and thermophilic Geobacillus stearothermophilus (formerly known as Bacillus PS3), were compared with the angular velocity of subunits D and F during power strokes of the A3B3DF-complex from the Methanococcus mazei Gö1 A-ATP synthase. Results provide a possible explanation for the difference in the rotary position of the ATP-binding dwells between F-type and A-type motors. This work was supported by National Institutes of Health Grant R01GM097510. doi:10.1016/j.bbabio.2016.04.377
On the structural possibility of pore-forming mitochondrial FoF1 ATP synthase Christoph Gerle Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Hyogo, Japan E-mail address: [email protected] (C. Gerle) The mitochondrial permeability transition is an inner mitochondrial membrane event involving the opening of the permeability transition pore concomitant with a sudden efflux of matrix solutes
e13
and breakdown of membrane potential. The mitochondrial FoF1 ATP synthase has been proposed as the molecular identity of the permeability transition pore. The likeliness of potential pore-forming sites in the mitochondrial FoF1 ATP synthase is discussed and a new model, the death finger model, is described. In this model, movement of a p-side density that connects the lipid-plug of the c-ring with the distal membrane bending Fo domain allows reversible opening of the c-ring and structural cross-talk with OSCP and the catalytic (αβ)3 hexamer. doi:10.1016/j.bbabio.2016.04.378
Sulfide-resistant O2 respiration: A new role for bacterial bd-type terminal oxidases Elena Fortea,1, Vitaliy B. Borisovb,1, Micol Falabellaa, Henrique G. Colaçoc, Mariana Tinajero-Trejod, Robert K. Poolee, João B. Vicentef, Paolo Sartia, Alessandro Giuffrèg, a Department of Biochemical Sciences and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy b Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation c Metabolism & Genetics Group, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Portugal d Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada e Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, United Kingdom f Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal g CNR Institute of Molecular Biology and Pathology, Rome, Italy1These authors contributed equally to this work. E-mail addresses: [email protected] (E. Forte), [email protected] (A. Giuffrè) Many prokaryotes produce H2S through orthologs of the mammalian H2S-synthesizing enzymes and dissimilatory sulfate reduction [1]. The human intestinal microbiota is a major source of H2S, particularly in the colon, where high levels of sulfide are reached. As sulfide is a potent inhibitor of respiratory terminal oxidases such as mitochondrial cytochrome c oxidase [2], we raised the hypothesis that in sulfide-rich environments, like the human colon, bacterial O2 respiration is enabled by sulfide-insensitive oxidases [3]. The hypothesis was tested on Escherichia coli, that encodes three respiratory oxidases, the hemecopper bo3 enzyme and two bd-type oxidases. The bd-type oxidases are prokaryotic enzymes, conferring resistance to oxidative/nitrosative stress and promoting bacterial virulence. Working on the enzymes isolated from E. coli, we found that, contrary to the bo3 oxidase, both bd oxidases are sulfide-insensitive. Accordingly, in E. coli respiratory mutants, aerobic respiration and growth were impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected even at high sulfide levels when either bd enzyme acted as the only terminal oxidase. Consistently, wild-type E. coli displayed sulfide-insensitive respiration and growth under O2-limiting conditions favoring the expression of bd oxidases. Altogether, these results show that bd-type oxidases enable sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. References 1. K. Shatalin, E. Shatalina, A. Mironov, E. Nudler, H2S: a universal defense against antibiotics in bacteria, Science 334 (2011) 986–990. 2. L.C. Petersen, The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase, Biochim. Biophys. Acta 460 (1977) 299–307.
Abstracts
mitochondrial population, elevated intramitochondrial NADH and dephosphorylated PDH insensitive to dichloroacetate, all consistent with a high resting mitochondrial calcium content. We propose that this reflects dysregulation of the thresholding of mitochondrial calcium uptake leading to a futile cycle, whereby import of Ca2 + at resting [Ca2 +]c is balanced by export in exchange for Na+ by the Na+/Ca2 + exchanger (NCX). This is balanced by Na+/H+ proton exchange, undermining oxidative phosphorylation. Blockade of the NCX with CGP37157 caused a rapid increase in intramitochondrial calcium in patient but not control cells associated with a small but significant increase in ATP content. Thus, MICU1 plays a key role in thresholding mitochondrial calcium uptake, protecting cells from futile calcium cycling, and serving as a signal/noise discriminator. References 1. Logan CV, Szabadkai G, et al. Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling. Nat Genet. 46(2) (2014) 188–93.
Seahorse metabolic analyzers are redefining disease relevant, cell-based assays. Basic cellular functions such as proliferation, differentiation, activation and stress create different metabolic demands for cells in terms of energy, biosynthesis and redox potential. The unique metabolic properties and liabilities of agerelated diseases are leading to many fresh perspectives of how to intervene in chronic, progressive disorders. More recently, a growing body of evidence points to a primary role for energy metabolism in cancer, immune function, cell fate and functional competence. The ability to explore these metabolic programs and liabilities has been enhanced by new tools that enable researchers to rapidly map the metabolic phenotype of any cell, regardless of its energy and metabolic status. This is accomplished by integrating real-time measures of mitochondrial respiration and glycolysis into a single powerful test. This talk will show how metabolism is key to understanding cancer and immune cell function. This knowledge is being employed to target cancer and enhance immune-based therapies, respectively.
doi:10.1016/j.bbabio.2016.04.039
doi:10.1016/j.bbabio.2016.04.374
Coenzyme Q redox status controls the dynamic configuration of the mitochondrial electron transport chain by reverse electron transport Jose Antonio Enriquez Centro Nacional de Invesitgaciones Cardiovasculares, Madrid, Spain E-mail address: [email protected] (J.A. Enriquez) The mitochondrial electron transport chain (mETC), composed of four multi-subunit complexes (complex I [CI]–complex IV [CIV]) and two electron carriers (coenzyme Q [CoQ, or ubiquinone] and cytochrome c [cyt c]), generates a transmembrane proton gradient that drives ATP synthesis by complex V (CV; ATP synthase). Freely moving respiratory complexes and mobile carriers co-exist in the inner mitochondrial membrane with larger structures called respiratory supercomplexes (SCs). Most CI occurs in SCs with CIII, with or without CIV (CI + CIII + CIV, known as the respirasome, and CI + CIII). The dynamic organization into SCs creates two functional CIII populations, defined by association or non-association with CI and functional pools of CoQ are defined for carrying electrons from NADH or FAD electrons (Lapuente-Brun et al., 2013). A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of harmful reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. CoQ redox status thus acts as a metabolic sensor that fine-tunes mETC configuration to match the prevailing substrate profile. doi:10.1016/j.bbabio.2016.04.373
Probing the energy metabolism of cancer and immunotherapy David A. Ferrick Seahorse Bioscience, A part of Agilent Technologies, N. Billerica, MA, USA E-mail address: [email protected] (D.A. Ferrick)
Chloroplast tomography allows revisiting diatoms photosynthesis Serena Flori, Dimitris Petroutsos, Denis Falconet, Giovanni Finazzi Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, Centre National de la Recherche Scientifique (CNRS), Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Université Grenoble Alpes, Institut National Recherche Agronomique (INRA), Institut de Recherche en Sciences et Technologies pour le Vivant (iRTSV), CEA Grenoble, F38054 Grenoble cedex 9, France E-mail address: giovanni.fi[email protected] (G. Finazzi) Diatoms are key ecological players in contemporary oceans, which actively contribute to CO2 sequestration via their photosynthesis. Although this process is similar to that of plants, it occurs in a different chloroplast environment with no apparent differentiation between appressed stacks (the grana, rich in photosystem-PS- 2) and non-appressed regions (the stroma lamellae, rich in PS1). This lack of segregation could affect photosynthesis, by promoting physical contacts between the two photosystems, and reduce the quantum yield of this process via direct energy spillover from PS2 to PS1. In this study, we combined in vivo spectroscopic studies with biochemical analysis and chloroplast tomography to reevaluate the structural/functional features of diatom chloroplasts in the model species Phaeodactylum tricornutum. We found [1] that photosynthetic complexes are structurally segregated in defined chloroplast compartments, which are connected by linking structures to ensure efficient diffusion of soluble electron flow carriers. This segregation optimizes light partitioning between the two photosystems, while introducing a need for active mechanisms of photoprotection. Refined electron microscopy analysis also reveals the existence of direct connections between the chloroplast and mitochondria, which likely funnel exchanges of reducing equivalents and ATP, as required for optimum carbon assimilation in diatoms [2]. Overall, the structural features of diatom chloroplasts disclose the sophisticated organization of chloroplasts derived from a secondary endosymbiosis event are “poorly” organized, and illustrate alternative solutions to optimize photosynthesis. References 1. S Flori et al., manuscript in preparation. 2. B. Bailleul, N.,Berne, O. Murik, D. Petroutsos, J. Prihoda, A. Tanaka, V. Villanova, R. Bligny, S. Flori, D. Falconet, A. Krieger-Liszkay, S; Santabarbara, F. Rappaport, P. Joliot, L. Tirichine, P.G. Falkowski, P. Cardol,
Abstracts
C. Bowler, G. Finazzi Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms, Nature, 524 (2015) :366–369. doi:10.1016/j.bbabio.2016.04.375
Shutting down the pore: The search for small molecule inhibitors of the mitochondrial permeability transition Michael Forte Vollum Institute, Oregon Health & Science University, Portland, OR, USA E-mail address: [email protected] (M. Forte) The mitochondrial permeability transition pore (PTP) is now recognized as playing a key role in a wide variety of human diseases whose common pathology may be based in mitochondrial dysfunction. Recently, PTP assays have been adapted to high-throughput screening approaches to identify small molecules specifically inhibiting the PTP. Following extensive secondary chemistry, the most potent inhibitors of the PTP described to date have been developed. We will provide an overview of each of these screening efforts, use of resulting compounds in animal models of PTP-based diseases, and problems that will require further study. doi:10.1016/j.bbabio.2016.04.376
Mechanisms of rotary ATP synthases revealed by single-molecule rotation studies Wayne D. Frasch Arizona State University, Tempe, AZ 85287-4501, USA E-mail address: [email protected] (W.D. Frasch) The effects of pH and site-directed mutations on the ability of the Fo motor to rotate clockwise against the counterclockwise rotation of the F1-ATPase motor from Escherichia coli support a model in which the Fo motor adopts alternate conformations that facilitate rotation in the ATP synthase direction. High resolution measurements of angular velocity of the γ-subunit as a function of rotational position during power strokes of F1-ATPases from the F-type ATP synthases of mesophilic E. coli and thermophilic Geobacillus stearothermophilus (formerly known as Bacillus PS3), were compared with the angular velocity of subunits D and F during power strokes of the A3B3DF-complex from the Methanococcus mazei Gö1 A-ATP synthase. Results provide a possible explanation for the difference in the rotary position of the ATP-binding dwells between F-type and A-type motors. This work was supported by National Institutes of Health Grant R01GM097510. doi:10.1016/j.bbabio.2016.04.377
On the structural possibility of pore-forming mitochondrial FoF1 ATP synthase Christoph Gerle Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Hyogo, Japan E-mail address: [email protected] (C. Gerle) The mitochondrial permeability transition is an inner mitochondrial membrane event involving the opening of the permeability transition pore concomitant with a sudden efflux of matrix solutes
e13
and breakdown of membrane potential. The mitochondrial FoF1 ATP synthase has been proposed as the molecular identity of the permeability transition pore. The likeliness of potential pore-forming sites in the mitochondrial FoF1 ATP synthase is discussed and a new model, the death finger model, is described. In this model, movement of a p-side density that connects the lipid-plug of the c-ring with the distal membrane bending Fo domain allows reversible opening of the c-ring and structural cross-talk with OSCP and the catalytic (αβ)3 hexamer. doi:10.1016/j.bbabio.2016.04.378
Sulfide-resistant O2 respiration: A new role for bacterial bd-type terminal oxidases Elena Fortea,1, Vitaliy B. Borisovb,1, Micol Falabellaa, Henrique G. Colaçoc, Mariana Tinajero-Trejod, Robert K. Poolee, João B. Vicentef, Paolo Sartia, Alessandro Giuffrèg, a Department of Biochemical Sciences and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy b Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation c Metabolism & Genetics Group, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Portugal d Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada e Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, United Kingdom f Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal g CNR Institute of Molecular Biology and Pathology, Rome, Italy1These authors contributed equally to this work. E-mail addresses: [email protected] (E. Forte), [email protected] (A. Giuffrè) Many prokaryotes produce H2S through orthologs of the mammalian H2S-synthesizing enzymes and dissimilatory sulfate reduction [1]. The human intestinal microbiota is a major source of H2S, particularly in the colon, where high levels of sulfide are reached. As sulfide is a potent inhibitor of respiratory terminal oxidases such as mitochondrial cytochrome c oxidase [2], we raised the hypothesis that in sulfide-rich environments, like the human colon, bacterial O2 respiration is enabled by sulfide-insensitive oxidases [3]. The hypothesis was tested on Escherichia coli, that encodes three respiratory oxidases, the hemecopper bo3 enzyme and two bd-type oxidases. The bd-type oxidases are prokaryotic enzymes, conferring resistance to oxidative/nitrosative stress and promoting bacterial virulence. Working on the enzymes isolated from E. coli, we found that, contrary to the bo3 oxidase, both bd oxidases are sulfide-insensitive. Accordingly, in E. coli respiratory mutants, aerobic respiration and growth were impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected even at high sulfide levels when either bd enzyme acted as the only terminal oxidase. Consistently, wild-type E. coli displayed sulfide-insensitive respiration and growth under O2-limiting conditions favoring the expression of bd oxidases. Altogether, these results show that bd-type oxidases enable sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. References 1. K. Shatalin, E. Shatalina, A. Mironov, E. Nudler, H2S: a universal defense against antibiotics in bacteria, Science 334 (2011) 986–990. 2. L.C. Petersen, The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase, Biochim. Biophys. Acta 460 (1977) 299–307.