- Email: [email protected]
Reversing Electron Transport in Ischemia and Beyond
4a
Saturday, February 11, 2017
sequence motif, DXXE, in the MCU channel. Our recent NMR data of the MCU channel indicate that the DXXE forms two parallel carboxylate rings at the channel entrance that appear to serve as the ion selectivity filter. We used paramagnetic probes to show that mutants with a single carboxylate ring can bind divalent cation specifically, while in the wild type, the two rings bind the ion cooperatively, resulting in drastically higher apparent affinity. Our new data, together with structural analysis of the DXXE motif, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter of the channel and that Ru360 directly blocks ion entry into the filter. 18-Subg Reversing Electron Transport in Ischemia and Beyond Paul Brookes. Univ Rochester, Rochester, NY, USA. Accumulation of the TCA cycle metabolite succinate in ischemic tissues has been reported for almost 50 years. However, the patho-physiologic importance of this event is still poorly understood. In 2014, it was reported that accumulated succinate is a key driver of Reactive Oxygen Species (ROS) generation at reperfusion [PMID: 25383517]. This has led to proposals that inhibiting ischemic succinate accumulation may afford therapeutic benefit in ischemia-reperfusion (IR) injury. However, the reversal of respiratory complex II (Cx-II, succinate dehydrogenase), which is the proposed route of ischemic succinate accumulation, may serve a beneficial role. Namely, by using fumarate as an alternate electron acceptor (in lieu of O2), reverse electron transfer (RET) at Cx-II allows complex I (Cx-I) to continue functioning. As such, Hþ pumping by Cx-I provides a potential source of mitochondrial membrane potential during ischemia, preventing the catabolism of glycolytic ATP by reversal of the ATP synthase. Consistent with a beneficial role, succinate accumulation is widely documented in hypoxic tissues of diving mammals. A prediction from this modified viewpoint, is that the timing of Cx-II inhibition relative to IR injury may yield differential outcomes. Namely prophylactic Cx-II inhibition may lose any benefit from RET at Cx-II during ischemia. Conversely, Cx-II inhibition only at reperfusion will be beneficial by preventing ROS generation. Data supporting this hypothesis will be discussed, along with compelling new data regarding the precise mechanism of succinate accumulation in ischemia, which may not be as straightforward as initially thought. 19-Subg Redox Regulation of Cytochrome C Oxidase Assembly Antoni Barrientos. Department of Neurology, University of Miami, Miami, FL, USA. Cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is the major oxygen consumer enzyme in the cell. COX biogenesis involves several redox-regulated steps. The process is highly regulated to prevent the formation of pro-oxidant intermediates. Regulation of COX assembly involves several reactive oxygen species and redox-regulated steps. One type of control targets the heme A-containing cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. We have recently reported that Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment. In vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation. We conclude that the redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production. 20-Subg Mitochondrial Production of ROS: Deviations from the ‘‘Standard Model’’ Anatoly Starkov. Brain and Mind Research Institute Weill Cornell Medicine, New York, NY, USA. Since the discovery of mitochondrial ROS production by P.K. Jensen in 1966, numerous mechanistic details of this process have been revealed. The mainstream interpretation of the obtained data has shaped a ‘‘standard model’’ of mitochondrial ROS production, which, although lacking a formal description,
is currently widely accepted. The ‘‘standard model’’ postulates that the major primary ROS generated in mitochondria is superoxide. This superoxide is predominantly generated by Complexes I and III of the respiratory chain, and possibly, a few other enzymes e.g., dihydrolipoamide dehydrogenase. Most of the data to support this concept have been obtained by inhibitor analysis with specific inhibitors of respiratory chain complexes and matrix enzymes. The biological ramifications of this model are tremendous, ranging from philosophical ‘‘mitochondrial theory of aging’’ to very practical and costly enterprises aimed at making mitochondria-targeted antioxidants that would ‘‘curb the production of ROS in mitochondria’’. However, there are reported cases, in which the results of inhibitor analysis did not fit the ‘‘standard model’’. We will discuss these cases in our presentation.
Biopolymers in Vivo Subgroup 21-Subg Organization of Nucleic Acids and Proteins by Lipid Membranes Sarah L. Keller. Dept of Chemistry, University of Washington, Seattle, WA, USA. On the early Earth, the first protocells were likely composed of biopolymers (e.g. RNA and proteins) encapsulated within a fatty acid membrane. A fundamental biophysical question is how the building blocks of RNA, namely the four nucleobases and ribose, were selected from a heterogeneous environment. We have found that nucleobases bind more strongly to fatty acid membranes than many similar bases. Furthermore, that binding may explain how the building blocks were concentrated, and how fatty acid membranes may have been stabilized in the salty environments of Earth’s early oceans (Black et al. PNAS 2013). Fast forwarding a few billion years, in modern cell membranes, biopolymers and lipids may work in concert to create membrane regions enriched in particular protein and lipid types. This talk will briefly summarize research projects inspired by questions inspired by both ancient and modern cells. 22-Subg Folding in the Cell - Ions, Crowders, Osmolytes Simon Ebbinghaus. Physical Chemistry II, Bochum, Germany. Proteins fold and function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of 40% with macromolecules. That under such conditions cells can keep their proteome folded and organized without uncontrollable aggregation is a remarkable aspect of biology. We first discuss how the different cosolutes in the cellular milieu such as ions, crowders and osmolytes govern the protein folding equilibrium. We thereby present a novel classification scheme of cosolute effects based on their thermodynamic fingerprints. This model is of fundamental importance to understand how the proteome stability is modulated by cellular processes, e.g. to understand how osmolytes or chaperones protect the proteome or how most destabilized proteins aggregate under different cell stresses. We further developed folding sensors that probe the different cosolute effects directly in cells. Thereby, we show that cell stress can significantly modulate the folding equilibrium. Remarkably, protective cellular mechanisms such as the heat shock response or the regulatory volume increase are highly adapted to minimize the impact on the proteome. 23-Subg RNA-Based Control of Cellular Phase Transitions Erin Langdon, Amy S. Gladfelter. Biology, UNC Chapel Hill, Chapel Hill, NC, USA. Intracellular phase transitions drive the formation of dynamic compartments for specific biochemistry and to localize molecules in time and space. How cells ensure that liquid-demixed structures remain distinct and control where phase transitions occur remain central open questions to the regulation of these compartments. The large cytoplasm of the filamentous fungus Ashbya gossypii is a powerful system for studying control of phase transitions because in this cell type nuclei divide asynchronously and multiple sites of cell polarity coexist. Both nuclear asynchrony and polarity establish require functional regionalization of an otherwise continuous cytoplasm. We have identified a polyQ-tract containing RNA-binding protein called Whi3 that undergoes RNA-dependent phase transitions and localizes mRNAs in the vicinity of nuclei and at incipient polarity sites. How the same protein forms spatially and functionally distinct bodies in the same cell is an interesting problem and a useful model for understanding the mechanisms of control of cellular phase transitions. We have found that different mRNAs can generate liquid droplets of Whi3 with distinct biophysical properties such as viscoelasticity and surface tension suggesting that RNAs may direct the fates of the phase transitioned compartments. We
Saturday, February 11, 2017
sequence motif, DXXE, in the MCU channel. Our recent NMR data of the MCU channel indicate that the DXXE forms two parallel carboxylate rings at the channel entrance that appear to serve as the ion selectivity filter. We used paramagnetic probes to show that mutants with a single carboxylate ring can bind divalent cation specifically, while in the wild type, the two rings bind the ion cooperatively, resulting in drastically higher apparent affinity. Our new data, together with structural analysis of the DXXE motif, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter of the channel and that Ru360 directly blocks ion entry into the filter. 18-Subg Reversing Electron Transport in Ischemia and Beyond Paul Brookes. Univ Rochester, Rochester, NY, USA. Accumulation of the TCA cycle metabolite succinate in ischemic tissues has been reported for almost 50 years. However, the patho-physiologic importance of this event is still poorly understood. In 2014, it was reported that accumulated succinate is a key driver of Reactive Oxygen Species (ROS) generation at reperfusion [PMID: 25383517]. This has led to proposals that inhibiting ischemic succinate accumulation may afford therapeutic benefit in ischemia-reperfusion (IR) injury. However, the reversal of respiratory complex II (Cx-II, succinate dehydrogenase), which is the proposed route of ischemic succinate accumulation, may serve a beneficial role. Namely, by using fumarate as an alternate electron acceptor (in lieu of O2), reverse electron transfer (RET) at Cx-II allows complex I (Cx-I) to continue functioning. As such, Hþ pumping by Cx-I provides a potential source of mitochondrial membrane potential during ischemia, preventing the catabolism of glycolytic ATP by reversal of the ATP synthase. Consistent with a beneficial role, succinate accumulation is widely documented in hypoxic tissues of diving mammals. A prediction from this modified viewpoint, is that the timing of Cx-II inhibition relative to IR injury may yield differential outcomes. Namely prophylactic Cx-II inhibition may lose any benefit from RET at Cx-II during ischemia. Conversely, Cx-II inhibition only at reperfusion will be beneficial by preventing ROS generation. Data supporting this hypothesis will be discussed, along with compelling new data regarding the precise mechanism of succinate accumulation in ischemia, which may not be as straightforward as initially thought. 19-Subg Redox Regulation of Cytochrome C Oxidase Assembly Antoni Barrientos. Department of Neurology, University of Miami, Miami, FL, USA. Cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is the major oxygen consumer enzyme in the cell. COX biogenesis involves several redox-regulated steps. The process is highly regulated to prevent the formation of pro-oxidant intermediates. Regulation of COX assembly involves several reactive oxygen species and redox-regulated steps. One type of control targets the heme A-containing cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. We have recently reported that Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment. In vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation. We conclude that the redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production. 20-Subg Mitochondrial Production of ROS: Deviations from the ‘‘Standard Model’’ Anatoly Starkov. Brain and Mind Research Institute Weill Cornell Medicine, New York, NY, USA. Since the discovery of mitochondrial ROS production by P.K. Jensen in 1966, numerous mechanistic details of this process have been revealed. The mainstream interpretation of the obtained data has shaped a ‘‘standard model’’ of mitochondrial ROS production, which, although lacking a formal description,
is currently widely accepted. The ‘‘standard model’’ postulates that the major primary ROS generated in mitochondria is superoxide. This superoxide is predominantly generated by Complexes I and III of the respiratory chain, and possibly, a few other enzymes e.g., dihydrolipoamide dehydrogenase. Most of the data to support this concept have been obtained by inhibitor analysis with specific inhibitors of respiratory chain complexes and matrix enzymes. The biological ramifications of this model are tremendous, ranging from philosophical ‘‘mitochondrial theory of aging’’ to very practical and costly enterprises aimed at making mitochondria-targeted antioxidants that would ‘‘curb the production of ROS in mitochondria’’. However, there are reported cases, in which the results of inhibitor analysis did not fit the ‘‘standard model’’. We will discuss these cases in our presentation.
Biopolymers in Vivo Subgroup 21-Subg Organization of Nucleic Acids and Proteins by Lipid Membranes Sarah L. Keller. Dept of Chemistry, University of Washington, Seattle, WA, USA. On the early Earth, the first protocells were likely composed of biopolymers (e.g. RNA and proteins) encapsulated within a fatty acid membrane. A fundamental biophysical question is how the building blocks of RNA, namely the four nucleobases and ribose, were selected from a heterogeneous environment. We have found that nucleobases bind more strongly to fatty acid membranes than many similar bases. Furthermore, that binding may explain how the building blocks were concentrated, and how fatty acid membranes may have been stabilized in the salty environments of Earth’s early oceans (Black et al. PNAS 2013). Fast forwarding a few billion years, in modern cell membranes, biopolymers and lipids may work in concert to create membrane regions enriched in particular protein and lipid types. This talk will briefly summarize research projects inspired by questions inspired by both ancient and modern cells. 22-Subg Folding in the Cell - Ions, Crowders, Osmolytes Simon Ebbinghaus. Physical Chemistry II, Bochum, Germany. Proteins fold and function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of 40% with macromolecules. That under such conditions cells can keep their proteome folded and organized without uncontrollable aggregation is a remarkable aspect of biology. We first discuss how the different cosolutes in the cellular milieu such as ions, crowders and osmolytes govern the protein folding equilibrium. We thereby present a novel classification scheme of cosolute effects based on their thermodynamic fingerprints. This model is of fundamental importance to understand how the proteome stability is modulated by cellular processes, e.g. to understand how osmolytes or chaperones protect the proteome or how most destabilized proteins aggregate under different cell stresses. We further developed folding sensors that probe the different cosolute effects directly in cells. Thereby, we show that cell stress can significantly modulate the folding equilibrium. Remarkably, protective cellular mechanisms such as the heat shock response or the regulatory volume increase are highly adapted to minimize the impact on the proteome. 23-Subg RNA-Based Control of Cellular Phase Transitions Erin Langdon, Amy S. Gladfelter. Biology, UNC Chapel Hill, Chapel Hill, NC, USA. Intracellular phase transitions drive the formation of dynamic compartments for specific biochemistry and to localize molecules in time and space. How cells ensure that liquid-demixed structures remain distinct and control where phase transitions occur remain central open questions to the regulation of these compartments. The large cytoplasm of the filamentous fungus Ashbya gossypii is a powerful system for studying control of phase transitions because in this cell type nuclei divide asynchronously and multiple sites of cell polarity coexist. Both nuclear asynchrony and polarity establish require functional regionalization of an otherwise continuous cytoplasm. We have identified a polyQ-tract containing RNA-binding protein called Whi3 that undergoes RNA-dependent phase transitions and localizes mRNAs in the vicinity of nuclei and at incipient polarity sites. How the same protein forms spatially and functionally distinct bodies in the same cell is an interesting problem and a useful model for understanding the mechanisms of control of cellular phase transitions. We have found that different mRNAs can generate liquid droplets of Whi3 with distinct biophysical properties such as viscoelasticity and surface tension suggesting that RNAs may direct the fates of the phase transitioned compartments. We