Репозиторий Университета

Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals.

Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals.

© 2020 The Authors Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.

© 2020 The Authors Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.

© 2020 The Authors Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.

© 2020 Elsevier Inc. Mitochondria are essential for neuronal function because they serve not only to sustain energy and redox homeostasis but also are harbingers of death. A dysregulated mitochondrial network can cascade until function is irreparably lost, dooming cells. TBI is most prevalent in the young and comes at significant personal and societal costs. Traumatic brain injury (TBI) triggers a biphasic and mechanistically heterogenous response and this mechanistic heterogeneity has made the development of standardized treatments challenging. The secondary phase of TBI injury evolves over hours and days after the initial insult, providing a window of opportunity for intervention. However, no FDA approved treatment for neuroprotection after TBI currently exists. With recent advances in detection techniques, there has been increasing recognition of the significance and roles of mitochondrial redox lipid signaling in both acute and chronic central nervous system (CNS) pathologies. Oxidized lipids and their downstream products result from and contribute to TBI pathogenesis. Therapies targeting the mitochondrial lipid composition and redox state show promise in experimental TBI and warrant further exploration. In this review, we provide 1) an overview for mitochondrial redox homeostasis with emphasis on glutathione metabolism, 2) the key mechanisms of TBI mitochondrial injury, 3) the pathways of mitochondria specific phospholipid cardiolipin oxidation, and 4) review the mechanisms of mitochondria quality control in TBI with consideration of the roles lipids play in this process.

© 2020 Elsevier Inc. Mitochondria are essential for neuronal function because they serve not only to sustain energy and redox homeostasis but also are harbingers of death. A dysregulated mitochondrial network can cascade until function is irreparably lost, dooming cells. TBI is most prevalent in the young and comes at significant personal and societal costs. Traumatic brain injury (TBI) triggers a biphasic and mechanistically heterogenous response and this mechanistic heterogeneity has made the development of standardized treatments challenging. The secondary phase of TBI injury evolves over hours and days after the initial insult, providing a window of opportunity for intervention. However, no FDA approved treatment for neuroprotection after TBI currently exists. With recent advances in detection techniques, there has been increasing recognition of the significance and roles of mitochondrial redox lipid signaling in both acute and chronic central nervous system (CNS) pathologies. Oxidized lipids and their downstream products result from and contribute to TBI pathogenesis. Therapies targeting the mitochondrial lipid composition and redox state show promise in experimental TBI and warrant further exploration. In this review, we provide 1) an overview for mitochondrial redox homeostasis with emphasis on glutathione metabolism, 2) the key mechanisms of TBI mitochondrial injury, 3) the pathways of mitochondria specific phospholipid cardiolipin oxidation, and 4) review the mechanisms of mitochondria quality control in TBI with consideration of the roles lipids play in this process.

© 2019 Elsevier B.V. Background: Skeletal muscle cells continuously generate reactive oxygen species (ROS). Excessive ROS can affect lipids resulting in lipid peroxidation (LPO). Here we investigated the effects of myotube intracellular calcium-induced signaling eliciting contractions on the LPO induction and the impact of LPO-product 4-hydroxynonenal (4-HNE) on physiology/pathology of myotubes using C2C12 myoblasts. Methods: C2C12 myoblasts were differentiated into myotubes, stimulated with caffeine and analyzed for the induction of LPO and formation of 4-HNE protein adducts. Further effects of 4-HNE on mitochondrial bioenergetics, NADH level, mitochondrial density and expression of mitochondrial metabolism genes were determined. Results: Short and long-term caffeine stimulation of myotubes promoted superoxide production, LPO and formation of 4-HNE protein adducts. Furthermore, low 4-HNE concentrations had no effect on myotube viability and cellular redox homeostasis, while concentrations from 10 μM and above reduced myotube viability and significantly disrupted homeostasis. A time and dose-dependent 4-HNE effect on superoxide production and mitochondrial NADH-autofluorescence was observed. Finally, 4-HNE had strong impact on maximal respiration, spare respiratory capacity, ATP production, coupling efficiency of mitochondria and mitochondrial density. Conclusion: Data presented in this work make evident for the first time that pathological 4-HNE levels elicit damaging effects on skeletal muscle cells while acute exposure to physiological 4-HNE induces transient adaptation. General significance: This work suggests an important role of 4-HNE on the regulation of myotube's mitochondrial metabolism and cellular energy production. It further signifies the importance of skeletal muscle cells hormesis in response to acute stress in order to maintain essential biological functions.

© 2019 Elsevier Inc. Loss of dopaminergic neurons in the substantia nigra is one of the pathogenic hallmarks of Parkinson's disease, yet the underlying molecular mechanisms remain enigmatic. While aberrant redox metabolism strongly associated with iron dysregulation and accumulation of dysfunctional mitochondria is considered as one of the major contributors to neurodegeneration and death of dopaminergic cells, the specific anomalies in the molecular machinery and pathways leading to the PD development and progression have not been identified. The high efficiency and relative simplicity of a new genome editing tool, CRISPR/Cas9, make its applications attractive for deciphering molecular changes driving PD-related impairments of redox metabolism and lipid peroxidation in relation to mishandling of iron, aggregation and oligomerization of alpha-synuclein and mitochondrial injury as well as in mechanisms of mitophagy and programs of regulated cell death (apoptosis and ferroptosis). These insights into the mechanisms of PD pathology may be used for the identification of new targets for therapeutic interventions and innovative approaches to genome editing, including CRISPR/Cas9.

© 2018 Elsevier B.V. Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CLOX, has been implicated in the cellular signaling events that initiate apoptosis. CLOX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ12-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CLOX, and treatment of cells with H2O2 increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.

© 2018 Conrad et al. Lipid peroxidation is the process by which oxygen combines with lipids to generate lipid hydroperoxides via intermediate formation of peroxyl radicals. Vitamin E and coenzyme Q10 react with peroxyl radicals to yield peroxides, and then these oxidized lipid species can be detoxified by glutathione and glutathione peroxidase 4 (GPX4) and other components of the cellular antioxidant defense network. Ferroptosis is a form of regulated nonapoptotic cell death involving overwhelming iron-dependent lipid peroxidation. Here, we review the functions and regulation of lipid peroxidation, ferroptosis, and the antioxidant network in diverse species, including humans, other mammals and vertebrates, plants, invertebrates, yeast, bacteria, and archaea. We also discuss the potential evolutionary roles of lipid peroxidation and ferroptosis.