scholarly journals Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

2006 ◽  
Vol 395 (2) ◽  
pp. 405-415 ◽  
Author(s):  
Fimmie Reinecke ◽  
Oksana Levanets ◽  
Yolanda Olivier ◽  
Roan Louw ◽  
Boitumelo Semete ◽  
...  
Keyword(s):  
Hela Cells ◽  
Complex I ◽  
Protective Role ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Ros Production ◽  
Complex I Deficiency ◽  
Overexpressing Cell ◽  
Dose Dependent Decrease ◽  
Complex I Activity

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.

Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency

2005 ◽  
Vol 289 (4) ◽  
pp. C881-C890 ◽  
Author(s):  
Werner J. H. Koopman ◽  
Henk-Jan Visch ◽  
Sjoerd Verkaart ◽  
Lambertus W. P. J. van den Heuvel ◽  
Jan A. M. Smeitink ◽  
...  
Keyword(s):  
Form Factor ◽  
Aspect Ratio ◽  
Complex I ◽  
Mitochondrial Morphology ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mitochondrial Network ◽  
Mitochondrial Structure ◽  
Complex I Deficiency ◽  
Complex I Activity ◽  
Human Complex

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

Abstract 224: Mitochondrial Complex I Deficiency Promotes Oxidative Stress During Ischemia Reperfusion Of Cardiac Myocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Guohua Gong ◽  
Georgios Karamanlidis ◽  
Chi Fung Lee ◽  
Rong Tian ◽  
Wang Wang
Keyword(s):  
Oxidative Stress ◽  
Cardiac Myocytes ◽  
Complex I ◽  
Ischemia Reperfusion ◽  
Mitochondrial Complex I ◽  
Ros Production ◽  
Mitochondrial Complex ◽  
Complex I Deficiency ◽  
Mitochondrial Ros ◽  
Complex I Activity

Normal cardiac function relies on highly coordinated intracellular events, such as calcium cycling and contraction, with adequate mitochondrial energy metabolism. However, mitochondrial respiration unavoidably produces reactive oxygen species (ROS) as electrons leak from the electron transport chain (ETC). Complex I of the ETC is believed to be the major site for ROS generation in the mitochondria. However, suppression of Complex I activity by chemical inhibitors leads to oxidative cell damage. In this study, we used a genetic model of Complex I deficiency, in which a key component of Complex I, Ndufs4, was deleted in the heart, to determine the causal role of Complex I in ischemia-reperfusion-induced oxidative stress in adult cardiac myocytes. Germline deletion of Ndufs4 in the heart (Ndufs4H-/-) leads to a ~75% decline of Complex I activity in cardiac mitochondria without obvious disease phenotype in the mice. As predicted, the mitochondrial respiration-coupled superoxide production events, superoxide flashes, were significantly decreased at baseline in the Ndufs4H-/- myocytes. Respiration substrate (pyruvate, 20 mM) failed to stimulate mitochondrial superoxide flash production in Ndufs4H-/- myocytes. This is accompanied by the slightly decreased steady state intracellular and mitochondrial ROS levels determined by the targeted H2O2 indicator, Hyper. The intracellular redox homeostasis is also tilted toward more reduced state, since the NADH/NAD ratio increased 67%. Surprisingly, ischemia reperfusion mimetic treatment of the myocytes caused dramatic increase in mitochondrial ROS production in Ndufs4H-/- groups, which contributed to the elevated overall cellular oxidative status. Overexpression of catalase in the mitochondria prevented these effects. Mechanistically, increased reducing equivalent (NADH) contributed to the dramatic ROS production during ischemia and reperfusion in Ndufs4H-/- myocytes. In summary, mitochondrial Complex I plays a critical role in controlling mitochondrial and cytosolic ROS homeostasis under normal conditions, and compromised Complex I function leads to accumulation of electron donors that paradoxically promote ROS production during ischemia reperfusion.

Mutants of Chlamydomonas reinhardtii Deficient in Mitochondrial Complex I: Characterization of Two Mutations Affecting the nd1 Coding Sequence

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1051-1060
Author(s):  
Claire Remacle ◽  
Denis Baurain ◽  
Pierre Cardol ◽  
René F Matagne
Keyword(s):  
Mitochondrial Genome ◽  
Chlamydomonas Reinhardtii ◽  
Complex I ◽  
Slow Growth ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Sequencing Analysis ◽  
Mitochondrial Complex ◽  
Genetical Analysis ◽  
Complex I Activity

Abstract The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 30 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components of complex I are coded for by mitochondrial genes. Three mutants deprived of complex I activity and displaying slow growth in the dark were isolated after mutagenic treatment with acriflavine. A genetical analysis demonstrated that two mutations (dum20 and dum25) affect the mitochondrial genome whereas the third mutation (dn26) is of nuclear origin. Recombinational analyses showed that dum20 and dum25 are closely linked on the genetic map of the mitochondrial genome and could affect the nd1 gene. A sequencing analysis confirmed this conclusion: dum20 is a deletion of one T at codon 243 of nd1; dum25 corresponds to a 6-bp deletion that eliminates two amino acids located in a very conserved hydrophilic segment of the protein.

scholarly journals Structural basis for a complex I mutation that blocks pathological ROS production

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhan Yin ◽  
Nils Burger ◽  
Duvaraka Kula-Alwar ◽  
Dunja Aksentijević ◽  
Hannah R. Bridges ◽  
...  
Keyword(s):  
Point Mutation ◽  
Complex I ◽  
Structural Changes ◽  
Ischemia Reperfusion ◽  
Single Point ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

scholarly journals β-Naphthoflavone and Ethanol Reverse Mitochondrial Dysfunction in A Parkinsonian Model of Neurodegeneration

2020 ◽  
Vol 21 (11) ◽  
pp. 3955
Author(s):  
Jesus Fernandez-Abascal ◽  
Elda Chiaino ◽  
Maria Frosini ◽  
Gavin P. Davey ◽  
Massimo Valoti
Keyword(s):  
Complex I ◽  
In Vitro Model ◽  
Inhibitory Effects ◽  
Cyp Induction ◽  
Cyp Isoforms ◽  
Vitro Model ◽  
Complex I Activity ◽  
Cyp 2D6

The 1-methyl-4-phenylpyridinium (MPP+) is a parkinsonian-inducing toxin that promotes neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it was reported that some Cytochrome P450 (CYP) isoforms, such as CYP 2D6 or 2E1, may be involved in the development of this neurodegenerative disease. In order to study a possible role for CYP induction in neurorepair, we designed an in vitro model where undifferentiated neuroblastoma SH-SY5Y cells were treated with the CYP inducers β-naphthoflavone (βNF) and ethanol (EtOH) before and during exposure to the parkinsonian neurotoxin, MPP+. The toxic effect of MPP+ in cell viability was rescued with both βNF and EtOH treatments. We also report that this was due to a decrease in reactive oxygen species (ROS) production, restoration of mitochondrial fusion kinetics, and mitochondrial membrane potential. These treatments also protected complex I activity against the inhibitory effects caused by MPP+, suggesting a possible neuroprotective role for CYP inducers. These results bring new insights into the possible role of CYP isoenzymes in xenobiotic clearance and central nervous system homeostasis.

Interaction of vaginal Lactobacillus strains with HeLa cells plasma membrane

2017 ◽  
Vol 8 (4) ◽  
pp. 625-633 ◽  
Author(s):  
N. Calonghi ◽  
C. Parolin ◽  
G. Sartor ◽  
L. Verardi ◽  
B. Giordani ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Physical Properties ◽  
Hela Cells ◽  
Protective Role ◽  
Vaginal Infections ◽  
Vaginal Lactobacilli ◽  
Cell Line Hela ◽  
Cervical Cells ◽  
Host Epithelium

Vaginal lactobacilli offer protection against recurrent urinary and vaginal infections. The precise mechanisms underlying the interaction between lactobacilli and the host epithelium remain poorly understood at the molecular level. Deciphering such events can provide valuable information on the mode of action of commensal and probiotic bacteria in the vaginal environment. We investigated the effects exerted by five Lactobacillus strains of vaginal origin (Lactobacillus crispatus BC1 and BC2, Lactobacillus gasseri BC9 and BC11 and Lactobacillus vaginalis BC15) on the physical properties of the plasma membrane in a cervical cell line (HeLa). The interaction of the vaginal lactobacilli with the cervical cells determined two kinds of effects on plasma membrane: (1) modification of the membrane polar lipid organisation and the physical properties (L. crispatus BC1 and L. gasseri BC9); (2) modification of α5β1 integrin organisation (L. crispatus BC2, L. gasseri BC11 and L. vaginalis BC15). These two mechanisms can be at the basis of the protective role of lactobacilli against Candida albicans adhesion. Upon stimulation with all Lactobacillus strains, we observed a reduction of the basal oxidative stress in HeLa cells that could be related to modifications in physical properties and organisation of the plasma membrane. These results confirm the strictly strain-specific peculiarities of Lactobacillus and deepen the understanding of the mechanisms underlying the health-promoting role of this genus within the vaginal ecosystem.

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+removal: aberrations in human complex I deficiency

2006 ◽  
Vol 291 (2) ◽  
pp. C308-C316 ◽  
Author(s):  
Henk-Jan Visch ◽  
Werner J. H. Koopman ◽  
Dimphy Zeegers ◽  
Sjenet E. van Emst-de Vries ◽  
Frank J. M. van Kuppeveld ◽  
...  
Keyword(s):  
Digital Imaging ◽  
Complex I ◽  
Removal Rate ◽  
Mitochondrial Matrix ◽  
Atp Production ◽  
Complex I Deficiency ◽  
Deficient Patient ◽  
Atp Supply ◽  
Human Complex

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca2+indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca2+concentration ([Ca2+]M) and, consequently, the Ca2+-stimulated increase in [ATP]M, thus allowing studies of how the increase in [ATP]Mand the cytosolic Ca2+removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca2+]Mand [ATP]Mby maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca2+indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca2+removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca2+removal during stimulation with a Ca2+-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca2+removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca2+pumps. This work reveals the usefulness of rhodamine-based Ca2+indicators in examining the role of intramitochondrial Ca2+in mitochondrial (patho) physiology.

scholarly journals Isoflurane Differentially Modulates Mitochondrial Reactive Oxygen Species Production via  Forward versus  Reverse Electron Transport Flow

2011 ◽  
Vol 115 (3) ◽  
pp. 531-540 ◽  
Author(s):  
Naoyuki Hirata ◽  
Yon Hee Shim ◽  
Danijel Pravdic ◽  
Nicole L. Lohr ◽  
Philip F. Pratt ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Electron Transport ◽  
Complex I ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
Submitochondrial Particles ◽  
Anesthetic Preconditioning ◽  
Cardioprotective Effects ◽  
Complex I Activity

Background Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles. Methods Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2 h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined. Results Isoflurane reduced myocardial infarct size (40 ± 9% = mean ± SD) compared with control experiments (60 ± 4%). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60 ± 9%). Isoflurane enhanced ROS generation in submitochondrial particles with nicotinamide adenine dinucleotide (reduced form), but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity. Conclusions The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

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