Mechanisms of the interaction of nitroxyl with mitochondria

It is now thought that NO• (nitric oxide) and its redox congeners may play a role in the physiological regulation of mitochondrial function. The inhibition of cytochrome c oxidase by NO• is characterized as being reversible and oxygen dependent. In contrast, peroxynitrite, the product of the reaction of NO• with superoxide, irreversibly inhibits several of the respiratory complexes. However, little is known about the effects of HNO (nitroxyl) on mitochondrial function. This is especially important, since HNO has been shown to be more cytotoxic than NO•, may potentially be generated in vivo, and elicits biological responses with some of the characteristics of NO and peroxynitrite. In the present study, we present evidence that isolated mitochondria, in the absence or presence of substrate, convert HNO into NO• by a process that is dependent on mitochondrial concentration as well as the concentration of the HNO donor Angeli's salt. In addition, HNO is able to inhibit mitochondrial respiration through the inhibition of complexes I and II, most probably via modification of specific cysteine residues in the proteins. Using a proteomics approach, extensive modification of mitochondrial protein thiols was demonstrated. From these data it is evident that HNO interacts with mitochondria through mechanisms distinct from those of either NO• or peroxynitrite, including the generation of NO•, the modification of thiols and the inhibition of complexes I and II.

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HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis

eLife ◽

10.7554/elife.46976 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 17

Author(s):

Shuping Zhang ◽

Alejandra Macias-Garcia ◽

Jacob C Ulirsch ◽

Jason Velazquez ◽

Vincent L Butty ◽

...

Keyword(s):

Iron Deficiency ◽

Mitochondrial Function ◽

Ribosomal Proteins ◽

Target Genes ◽

Mitochondrial Protein ◽

Erythroid Differentiation ◽

Protein Translation ◽

Ribosome Profiling ◽

Underlying Mechanisms

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.

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Oxidative modification of hepatic mitochondria protein thiols: effect of chronic alcohol consumption

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00399.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. G521-G527 ◽

Cited By ~ 60

Author(s):

Aparna Venkatraman ◽

Aimee Landar ◽

Ashley J. Davis ◽

Elena Ulasova ◽

Grier Page ◽

...

Keyword(s):

Aldehyde Dehydrogenase ◽

Nitrosative Stress ◽

Mitochondrial Protein ◽

Oxidative Modification ◽

Mitochondrial Proteins ◽

In Vivo Model ◽

Chronic Alcohol ◽

In Vivo Models ◽

Protein Thiols

Redox modification of mitochondrial proteins is thought to play a key role in regulating cellular function, although direct evidence to support this hypothesis is limited. Using an in vivo model of mitochondrial redox stress, ethanol hepatotoxicity, the modification of mitochondrial protein thiols was examined using a proteomics approach. Specific labeling of reduced thiols in the mitochondrion from the livers of control and ethanol-fed rats was achieved by using the thiol reactive compound (4-iodobutyl)triphenylphosphonium (IBTP). This molecule selectively accumulates in the organelle and can be used to identify thiol-containing proteins. Mitochondrial proteins that have been modified are identified by decreased labeling with IBTP using two-dimensional SDS-PAGE followed by immunoblotting with an antibody directed against the triphenylphosphonium moiety of the IBTP molecule. Analyses of these data showed a significant decrease in IBTP labeling of thiols present in specific mitochondria matrix proteins from ethanol-fed rats compared with their corresponding controls. These proteins were identified as the low- Km aldehyde dehydrogenase and glucose-regulated protein 78. The decrease in IBTP labeling in aldehyde dehydrogenase was accompanied by a decrease in specific activity of the enzyme. These data demonstrate that mitochondrial protein thiol modification is associated with chronic alcohol intake and might contribute to the pathophysiology associated with hepatic injury. Taken together, we have developed a protocol to chemically tag and select thiol-modified proteins that will greatly enhance efforts to establish posttranslational redox modification of mitochondrial protein in in vivo models of oxidative or nitrosative stress.

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The human mitochondrial 12S rRNA m4C methyltransferase METTL15 is required for mitochondrial function

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012127 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8505-8513 ◽

Cited By ~ 5

Author(s):

Hao Chen ◽

Zhennan Shi ◽

Jiaojiao Guo ◽

Kao-jung Chang ◽

Qianqian Chen ◽

...

Keyword(s):

Mitochondrial Function ◽

Mitochondrial Protein ◽

Nuclear Gene ◽

Small Subunit ◽

12S Rrna ◽

Mitochondrial Rna ◽

Small Subunit Rrna ◽

Mitochondrial Ribosomes

Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.

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Aerobic metabolism of human quadriceps muscle: in vivo data parallel measurements on isolated mitochondria

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.280.2.e301 ◽

2001 ◽

Vol 280 (2) ◽

pp. E301-E307 ◽

Cited By ~ 42

Author(s):

Ulla F. Rasmussen ◽

Hans N. Rasmussen ◽

Peter Krustrup ◽

Bjørn Quistorff ◽

Bengt Saltin ◽

...

Keyword(s):

Work Performance ◽

Citrate Synthase ◽

Mitochondrial Protein ◽

Knee Extensor ◽

Atp Synthesis ◽

Quadriceps Muscle ◽

Isolated Mitochondria ◽

Respiratory Chain Activity

The aim of the present study was to examine whether parameters of isolated mitochondria could account for the in vivo maximum oxygen uptake (V˙o 2 max) of human skeletal muscle.V˙o 2 max and work performance of the quadriceps muscle of six volunteers were measured in the knee extensor model (range 10–18 mmol O2 · min−1 · kg−1at work rates of 22–32 W/kg). Mitochondria were isolated from the same muscle at rest. Strong correlations were obtained betweenV˙o 2 max and a number of mitochondrial parameters (mitochondrial protein, cytochrome aa 3, citrate synthase, and respiratory activities). The activities of citrate synthase, succinate dehydrogenase, and pyruvate dehydrogenase, measured in isolated mitochondria, corresponded to, respectively, 15, 3, and 1.1 times the rates calculated from V˙o 2 max. The respiratory chain activity also appeared sufficient. Fully coupled in vitro respiration, which is limited by the rate of ATP synthesis, could account for, at most, 60% of theV˙o 2 max. This might be due to systematic errors or to loose coupling of the mitochondrial respiration under intense exercise.

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PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis

Protein & Cell ◽

10.1007/s13238-021-00888-x ◽

2021 ◽

Author(s):

Weili Yang ◽

Xiangyu Guo ◽

Zhuchi Tu ◽

Xiusheng Chen ◽

Rui Han ◽

...

Keyword(s):

Protein Expression ◽

Mitochondrial Function ◽

Kinase Activity ◽

Mitochondrial Protein ◽

Neuronal Function ◽

Mitochondrial Homeostasis ◽

Primate Brain

AbstractIn vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson’s disease (PD). However, difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1. Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains. CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology. Importantly, PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival. Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.

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Mitochondrial and redox modifications in early stages of Huntington disease

10.1101/2022.01.14.476381 ◽

2022 ◽

Author(s):

Carla Lopes ◽

Ildete Luisa Ferreira ◽

Carina Maranga ◽

Margarida Beatriz ◽

Sandra Mota ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Mouse Brain ◽

Mitochondrial Function ◽

Huntington Disease ◽

Skin Fibroblasts ◽

Isolated Mitochondria ◽

Early Stages ◽

Yac128 Mouse ◽

Human And Mouse

Defects in mitochondrial function and mitochondrial-related redox deregulation have been attributed to Huntington disease (HD), a genetic neurodegenerative disorder largely affecting the striatum. However, whether these changes occur in early stages of the disease and can be detected in vivo is still unclear. Thus, in the present study, we analyzed changes in mitochondrial function and overreduced states associated with production of reactive oxygen species (ROS) at early stages and along disease progression in vivo in the brain by positron emission tomography (PET) and in skin fibroblasts of premanifest/early and manifest HD patients, and in YAC128 transgenic mouse brain (striatum and cortex) at early-symptomatic (3 month-old, mo) and symptomatic (6 to 12 mo) stages. In vivo human and mouse brain PET imaging was assessed using [64Cu]-ATSM; analysis of oxygen consumption rates was assessed by Seahorse analysis, hydrogen peroxide levels were determined using fluorescent probes and mitochondrial morphology by transmission electron microscopy in human skin fibroblasts and mouse striatal and cortical isolated mitochondria. Premanifest and prodromal HD carriers exhibited enhanced whole-brain (with exception of caudate) [64Cu]-ATSM labelling, correlating with CAG repeat number, concomitantly with enhanced basal and maximal respiration, proton (H+) leak and increased hydrogen peroxide levels, the later progressing to advanced HD stage, in human fibroblasts. Mitochondria from fibroblasts of premanifest HD carriers also showed reduced roundness, while higher number of mitochondrial DNA copies correlated with maximal respiratory capacity. In vivo animal PET analysis showed increased accumulation of [64Cu]-ATSM in YAC128 mouse striatum. Pre/early-symptomatic YAC128 mouse striatal, but not cortical, isolated mitochondria exhibited a rise in basal and maximal mitochondrial respiration and in ATP production along with increased complex II and III activities, enhanced mitochondrial hydrogen peroxide and roundness, as revealed by brain ultrastructure analysis, further presenting defects in Ca2+ handling, supporting increased striatal susceptibility in the YAC128 mouse model. Data demonstrate both human and mouse mitochondrial overactivity and altered morphology at early HD stages, facilitating redox unbalance, the latter extending over late disease stages.

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Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function

AJP Cell Physiology ◽

10.1152/ajpcell.00200.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. C1136-C1143 ◽

Cited By ~ 30

Author(s):

N. M. A. van den Broek ◽

J. Ciapaite ◽

K. Nicolay ◽

J. J. Prompers

Keyword(s):

Skeletal Muscle ◽

Oxygen Consumption ◽

Mitochondrial Dysfunction ◽

Mitochondrial Function ◽

Atp Synthesis ◽

Resonance Spectroscopy ◽

Isolated Mitochondria ◽

Significant Difference

31P magnetic resonance spectroscopy (MRS) has been used to assess skeletal muscle mitochondrial function in vivo by measuring 1) phosphocreatine (PCr) recovery after exercise or 2) resting ATP synthesis flux with saturation transfer (ST). In this study, we compared both parameters in a rat model of mitochondrial dysfunction with the aim of establishing the most appropriate method for the assessment of in vivo muscle mitochondrial function. Mitochondrial dysfunction was induced in adult Wistar rats by daily subcutaneous injections with the complex I inhibitor diphenyleneiodonium (DPI) for 2 wk. In vivo 31P MRS measurements were supplemented by in vitro measurements of oxygen consumption in isolated mitochondria. Two weeks of DPI treatment induced mitochondrial dysfunction, as evidenced by a 20% lower maximal ADP-stimulated oxygen consumption rate in isolated mitochondria from DPI-treated rats oxidizing pyruvate plus malate. This was paralleled by a 46% decrease in in vivo oxidative capacity, determined from postexercise PCr recovery. Interestingly, no significant difference in resting, ST-based ATP synthesis flux was observed between DPI-treated rats and controls. These results show that PCr recovery after exercise has a more direct relationship with skeletal muscle mitochondrial function than the ATP synthesis flux measured with 31P ST MRS in the resting state.

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Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments

AJP Renal Physiology ◽

10.1152/ajprenal.00031.2020 ◽

2020 ◽

Vol 318 (5) ◽

pp. F1237-F1245

Author(s):

Allison McCrimmon ◽

Mark Domondon ◽

Regina F. Sultanova ◽

Daria V. Ilatovskaya ◽

Krisztian Stadler

Keyword(s):

Oxygen Consumption ◽

Mitochondrial Function ◽

Respiratory Function ◽

Rapid Assessment ◽

Acute Injury ◽

Mitochondrial Oxygen Consumption ◽

Isolated Mitochondria ◽

Proximal Tubular ◽

Mitochondrial Respiratory

Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/ db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator’s choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.

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Proteolysis of the products of mitochondrial protein synthesis in yeast mitochondria and submitochondrial particles

Biochemical Journal ◽

10.1042/bj1820195 ◽

1979 ◽

Vol 182 (1) ◽

pp. 195-202 ◽

Cited By ~ 28

Author(s):

S L Kalnov ◽

L A Novikova ◽

A S Zubatov ◽

V N Luzikov

Keyword(s):

Mitochondrial Protein ◽

Mitochondrial Translation ◽

Submitochondrial Particles ◽

Isolated Mitochondria ◽

Saccharomyces Cerevisiae Mitochondria ◽

Mitochondrial Inner Membrane ◽

Culture Growth ◽

Inside Out ◽

Culture Growth Phase

Degradation of mitochondrial translation products in Saccharomyces cerevisiae mitochondria was studied by selectively labelling these entities in vivo in the presence of cycloheximide and following their fate in isolated mitochondria. One-third to one-half of the mitochondrial translation products are shown to be degraded, depending on the culture growth phase, with an approximate half-life of 35 min. This process is shown to be ATP-dependent, enhanced in the presence of puromycin and inhibited by chloramphenicol. Further, the proteolysis is suppressed by detergents and is insensitive to antisera against yeast proteinases A and B when measured in mitochondria or ‘inside-out’ submitochondrial particles. It is concluded that the breakdown of mitochondrial translation products is most probably due to the action of endogenous proteinase(s) associated with the mitochondrial inner membrane. This proteinase is inhibited by phenylmethanesulphonyl fluoride, leupeptin, antipain and chymostatin.

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Diet-Induced Vitamin D Deficiency Results in Reduced Skeletal Muscle Mitochondrial Respiration in C57BL/6J Mice

10.1101/2020.05.15.098087 ◽

2020 ◽

Author(s):

Stephen P. Ashcroft ◽

Gareth Fletcher ◽

Ashleigh M. Philp ◽

Philip J. Atherton ◽

Andrew Philp

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Vitamin D Deficiency ◽

Mitochondrial Function ◽

Dietary Intervention ◽

Mitochondrial Protein ◽

Current Evidence ◽

Performance Deficits

AbstractVitamin D deficiency is known to be associated with symptoms of skeletal muscle myopathy including muscle weakness and fatigue. Recently, vitamin D related metabolites have been linked to the maintenance of mitochondrial function within skeletal muscle. However, current evidence is limited to in vitro models and the effects of diet-induced vitamin D deficiency upon skeletal muscle mitochondrial function in vivo have received little attention. In order to examine the role of vitamin D in the maintenance of mitochondrial function in vivo, we utilised an established model of diet-induced vitamin D deficiency in C57BL/6J mice. Mice were fed either a control (2,200 IU/kg) or a vitamin D deplete (0 IU/kg) diet for periods of 1-, 2- and 3-months. Skeletal muscle mitochondrial function and ADP sensitivity were assessed via high-resolution respirometry and mitochondrial protein content via immunoblotting. As a result of 3-month of diet-induced vitamin D deficiency, respiration supported via CI+IIP and ETC were 35% and 37% lower when compared to vitamin D replete mice (P < 0.05). Despite functional alterations, the protein expression of electron transfer chain subunits remained unchanged in response to dietary intervention (P > 0.05). In conclusion, we report that 3-months of diet-induced vitamin D deficiency reduced skeletal muscle mitochondrial function in C57BL/6J mice. Our data, when combined with previous in vitro observations, suggests that vitamin D mediated regulation of mitochondrial function may underlie the exacerbated muscle fatigue and performance deficits observed during vitamin D deficiency.

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