Electrophoresis techniques to investigate defects in oxidative phosphorylation

Defects in mitochondrial oxidative phosphorylation (OXPHOS) are a frequent cause of severe inherited metabolic disorders and also contribute to aging. The OXPHOS system constitutes five multi-subunit complexes embedded in the mitochondrial inner membrane. Correct function of this system requires proper assembly of the 80 proteins in the complexes, as well as numerous assembly factors. Blue native electrophoresis has become a crucial tool to investigate OXPHOS-related defects in mitochondrial disease patients. In addition, OXPHOS-assembly profiles can be obtained by two dimensional blue native/SDS gel electrophoresis, which provides additional information for identifying disease-causing mutations and insight in the role of specific proteins in the biogenesis of the OXPHOS system. Here we provide a practical guide on how to set-up the basic technique to study OXPHOS defects in patient-derived cells and tissues.     

The use of lymphocytes to screen for oxidative phosphorylation disorders

Biochemical analysis of oxidative phosphorylation (OXPHOS) disorders is traditionally carried out on muscle biopsies, cultured fibroblasts, and transformed lymphocytes. Here we present a new screening technique using lymphocytes to identify OXPHOS dysfunction and initially avoid an invasive diagnostic procedure. Lymphocytes represent an easily obtainable source of tissue that presents advantages over the use of fibroblasts or lymphoblast cell lines. The time delay in culturing skin fibroblasts and the interactions between cell transformation and mitochondrial activity are avoided in this methodology. The method requires a small amount of blood (<5 mL); can be completed in a few hours, and allows for repeated measurements. Our assay has been adapted from published methods utilizing cultured fibroblasts and transformed lymphocytes, and our data suggest that measurement of ATP synthesis in lymphocytes is an effective screening tool for diagnosing OXPHOS disorders. This method may also provide an objective tool for monitoring response to treatment and evaluating progression of disease.     

Antibody-based approaches to diagnosis and characterization of oxidative phosphorylation diseases

Mitochondrial disorders caused by defects in oxidative phosphorylation function are difficult to diagnose. Here we review the emerging use of antibody-based approaches for this diagnosis. Novel methods involving immunohistochemistry and immunocapture of defective enzymes for characterization are described that add to the arsenal of approaches available.     

Role of energy in oxidative phosphorylation

This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and P_i and for the release of ATP bound at the catalytic sites of F₁-ATPase. The process of ATP synthesis on the surface of F₁ from F₁-bound ADP and P_i appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a lowK _m (for ADP and P_i)-lowV _max mode and a highK _m -highV _max mode. TheK _m extremes for ADP are 2–3 µM and 120–150 µM, andV _max for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 s^–1 (mole F₁)^–1 at 30°C, which corresponds to 11 µmol ATP (min · mg of protein)^–1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunitc) of the membrane sector (F₀) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F₁, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F₁ in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential () and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions is exquisitely sensitive to changes in the rates of energy production and consumption.     

Patients with Leber hereditary optic neuropathy fail to compensate impaired oxidative phosphorylation

Ninety-five percent of Leber hereditary optic neuropathy (LHON) patients carry a mutation in one out of three mtDNA-encoded ND subunits of complex I. Penetrance is reduced and more male than female carriers are affected. To assess if a consistent biochemical phenotype is associated with LHON expression, complex I- and complex II-dependent adenosine triphosphate synthesis rates (CI-ATP, CII-ATP) were determined in digitonin-permeabilized peripheral blood mononuclear cells (PBMCs) of thirteen healthy controls and for each primary mutation of a minimum of three unrelated patients and of three unrelated carriers with normal vision and were normalized per mitochondrion (citrate synthase activity) or per cell (protein content). We found that in mitochondria, CI-ATP and CII-ATP were impaired irrespective of the primary LHON mutation and clinical expression. An increase in mitochondrial density per cell compensated for the dysfunctional mitochondria in LHON carriers but was insufficient to result in a normal biochemical phenotype in early-onset LHON patients.     

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.     

Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles

The Central Nervous System (CNS) function was shown to be fueled exclusively by oxidative phosphorylation (OXPHOS). This is in line with the sensitivity of brain to hypoxia, but less with the scarcity of the mitochondria in CNS. Consistently with the ectopic expression of F_oF₁-ATP synthase and the electron transfer chain in myelin, we have reported data demonstrating that isolated myelin vesicles (IMV) conduct OXPHOS. It may suggest that myelin sheath could be a site for the whole aerobic degradation of glucose.     

Relationship between chemiosmotic flows and thermodynamic forces in oxidative phosphorylation

A set of equations has been derived, describing quantitatively the relationships between flows and thermodynamic forces in the chemiosmotic model of oxidative phosphorylation.Experimental tests of these equations give information on the stoichiometric coupling constants between the different flows.     

The oxidative phosphorylation (OXPHOS) system: nuclear genes and human genetic diseases

The ubiquitous nature of mitochondria, the dual genetic foundation of the respiratory chain in mitochondrial and nuclear genome, and the peculiar rules of mitochondrial genetics all contribute to the extraordinary heterogeneity of clinical disorders associated with defects of oxidative phosphorylation (mitochondrial encephalomyopathies). Here, we review recent findings about nuclear gene defects in isolated OXPHOS enzyme complex deficiency. This information should help in identifying patients with mitochondrial disease and defining a biochemical and molecular basis of the disorder found in each patient. This knowledge is indispensable for accurate genetic counseling and prenatal diagnosis, and is a prerequisite for the development of rational therapies, which are still, at present, woefully inadequate.     

Effective coupling of four independent membrane systems in steady-state oxidative phosphorylation

Alexandre et al. have proposed a four-system model of oxidative phosphorylation. The thermodynamic consequences of this model are explored, assuming as a first approximation that these four systems can be treated as a self-contained group. The method can be generalized, in higher approximations, to include further systems and other complications. Respiratory control is considered from the point of view of the model. Self-consistent numerical examples are given to represent mitochondrial activity in state 3 and in state 4.     

Mitogenome evolution in Cephini (Hymenoptera: Cephidae): Evidence for parallel adaptive evolution

Two mitogenomes of remarkable size of the stem-boring sawflies, Trachelus tabidus (18539 bp) and T. iudaicus (20730 bp), were characterized and compared with previously known mitogenomes of Cephini. A rearrangement in the IQM gene cluster, an unusual elongation in rrnS gene and supposedly functional tandem repeat sequences in the A+T-rich region are synapomorphies of Cephini. Mitogenome evolution of the Cephini was investigated in a dataset of seven species representing all genera. The noticeably divergent mitogenomes of the Cephini both at nucleotide and amino acid level, and the variable rates of ω values, indicate the effects of different selective forces, rather than neutral evolution. The effect of positive selection is revealed by radical amino acid changes with ten physicochemical properties (P_α, B_r, pK′, h, E_l, H_nc, α_c, α_n, R_a and H_p) throughout each branch of the tree. The radical changes in the ND and ATP complexes are mostly related to chemical and energetic properties due to different energy requirements among species. The phenology of Cephini species and their host plants, together with habitat structure, rather than host plant preferences or phylogenetic constraint, are suggested to explain the role of adaptive evolution in shaping Cephini mitogenomes.     

Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation

Oxidative phosphorylation (OXPHOS) is the main source of energy in eukaryotic cells. This process is performed by means of electron flow between four enzymes, of which three are proton pumps, in the inner mitochondrial membrane. The energy accumulated in the proton gradient over the inner membrane is utilized for ATP synthesis by a fifth OXPHOS complex, ATP synthase. Four of the OXPHOS protein complexes associate into stable entities called respiratory supercomplexes. This review summarises the current view on the arrangement of the electron transport chain in mitochondrial cristae. The functional role of the supramolecular organisation of the OXPHOS system and the factors that stabilise such organisation are highlighted. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes

The organization of the oxidative phosphorylation (OXPHOS) system within the inner mitochondrial membrane appears to be far more complicated than previously thought. In particular, the individual protein complexes of the OXPHOS system (complexes I to V) were found to specifically interact forming defined supramolecular structures. Blue-native polyacrylamide gel electrophoresis and single particle electron microscopy proved to be especially valuable in studying the so-called "respiratory supercomplexes". Based on these procedures, increasing evidence was presented supporting a "solid state" organization of the OXPHOS system. Here, we summarize results on the formation, organisation and function of the various types of mitochondrial OXPHOS supercomplexes.     

Altered O-GlcNAc modification and phosphorylation of mitochondrial proteins in myoblast cells exposed to high glucose

Hyperglycemia induced increased posttranslational modification of proteins by O-linked-β-N-acetyl glucosamine (O-GlcNAcylation) and mitochondrial dysfunction has been independently implicated in the development of insulin resistance. It is not known whether repertoire of O-GlcNAcylated proteins includes mitochondrial proteins and their altered O-GlcNAcylation impinges on their phosphorylation mediated normal functioning thus contribute to mitochondrial dysfunction and insulin resistance. We have explored the O-GlcNAcylation of mitochondrial proteins from myoblast cells under basal (4 mM) and high glucose (30 mM) conditions using a combination of proteomic approaches. Furthermore, we have assessed the accompanied changes in the phosphorylation of mitochondrial proteins. We report that a number of mitochondrial proteins are O-GlcNAcylated under basal condition which is altered under high glucose condition. In addition, we report that exposure to high glucose not only changes the O-GlcNAcylation of mitochondrial proteins but also changes their phosphorylation profiles. The dynamic and complex interplay between O-GlcNAcylation and phosphorylation of mitochondrial proteins was further validated by immunoblot analysis of HSP60, prohibitin, and voltage-dependent anion channel 1 as candidate proteins. O-GlcNAcylation of mitochondrial proteins may play a role in normal functioning of mitochondria. High glucose induced changes in O-GlcNAcylation and phosphorylation of mitochondrial proteins may be associated with mitochondrial dysfunction and insulin resistance.     

Oxidative phosphorylation supercomplexes and respirasome reconstitution of the colorless alga Polytomella sp.

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F₁F_O-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V₂ and V₄) and different respiratory supercomplexes (I/IV₆, I/III₄, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V₄ ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called “respirasome” was able to perform in-vitro oxygen consumption.     

Circadian rhythms of oxidative phosphorylation: effects of rotenone and melatonin on isolated rat brain mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation

The effects of three tetrachlorobiphenylols [2′,3′,4′,5′-tetrachloro-2-biphenylol (1); 2′,3′,4′,5′-tetrachloro-4-biphenylol (2); and 2′,3′,4′,5′-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3′,5,5′-tetrachloro-4,4′-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase)] activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1–3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5–7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (≥0.4 μM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1–7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.