Journal of bioenergetics

Ubiquinone, commonly called coenzyme Q10 (CoQ), is a lipophilic electron carrier and endogenous antioxidant found in all cellular membranes. In the mitochondrial inner membrane it transfers electrons to complex III of the electron transport chain. The short chain CoQ analogue idebenone is in clinical trials for a number of diseases that exhibit a mitochondrial etiology. Nevertheless, evidence that idebenone ameliorates neurological symptoms in human disease is inconsistent. Although championed as an antioxidant, idebenone can also act as a pro-oxidant by forming an unstable semiquinone at complex I. The antioxidant function of idebenone is critically dependent on two-electron reduction to idebenol without the creation of unstable intermediates. Recently, cytoplasmic NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as a major enzyme catalyzing idebenone reduction. While reduction allows idebenone to act as an antioxidant, evidence also suggests that NQO1 enables idebenone to shuttle reducing equivalents from cytoplasmic NAD(P)H to mitochondrial complex III, bypassing any upstream damage to the electron transport chain. In this mini-review we discuss how idebenone can influence mitochondrial function within the context of cytoprotection. Importantly, in the brain NQO1 is expressed primarily by glia rather than neurons. As NQO1 is an inducible enzyme regulated by oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway, optimizing NQO1 expression in appropriate cell types within a specific disease context may be key to delivering on idebenone’s therapeutic potential.

The protective effect of ADP on unspecific Ca2+ release and collapse of the transmembrane potential was analyzed in mitochondria from kidneys of rats. The presence of ADP in the incubation mixture prevents Ca2+ leakage and collapse of δω in sucrose-containing medium, but fails to do so in KCl medium. The effect of the adenine nucleotide in sucrose media correlates with an increase in the level of reduced pyridine nucleotides; the increase was due to a stimulatory effect on the activity of glutamic dehydrogenase. It also was observed that in KCl media, in the presence and in the absence of ADP the rate of NADH oxidation through the respiratory chain was higher than in sucrose; in this latter medium a high level of reduced pyridine nucleotides was found, in comparison to KCl media. It is proposed that the role of ADP is to increase glutamic dehydrogenase activity and in consequence to provoke a higher rate of formation of NADH which in turn controls Ca2+ release.

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (K d) of 0.3–0.5 and 23–92 μM. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a K d of greater than 100 μM, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-14C] ATP and [8-14C] ADP with K ds of 4–5 and 16–18 μM, respectively. Mg2+ (2 mM) reduced the apparent ATP affinity to 53 μM, an effect entirely explained by chelation of ATP; with Mg2+, K d using calculated free ATP was 3 μM. A combination of gel filtration, Cu2+-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg2+-free nucleotides: the K d for ATP is about 3–5 μM and for Mant-ATP it is about 10 times lower; and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form.

The favorable effect of simvastatin on pulmonary arterial hypertension (PAH) has been well defined despite the unknown etiology of PAH. However, whether simvastatin exerts similar effects on PAH induced right heart failure (RHF) remains to be determined. We aimed to investigate the function of simvastatin in PAH induced RHF. Rats in the RHF and simvastatin groups were injected intraperitoneally with monocrotaline to establish PAH-induced RHF model. The expression of miR-21-5p in rat myocardium was detected and miR-21-5p expression was inhibited using antagomiRNA. The effect of simvastatin on hemodynamic indexes, ventricular remodeling of myocardial tissues, myocardial energy metabolism, and calmodulin was explored. Dual-luciferase reporter system was used to verify the binding relationship between miR-21-5p and Smad7. In addition, the regulatory role of simvastatin in Smad7, TGFBR1 and Smad2/3 was investigated. Simvastatin treatment improved hemodynamic condition, myocardial tissue remodeling, and myocardial energy metabolism, as well as increasing calmodulin expression in rats with PAH-induced RHF. After simvastatin treatment, the expression of miR-21-5p in myocardium of rats was decreased significantly. miR-21-5p targeted Smad7 and inhibited the expression of Smad7. Compared with RHF rats, the expressions of TGFBR1 and Smad2/3 in myocardium of simvastatin-treated rats were decreased significantly. Collectively, we provided evidence that simvastatin can protect ATPase activity and maintain myocardial ATP energy reserve through the miR-21-5p/Smad/TGF-β axis, thus ameliorating PAH induced RHF.

The disease Cystic Fibrosis (CF) is caused by mutations in the protein called CFTR, cystic fibrosis transmembrane conductance regulator, an ABC-transporter–like protein found in the plasma membrane of animal cells. CFTR is believed to function primarily as a Cl− channel, but evidence is mounting that this protein has other roles as well. Structurally, CFTR consists of a single polypeptide chain (1480 amino acids) that folds into 5 distinct domains. These include 2 transmembrane domains that are involved in channel formation; 2 nucleotide-binding domains (NBF1 and NBF2), the first of which clearly binds and hydrolyzes ATP; and 1 regulatory domain (R) that is phosphorylated in a cAMP-dependent process. Currently, the 3D structure of neither CFTR nor its domains has been elucidated, although both nucleotide domains have been modeled in 3D, and solution structures in 3D have been obtained for peptide segments of NBF1. The most common mutation causing CF is the deletion (Δ) of a single phenylalanine (F) in position 508 within a putative helix located in NBF1. CF patients bearing this ΔF508 mutation frequently experience chronic lung infections, particularly by Pseudomonas aeruginosa, and have a life span that rarely exceeds the age of 30. Since the CFTR gene was cloned and sequenced in 1989, there has been over a decade of research focused on understanding the molecular basis of CF caused by the ΔF508 mutation, with the ultimate objective of using the knowledge gained to carry out additional research designed to correct the underlying defect. In general, this pioneering or “ground roots” research has succeeded according to plan. This brief review summarizes some of the highlights with a focus on those studies conducted in the authors' laboratory. For us, this research has been both exciting and rewarding mainly because the results obtained, despite very limited funding, have provided considerable insight, not only into the chemical, molecular, and pathogenic basis of CF, but have made it possible for us and others to now develop novel, chemically rational, and “cost effective” strategies to identify agents that correct the structural defect in the Δ F508 CFTR protein causing most cases of CF.

Defects of cytochromec oxidase (COX) show remarkable clinical, biochemical, and genetic heterogeneity. Clinically, there are two main groups of disorders, one dominated by muscle involvement, the other by brain dysfunction. Biochemically, the enzyme defect may be confined to one or a few tissues (reflecting the existence of tissue-specific isozymes) or affect all tissues. Immunologically reactive enzyme protein is decreased in some forms of COX deficiency but not in others. Because COX is encoded both by nuclear and by mitochondrial genes, COX deficiencies may be due to mutations of either genome and may offer useful models to study the communication between nuclei and mitochondria. We have isolated full-length cDNA clones encoding human COX subunits IV, Vb, and VIII and a partial-length clone for subunit Va. These clones are being used as probes to analyze the DNA and RNA of patients with COX deficiency.

Guanidinoacetic acid (GAA) is a natural precursor of creatine, and a possible substrate for the creatine kinase (CK) enzyme system, serving as a creatine mimetic. Its direct role in cellular bioenergetics has been confirmed in several studies, however GAA utilization by CK seems to be a second-rate as compared to creatine, and compartment-dependent. Here we discuss various factors that might affect GAA use in high-energy phosphoryl transfer in the cytosol and mitochondria.

The C-terminus (CT) of rCx46 consists of 186 residues (H230-I416). Recent studies showed that rCx4628.2, truncated after H243, altered the formation of functional hemichannels when expressed in Xenopus oocytes, while rCx4637.7, truncated after A333 formed gap junction hemichannels similarly to rCx46wt. To analyze the role of the CT up to A333 in functional expression with cell imaging and dye-transfer techniques, different mutants were generated by C-terminal truncation between H243-A333, labeled with EGFP and expressed in HeLa cells. These rCx46 variants were characterized according to their compartmentalization in organelles, their presence in microscopic detectable vesicles and their ability to form gap junction plaques. rCx46 truncated after A311 (rCx4635.3) was compartmentalized, was found in vesicles and formed functional gap junction plaques similarly to rCx46wt. With a truncation after P284 (rCx4632.6), the protein was not compartmentalized and the amount of vesicles containing the protein were reduced; however, functional gap junction plaque formation was not affected as compared to rCx4635.3. rCx4628.2 did not form functional gap junction plaques; it was not found in vesicles or in cellular compartments. Live-cell imaging and detection of annular junctions for rCx4632.6 and rCx4635.3 revealed that the truncation after P284 reduced the frequency of vesicle budding from gap junction plaques and the formation of annular junctions. These results suggest that the C-terminal region of rCx46 up to A311 (rCx4635.3) is necessary for its correct compartmentalization and internalization in the form of annular junctions, while the H230-P284 C-terminal region (rCx4632.6) is sufficient for the formation of dye coupled gap junction channels.