Lung-specific RNA interference of coupling factor 6, a novel peptide, attenuates pulmonary arterial hypertension in rats.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease associated with high morbidity and mortality rates. However, the exact regulatory mechanism of PAH is unknown. Although coupling factor 6 (CF6) is known to function as a repressor, its role in PAH has not been explored. Here, we investigated the involvement of endogenous CF6 in the development of PAH. METHODS: PAH was induced with monocrotaline (MCT), as demonstrated by significant increases in pulmonary artery pressure and vessel wall thickness. The adeno-associated virus (AAV) carrying CF6 short hairpin RNA (shRNA) or control vector (2×10(10) gp) was intratracheally transfected into the lungs of rats 2 weeks before or after MCT injection. RESULTS: A 2-6-fold increase in CF6 was observed in the lungs and circulation of the MCT-injected rats as confirmed by qRT-PCR and ELISA. Immunohistochemistry analysis revealed a small quantity of CF6 localized to endothelial cells (ECs) under physiological conditions spread to surrounding tissues in a paracrine manner in PAH lungs. Notably, CF6 shRNA effectively inhibited CF6 expression, abolished lung macrophage infiltration, reversed endothelial dysfunction and vascular remodeling, and ameliorated the severity of pulmonary hypertension and right ventricular dysfunction at 4 weeks both as a pretreatment and rescue intervention. In addition, the circulating and lung levels of 6-keto-PGF1a, a stable metabolite of prostacyclin, were reversed by CF6 inhibition, suggesting that the effect of CF6 inhibition may partly be mediated through prostacyclin. CONCLUSIONS: CF6 contributes to the pathogenesis of PAH, probably in association with downregulation of prostacyclin. The blockage of CF6 might be applied as a novel therapeutic approach for PAH and PA remodeling.

Mitochondrial Inhibitory Factor Protein 1 Functions as an Endogenous Inhibitor for Coupling Factor 6.

Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex unlike a proton-mediated clockwise rotation in the mitochondria, resulting in ATP hydrolysis, proton import, and apoptosis. Inhibitory peptide 1 (IF1) inhibits a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis by a clockwise rotation. We tested the hypothesis that IF1 may antagonize the biological action of CF6 in human embryonic kidney 293 cells. We generated mature and immature IF1 expression vectors and those labeled with GFP at the C-terminus. In the immature IF1-GFP overexpressing cells, the mitochondrial network of IF1-GFP was newly found at the plasma membrane after peripheral translocation, whereas in mature IF1-GFP transfected cells, a less punctuate rather homogenous pattern was found in the cytoplasm. IF1 protein was detected in the exosome fraction of culture media, and it was enhanced by mature or immature IF1 transfection. Extracellular ATP hydrolysis was enhanced by CF6, whereas immature or mature IF1 transfection suppressed ATP hydrolysis in response to CF6. Intracellular pH was decreased by CF6 but was unchanged after immature IF1 transfection. CF6-induced increase in apoptotic cells was blocked by immature or mature IF1, being accompanied by protein kinase B (PKB) phosphorylation. IF1 antagonizes the pro-apoptotic action of CF6 by relief of intracellular acidification and resultant phosphorylation of PKB. Given the widespread biological actions of CF6, the physiological and pathological functions of IF1 may be expected to be complex. J. Cell. Biochem. 117: 1680-1687, 2016. © 2015 Wiley Periodicals, Inc.

The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit.

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).

Further studies on F1-ATPase inhibition by local anesthetics.

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.

Inhibition of coupling factor B activity by cadmium ion, arsenite-2,3-dimercaptopropanol, and phenylarsine oxide, and preferential reactivation by dithiols.

Coupling factor B activity was measured by the stimulation of the ATP-driven NAD+ reduction by succinate or the 32Pi-ATP exchange activity of Factor B-depleted submitochondrial particles. Half-maximal coupling activity was inhibited by 30 microM cadmium, 5 microM phenylarsine oxide, or 0.3 mM arsenite-2,3-dimercaptopropanol. The inhibition was relieved by slight excess of dithiol but not by a 10-fold molar excess of 2-mercaptoethanol. Inhibition of coupling activity by phenylarsine oxide or cadmium was not due to interference in binding of Factor B to depleted particles. Isolated Factor B binds phenylarsine oxide resulting in loss of ability to stimulate depleted submitochondrial particles. The inhibition was largely overcome by dithiol but not by monothiols. The residual coupling activity of depleted submitochondrial particles was highly resistant to cadmium or arsenical. Moreover, binding of arsenical to the depleted particles per se, did not result in inhibition of Factor B-stimulated activity. Furthermore, the addition of phenylarsine oxide to H+-ATPase resulted in loss of Pi-ATP exchange and stimulation of oligomycin-sensitive ATPase activities. Both effects were further potentiated by 2-mercaptoethanol and reversed by dithiols. These effects parallel uncoupling of oxidative phosphorylation in mitochondria by these inhibitors and point to Factor B as the probable component sensitive to these inhibitors.

Effects of inhibitors on mitochondrial adenosine triphosphatase of Tetrahymena pyriformis ST.

Mitochondrial adenosine triphosphatase (ATPase) of the ciliate protozoon Tetrahymena pyriformis ST is completely inhibited by antiserum prepared against F1-ATPase purified from Schizosaccharomyces pombe, and by naturally occurring inhibitor proteins from this yeast and from bovine heart mitochondria. An ATPase inhibitor protein is also present in extracts of T. pyriformis. Mitochondrial ATPase of T. pyriformis is only partially inhibited by the F0-ATPase inhibitors N,N'-dicyclohexylcarbodiimide, oligomycin, leucinostatin, triethyltin sulphate and venturicidin, and (at high titres) by the F1-ATPase inhibitors Dio-9, efrapeptin, 4-chloro-7-nitrobenzofurazan and spegazzinine. Aurovertin, citreoviridin and quercetin were not inhibitory. Resistance to inhibitors distinguishes this mitochondrial ATPase from all those previously examined.

Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit.

When the bovine mitochondrial F1-ATPase is inactivated with dicyclohexyl[14C]carbodiimide and then gel-filtered, from 2 to 3 g atoms of 14C are incorporated/mol of enzyme. Prior inactivation of the enzyme by the modification of an essential tyrosine residue with 4-chloro-7-nitrobenzofurazan, a reaction that can be reversed by thiols, does not affect the irreversible inactivation of the ATPase by dicyclohexyl[14C]carbodiimide. During the large scale modification of the F1-ATPase by dicyclohexyl[14C]carbodiimide which led to 70% inactivation, 1.9 g atoms of 14C were incorporated/mol of enzyme. Isolation of the alpha, beta, and gamma subunits from this large scale inactivation revealed that the gram atoms of 14C bound per mol of each of the subunits was: alpha, 0.04; beta, 0.56; and gamma, 0.04. The majority of the radioactivity in a cyanogen bromide digest of the 14C-labeled beta subunit was isolated in a fragment that has the following amino acid sequence: Glu-Leu-Ile-Asn-Asn-Val-Ala-Lys-Ala-His-Gly-Gly-Tyr-Ser-Val-Phe-Ala-Gly-Val-Gly -Glu-Arg-Thr-Arg-Glu-Gly-Asn-Asp-Leu-Tyr-Glu*-His-Met; where Glu* represents the N gamma-glutamyl derivative of dicyclohexyl[14C]urea.

Inhibition of the links between electron transfer and proton translocation in mitochondria.

The mechanism by which proton extrusion is linked to electron transfer in mitochondria was investigated by means of the primary amine-specific reagent fluorescamine, and of compounds obtained from the reaction of fluorescamine with simple amines (e.g. benzylamine) and with the mycosamine-containing antibiotic amphotericin B. The effect of these 'modifiers' (i.e. fluorescamine transfer chain were assayed separately using specific inhibitors to block the action associated with the other site. Both types of modifiers inhibited the proton extrusion across the membrane to a significantly greater extent than the electron transfer process in both sites II and III. In contrast, the lactone derivative (or cyclic form) of the amine-fluorescamine compounds had no significant inhibitory effect on the proton extrusion and its associated electron transfer. These results are consistent with the hypothesis that the link between proton extrusion and electron transfer in mitochondria is indirect in nature. The results show that: (a) the links involved in sites II and III are identical or very similar in nature; (b) a covalent modification of primary amino groups in the inner membrane is not essential for the expression of these differential inhibitory effects; (c) specific structural features in the amine-fluorescamine compounds, and in the mitochondria-fluorescamine derivatives, are crucial for the expression of the inhibitory effects. Our results contradict the 'redox loop' model of Mitchell, and are compatible with the proton pump concept for the linked proton translocation in oxidative phosphorylation.

Energy coupling factor as target of colicin K: characterization of a colicin K-insensitive ecf mutant of Escherichia coli.

We isolated a colicin K-insensitive energy uncoupled mutant of Escherichia coli. This mutant was presumed to be an ecf mutant as evidenced by its similarity to a known ecf mutant (M. A. Lieberman and J.-S. Hong, 1974) with respect to the mutational site, reversion pattern, and defects in transport and growth. The mutation conferring the colicin K-insensitivity resided in the ecf gene as the majority of the secondary mutations overcoming the ecf phenotype reverted the colicin K-insensitive phenotype to colicin K-sensitive. The insensitivity of the mutant to colicin K was not due to either a defect in adsorption or to a lack of the energized membrane state. The defect was most probably due to the inability of colicin K molecules to interact with their target. Our previous studies concerning the role of the ecf gene product in energy coupling to active transport and oxidative phosphorylation support the contention that the ECF protein is itself the direct target of colicin K.