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ABSTRACT: Growing evidence shows that, among triiodothyronine derivatives, 3,5 diiodo-L-thyronine (T(2)) plays an important role in energy metabolism and fat storage. In the present study, short-term effects of T(2) administration to hypothyroid rats on fatty acid oxidation rate and bioenergetic parameters were investigated. Within 1 h following T(2) injection, state 3 and state 4 respiration rates, which were reduced in hypothyroid mitochondria, were noticeably increased particularly in succinate- with respect to glutamate/malate-energized mitochondria. Maximal respiratory activity, observed when glutamate/malate/succinate were simultaneously present in the respiratory medium, was significantly stimulated by T(2) treatment. A T(2)-induced increase in respiratory rates was also observed when palmitoyl-CoA or L-palmitoylcarnitine were used as substrates. No significant change in respiratory control index and ADP/O ratio was observed. The activities of the mitochondrial respiratory chain complexes, especially Complex II, were increased in T(2)-treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and hydrolysis direction, were enhanced. The rate of fatty acid oxidation, followed by conversion of [(14)C]palmitate to CO(2) and ketone bodies, was higher in hepatocytes isolated from T(2)-treated rats. This increase occurs in parallel with the raise in the activity of carnitine palmitoyltransferase-I, the rate limiting enzyme of fatty acid β-oxidation, assayed in situ in digitonin-permeabilized hepatocytes. Overall, these results indicate that T(2) rapidly increases the ability of mitochondria to import and oxidize fatty acids. An emerging idea in the literature is the ability of T(2) to reduce adiposity and dyslipidemia and to prevent the development in liver steatosis. The results of the present study, showing a rapid T(2)-induced increase in the ability of mitochondria to import and oxidize fatty acids, may contribute to understand the biochemical mechanisms of T(2)-metabolic effects.
PLoS ONE 01/2013; 8(1):e52328. · 4.09 Impact Factor
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ABSTRACT: Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.
Journal of proteomics 02/2012; 75(8):2331-41. · 5.07 Impact Factor
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ABSTRACT: Short-term effects of 3,5-L-diiodothyronine (T(2)) administration to hypothyroid rats on F(o)F(1)-ATP synthase activity were investigated in liver mitochondria. One hour after T(2) injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. F(o)F(1)-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T(2) administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of F(o)F(1)-ATP synthase was found, ruling out a T(2) genomic effect. In T(2)-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T(2) administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T(2)-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced F(o)F(1)-ATP synthase activity observed following injection of T(2) to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.
Journal of Bioenergetics 08/2011; 43(4):349-57. · 2.81 Impact Factor
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ABSTRACT: Besides triiodothyronine (T3), 3,5-diiodo-L-thyronine (T2) has been reported to affect mitochondrial bioenergetic parameters. T2 effects have been considered as independent of protein synthesis. Here, we investigated the effect of in vivo chronic T2 administration to hypothyroid rats on liver mitochondrial F(o)F(1)-ATP synthase activity and expression. T2 increased state 4 and state 3 oxygen consumption and raised ATP synthesis and hydrolysis, which were reduced in hypothyroid rats. Immunoblotting analysis showed that T2 up-regulated the expression of several subunits (alpha, beta, F(o)I-PVP and OSCP) of the ATP synthase. The observed increase of beta-subunit mRNA accumulation suggested a T2-mediated nuclear effect. Then, the molecular basis underlying T2 effects was investigated. Our results support the notion that the beta-subunit of ATP synthase is indirectly regulated by T2 through, at least in part, the activation of the transcription factor GA-binding protein/nuclear respiratory factor-2. These findings provide new insights into the T2 role on bioenergetic mechanisms.
Biochimica et Biophysica Acta 10/2009; 1797(2):233-40. · 4.66 Impact Factor
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ABSTRACT: The H(+) F(o)F(1)-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F(1) moiety of the complex protrudes at the inner side of the membrane, the F(o) sector spans the membrane reaching the outer side. The IF(1) component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F(o)F(1)-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF(1) (residue 42-58) to the F(1)-alpha/beta subunits. We have analyzed the effect of native purified IF(1) the IF(1)-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K(+)] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F(o)F(1) complex. The results show that IF(1), and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F(1) to the F(o) side and in the opposite direction. Cross-linking of IF(1) to F(1)-alpha/beta subunits inhibits the ATP-driven H(+) translocation but enhances H(+) conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F(o)F(1) complex.
Biochemical and Biophysical Research Communications 05/2009; 384(1):43-8. · 2.48 Impact Factor
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ABSTRACT: Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.
Biochimica et Biophysica Acta 09/2008; 1777(10):1326-35. · 4.66 Impact Factor
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ABSTRACT: Nonomuraea sp. ATCC 39727 is an aerobic actinomycete, industrially important as a producer of the glycopeptide A40926, which is used as a precursor of the semi-synthetic antibiotic dalbavancin. Previous studies showed that the production of A40926 is depressed by calcium, but promoted by l-glutamine or l-asparagine. In this study, the protein expression changes of Nonomuraea sp. ATCC 39727 in these two different growth and antibiotic-production conditions have been investigated by two-dimensional electrophoresis and mass spectrometry (MS) analysis. Few protein spots show statistically significant expression changes, and, among this group of proteins, malate dehydrogenase (MDH) shows a significant decrease in the overproduction condition. The decrease of MDH is of particular interest because it is the first described significant change in the expression levels of enzymes of the central metabolism related with A40926 overproduction.
FEMS Microbiology Letters 10/2007; 274(1):35-41. · 2.04 Impact Factor
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ABSTRACT: Using isolated polypeptides of the F0 sector of bovine heart mitochondrial H+-ATPase, antisera were developed detecting specifically two components of F0. These two components were identified as F0I and oligomycin-sensitivity-conferring protein (OSCP) respectively. Both F0I and OSCP were digested by mild trypsin treatment of submitochondrial particles depleted of the catalytic part of H+-ATPase (USMP). Proteolysis was largely prevented by binding of F1 to F0. Proteolysis of F0I resulted in the formation of three immunoreactive. membrane-bound fragments of apparently 26 kDa, 25.5 kDa and 18 kDa, respectively, indicating that F0I contains trypsin-accessible Arg or Lys residues located close to the end and the middle part of the protein, respectively, which are in intimate contact with F1. Digestion of USMP with trypsin resulted in depression of passive H+ conduction through F0 which could be ascribed to proteolysis of F0I.
European Journal of Biochemistry. 03/2005; 173(1):1 - 8.
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ABSTRACT: To characterize the role of supernumerary subunits of the mammalian FoF1 ATP synthase, crossreconstitution of mitochondrial and bacterial FoF1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity. Subunits of the mammalian Fo, in particular FoI-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N, N′-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N, N′-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial FoI-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. FoI-PVP and OSCP added to UPEc, promoted inhibition by N, N′-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli Fo. The presence of FoI-PVP and OSCP also promoted inhibition by N, N′-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.
European Journal of Biochemistry. 03/2005; 222(3):733 - 741.
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ABSTRACT: The inhibitor protein IF1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial FoF1-ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact FoF1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF1 (residues 42-58) binds to the alpha and beta subunits of F1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF1 binds, simultaneously, to the OSCP subunit of Fo in a pH-independent process. This binding keeps IF1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.
Journal of Bioenergetics 11/2004; 36(5):447-57. · 2.81 Impact Factor
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ABSTRACT: The protein IF(1) is a natural inhibitor of the mitochondrial F(o)F(1)-ATPase. Many investigators have been prompted to identify the shortest segment of IF(1), retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF(1)-(42-58) and IF(1)-(22-46) is correlated to their structure and conformational plasticity determined by CD and [1H]-NMR spectroscopy. Among all the IF(1) segments tested, IF(1)-(42-58) exerts the most potent, pH and temperature dependent activity on the F(o)F(1) complex. The results suggest that, due to its flexible structure, it can fold in helical and/or beta-spiral arrangements that favor the binding to the F(o)F(1) complex, where the native IF(1) binds. IF(1)-(22-46), instead, as it adopts a rigid alpha-helical conformation, it inhibits ATP hydrolysis only in the soluble F(1) moiety.
Peptides 01/2003; 23(12):2127-41. · 2.43 Impact Factor
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ABSTRACT: We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated.The results obtained show that the α subunit N-terminus is essential for the correct, functional connection of F1 to F0. The α-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit α, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits γ and δ which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit α, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the γ subunit uncovered, surround the γ and the δ subunits located in the central stalk.
European Journal of Biochemistry. 12/2001; 267(14):4445 - 4455.
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ABSTRACT: Diamide treatment of the FoF1-ATP synthase in “inside out” submitochondrial particles (ESMP) in the absence of a respiratory ΔμH+ as well as of isolated Fo reconstituted with F1 or F1-γ subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the FoI-PVP(b) subunit and cysteine 91 at the carboxyl end of a small α-helix of subunit F1-γ, both located in the stalk. The FoI-PVP(b) and F1-γ cross-linking cause dramatic enhancement of oligomycin-sensitive decay of ΔμH+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that FoI-PVP(b) and F1-γ are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of FoI-PVP(b) and the short α-helix of F1-γ can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated Fo, diamide also induces cross-linking of OSCP with another subunit of Fo, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of ΔμH+ generated by respiration, neither cross-linking between FoI-PVP(b) and F1-γ subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by ΔμH+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by ΔμH+ decay through Fo induces a relative motion and/or a separation of the FoI-PVP(b) subunit and F1-γ which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.
11/1998;
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ABSTRACT: A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H+-ATP synthase.Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi—ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the γ subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of γ subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified γ subunit was added back. The addition to F0 liposomes of γ subunit, but not that of α and β subunits, caused per se inhibition of H+ conduction.It is concluded that F0I-PVP and γ subunits are directly involved in the gate of the F0F1 H+-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.
European Journal of Biochemistry. 07/1992; 208(1):9 - 16.
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ABSTRACT: A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in ‘inside out’ submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes.The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2′-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N′-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N′-decyclo[14C]hexylcarbodiimide to the 9-kDa protein.
European Journal of Biochemistry. 04/1987; 164(3):517 - 523.
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ABSTRACT: Besides triiodothyronine (T3), 3,5-diiodo-L-thyronine (T2) has been reported to affect mitochondrial bioenergetic parameters. T2 effects have been considered as independent of protein synthesis. Here, we investigated the effect of in vivo chronic T2 administration to hypothyroid rats on liver mitochondrial FoF1-ATP synthase activity and expression. T2 increased state 4 and state 3 oxygen consumption and raised ATP synthesis and hydrolysis, which were reduced in hypothyroid rats. Immunoblotting analysis showed that T2 up-regulated the expression of several subunits (α, β, FoI-PVP and OSCP) of the ATP synthase. The observed increase of β-subunit mRNA accumulation suggested a T2-mediated nuclear effect. Then, the molecular basis underlying T2 effects was investigated. Our results support the notion that the β-subunit of ATP synthase is indirectly regulated by T2 through, at least in part, the activation of the transcription factor GA-binding protein/nuclear respiratory factor-2. These findings provide new insights into the T2 role on bioenergetic mechanisms.
Biochimica et Biophysica Acta (BBA) - Bioenergetics.
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ABSTRACT: Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-FoF1-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F1 (α/β and γ) and Fo (FoI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF1 is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H+ and are inhibited by F1 and Fo-targeting inhibitors. We hypothesise that ecto-FoF1-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.
Biochimica et Biophysica Acta (BBA) - Bioenergetics.
