Stephanie French

Metabolic network control of oxidative phosphorylation: Multiple roles of inorganic phosphate

Phosphate (Pi) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that Pi controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F1F0-ATPase in heart and skeletal muscle mitochondria. In these studies the effect of Pi was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. Pi enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. Pi oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of Pi was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [Pi]. These results demonstrate that Pi signaling results in the balanced modulation of oxidative phosphorylation, by influencing both ΔGH+ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems.

The Mammalian Longevity-associated Gene Product p66shc Regulates Mitochondrial Metabolism

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66shc have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66shc localizes to the mitochondria and that p66shc-/- fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66shc-/- mouse embryonic fibroblasts were lower than similarly maintained wild type cells. Differences in oxygen consumption were particularly evident under chemically uncoupled conditions, demonstrating that p66shc-/- cells have a reduction in both their resting and maximal oxidative capacity. We further demonstrate that reconstitution of p66shc expression in p66shc-/- cells increases oxygen consumption. The observed defect in oxidative capacity seen in p66shc-/- cells is partially offset by augmented levels of aerobic glycolysis. This metabolic switch is manifested by p66shc-/- cells exhibiting an increase in lactate production and a stricter requirement for extracellular glucose in order to maintain intracellular ATP levels. In addition, using an in vivo NADH photobleaching technique, we demonstrate that mitochondrial NADH metabolism is reduced in p66shc-/- cells. These results demonstrate that p66shc regulates mitochondrial oxidative capacity and suggest that p66shc may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.

Quantitative Mitochondrial Phosphoproteomics Using iTRAQ on an LTQ-Orbitrap with High Energy Collision Dissociation

With the use of iTRAQ labeling and mass spectrometry on an LTQ-Orbitrap with HCD capability, we assessed relative changes in protein phosphorylation in the mitochondria upon physiological perturbation. As a reference reaction, we monitored the well-characterized regulation of pyruvate dehydrogenase (PDH) activity via phosphorylation/dephosphorylation by pyruvate dehydrogenase kinase/pyruvate dehydrogenase phosphatase in response to dichloroacetate, de-energization and Ca2+. Relative quantification of phosphopeptides of PDH-E1alpha subunit from porcine heart revealed dephosphorylation at three serine sites (Ser231, Ser292 and Ser299). Dephosphorylation at Ser292 (i.e., the inhibitory site) with DCA correlated with an activation of PDH activity as previously reported, consistent with our de-energization data. Calcium also dephosphorylated (i.e., activated) PDH, thus, confirming calcium activation of PDP. With this approach, we successfully monitored other phosphorylation sites of mitochondrial proteins including adenine nucleotide translocase, malate dehydrogenase and mitochondrial creatine kinase. Among them, four proteins exhibited phosphorylation changes with these physiological stimuli: (1) BCKDH-E1alpha subunit increased phosphorylation at Ser337 with DCA and de-energization; (2) apoptosis-inducing factor phosphorylation was elevated at Ser345 with calcium; (3) ATP synthase F1 complex alpha subunit and (4) mitofilin dephosphorylated at Ser65 and Ser264 upon de-energization. This screening validated the iTRAQ/HCD technology as a method for functional quantitation of mitochondrial protein phosphorylation as well as providing insight into the regulation of mitochondria via phosphorylation.

Succinyl-CoA Synthetase Is a Phosphate Target for the Activation of Mitochondrial Metabolism

Succinyl-CoA synthetase (SCS) is the only mitochondrial enzyme capable of ATP production via substrate level phosphorylation in the absence of oxygen, but it also plays a key role in the citric acid cycle, ketone metabolism, and heme synthesis. Inorganic phosphate (P(i)) is a signaling molecule capable of activating oxidative phosphorylation at several sites, including NADH generation and as a substrate for ATP formation. In this study, it was shown that P(i) binds the porcine heart SCS alpha-subunit (SCSalpha) in a noncovalent manner and enhances its enzymatic activity, thereby providing a new target for P(i) activation in mitochondria. Coupling 32P labeling of intact mitochondria with SDS gel electrophoresis revealed that 32P labeling of SCSalpha was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS), we showed that this enhanced 32P labeling resulted from a simple binding of 32P, not covalent protein phosphorylation. The ability of SCSalpha to retain its 32P throughout the SDS denaturing gel process was unique over the entire mitochondrial proteome. In vitro studies also revealed a P(i)-induced activation of SCS activity by more than 2-fold when mitochondrial extracts and purified BSCS were incubated with millimolar concentrations of P(i). Since the level of 32P binding to SCSalpha was increased in substrate-depleted mitochondria, where the matrix P(i) concentration is increased, we conclude that SCS activation by P(i) binding represents another mitochondrial target for the P(i)-induced activation of oxidative phosphorylation and anaerobic ATP production in energy-limited mitochondria.

Use of 32P to Study Dynamics of the Mitochondrial Phosphoproteome

Protein phosphorylation is a well-characterized regulatory mechanism in the cytosol, but remains poorly defined in the mitochondrion. In this study, we characterized the use of (32)P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The (32)P labeling technique was compared and contrasted to Phos-tag protein phosphorylation fluorescent stain and 2D isoelectric focusing. Of the 64 proteins identified by MS spectroscopy in the Phos-Tag gels, over 20 proteins were correlated with (32)P labeling. The high sensitivity of (32)P incorporation detected proteins well below the mass spectrometry and even 2D gel protein detection limits. Phosphate-chase experiments revealed both turnover and phosphate associated protein pool size alterations dependent on initial incubation conditions. Extensive weak phosphate/phosphate metabolite interactions were observed using nondisruptive native gels, providing a novel approach to screen for potential allosteric interactions of phosphate metabolites with matrix proteins. We confirmed the phosphate associations in Complexes V and I due to their critical role in oxidative phosphorylation and to validate the 2D methods. These complexes were isolated by immunocapture, after (32)P labeling in the intact mitochondria, and revealed (32)P-incorporation for the alpha, beta, gamma, OSCP, and d subunits in Complex V and the 75, 51, 42, 23, and 13a kDa subunits in Complex I. These results demonstrate that a dynamic and extensive mitochondrial matrix phosphoproteome exists in heart and liver.

Proteomic changes associated with diabetes in the BB-DP rat

These studies were structured with the aim of utilizing emerging technologies in two-dimensional (2D) gel electrophoresis and mass spectrometry to evaluate protein expression changes associated with type 1 diabetes. We reasoned that a broad examination of diabetic tissues at the protein level might open up novel avenues of investigation of the metabolic and signaling pathways that are adversely affected in type 1 diabetes. This study compared the protein expression of the liver, heart, and skeletal muscle of diabetes-prone rats and matched control rats by semiquantitative liquid chromatography-mass spectrometry and differential in-gel 2D gel electrophoresis. Differential expression of 341 proteins in liver, 43 in heart, and 9 (2D gel only) in skeletal muscle was detected. These data were assembled into the relevant metabolic pathways affected primarily in liver. Multiple covalent modifications were also apparent in 2D gel analysis. Several new hypotheses were generated by these data, including mechanisms of net cytosolic protein oxidation, formaldehyde generation by the methionine cycle, and inhibition of carbon substrate oxidation via reduction in citrate synthase and short-chain acyl-CoA dehydrogenase.

Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH

Fluorescent determinations of NADH in porcine heart mitochondria were subject to significant errors caused by alterations in inner filter effects during numerous metabolic perturbations. These inner filter effects were primarily associated with changes in mitochondrial volume and accompanying light scattering. The observed effects were detected in a standard commercial fluorometer with emission orthogonal to the excitation light path and, to a lesser extent, in a light path geometry detecting only the surface fluorescence. A method was developed to detect and correct for inner filter effects on mitochondrial NADH fluorescence measurements that were independent of the optical path geometry using an internal fluorescent standard and linear least-squares spectral analysis. A simple linear correction with the inner fluorescence reference was found to adequately correct for inner filter effects. This approach may be useful for other fluorescence probes in isolated mitochondria or other light-scattering media.

32P Labeling of Protein Phosphorylation and Metabolite Association in the Mitochondria Matrix

Protein phosphorylations, as well as phosphate metabolite binding, are well characterized post-translational mechanisms that regulate enzyme activity in the cytosol, but remain poorly defined in mitochondria. Recently extensive matrix protein phosphorylation sites have been discovered but their functional significance is unclear. Herein we describe methods of using (32)P labeling of intact mitochondria to determine the dynamic pools of protein phosphorylation as well as phosphate metabolite association. This screening approach may be useful in not only characterizing the dynamics of these pools, but also provide insight into which phosphorylation sites have a functional significance. Using the mitochondrial ATP synthetic capacity under appropriate conditions, inorganic (32)P was added to energized mitochondria to generate high specific activity gamma-P(32)-ATP in the matrix. In general, SDS denaturing and gel electrophoresis was used to primarily follow protein phosphorylation, whereas native gel techniques were used to observe weaker metabolite associations since the structure of mitochondrial complexes was minimally affected. The protein phosphorylation and metabolite association within the matrix was found to be extensive using these approaches. (32)P labeling in 2D gels was detected in over 40 proteins, including most of the complexes of the cytochrome chain and proteins associated with intermediary metabolism, biosynthetic pathways, membrane transport, and reactive oxygen species metabolism. (32)P pulse-chase experiments further revealed the overall dynamics of these processes that included phosphorylation site turnover as well as phosphate-protein pool size alterations. The high sensitivity of (32)P resulted in many proteins being intensely labeled, but not identified due to the sensitivity limitations of mass spectrometry. These low concentration proteins may represent signaling proteins within the matrix. These results demonstrate that the mitochondrial matrix phosphoproteome is both extensive and dynamic. The use of this, in situ, labeling approach is extremely valuable in confirming protein phosphorylation sites as well as examining the dynamics of these processes under near physiological conditions.

In Vivo Microscopy Reveals Extensive Embedding of Capillaries within the Sarcolemma of Skeletal Muscle Fibers

To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice.

Synthesis and inhibitory activity of difluoroketone substrate analogs of N-myristoyltransferase.

Abstract Two fluorinated nonhydrolyzable analogs of myristoyl-coenzyme A were synthesized and tested for inhibitory activity against N-myristoyltransferase (NMT).S-(2,2-Difluoro-3-oxohexadecyl)-coenzyme A (3) and S-(3,3-difluoro-2-oxopentadecyl)-coenzyme A (2) were prepared by alkylation of coenzyme A and were purified by reverse phase chromatography. Inhibition of NMT was observed with 3 and 2, with IC50s of 110 nM and 80 nM, respectively, in an in vitro assay developed in our laboratory. The known unfluorinated analog S-(2-oxopentadecyl)-coenzyme A (1) was found to have an IC50 of 7 nM. At 100 μM in D2O, 3 was 59% hydrated and 2 was 88% hydrated.