Bong-Hyun Ahn

A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis

Here, we demonstrate a role for the mitochondrial NAD-dependent deacetylase Sirt3 in the maintenance of basal ATP levels and as a regulator of mitochondrial electron transport. We note that Sirt3−/− mouse embryonic fibroblasts have a reduction in basal ATP levels. Reconstitution with wild-type but not a deacetylase-deficient form of Sirt3 restored ATP levels in these cells. Furthermore in wild-type mice, the resting level of ATP correlates with organ-specific Sirt3 protein expression. Remarkably, in mice lacking Sirt3, basal levels of ATP in the heart, kidney, and liver were reduced >50%. We further demonstrate that mitochondrial protein acetylation is markedly elevated in Sirt3−/− tissues. In addition, in the absence of Sirt3, multiple components of Complex I of the electron transport chain demonstrate increased acetylation. Sirt3 can also physically interact with at least one of the known subunits of Complex I, the 39-kDa protein NDUFA9. Functional studies demonstrate that mitochondria from Sirt3−/− animals display a selective inhibition of Complex I activity. Furthermore, incubation of exogenous Sirt3 with mitochondria can augment Complex I activity. These results implicate protein acetylation as an important regulator of Complex I activity and demonstrate that Sirt3 functions in vivo to regulate and maintain basal ATP levels.

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.

α-Synuclein Interacts with Phospholipase D Isozymes and Inhibits Pervanadate-induced Phospholipase D Activation in Human Embryonic Kidney-293 Cells

α-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinsons disease and Alzheimers disease. Although the function of α-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of α-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type α-synuclein or the mutant forms of α-synuclein (A53T, A30P) associated with Parkinsons disease. Tyrosine phosphorylation of α-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr125 to Phe slightly increases inhibitory effect of α-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing α-synuclein than in control cells. Binding of α-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Aβ component of α-synuclein. Although biologically important, co-transfection studies indicate that the interaction of α-synuclein with PLD does not influence the tendency of α-synuclein to form pathological inclusions. These results suggest that the association of α-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of α-synuclein.

Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation

ABSTRACT Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Overexpression of phospholipase D enhances matrix metalloproteinase-2 expression and glioma cell invasion via protein kinase C and protein kinase A/NF-κB/Sp1-mediated signaling pathways

Glioblastoma is a severe type of primary brain tumor, and its highly invasive character is considered to be a major therapeutic obstacle. Phospholipase D (PLD) isozyme is overexpressed in various human tumor tissues and involved in tumorigenesis. However, the molecular mechanisms by which PLD enhances glioma invasion are unknown. In this study, we demonstrate that the increased expression of PLD and its enzymatic activity in the glioma stimulate the secretion and expression of matrix metalloproteinase (MMP)-2 and induce the invasiveness of glioma cells. The upregulation of MMP-2 induced by phosphatidic acid (PA), the product of PLD, was mediated by protein kinase C (PKC), protein kinase A (PKA), nuclear factor-kappaB (NF-kappaB) and Sp1 and it enhanced glioma cell invasion. PA activated PKC and PKA and induced the nuclear translocation and transactivation of NF-kappaB. PA also increased the binding of NF-kappaB and Sp1 to the MMP-2 promoter. Mutation of the NF-kappaB- or Sp1-binding sites significantly attenuated MMP-2 promoter activity. This is the first report to show that NF-kappaB and Sp1 are essential transcriptional factors linking PLD to MMP-2 upregulation, providing evidence that PLD contributes to glioma progression by enhancing MMP-2 expression and tumor cell invasion via PKC/PKA/NF-kappaB/Sp1-mediated signaling pathways.

Differential regulation of apoptosis by caspase-mediated cleavage of phospholipase D isozymes.

Phospholipase D (PLD) has been implicated in survival and anti-apoptosis, but the molecular mechanism by which it responds to apoptotic stimuli is poorly unknown. Here, we demonstrate that cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site (DDVD(545)S) and PLD2 is cleaved at two or three sites (PTGD(13)ELD(16)S and DEVD(28)T) in the front of N-terminus. Cleavage of PLD was endogenously detected in post-mortem Alzheimer brain together with activated caspase-3, suggesting the physiological relevance. The cleavage of PLD1 but not PLD2 might act as an inactivating process since PLD1 but not PLD2 activity is significantly decreased during apoptosis, suggesting that differential cleavage of PLD isozymes could affect its enzymatic activity. Moreover, caspase-resistant mutant of PLD1 showed more potent anti-apoptotic capacity than that of wild type PLD1, whereas PLD2 maintained anti-apoptotic potency in spite of its cleavage during apoptosis. Moreover, PLD2 showed more potent anti-apoptotic effect than that of PLD1 in overexpression and knockdown experiments, suggesting that difference in anti-apoptotic potency between PLD1 and PLD2 might be due to its intrinsic protein property. Taken together, our results demonstrate that differential cleavage pattern of PLD isozymes by caspase might affect its enzymatic activity and anti-apoptotic function.

Phorbol myristate acetate-induced Egr-1 expression is suppressed by phospholipase D isozymes in human glioma cells

Early growth response‐1 (Egr‐1) is involved in the regulation of cell growth. Here, we found that overexpression of phospholipase D (PLD) isozymes decreased tumor promoter phorbol myristate acetate (PMA)‐induced Egr‐1 expression and transactivation in glioma cells. Suppression of PMA‐induced Egr‐1 was dependent on the expression level of PLD isozymes. Overexpression of catalytically inactive PLD, treatment with PA, and prevention of PA dephosphorylation by 1‐propranolol significantly suppressed PMA‐induced Egr‐1 expression. PLD‐induced suppression of Egr‐1 was reversed by inhibition of phosphatidylinositol 3‐kinase (PI3K). Taken together, these results suggest that elevated expression and activity of PLD attenuate PMA‐induced Egr‐1 expression via PI3K pathway.