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Query: EC:2.7.11.31 (
AMP-activated protein kinase
)
13,065
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Insulin is a promising drug for the treatment of diseases associated with brain damage. However, the mechanism of its neuroprotective action is far from being understood. Our aim was to study the insulin-induced protection of cortical neurons in oxidative stress and its mechanism. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. The insulin neuroprotection was shown to depend on insulin concentration in the nanomolar range. Insulin decreased the reactive oxygen species formation in neurons. The insulin-induced modulation of various protein kinase activities was studied at eight time-points after neuronal exposure to prooxidant (hydrogen peroxide). In prooxidant-exposed neurons, insulin increased the phosphorylation of GSK-3beta at Ser
9
(thus inactivating it), which resulted from Akt activation. Insulin activated ERK1/2 in neurons 5-30 min after cell exposure to prooxidant.
Hydrogen peroxide
markedly activated
AMPK
, while it was for the first time shown that insulin inhibited it in neurons at periods of the most pronounced activation by prooxidant. Insulin normalized Bax/Bcl-2 ratio and mitochondrial membrane potential in neurons in oxidative stress. The inhibitors of the PI3K/Akt and MEK1/2/ERK1/2 signaling pathways and the
AMPK
activator reduced the neuroprotective effect of insulin. Thus, the protective action of insulin on cortical neurons in oxidative stress appear to be realized to a large extent through activation of Akt and ERK1/2, GSK-3beta inactivation, and inhibition of
AMPK
activity increased by neuronal exposure to prooxidant.
...
PMID:The Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on the Modulation of Akt, GSK-3beta, ERK1/2, and AMPK Activities. 3136 43
ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria, but it is associated with the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of
H2O2
, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage
AMPK
-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as a result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells.
...
PMID:DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism. 3198 24
Hydrogen peroxide
(H
2
O
2
) modulates critical phosphorylation pathways in vascular endothelial cells, many of which affect endothelial nitric oxide synthase (eNOS) signal transduction. Both intracellular and extracellular sources of H
2
O
2
have been implicated in eNOS regulation, yet the specific endothelial pathways remain incompletely understood. Here we exploited chemogenetic approaches and live-cell imaging methods to both generate and detect H
2
O
2
in different subcellular compartments (cytosol, nucleus, and caveolae) of cultured EA.hy926 human endothelial cells. We developed novel recombinant constructs encoding differentially-targeted yeast d-amino acid oxidase (DAAO), which generates H
2
O
2
only when its d-amino acid substrate is provided. DAAO was expressed as a fusion protein with the new H
2
O
2
biosensor HyPer7.2, which allowed us to quantitate intracellular H
2
O
2
levels by ratiometric imaging in living endothelial cells following the activation of DAAO by d-alanine. The addition of extracellular H
2
O
2
to the HyPer-DAAO-transfected cells led to increases in H
2
O
2
throughout different regions of the cell, as measured using the differentially-targeted HyPer biosensor for H
2
O
2
. The sensor response to extracellular H
2
O
2
was more rapid than that quantitated following the addition of d-alanine to transfected cells to activate differentially-targeted DAAO. The maximal intracellular levels of H
2
O
2
observed in response to the addition of extracellular H
2
O
2
vs. intracellular (DAAO-generated) H
2
O
2
were quantitatively similar. Despite these similarities in the measured levels of intracellular H
2
O
2
, we observed a remarkable quantitative difference in the activation of endothelial phosphorylation pathways between chemogenetically-generated intracellular H
2
O
2
and the phosphorylation responses elicited by the addition of extracellular H
2
O
2
to the cells. Addition of extracellular H
2
O
2
had only a nominal effect on phosphorylation of eNOS, kinase Akt or
AMP-activated protein kinase
(
AMPK
). By contrast, intracellular H
2
O
2
generation by DAAO caused striking increases in the phosphorylation of these same key signaling proteins. We also found that the
AMPK
inhibitor Compound C completely blocked nuclear H
2
O
2
-promoted eNOS phosphorylation. However, Compound C had no effect on eNOS phosphorylation following H
2
O
2
generation from cytosol- or caveolae-targeted DAAO. We conclude that H
2
O
2
generated in the cell nucleus activates
AMPK
, leading to eNOS phosphorylation; in contrast,
AMPK
activation by cytosol- or caveolae-derived H
2
O
2
does not promote eNOS phosphorylation via
AMPK
. These findings indicate that H
2
O
2
generated in different subcellular compartments differentially modulates endothelial cell phosphorylation pathways, and suggest that dynamic subcellular localization of oxidants may modulate signaling responses in endothelial cells.
...
PMID:Differential endothelial signaling responses elicited by chemogenetic H
2
O
2
synthesis. 3259 Mar 30
Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyze the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using WRL68 (HeLa derivate) cells, we previously showed that PARP-1 activation induced by oxidative stress after
H2O2
treatment lead to depletion of cellular NAD+ and ATP, which promoted cell death. In this work, LC-MS/MS-based phosphoproteomics in WRL68 cells showed that the oxidative damage induced by
H2O2
increased the phosphorylation of YAP1, a transcriptional co-activator involved in cell survival, and modified the phosphorylation of other proteins involved in transcription. Genetic or pharmacological inhibition of PARP-1 in
H2O2
-treated cells reduced YAP1 phosphorylation and degradation and increased cell viability. YAP1 silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells exposed to oxidative damage. Supplementation of NAD+ also reduced YAP1 phosphorylation, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of the metabolic sensor
AMPK
. Since NAD+ supplementation reduced the phosphorylation of some
AMPK
substrates, we hypothesized that the loss of cellular NAD+ after PARP-1 activation may induce an energy stress that activates
AMPK
. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in which PARP-1 activation reduced cell viability by promoting the phosphorylation and degradation of YAP1 through a mechanism that involves the depletion of NAD+.
...
PMID:PARP-1 activation after oxidative insult promotes energy stress-dependent phosphorylation of YAP1 and reduces cell viability. 3314 86
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