Dataset Information

Effects of ethanol on conformational changes of Akt studied by chemical cross-linking, mass spectrometry, and (18)O labeling.

ABSTRACT: Although PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system, underlying molecular mechanisms have not been fully elucidated. Akt-membrane interaction is a prerequisite step for Akt activation since it induces interdomain conformational changes to an open conformer that allows Akt phosphorylation by upstream kinases. In this study, we investigated the effect of ethanol on Akt activation by quantitatively probing Akt conformation using chemical cross-linking, (18)O labeling and mass spectrometry. We found that ethanol at pharmacologically relevant concentrations (20 or 170 mM) directly interacts with Akt and alters the local pleckstrin homology domain configuration near the PIP(3)-binding site. We also found that ethanol significantly impairs subsequent membrane-induced interdomain conformational changes needed for Akt activation. The observed alteration of Akt conformation caused by ethanol during the activation sequence provides a new molecular basis for the effects of ethanol on Akt signaling. The in vitro conformation-based approach employed in this study should also be useful in probing the molecular mechanisms for the action of ethanol or drugs on other signaling proteins, particularly for those undergoing dramatic conformational change during activation processes such as members of AGC kinase super family.

SUBMITTER: Huang BX

PROVIDER: S-EPMC3475205 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effects of ethanol on conformational changes of Akt studied by chemical cross-linking, mass spectrometry, and (18)O labeling.

Huang Bill X BX Kim Hee-Yong HY

ACS chemical biology 20111207 2

Although PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system, underlying molecular mechanisms have not been fully elucidated. Akt-membrane interaction is a prerequisite step for Akt activation since it induces interdomain conformational changes to an open conformer that allows Akt phosphorylation by upstream kinases. In this study, we investigated the effect of ethanol on Akt activation by quantitatively prob ...[more]

PMID: 22129086

Similar Datasets

Project description:Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze-thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical isotope labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray for analysis. As a proof-of-principle, we used dansylation labeling to analyze the amine- and phenol-submetabolomes in two sets of exosome samples isolated from the blood samples of five pancreatic cancer patients before and after chemotherapy treatment. The average number of peak pairs or metabolites detected was 1964 ± 60 per sample for a total of 2446 peak pairs ( n = 10) in the first set and 1948 ± 117 per sample for a total of 2511 peak pairs ( n = 10) in the second set. There were 101 and 94 metabolites positively identified in the first and second set, respectively, and 1580 and 1590 peak pairs with accurate masses matching those of metabolites in the MyCompoundID metabolome database. Analyzing the mixtures of 12C-labeled individual exosome samples spiked with a 13C-labeled pooled sample which served as an internal standard allowed relative quantification of metabolomic changes of exosomes of blood samples collected before and after treatment.

Dataset Information

Effects of ethanol on conformational changes of Akt studied by chemical cross-linking, mass spectrometry, and (18)O labeling.

Publications

Effects of ethanol on conformational changes of Akt studied by chemical cross-linking, mass spectrometry, and (18)O labeling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets