We have previously demonstrated that DHA promotes neuronal survival under adverse conditions by increasing membrane phosphatidylserine (PS) content and facilitating Akt signaling through its specific interaction with membrane PS. We found a novel molecular mechanism for Akt activation where Akt interaction with PS-binding residues, particularly in the regulatory domain, is critical for Akt phosphorylation at S473 by mTORC2. Since phosphorylation at S473 is an important modulatot for Akt activation, S473 phosphorylation can serve as a new target for drug development which is different from well-recognized PIP3- or ATP-binding site. As an effort to identify selective Akt inhibitors based on this new target, we developed a cell-based high throughput screening (HTS) assay using homogeneous time-resolved fluorescence (HTRF) detection. We subsequently tested approximately 400,000 MLSCN (Molecular Libraries Screening Center Network) compound collections in collaboration with NCATS. Among approximately 1800 compounds screened as positive hits, tertiary assays were performed for about 100 selected compounds during this period. Among high probability hits from a automated HTRF screen, only compounds that showed substantial and reproducible inhibition of Akt S473 phosphorylation confirmed by western blot analysis, without an effect on PI3K, PDK1, and SGK1 were selected. This endeavor enabled us to narrow down the list of potential interest to four which were found to share a common structural moiety. Further testing is in progress to determine the best candidate for lead optimization. The small molecules that inhibit specifically Akt S473 phosphorylation/mTORC2 activity thus identified will not only serve as valuable research tools but also may have significant therapeutic potential with fewer side effects, especially for conditions involving hyperactive Akt signaling such as cancer and Alzheimers disease. As PS accumulation is an important aspect of DHA- or ethanol-mediated modulation of Akt signaling, we continued to investigate the characteristics of neuronal PS synthesis. While it is postulated that the conformational status of PS synthases (PSS) is intimately coupled to its activity, the crystal structure of this integral membrane protein is not available at present. To provide molecular insight for the observed functional selectivity of PSS towards DHA-containing phospholipids, we probed the conformation of purified PSS2 by an integrated approach combining chemical cross-linking and quantitative mass spectrometry developed in our laboratory. Immunopurified PSS2 was incubated with disuccinimidyl suberate (DSS), a lysine specific cross-linker. The monomeric cross-linked PSS2 was isolated with SDS-PAGE and subjected to tryptic digestion and nanoLC-ESI-MS/MS analysis. The cross-linked peptides were identified by xQuest software based on accurate molecular weight and fragmentation pattern in the MS/MS spectra. Changes in the intensity of cross-linked peptides derived from PSS2 in different membrane matrices were quantified by label-free protein quantitation. Our data revealed the proximity of five lysine pairs in PSS structure, providing for the first time the experimental evidence on the three-dimensional structure of this integral membrane protein. Furthermore, we found that PSS2 in the DHA-rich matrix has a more folded structure between the putative TM5 and TM6 regions in comparison to the monounsaturated lipid matrix while exhibiting higher catalytic activity. This finding may provide a molecular basis of the DHA-dependent substrate specificity of PSS2 for controlling membrane production of PS, an important modulator of many signaling pathways such as PI3K/Akt. We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant mechanism for hippocampal neuritogenesis, synaptogensis and glutamatergic synaptic activity. During this period, we investigated signaling mechanisms for the synaptamide effects. We found that synaptamide induces cAMP accumulation, CREB phosphorylation, transcriptional activity of CRE and neurite growth in cortical neurons at low nM concentrations. The synaptamide-induced neurite growth in cortical neurons as well as neurogenic differentiation of NSCs was abolished when treated with an adenyl cyclase (AC) inhibitor, SQ22,536. These data suggested that a G protein-coupled receptor (GPCR) mediates the observed synaptamide-induced bioactivities. Subsequent investigation on the effects of ethanol on synaptamide-induced signaling processes leading to neurite growth and neurogenic differentiation is in progress. We continued to develop an approach for identification of novel metabolites derived from uniformly 13C-labeled substrates using HPLC/ESI-MS/MS and software-assisted peak finding routine. Using platelet and leukocyte metabolites formed from 13C20-arachidonic acid (AA) and DHA as model systems. Based on the unique isotope profile of 13C20-AA- or 13C22-DHA-derived metabolites together with the predicted mass differences from the corresponding 13C20-AA- or 13C22-DHA-counterparts, an automatic peak finding algorithm has been developed. This approach allowed simultaneous identification and relative quantification of novel metabolites under differing biological conditions. We have validated the method using phospholipid extracts from rat NSC cultures that were incubated with a mixture of unlabeled and labeled DHA (13C22-DHA) under conditions of either proliferation or differentiation. Automated analysis of negative ion mode MS data revealed that at least 59 unique DHA-derived phospholipid compounds were differentially produced in the NSC cultures under these conditions. We will subsequently use this method to search for biologically active metabolites of DHA and synaptamide. This metabolite search strategy will also be extended to other classes of bioactive compounds for which uniformly labeled 13C-isotopes are available. During this review period, we have developed a novel two-step chemical cross-linking strategy for the identification of Akt-interacting proteins by co-IP coupled to tandem mass spectrometry. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, a cleavable crosslinker, dithiobissuccinimidylpropionate, was introduced to produce stable complexes between Akt and binding partners before SDS-PAGE and nanoLC-MS/MS analysis. This two-step cross-linking strategy successfully overcomes the loss of interacting proteins and antibody contamination, both of which are problematic in conventional co-IP/MS methods, thus dramatically improving the identification of Akt partners. The simple yet effective method can be applied to other protein-protein interactions, particularly for identifying low abundant or weakly bound interacting proteins, or when the quantity of biological samples is limited.
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