Functional proteomics analysis of non-vesicular release mechanisms in stress exposed neuron-role of stress-induced non-vesicular release Prion, NDI and FGF-1 in neuron

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The purpose of this research is to demonstrate the stress-induced mechanism of non-vesicular release FGF-1, NDI and STI, as prion (PrP^c) associated protein using the proteomics analyses. We attempted to characterize the non-vesicular mode of FGF-1 and NDI release in the analyses using immunocytochemistry and immunoblot of conditioned medium (CM) from astrocytes. FGF-1 was completely released from astrocytes upon serum-deprivation stress in a Brefeldin A-insensitive manner. In the immunoprecipitation study using anti-FGF-1 IgG, S100A13 was identified to be the major protein co-eluted with FGF-1. The interaction between GST-FGF-1 and Strep-tag II S100A13 was found to be Ca^<2+>-sensitive, and to require the C-terminal 11 amino acid peptide sequence of S100A13. The overexpression of D88-98 mutant of S100A13 selectively inhibited the serum-deprivation stress-induced release of FGF-1, but not the release of S100A13 mutant from C6 glioma cells. However, amlexanox, anti-allergic drug whose t … More arget is S100A13, completely inhibited the stress-induced release of FGF-1 as well as S100A13. The stress-induced release of both proteins was also abolished by BAPTA-AM, an intracellular Ca^<2+> chelating agent. The serum-deprivation caused Ca^<2+> spikes in v-conotoxin GVIA and thapsigargin-sensitive manner. These same results observed NDI protein experiments. These results suggest that S100A13 is a cargo molecule for the serum-deprivation stress-induced non-vesicular release of FGF-1 and NDI, and that its driving force of protein-protein interaction and release is possibly mediated by Ca^<2+>-induced Ca^<2+> release (CICR) coupled to N-type Ca^<2+> channel activity. On the other hand, STI have neuronal survival activity. STI inhibitor, as a PrP^c c terminal peptide (113-132), treated neuron was enhanced decrease in survival activity under the oxygen-glucose deprivation. Moreover, STI inhibitor induced neuronal cell death under the serum containing normal condition. These results suggested that PrP^c have increase in neuronal survival activity under the normal condition. Less