VI Membrane targets for 15-deoxy Δ^12,14-Prostaglandin J₂
[³H]15d-PGJ₂ bound specifically to plasma membranes of cortical neurons  (Figure 13). Among PGD₂ metabolites, 15d-PGJ₂ exhibited the highest affinity for the specific binding sites (Figure 14). Other eicosanoids and PPAR agonists did not affect the specific binding sites. The affinities of 15d-PGJ₂–related compounds for SBJ was 15d-PGJ₂ > Δ¹²-PGJ₂ > PGJ₂ > PGA₂ >> PGD₂ > 15d-PGD₂ in sequence, indicating the apoptotic effect of 15d-PGJ₂-related compounds was correlated to their affinities for SBJ, but not to those for DP1 or DP2 (Table 3).
A proteomic approach by using biotinylated 15d-PGJ₂ was used to identify protein targets for 15d-PGJ₂ in the plasma membrane. In a similar fashion to 15d-PGJ₂, biotinylated 15d-PGJ₂ also induced neuronal cell death in a concentration- dependent manner (Figure 15). Biotinylated 15d-PGJ₂-modified proteins were western blotted with anti-biotin antibody. We have succeeded in indentifying 22 spots (Figure 16). Spot 8 corresponding to a 50 kDa 15d-PGJ₂-protein conjugate was one of the targets of the modification by biotinylated 15d-PGJ₂ (Figure 17). Using MASCOT, the probability based MOWSE score was 267 for GFAP (p < 0.05), with 28 peptide matches (error ± 0.02%), which represents 56% sequence coverage (Figure 18). Table 4 lists the identity of 22 protein spots, which could be identified in three independent experiments.
Eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). These proteins include factors associated with the two remarks of AD, amyloid plaque and neurofibrillary tangle (Figure 19). For example, amyloid plaque interacts with GAPDH and tubulin β. Amyloid precursor protein interacts with GAPDH and HSPa8. , fAβ interacts with PKM1/M2, Enolase2 and GAPDH. Beyond classical roles as glycolytic enzymes and molecular chaperones, Enolase2, GAPDH and Hspa8 might form the complex of trans-plasma-membrane oxidoreductases; (PMOs) and can contribute to the generation of reacting oxygen species and neuronal apoptosis.