Subcellular Proteomics Of PSI-induced Inclusions Isolated From PC12 Cells

Background: Lewy bodies (LBs), the spherical, intracytoplasmic inclusions which are eosinophilic and alpha-synuclein (alpha-SYN) immunostaining-positive, are localized in neuronal perikaryon, and are described as pathological hallmark of dopaminergic neurons degenerating in substantia nigra pars compacta (SNpc) of Parkinson's disease (PD) patients. Constitutive protein feature of LBs is mainly characterized by multiple protein content which has also been detected in LB-like inclusions in vitro cellular PD models. In the development of LBs, chaperone proteins which are involved in protein folding and transport are significant but their profile is incompletely unraveled. One strategy for better understanding protein content of LBs is proteomic investigation of LBs. Aim: Here, to characterize constitutive protein feature of artificially synthesized proteasome inhibitor (PSI)-induced inclusions and thereby provide with new clues of alternative and potential candidates of protein component of LBs for further investigation, we performed a proteomic analysis of PSI-induced inclusions. Methods: Mainly using the approaches of subcellular biochemical fractionation, two-dimensional electrophoresis (2-D electrophoresis) and protein identification via peptide mass fingerprints (PMF), we were able to isolate pure intact PSI-induced inclusions from PC12 cells exposed to PSI (10μmol/L) for 48 hours (h), and then established the proteomic profile of these intermediate organelles. Results: 1, Of the 56 identified proteins, first of all, 28 have not been reported as protein components of LBs, 20 have been reported as protein components of LBs, and 8 are putative novel proteins based on the up to date protein databases. 2 Furthermore, the 10 newly identified chaperone proteins and (or) chaperone-like proteins in the PSI-induced inclusions were found including 58 kDa glucose regulated protein(GRP58), 75 kDa glucose regulated protein (GRP75), 105 kDa heat shock protein 1 (HSP105), 150 kDa oxygen regulated protein (ORP150), calcium-binding protein 1 (CaBP1), CBP-50 protein (CBP-50), mitochondrial matrix protein P1(or HSP60), prolyl-4-hydroxylase beta polypeptide (P4HB), stress-induced- phosphoprotein 1 (STI1), and T-complex polypeptide 1 (TCP-1) epsilon subunit (TCP-1 epsilon). P4HB, a member of the protein disulfide isomerase (PDI) family, was validated by immunostaining method. Conclusion: At this stage of investigation, these findings suggest that P4HB and the other proteins may participate in the formation of PSI-induced inclusions when proteasome inhibition occurs to PC12 cells. In addition, P4HB and the other newly identified chaperone proteins and (or) chaperone-like proteins maybe provide with useful clues to revealing new candidates of protein component of LBs for further investigation.