Study On Proteomic Pattern Of Molecular Chaperones Identified From Sporadic Parkinson’s Disease Model In SH-SY5Y Cell Under Condition Of Proteasome Inhibition

Background:Molecular chaperones found to be up-regulated in the ubiquitin-proteasome system (UPS) impairment as one of pathomechanistic sporadic Parkinson’s disease (sPD) may protect against protein degradation stress response. Objective:To reveal in vitro pattern of chaperone proteins expressed during UPS impairment in the pathogenesis of sporadic Parkinson’s disease (sPD), a proteomic analysis of molecular chaperone was made from sPD model in SH-SY5Y cell under condition of proteasome inhibition. Methods:SH-SY5Y cell was treated with trans retinoic acid(10 μmol/L) and tetradecanoylphorbol-13-acetate (80 nmol/L) for 72 hours in succession, thus inducing neuron-like differentiation and phenotype. Differentiated cell (control) was treated with proteasome inhibitor (10 μmol/L) for 24 hours, thus establishing sPD model in SH-SY5Y cell under condition of proteasome inhibition (experimental group). Two-dimensional difference in-gel electrophoresis was used to obtain 106 protein spots expressed dimensionally between control and experimental group, among which differential expression of 17 protein spots was shown most obviously and appointed as target proteins. The targets were only obtained through preparative gel of experimental group. Matrix assisted laser desorption ionization-time of flight mass spectrometry was used to obtain peptide mass fingerprinting of trypsin products of the targets from protein digestion. Candidate identification via the raw data was initially accomplished against SwissProt database and further enhanced against NCBInr database. Candidates were generalized both with biological function and as categories, based on protein bioinformatics datasets of homologous protein. One of the candidates was validated at the level of gene transcript throuth relative quantitative determination. Results:First of all,17 candidates were identified as follows:27-kDa heat shock protein (HSP-27),32-kDa heat shock protein (HSP-32),58-kDa glucose regulated protein (GRP-58),70-kDa heat shock protein (HSP-70),70-kDa heat shock cognate protein (HSC-70),75-kDa glucose regulated protein (GRP-75),105-kDa heat shock protein (HSP-105),150-kDa oxygen regulated protein (ORP-150),calcium-binding protein 1 (CaBP-1),CBP-50 protein (CBP-50),60-kDa mitochondrial heat shock protein 60 (mHSP-60),prolyl-4-hydroxylase beta polypeptide (P4HB),stress induced phosphoprotein-1或stress inducible phosphoprotein-1 (STIP-1), 14-3-3zeta (14-3-3ξ),T-complex polypeptide 1 beta subunit (TCP-1β),T-complex polypeptide 1 epsilon subunit (TCP-1ε),Valosin-containing protein (VCP). Furthermore, they were generalized as 4 categories of molecular chaperones as follows:HSP-27、HSP-32、HSP-70、HSC-70、HSP-105、GRP-58、GRP-75、ORP150and VCP are ascribed to chaperon proteins; P4HB、14-3-3ξ、CaBP-1and CBP-50 are ascribed to chaperon-like protein; STIP-1 is ascribed to co-chaperone protein;HSP-60、TCP-1β、�'� TCP-1εare ascribed to chaperonins. Conclusion:It is suggested that 17 molecular chaperones might incorporated into protein degredation stress response during UPS pathway in the pathogenesis of sPD.