An In Vitro Study Of Interaction Between α-synuclein And Mortalin: A Stress Protein Relevance With Parkinson's Disease

BackgroundParkinson's disease(PD),one of the most common neurodegenerative diseases, is characterized pathologically by the relatively preferential loss of dopaminergic neurons with resultant depletion of striatal dopamine and presence of Lewy bodies mainly composed by alpha-synuclein(α-SYN)in the remaining neurons in the substantia nigra.A lot of evidence suggests that the aggregation ofα-SYN play an essential role in the pathogenesis of PD and formation of Lewy body.DJ-1,another key protein involving in the PD pathogenesis,has been found to play a role in the inhibition of theα-SYN aggregation.However,it is still controversial whether there is directe interaction betweenα-SYN and DJ-1.Mortalin,a mitochondrial stress protein,is identified to be associated both withα-synuclein and DJ-1 by using a quantitative approach.It is demonstrated that mortalin decreased in the mitochondria-enriched fraction of PD nigral tissue as compared to controls,while increased in cytosol-enriched fraction.Overexpression of mortalin rendered DAergic cells more vulnerable to rotenone-induced neurotoxicity (Rotenone is a widely used parkinsonian toxicant which is a potent mitochondrial inhibitor),whereas inhibition of mortalin expression with siRNA led to opposite effects,suggesting that mortalin is a critical mediator of neurotoxicity induced by rotenone.Moreover,a latest study illustrated that mortalin may be involed in the progression of Lewy body.Although all of these results suggest that mortalin may play a role in the PD pathogenesis,the mechanisms of mortalin in the PD pathogenesis remain elusive.Objectives(1)To study whether mortalin interacts withα-SYN in vitro.(2)To investigate whether mutantα-SYN(A53T and A30P)have an effect on the interaction between mortalin andα-SYN in vitro.Methods(1)Construct plasmids of WT and mutantα-SYN(A53T and A30P)expressing GST fusion proteins.(2)Express and purify GST fusion proteins with GST beads.(3)Investigate the interaction betweenα-SYN and mortalin by GST Pull-down assay.ResultsPartⅠMortalin was precipitated by GST -WTα-SYNPartⅡMortalin was precipitated by GST -mutantα-SYN(A53T and A30P) while the amount of precipitated mortalin is much less than that by GST -WTα-SYN.ConclusionPartⅠMortalin is interacted with WTα-SYN in vitro evaluated by GST Pull-down assay.PartⅡMutation ofα-SYN(A53T and A30P)interferes the interaction between mortalin andα-SYN in vitro.