Protein array analysis of oligomerization-induced changes in alpha-synuclein protein-protein interactions points to an interference with Cdc42 effector proteins.
Aggregation of alpha-synuclein may contribute to neuropathology in Parkinson's disease patients and in transgenic animal models. Natively unfolded alpha-synuclein binds to various proteins and conformational changes due to alpha-synuclein misfolding may alter physiological interactions. In the present study, we used protein arrays spotted with 5000 recombinant human proteins for a large scale interaction analysis of monomeric versus oligomeric alpha-synuclein. Monomeric alpha-synuclein bound to arrayed cAMP regulated phosphoprotein 19 and binding appears to be disrupted by alpha-synuclein oligomerization. Incubation with recombinant alpha-synuclein oligomers lead to the identification of several GTPase activating proteins and Cdc42 effector proteins as binding partners. Protein database searches revealed a Cdc42/Rac interactive binding domain in some interactors. To demonstrate in vivo relevance, we analyzed brainstem protein extracts from alpha-synuclein(A30P) transgenic mice. Pull-down assays using beads conjugated with a Cdc42/Rac interactive binding domain lead to an enrichment of endogenous alpha-synuclein oligomers. Cdc42 effector proteins were also co-immunoprecipitated with alpha-synuclein from brainstem lysates and were colocalized with alpha-synuclein aggregates in brain sections by double immunostaining. By two-dimensional gel electrophoretic analysis of synaptosomal fractions from transgenic mouse brains we detected additional isoforms of septin 6, a downstream target of Cdc42 effector proteins. Small GTPases have recently been identified in a genetic modifier screen to suppress alpha-synuclein toxicity in yeast. Our data indicate that components of small GTPase signal transduction pathways may be directly targeted by alpha-synuclein oligomers which potentially leads to signaling deficits and neurodegeneration.
Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riss, Germany.
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Schnack
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Danzer
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Hengerer
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Gillardon
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D008822:Mice, Transgenic; D018625:Microscopy, Atomic Force; D010300:Parkinson Disease / Q000378:metabolism / Q000473:pathology; D040081:Protein Array Analysis; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry* / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D051844:alpha-Synuclein; D020764:cdc42 GTP-Binding Protein