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.
Mesh Terms:
Animals, Blotting, Western, Brain, Electrophoresis, Gel, Two-Dimensional, Humans, Immunohistochemistry, Immunoprecipitation, Mice, Mice, Transgenic, Microscopy, Atomic Force, Parkinson Disease, Protein Array Analysis, Protein Conformation, Protein Folding, Recombinant Proteins, alpha-Synuclein, cdc42 GTP-Binding Protein
Animals, Blotting, Western, Brain, Electrophoresis, Gel, Two-Dimensional, Humans, Immunohistochemistry, Immunoprecipitation, Mice, Mice, Transgenic, Microscopy, Atomic Force, Parkinson Disease, Protein Array Analysis, Protein Conformation, Protein Folding, Recombinant Proteins, alpha-Synuclein, cdc42 GTP-Binding Protein
Neuroscience
Date: Jul. 17, 2008
PubMed ID: 18541383
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