Validation of monomer and oligomer preference of αSN interacting proteins.

<p>Proteins pulled down by monomer αSN (M), oligomer αSN (O), and buffer control (B) from porcine (A-F) and human (G-I) brain extracts were analyzed by immunoblotting using antibodies against antigens selected among the monomer and oligomer binding proteins. Monomer binding antigens were myelin proteolipid protein (mPLP) and Abl interactor 1 (Abl1) and oligomer binding proteins were glial fibrillary acidic protein (GFAP), glutamate decarboxylase 2 (GAD2), and synapsin 1 (Syn1). VAMP-2 was tested because it has been reported to bind αSN, although it was not detected in our proteomic analysis. One representative of three experiments is presented for porcine αSN binding proteins (A, C, E), and the quantification of the three experiments is presented in panels B, D, F. The quantification of bands was performed after subtracting the non-specific signal in the buffer control from the specific bands in monomer and oligomer samples. Bars represent mean ratio between monomer and oligomer ± S.D. of the three replicates. The values for binding to monomer and oligomer were compared by Student’s t-test and the resulting p-values are listed above the bars. * Indicates that the band intensity from oligomer did not differ significantly from background making quantifications impracticable. In order to ensure that the interaction were not due to species differences between human and porcine proteins we conducted validations in human brain extracts. One representative of two experiments is presented for each validated protein. The validation for both porcine and human of mPLP, Abl1, Syn1 and VAMP-2 was conducted in the LP2 fraction enriched in synaptic vesicle and the validation to GFAP and GAD2 in the LS1 fraction of synaptosomal lysate.</p

Possible molecular pathways initiated by αSN in disease.

<p>Under normal conditions αSN is predominantly located in nerve terminals (blue). During disease αSN undergo aggregation and this lead to novel conformation-dependent interactions (red), which represents a gain of function. In addition, αSN species (monomeric and oligomeric) are concentrated at abnormal sites, like axons and the cell body, or in astrocytes and oligodendrocytes, which give rise to novel interactions because new partners are introduced (green). Finally, an abnormal sorting and aggregation leads to a loss of, or reduced normal αSN concentration in nerve terminals where critical monomer specific interactions will be compromised thus representing a loss of function.</p

Subcellular localization of the αSN interacting proteins.

<p>A total of 178 proteins were identified as αSN interacting proteins and they were grouped in proteins preferentially binding monomer αSN (MP, N = 10, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116473#pone.0116473.t001" target="_blank">Table 1</a>), oligomers (OP, N = 76, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116473#pone.0116473.t002" target="_blank">Table 2</a>) and proteins not displaying any preferences (NPB, N = 92, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116473#pone.0116473.t003" target="_blank">Table 3</a>). They were grouped based on their subcellular localization as described by their principal localization in the Uniprot database to demonstrate the aggregation state of αSN have potential for significantly changing its cellular targets.</p

Preferential αSN monomer interacting proteins.

<p>Preferential αSN monomer interacting proteins.</p