Identification of Novel Proteins Associated with Both -Synuclein and DJ-1*□S

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that -synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether -synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with -synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the -synucleinand DJ-1-associated protein complexes, respectively. Among -synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between -synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both -synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with -synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both -synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between -synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both -synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1 modulates the function of -synuclein, and vice versa, in the setting of PD. Molecular & Cellular Proteomics 6:845–859, 2007.