Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by

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 ␣-synuclein-and 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.

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Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson’s disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.

Background: SMURF1 ubiquitin ligase controls ubiquitination and stability of diverse cellular protein substrates. Results: Deubiquitinase USP9X interacts with SMURF1 and stabilizes SMURF1 through deubiquitination. Conclusion: USP9X is novel regulator of SMURF1 and is required for SMURF1-dependent cellular physiology. Significance: Association between deubiquitinase and ubiquitin ligase may serve as a common strategy to control the cellular protein dynamics through modulating ubiquitin ligase activity.