title: Identification of novel proteins associated with both alpha-synuclein and DJ-1.
abstract: The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that alpha-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether alpha-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with alpha-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 alpha-synuclein- and DJ-1-associated protein complexes, respectively. Among alpha-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 alpha-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 alpha-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with alpha-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 alpha-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between alpha-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both alpha-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 alpha-synuclein, and vice versa, in the setting of PD.
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington 98104, USA.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Jin
authors.forename: G Jane
authors.initials: GJ
authors.lastname: Li
authors.forename: Jeanne
authors.initials: J
authors.lastname: Davis
authors.forename: David
authors.initials: D
authors.lastname: Zhu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Catherine
authors.initials: C
authors.lastname: Pan
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D010300:Parkinson Disease / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D051381:Rats; D012402:Rotenone; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D051844:alpha-Synuclein; D012402:Rotenone