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

      The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that α-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether α-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with α-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 α-synuclein- and DJ-1-associated protein complexes, respectively. Among α-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 α-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 α-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with α-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 α-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between α-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both α-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 α-synuclein, and vice versa, in the setting of PD.
      Parkinson disease (PD),
      The abbreviations used are: PD, Parkinson disease; AD, Alzheimer disease; SILAC, stable isotope labeling by amino acids in cell culture; MES, rat mesencephalic neuronal cell line; ER, endoplasmic reticulum; IP, immunoprecipitation; WB, Western blot; RP, reverse-phase; SCX, strong cation-exchange; PSF, pyrimidine tract-binding protein-associated splicing factor; ASAPRatio, automated statistical analysis of protein ratio.
      1The abbreviations used are: PD, Parkinson disease; AD, Alzheimer disease; SILAC, stable isotope labeling by amino acids in cell culture; MES, rat mesencephalic neuronal cell line; ER, endoplasmic reticulum; IP, immunoprecipitation; WB, Western blot; RP, reverse-phase; SCX, strong cation-exchange; PSF, pyrimidine tract-binding protein-associated splicing factor; ASAPRatio, automated statistical analysis of protein ratio.
      the second most common neurodegenerative disorder after Alzheimer disease (AD), presents with bradykinesia, resting tremor, rigidity, and postural instability (
      • Stacy M.
      Managing late complications of Parkinson’s disease.
      ). Pathological hallmarks of PD include relatively selective loss of brainstem neurons, including dopaminergic neurons in the substantia nigra pars compacta, and formation of round eosinophilic intracytoplasmic proteinaceous inclusions called Lewy bodies in surviving neurons and dystrophic Lewy neurites in the neuropil (
      • Forno L.S.
      • DeLanney L.E.
      • Irwin I.
      • Langston J.W.
      Electron microscopy of Lewy bodies in the amygdala-parahippocampal region. Comparison with inclusion bodies in the MPTP-treated squirrel monkey.
      ).
      Much of what has been learned about PD over the past 2 decades has come about through the study of a protein called α-synuclein whose mutations, duplications, and triplications have been associated with early onset of autosomal dominant familial PD (
      • Trojanowski J.Q.
      • Lee V.M.
      Aggregation of neurofilament and α-synuclein proteins in Lewy bodies: implications for the pathogenesis of Parkinson disease and Lewy body dementia.
      ,
      • Polymeropoulos M.H.
      • Lavedan C.
      • Leroy E.
      • Ide S.E.
      • Dehejia A.
      • Dutra A.
      • Pike B.
      • Root H.
      • Rubenstein J.
      • Boyer R.
      • Stenroos E.S.
      • Chandrasekharappa S.
      • Athanassiadou A.
      • Papapetropoulos T.
      • Johnson W.G.
      • Lazzarini A.M.
      • Duvoisin R.C.
      • Di I.G.
      • Golbe L.I.
      • Nussbaum R.L.
      Mutation in the α-synuclein gene identified in families with Parkinson’s disease.
      ,
      • Kruger R.
      • Kuhn W.
      • Muller T.
      • Woitalla D.
      • Graeber M.
      • Kosel S.
      • Przuntek H.
      • Epplen J.T.
      • Schols L.
      • Riess O.
      Ala30Pro mutation in the gene encoding α-synuclein in Parkinson’s disease.
      ). What is more important is the realization that aggregation of α-synuclein plays critical roles in sporadic PD (
      • Trojanowski J.Q.
      • Lee V.M.
      Aggregation of neurofilament and α-synuclein proteins in Lewy bodies: implications for the pathogenesis of Parkinson disease and Lewy body dementia.
      ,
      • El-Agnaf O.M.
      • Salem S.A.
      • Paleologou K.E.
      • Curran M.D.
      • Gibson M.J.
      • Court J.A.
      • Schlossmacher M.G.
      • Allsop D.
      Detection of oligomeric forms of α-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease.
      ,
      • Berg D.
      • Niwar M.
      • Maass S.
      • Zimprich A.
      • Moller J.C.
      • Wuellner U.
      • Schmitz-Hubsch T.
      • Klein C.
      • Tan E.K.
      • Schols L.
      • Marsh L.
      • Dawson T.M.
      • Janetzky B.
      • Muller T.
      • Woitalla D.
      • Kostic V.
      • Pramstaller P.P.
      • Oertel W.H.
      • Bauer P.
      • Krueger R.
      • Gasser T.
      • Riess O.
      α-Synuclein and Parkinson’s disease: implications from the screening of more than 1,900 patients.
      ). The precise mechanisms by which α-synuclein mediates PD development remain to be defined, although it appears that the following processes are involved at least partially: increased oxidative stress, mitochondrial dysfunction, and abnormal protein aggregation (α-synuclein in particular) as well as failure of the ubiquitin-proteasome system and lysosomal system (
      • Gandhi S.
      • Wood N.W.
      Molecular pathogenesis of Parkinson’s disease.
      ,
      • Hsu L.J.
      • Sagara Y.
      • Arroyo A.
      • Rockenstein E.
      • Sisk A.
      • Mallory M.
      • Wong J.
      • Takenouchi T.
      • Hashimoto M.
      • Masliah E.
      α-Synuclein promotes mitochondrial deficit and oxidative stress.
      ).
      DJ-1 is a relatively new protein that has been linked to autosomal recessive familial PD (
      • Bonifati V.
      • Rizzu P.
      • Squitieri F.
      • Krieger E.
      • Vanacore N.
      • van Swieten J.C.
      • Brice A.
      • van Duijn C.M.
      • Oostra B.
      • Meco G.
      • Heutink P.
      DJ-1(PARK7), a novel gene for autosomal recessive, early onset parkinsonism.
      ,
      • Macedo M.G.
      • Anar B.
      • Bronner I.F.
      • Cannella M.
      • Squitieri F.
      • Bonifati V.
      • Hoogeveen A.
      • Heutink P.
      • Rizzu P.
      The DJ-1L166P mutant protein associated with early onset Parkinson’s disease is unstable and forms higher-order protein complexes.
      ). The physiological function of DJ-1 is unclear although increasing evidence suggests that DJ-1 may function as an antioxidative protein or as a sensor of oxidative stress (
      • Mitsumoto A.
      • Nakagawa Y.
      • Takeuchi A.
      • Okawa K.
      • Iwamatsu A.
      • Takanezawa Y.
      Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat.
      ,
      • Kinumi T.
      • Kimata J.
      • Taira T.
      • Ariga H.
      • Niki E.
      Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide-mediated oxidation in vivo in human umbilical vein endothelial cells.
      ,
      • Taira T.
      • Saito Y.
      • Niki T.
      • Iguchi-Ariga S.M.
      • Takahashi K.
      • Ariga H.
      DJ-1 has a role in antioxidative stress to prevent cell death.
      ). DJ-1 has also been reported to confer protection against endoplasmic reticulum (ER) stress, proteasomal inhibition, and toxicity induced by overexpression of Pael-R (
      • Yokota T.
      • Sugawara K.
      • Ito K.
      • Takahashi R.
      • Ariga H.
      • Mizusawa H.
      Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition.
      ). Indeed overexpression of DJ-1 clearly antagonizes much of the deleterious effects of α-synuclein, including α-synuclein aggregation (
      • Shendelman S.
      • Jonason A.
      • Martinat C.
      • Leete T.
      • Abeliovich A.
      DJ-1 is a redox-dependent molecular chaperone that inhibits α-synuclein aggregate formation.
      ,
      • Zhou W.
      • Freed C.R.
      DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T α-synuclein toxicity.
      ,
      • Zhou W.
      • Zhu M.
      • Wilson M.A.
      • Petsko G.A.
      • Fink A.L.
      The oxidation state of DJ-1 regulates its chaperone activity toward α-synuclein.
      ). The fact that DJ-1 modulates multiple cellular insults mediated by α-synuclein has led to investigations on whether DJ-1 directly interacts with α-synuclein (
      • Meulener M.C.
      • Graves C.L.
      • Sampathu D.M.
      • Armstrong-Gold C.E.
      • Bonini N.M.
      • Giasson B.I.
      DJ-1 is present in a large molecular complex in human brain tissue and interacts with α-synuclein.
      ,
      • Betarbet R.
      • Canet-Aviles R.M.
      • Sherer T.B.
      • Mastroberardino P.G.
      • McLendon C.
      • Kim J.H.
      • Lund S.
      • Na H.M.
      • Taylor G.
      • Bence N.F.
      • Kopito R.
      • Seo B.B.
      • Yagi T.
      • Yagi A.
      • Klinefelter G.
      • Cookson M.R.
      • Greenamyre J.T.
      Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, α-synuclein, and the ubiquitin-proteasome system.
      ). Several groups, including us, have demonstrated that DJ-1 is present immunohistologically in the halo part of Lewy bodies, which contain insoluble α-synuclein, in sporadic PD patients (
      • Neumann M.
      • Muller V.
      • Gorner K.
      • Kretzschmar H.A.
      • Haass C.
      • Kahle P.J.
      Pathological properties of the Parkinson’s disease-associated protein DJ-1 in α-synucleinopathies and tauopathies: relevance for multiple system atrophy and Pick’s disease.
      ,
      • Jin J.
      • Meredith G.E.
      • Chen L.
      • Zhou Y.
      • Xu J.
      • Shie F.S.
      • Lockhart P.
      • Zhang J.
      Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson’s disease.
      ). Neumann et al. (
      • Bandopadhyay R.
      • Kingsbury A.E.
      • Cookson M.R.
      • Reid A.R.
      • Evans I.M.
      • Hope A.D.
      • Pittman A.M.
      • Lashley T.
      • Canet-Aviles R.
      • Miller D.W.
      • McLendon C.
      • Strand C.
      • Leonard A.J.
      • Abou-Sleiman P.M.
      • Healy D.G.
      • Ariga H.
      • Wood N.W.
      • de Silva R.
      • Revesz T.
      • Hardy J.A.
      • Lees A.J.
      The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson’s disease.
      ), however, reported negative results. Similar to immunohistochemical studies, the results obtained in direct investigation of the relationship between soluble α-synuclein and DJ-1 are also controversial. Although some reported that DJ-1 could be immunoprecipitated along with soluble α-synuclein (
      • Meulener M.C.
      • Graves C.L.
      • Sampathu D.M.
      • Armstrong-Gold C.E.
      • Bonini N.M.
      • Giasson B.I.
      DJ-1 is present in a large molecular complex in human brain tissue and interacts with α-synuclein.
      ), others have not been able to replicate the result (
      • Zhou W.
      • Zhu M.
      • Wilson M.A.
      • Petsko G.A.
      • Fink A.L.
      The oxidation state of DJ-1 regulates its chaperone activity toward α-synuclein.
      ). In a proteomics study conducted in our laboratory previously, where ∼250 candidate proteins were identified in the soluble α-synuclein protein complex, DJ-1 was not one of them (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ). It should be noted, however, that a negative proteomics study does not necessarily mean that the protein of interest is not present in the protein complex. This is largely because current mass spectrometric methods do not identify all the proteins when the protein mixture is modestly complex, i.e. with great dynamic ranges (
      • Corthals G.L.
      • Wasinger V.C.
      • Hochstrasser D.F.
      • Sanchez J.C.
      The dynamic range of protein expression: a challenge for proteomic research.
      ). In addition, proteins of interest could undergo post-translational modification, thereby making them difficult to be detected by a mass spectrometer. Finally false negative results could result from a typical affinity purification procedure, which is usually designed to minimize false positive results. In other words, it is possible that weak protein-protein interaction, whether directly or indirectly, could disappear secondarily to an artificial biochemical purification of the protein complex.
      The goal of the study was to further investigate proteins potentially associated with soluble α-synuclein, DJ-1, or both with a more advanced proteomics technology called stable isotope labeling by amino acids in cell culture (SILAC). To increase the likelihood of identifying bona fide proteins associated with α-synuclein and/or DJ-1 that likely relate to PD pathogenesis and Lewy body formation, we compared the protein profiles of these two protein complexes in a dopaminergic cell line (MES cells) exposed to rotenone, a pesticide that produces parkinsonism in animals and induces Lewy body-like inclusions in the remaining dopaminergic neurons (
      • Betarbet R.
      • Sherer T.B.
      • MacKenzie G.
      • Garcia-Osuna M.
      • Panov A.V.
      • Greenamyre J.T.
      Chronic systemic pesticide exposure reproduces features of Parkinson’s disease.
      ) versus controls. Among the 324 and 306 proteins identified to be associated with α-synuclein and DJ-1, respectively, 141 and 119 proteins displayed significant changes in relative abundance in α-synuclein and DJ-1 complexes after rotenone treatment. Although we did not observe direct interaction between α-synuclein and DJ-1, either via proteomics analysis or subsequent co-immunoprecipitation (IP) studies, we identified 144 proteins associated with both α-synuclein and DJ-1. Among these common proteins, 114 showed significant changes in relative abundance after rotenone treatment in at least one of the complexes; a subset of these proteins was further validated using alternative means as to their true association with both α-synuclein and DJ-1.

      EXPERIMENTAL PROCEDURES

       Reagents and Antibodies—

      All reagents were purchased from Sigma unless specified. Antibodies used include: α-synuclein for Western blot (WB) (BD Pharmingen), α-synuclein for IP (Cell Signaling Technology, Beverly, MA), α-synuclein for immunofluorescent staining (Chemicon, Temecula, CA), DJ-1 (Novus Biologicals, Littleton, CO), mortalin/grp75 (Stressgen, San Diego, CA), nucleolin (Novus Biologicals), calnexin (BD Pharmingen), grp94 (Stressgen), and clathrin (Chemicon).

       Cell Culture and Treatment—

      A dopaminergic neuron cell line, MES, a gift from Dr. Le at the Baylor College of Medicine, has been widely used in PD-related experiments (
      • Crawford G.C.
      • Le W.
      • Smith R.G.
      • Xie W.J.
      • Stefani E.
      • Appel S.H.
      A novel N18TG2X mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin.
      ,
      • Zhang J.
      • Price J.O.
      • Graham D.G.
      • Montine T.J.
      Secondary excitotoxicity contributes to dopamine-induced apoptosis of dopaminergic neuronal cultures.
      ,
      • Zhang J.
      • Kravtsov V.
      • Amarnath V.
      • Picklo M.J.
      • Graham D.G.
      • Montine T.J.
      Enhancement of dopaminergic neurotoxicity by the mercapturate of dopamine: relevance to Parkinson’s disease.
      ). Detailed methods for culturing MES cells have been described previously by us (
      • Zhang J.
      • Kravtsov V.
      • Amarnath V.
      • Picklo M.J.
      • Graham D.G.
      • Montine T.J.
      Enhancement of dopaminergic neurotoxicity by the mercapturate of dopamine: relevance to Parkinson’s disease.
      ). To identify the proteins specifically associated with α-synuclein and DJ-1, a newly developed quantitative proteomics technique, SILAC, was used (
      • Ong S.E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      ,
      • McLaughlin P.
      • Zhou Y.
      • Ma T.
      • Liu J.
      • Zhang W.
      • Hong J.S.
      • Kovacs M.
      • Zhang J.
      Proteomic analysis of microglial contribution to mouse strain-dependent dopaminergic neurotoxicity.
      ). Two groups of MES cells were grown in identical culture media except for one essential amino acid, l-arginine: the first medium contains the “light” form (l-[12C6]Arg isotope), and the second medium contains the “heavy” form (l-[13C6]Arg isotope; Cambridge Isotope Laboratories, Andover, MA). After being cultured for at least five generations (to achieve near 100% incorporation of arginine (
      • Ong S.E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      )), l-[13C6]Arg- and l-[12C6]Arg-labeled MES cells were treated with 20 nm rotenone or DMSO, respectively, for 3 days.

       Isolation of α-Synuclein- or DJ-1-associated Protein Complex—

      Anti-α-synuclein or -DJ-1 antibodies was covalently bound to an N-hydroxysuccinimide affinity column following the manufacturer's instructions (Amersham Biosciences). SILAC-labeled MES cells (l-[12C6]Arg for DMSO and l-[13C6]Arg for rotenone, respectively) were lysed in Nonidet P-40 lysis buffer (150 mm NaCl, 0.5% Nonidet P-40, 50 mm Tris, pH 8.0, protease inhibitor mixture, 1 mm PMSF) by gentle Dounce homogenization followed by centrifugation at 14,000 × g to remove debris. Protein concentration was determined by standard BCA assay. Equal amount of proteins from DMSO- and rotenone-treated cells were combined and loaded onto the affinity column (0.2 ml/min) and washed with binding buffer (75 mm Tris-HCl, pH 7.4). Bound associated proteins were eluted with 100 mm glycine-HCl and 0.5 m NaCl at pH 2.7 and desalted with a Hi-Trap desalting column (Amersham Biosciences).

       In-solution Digestion of α-Synuclein- or DJ-1-associated Proteins Followed by Protein Identification by LCQ-MS/MS—

      The desalted α-synuclein- or DJ-1-associated proteins were concentrated in a SpeedVac (Thermo, Waltham, MA) and then digested directly with trypsin (Promega, Madison, WI) overnight at 37 °C in 50 mm Tris-HCl (pH 7.8). The digested peptides were desalted with a reverse-phase (RP) Atlantis dC18 column (Waters, Milford, MA) and were further separated by a two-dimensional microcapillary high performance LC system, which integrated a strong cation-exchange (SCX) column (100 mm in length × 0.32-mm inner diameter; particle size, 5 μm) with two alternating RP C18 columns (100 mm in length × 0.18-mm inner diameter), followed by analysis of each peptide with MS/MS using an LCQ DECA XP PLUS ion trap (ThermoElectron, San Jose, CA). Settings for the LC-MS/MS were the following: Six fractions were eluted from SCX using a binary gradient of 2–90% solvent D (1.0 m ammonium chloride and 0.1% formic acid in 5% acetonitrile) versus solvent C (0.1% formic acid in 5% acetonitrile). Each fraction was injected onto an RP column automatically, and the peptides were resolved using a 300-min binary gradient of 5–80% solvent B (acetonitrile and 0.1% formic acid) versus solvent A (0.1% formic acid in water). A flow rate of 160 μl/min with a split ratio of 1:80 was used. Peptides were eluted directly into the ESI ion trap mass spectrometer capable of data-dependent acquisition. Each full MS scan was followed by two MS/MS scans of the two most intense peaks in the full MS spectrum with dynamic exclusion enabled to allow detection of less abundant peptide ions. MS scan events and HPLC solvent gradients were controlled by the Xcalibur software (ThermoElectron).

       MS/MS Data Analysis—

      The α-synuclein- or DJ-1-associated proteins were later identified automatically using the computer program Sequest™, which searched the MS/MS spectra against the rat + mouse International Protein Index (IPI, version 3.01, 43,175 entries) database (
      • Jin J.
      • Meredith G.E.
      • Chen L.
      • Zhou Y.
      • Xu J.
      • Shie F.S.
      • Lockhart P.
      • Zhang J.
      Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson’s disease.
      ,
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ,
      • Zhang J.
      • Goodlett D.R.
      • Quinn J.F.
      • Peskind E.
      • Kaye J.A.
      • Zhou Y.
      • Pan C.
      • Yi E.
      • Eng J.
      • Wang Q.
      • Aebersold R.H.
      • Montine T.J.
      Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease.
      ,
      • Zhang J.
      • Goodlett D.R.
      • Peskind E.R.
      • Quinn J.F.
      • Zhou Y.
      • Wang Q.
      • Pan C.
      • Yi E.
      • Eng J.
      • Aebersold R.H.
      • Montine T.J.
      Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid.
      ). Search parameters for the SILAC-labeled samples used in this study were the following: +6 Da for 13C isotopically labeled arginine, +16 Da for oxidized methionine, and +57 Da for carbamidomethyl; mass tolerance, ±3 Da. Potential peptides and proteins were further analyzed with two newly developed computer software programs, PeptideProphet™ and ProteinProphet™, based on statistical models (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ,
      • Keller A.
      • Nesvizhskii A.I.
      • Kolker E.
      • Aebersold R.
      Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.
      ,
      • Keller A.
      • Purvine S.
      • Nesvizhskii A.I.
      • Stolyar S.
      • Goodlett D.R.
      • Kolker E.
      Experimental protein mixture for validating tandem mass spectral analysis.
      ). PeptideProphet uses various Sequest scores and a number of other parameters to calculate a probability score for each identified peptide. The peptides are then assigned a protein identification using ProteinProphet. ProteinProphet allows filtering of large scale data sets with assessment of predictable sensitivity and false positive identification error rates. In our study, only proteins with a high probability of accuracy (<5% error rate as determined by ProteinProphet) were selected. Quantification of the ratio of each protein (isotopically light [control] versus heavy [rotenone treatment]) was calculated using the automated statistical analysis of protein ratio (ASAPRatio) program (
      • Li X.J.
      • Zhang H.
      • Ranish J.A.
      • Aebersold R.
      Automated statistical analysis of protein abundance ratios from data generated by stable-isotope dilution and tandem mass spectrometry.
      ). All of these methods, freely accessible at the website of the Institute of Systems of Biology, are used routinely in our laboratory (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ,
      • Jin J.
      • Hulette C.
      • Wang Y.
      • Zhang T.
      • Pan C.
      • Wadhwa R.
      • Zhang J.
      Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: relevance to Parkinson disease.
      ,
      • Jin J.
      • Meredith G.
      • Chen L.
      • Zhou Y.
      • Xu J.
      • Xie F.
      • Lockhart P.
      • Zhang J.
      Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson’s disease.
      ,
      • Zhou Y.
      • Wang Y.
      • Kovacs M.
      • Jin J.
      • Zhang J.
      Microglial activation induced by neurodegeneration: a proteomic analysis.
      ).

       Triple Immunofluorescent Staining and Confocal Analysis as Well as WB Analyses of Candidate Proteins—

      MES cells were seeded on chambered glass slides (Nalge Nunc, Naperville, IL) and then fixed in 4% paraformaldehyde followed by overnight incubation with primary antibody (α-synuclein, DJ-1, and one of the candidate proteins (mortalin, nucleolin, grp94, calnexin, and clathrin)) and then incubated with secondary antibody (1:200 Flex Fluor® 633 goat anti-mouse IgG, 1:200 Flex Fluor 568 goat anti-sheep IgG, and 1:200 Flex Fluor 488 goat anti-rabbit IgG; Molecular Probes, Eugene, OR). A laser scanning confocal microscope (Bio-Rad LS2000) was used to capture images.
      To further evaluate the association of mortalin, nucleolin, grp94, calnexin, and clathrin with both α-synuclein and DJ-1, proteins purified by the affinity columns were also blotted against antibodies to each of the five proteins. Finally the relative changes after rotenone treatment as determined by WB analysis were compared with those obtained via SILAC. The intensity of the corresponding bands was quantified with Quantity One (Bio-Rad). At least three independent experiments were performed for each candidate protein.

       Immunoprecipitation of DJ-1, α-Synuclein, and Mortalin—

      Co-IP of DJ-1, α-synuclein, and mortalin was performed using the ExactaCruz™ IP/WB kit following the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA).

      RESULTS

       Identification of the Proteins Associated with α-Synuclein and DJ-1—

      Affinity purification of proteins associated with α-synuclein and DJ-1 followed by MS identification of proteins, although a very powerful approach to analyzing protein-protein interaction, is well known to yield false positive results. This is because even trace amounts of proteins nonspecifically bound to the column or the antibodies can be identified by sensitive MS. To robustly select candidate proteins likely related to PD pathogenesis or Lewy body formation, we took advantage of quantitative proteomics as it is believed that proteins modulated by a biological process in a protein complex are less likely to be contaminants (
      • Gingras A.C.
      • Aebersold R.
      • Raught B.
      Advances in protein complex analysis using mass spectrometry.
      ,
      • Blagoev B.
      • Kratchmarova I.
      • Ong S.E.
      • Nielsen M.
      • Foster L.J.
      • Mann M.
      A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
      ). The basic principle of this study is illustrated in Fig. 1. Essentially MES cells were labeled with [12C6]- or [13C6]-l-arginine and treated with the vehicle DMSO or the parkinsonian toxicant rotenone for 3 days, respectively. Next the proteins associated with α-synuclein and DJ-1 were isolated with affinity columns using a method described by our laboratory previously (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ) before MS identification of proteins.
      Figure thumbnail gr1
      Fig. 1Approaches to identifying proteins associated with α-synuclein and DJ-1 with affinity purification. MES cells were labeled with [12C6]- or [13C6]-l-arginine and treated with DMSO or rotenone for 3 days, respectively. Next equal amounts of proteins from each cell lysate were combined before affinity purification using columns mounted with α-synuclein or DJ-1 antibody. The proteins eluted from the affinity column were digested with trypsin, and the resulting peptides were separated by on-line SCX and reverse-phase chromatography consecutively. The arginine-containing peptides from two settings occurred in doublets, separated by 6 Da, and were identified and quantified by mass spectrometry.
      With this approach, we identified 324 and 306 α-synuclein- and DJ-1-associated proteins with more than two peptides, respectively, using a 5% error rate as defined by ProteinProphet (Supplemental Appendices I and II, respectively). To further validate the results obtained by ProteinProphet filtering, we also searched the same α-synuclein MS data against the randomized decoy database, identifying three proteins that were common to both database. Thus, the actual error rate was less than 1% if judged by the search results of the decoy database. Of the 324 α-synuclein-associated proteins, 82 proteins increased and 59 proteins decreased significantly in the relative abundance after rotenone treatment (Supplemental Appendix III). Several aspects of the results are noteworthy. First, many of the identified proteins are novel; this is true even when only the proteins with relative abundance changes, i.e. proteins regulated by rotenone and thus potentially related to PD pathogenesis (Supplemental Appendix III), are considered. Second, in comparison with our previous proteomics investigation where ICAT technology was used and ∼250 proteins associated with α-synuclein were identified (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ), 62 proteins were identified in both experiments, i.e. 262 new proteins were identified. Of the overlapped proteins, many displaying quantitative changes in the previous investigation were also among those with quantitative changes in the same direction in the current study. These proteins include: tubulin α chain, aldolase 1, and hypothetical RNA-binding protein. Finally among the new proteins identified, there were more than 100 proteins with quantitative changes, and it should be noted that several of these, e.g. calmodulin and septin, have been reported to be associated with α-synuclein or co-localized to Lewy bodies in PD patients (
      • Ihara M.
      • Tomimoto H.
      • Kitayama H.
      • Morioka Y.
      • Akiguchi I.
      • Shibasaki H.
      • Noda M.
      • Kinoshita M.
      Association of the cytoskeletal GTP-binding protein Sept4/H5 with cytoplasmic inclusions found in Parkinson’s disease and other synucleinopathies.
      ).
      Of the 306 DJ-1-associated proteins identified with more than two peptides (Supplemental Appendix II), 63 proteins increased and 56 proteins decreased significantly in the relative abundance after rotenone treatment (Supplemental Appendix IV). In contrast to α-synuclein, where protein-protein interactions have been studied much more extensively, identification of proteins interacting with DJ-1 has yielded much less success with either conventional biochemistry or a proteomics experiment conducted by another group of investigators (
      • Xu J.
      • Zhong N.
      • Wang H.
      • Elias J.E.
      • Kim C.Y.
      • Woldman I.
      • Pifl C.
      • Gygi S.P.
      • Geula C.
      • Yankner B.A.
      The Parkinson’s disease-associated DJ-1 protein is a transcriptional co-activator that protects against neuronal apoptosis.
      ,
      • Li H.M.
      • Niki T.
      • Taira T.
      • Iguchi-Ariga S.M.
      • Ariga H.
      Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress.
      ,
      • Junn E.
      • Taniguchi H.
      • Jeong B.S.
      • Zhao X.
      • Ichijo H.
      • Mouradian M.M.
      Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death.
      ). Among all previously reported proteins, we identified two of them, mortalin (mthsp70/grp75) and pyrimidine tract-binding protein-associated splicing factor (PSF), both of which demonstrated relative changes after rotenone treatment.

       Common Proteins Associated with Both α-Synuclein and DJ-1—

      As mentioned earlier, one of the controversial issues is whether DJ-1 interacts with α-synuclein directly, thereby executing its function (
      • Zhou W.
      • Zhu M.
      • Wilson M.A.
      • Petsko G.A.
      • Fink A.L.
      The oxidation state of DJ-1 regulates its chaperone activity toward α-synuclein.
      ,
      • Meulener M.C.
      • Graves C.L.
      • Sampathu D.M.
      • Armstrong-Gold C.E.
      • Bonini N.M.
      • Giasson B.I.
      DJ-1 is present in a large molecular complex in human brain tissue and interacts with α-synuclein.
      ). In our proteomics studies shown in Supplemental Appendices I and II, α-synuclein was not among the list of DJ-1-associated proteins and vise versa. Notably DJ-1 is not seen in association with α-synuclein in our previous ICAT analysis when a different proteomics approach was taken (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ). The obvious question then becomes whether there are intermediate proteins that might connect the functions of α-synuclein and DJ-1. Comparing the list of proteins associated with α-synuclein and DJ-1, we identified 144 common proteins. Although all of these proteins can be genuine proteins interacting with both α-synuclein and DJ-1, some could also be false positive results for the reasons discussed earlier. Thus, as the initial step to identifying potential candidate proteins, only those with significant relative abundance changes in either α-synuclein or DJ-1 complexes (a total of 114) after rotenone treatment were considered. We separated the common proteins into four groups (Table I): 1) changes with the same directions in α-synuclein- and DJ-1-associated proteins, 2) changes with different directions in α-synuclein- and DJ-1-associated proteins, 3) changes only in α-synuclein- but not in DJ-1-associated proteins, and 4) changes only in DJ-1- but not in α-synuclein-associated proteins. Figs. 2 and 3 show pie charts summarizing the functions of the common proteins and the functions of the common proteins with changes, respectively. Major classes of proteins included those related to chaperone, cytoskeleton, and ribosome, all of which have been implicated in rotenone-induced pathology (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ,
      • Diaz-Corrales F.J.
      • Asanuma M.
      • Miyazaki I.
      • Miyoshi K.
      • Ogawa N.
      Rotenone induces aggregation of γ-tubulin protein and subsequent disorganization of the centrosome: relevance to formation of inclusion bodies and neurodegeneration.
      ,
      • Hsuan S.L.
      • Klintworth H.M.
      • Xia Z.
      Basic fibroblast growth factor protects against rotenone-induced dopaminergic cell death through activation of extracellular signal-regulated kinases 1/2 and phosphatidylinositol-3 kinase pathways.
      ). Notably a significant number of proteins listed in Table I were novel and without known functions. It should be noted that common proteins within some categories, e.g. signaling pathways, were found to exhibit changes in relative abundance after rotenone treatment in α-synuclein or DJ-1 complex but not both. On the other hand, some proteins within other categories, such as ribosomal proteins, seemed to exhibit changes in relative abundance after rotenone treatment in both α-synuclein and DJ-1 complexes (although not always in the same direction).
      Table IProteins associated with both α-synuclein and DJ-1
      Figure thumbnail gr2
      Fig. 2The functional classification of common proteins. The proteins associated with both α-synuclein and DJ-1 were classified into the following categories: chaperone, cytoskeleton, DNA/RNA-binding protein, metabolism, mitochondrial protein, protein synthesis, ribosomal protein, protein related to signal transduction, and unknown functions. For a protein with multiple functions, it was assigned to the one that is best known.
      Figure thumbnail gr3
      Fig. 3The functional classification of α-synuclein- and DJ-1-associated proteins listed in with relative abundance changes after rotenone treatment. The classification scheme is identical to those used for . Panel I, changes of proteins with the same directions in α-synuclein- and DJ-1-associated proteins; Panel II, changes of proteins with different directions in α-synuclein- and DJ-1-associated proteins; Panel III, changes of proteins only in α-synuclein- but not in DJ-1-associated proteins; Panel IV, changes of proteins only in DJ-1- but not in α-synuclein-associated proteins.

       Validation of the Common Proteins Associated with α-Synuclein and DJ-1—

      It cannot be overemphasized that the high throughput proteomics analysis only provides a list of candidate proteins for further study, i.e. the proteins identified by this approach, including those with quantitative changes after rotenone treatment, require validation before their biological roles are pursued. It is obviously impractical to verify all of the proteins listed in Table I; thus we selected five proteins for further validation as a first step toward verifying proteins most likely to have biological importance. The criteria used in selecting candidate proteins included: 1) the identification and quantification of each candidate are based on multiple peptides; 2) there is an antibody available commercially; and 3) either (a) the candidate has to be an important protein in PD (
      • Jin J.
      • Hulette C.
      • Wang Y.
      • Zhang T.
      • Pan C.
      • Wadhwa R.
      • Zhang J.
      Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: relevance to Parkinson disease.
      ), but its relationship to α-synuclein or DJ-1 has not been studied yet, or (b) the protein belongs to a process important in PD, although its direct involvement in PD has not been established previously. With these caveats in mind, we selected mortalin (grp75/stress-70), a mitochondrial stress protein, which has been demonstrated recently by our group to play key roles in PD pathogenesis. Its relative abundance increased in DJ-1-associated proteins after rotenone treatment but did not change in α-synuclein-associated proteins. The other four proteins used were nucleolin, calnexin, grp94, and clathrin. Nucleolin is a protein with potential roles in AD (
      • Reiser G.
      • Bernstein H.G.
      Altered expression of protein p42IP4/centaurin-α1 in Alzheimer’s disease brains and possible interaction of p42IP4 with nucleolin.
      ,
      • Dranovsky A.
      • Vincent I.
      • Gregori L.
      • Schwarzman A.
      • Colflesh D.
      • Enghild J.
      • Strittmatter W.
      • Davies P.
      • Goldgaber D.
      Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer’s disease pathology.
      ) but with unknown functions in PD. Its relative abundance decreased significantly in α-synuclein protein complex after rotenone treatment but did not change in DJ-1-associated protein complex after rotenone treatment. Both calnexin and grp94 are ER stress proteins (
      • Argon Y.
      • Simen B.B.
      GRP94, an ER chaperone with protein and peptide binding properties.
      ,
      • Cribb A.E.
      • Peyrou M.
      • Muruganandan S.
      • Schneider L.
      The endoplasmic reticulum in xenobiotic toxicity.
      ,
      • Bedard K.
      • Szabo E.
      • Michalak M.
      • Opas M.
      Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57.
      ), and grp94 increased in α-synuclein protein complex but did not change in DJ-1 protein complex, whereas calnexin increased in both α-synuclein and DJ-1 protein complexes. Clathrin, a protein involved in endocytosis, increased in α-synuclein protein complex but decreased in DJ-1 protein complex after rotenone treatment. Notably despite the fact that none of these three proteins have been associated with PD or even in neurodegeneration thus far, both ER stress and dysfunction of vesicular trafficking have been clearly demonstrated to be important in PD pathogenesis (
      • Lindholm D.
      • Wootz H.
      • Korhonen L.
      ER stress and neurodegenerative diseases.
      ,
      • Ryu E.J.
      • Harding H.P.
      • Angelastro J.M.
      • Vitolo O.V.
      • Ron D.
      • Greene L.A.
      Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson’s disease.
      ).
      The validation process was conducted in three steps: 1) affinity purification of α-synuclein- and DJ-1-associated proteins followed by WB for all five candidate proteins; 2) confocal analysis of co-localization of all five proteins, which provided a fast and effective way of confirming the association of protein-protein interactions; and 3) reverse IP of one of the candidate proteins (mortalin), which is a more extensive validation of protein-protein interactions. For Western confirmation, the α-synuclein and DJ-1 complexes were isolated in the same manner as those for SILAC experiments followed by probing the elutes using antibodies against all five candidate proteins along with antibodies against α-synuclein and DJ-1, respectively. The results showed that all of the candidate proteins could be detected in α-synuclein- and DJ-1-associated protein complexes, respectively (Fig. 4). Furthermore we performed quantitative analyses of the corresponding bands for all five candidate proteins, comparing the concordance between SILAC results (ASAPRatio) and WB results (WB ratio). The quantitative analyses showed that SILAC results of four of the five candidate proteins were confirmed by WB, i.e. the change in direction of the WB experiments corroborated those of the SILAC experiments. The only exception was clathrin; the ASAPRatio of this protein was 0 and 999 in α-synuclein and DJ-1 protein complexes, respectively, after rotenone treatment, whereas the WB ratio was 1.45 ± 0.5 and 1.17 ± 0.21, respectively (Table II). Overall the WB data largely validated quantitative proteomics results obtained via SILAC experiments. It should be noted, however, that the dynamic range of WB analysis is not necessarily linear in the absence of a standard protein to construct a standard curve for each candidate protein.
      Figure thumbnail gr4
      Fig. 4Affinity purification and WB validation of mortalin, grp94, calnexin, clathrin, and nucleolin in α-synuclein and DJ-1 protein complexes. MES cells were treated with 20 nm rotenone (R) or DMSO (D) for 3 days and then lysed in Nonidet P-40 lysis buffer, respectively. The total cell lysate was purified with α-synuclein or DJ-1 affinity column. Afterward equal amounts of protein (20 μg) from the total cell lysate (input) and elutes were analyzed by WB with antibodies against α-synuclein, DJ-1, mortalin, grp94, calnexin, clathrin, or nucleolin, respectively.
      Table IIComparison of ASAPRatio versus WB ratio for mortalin, grp94, calnexin, clathrin, and nucleolin in α-synuclein (α-Syn) and DJ-1 protein complexes after rotenone treatment
      ProteinsASAPRatioWB ratio
      WB ratios are presented as mean ± S.D. obtained from three independent experiments.
      α-SynDJ-1α-SynDJ-1
      Mortalin0.97 ± 0.20.55 ± 0.30.92 ± 0.020.80 ± 0.07
      Grp940.39 ± 0.131.01 ± 0.10.65 ± 0.081.40 ± 0.37
      Calnexin0.36 ± 0.050.36 ± 0.060.87 ± 0.150.67 ± 0.23
      Clathrin09991.45 ± 0.501.17 ± 0.21
      Nucleolin2.12 ± 0.991.1 ± 0.141.88 ± 0.401.09 ± 0.05
      a WB ratios are presented as mean ± S.D. obtained from three independent experiments.
      Next we adopted confocal analysis as an alternative way to confirm the association of protein-protein interaction. Fig. 5 shows the triple staining results of α-synuclein and DJ-1 with mortalin, nucleolin, calnexin, grp94, and clathrin, respectively. DJ-1 was localized diffusely in the nucleus and cytoplasm, whereas α-synuclein was mostly distributed in the cytoplasm. Among the five candidate proteins, clathrin, grp94, and calnexin mostly localized in the cytoplasm, whereas the staining of mortalin, which displayed a perinuclear pattern, and nucleolin demonstrated localization in both the nucleolus and cytoplasm. Remarkably although the staining patterns of these proteins were different from each other, all of them were co-localized with DJ-1 and α-synuclein to some degree in the cytoplasm.
      Figure thumbnail gr5
      Fig. 5Co-localization of mortalin, nucleolin, calnexin, grp94, and clathrin with α-synuclein and DJ-1 in MES cells. MES cells were fixed and triple stained with antibodies against α-synuclein (Column A, red); DJ-1 (Column B, green); and mortalin, nucleolin, calnexin, grp94, or clathrin (Column C, blue) simultaneously. The images were visualized with a confocal microscope. Merged images are shown in white when three antibodies are co-localized (Column D).
      After confirming the protein-protein associations of these five candidate proteins with both α-synuclein and DJ-1, we further validated the associations using reverse IP for mortalin whose relative abundance changes significantly after rotenone treatment as well as in human PD in our earlier investigation (
      • Jin J.
      • Hulette C.
      • Wang Y.
      • Zhang T.
      • Pan C.
      • Wadhwa R.
      • Zhang J.
      Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: relevance to Parkinson disease.
      ). In this set of experiments, samples were prepared in the same manner as those for the affinity purification before MS analysis with the exception that the MES cell lysates were immunoprecipitated with mortalin, DJ-1, and α-synuclein antibodies, respectively, and then immunoblotted with anti-mortalin, -DJ-1, and -α-synuclein, respectively. The results, shown in Fig. 6, demonstrated that mortalin was not only present in anti-mortalin-precipitated samples but also in anti-DJ-1- and anti-α-synuclein-precipitated samples. We also used the same batch of the precipitated sample to blot with anti-DJ-1 and anti-α-synuclein, respectively, and found that both DJ-1 and α-synuclein were present in mortalin complexes. Finally DJ-1 was absent in the α-synuclein complex and vice versa.
      Figure thumbnail gr6
      Fig. 6Mortalin was co-immunoprecipitated with both DJ-1 and α-synuclein. MES cells were lysed with Nonidet P-40 lysis buffer, and the total cell lysates were immunoprecipitated with anti-mortalin, α-synuclein (α-Syn), or DJ-1 antibody followed by immunoblotting with antibodies against mortalin, α-synuclein, or DJ-1, respectively.

      DISCUSSION

      The major goal of this study was to identify potential proteins interacting with α-synuclein, DJ-1, or both, thereby further elucidating the mechanisms by which these two critical proteins participate in cellular functions and PD pathogenesis. Several major findings have resulted from this investigation, including the following. 1) 324 and 306 candidate proteins were identified to be associated with α-synuclein and DJ-1, respectively. 2) Among these candidate proteins, 144 proteins were associated with both α-synuclein and DJ-1. Furthermore 114 of the common proteins displayed significant changes in relative abundance in α-synuclein and/or DJ-1 complexes after rotenone treatment and were consequently judged to be more likely to be truly associated proteins and to have relevant biological functions related to PD. 3) Five proteins that were associated with both α-synuclein and DJ-1 as determined by proteomics analysis were further validated with additional means.
      Identification of a total of 324 proteins associated with α-synuclein represents a significant improvement over what has been reported in the literature (
      • Shimura H.
      • Schlossmacher M.G.
      • Hattori N.
      • Frosch M.P.
      • Trockenbacher A.
      • Schneider R.
      • Mizuno Y.
      • Kosik K.S.
      • Selkoe D.J.
      Ubiquitination of a new form of α-synuclein by parkin from human brain: implications for Parkinson’s disease.
      ,
      • Engelender S.
      • Kaminsky Z.
      • Guo X.
      • Sharp A.H.
      • Amaravi R.K.
      • Kleiderlein J.J.
      • Margolis R.L.
      • Troncoso J.C.
      • Lanahan A.A.
      • Worley P.F.
      • Dawson V.L.
      • Dawson T.M.
      • Ross C.A.
      Synphilin-1 associates with α-synuclein and promotes the formation of cytosolic inclusions.
      ,
      • Jensen P.H.
      • Hager H.
      • Nielsen M.S.
      • Hojrup P.
      • Gliemann J.
      • Jakes R.
      α-Synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356.
      ), including our own (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ). Among the 34 proteins reported by others (
      • Zhou Y.
      • Gu G.
      • Goodlett D.R.
      • Zhang T.
      • Pan C.
      • Montine T.J.
      • Montine K.S.
      • Aebersold R.H.
      • Zhang J.
      Analysis of α-synuclein-associated proteins by quantitative proteomics.
      ), 12 were identified in our current study. It is noteworthy that 62 of the proteins identified in our previous proteomics study were seen again in the current investigation when a different approach was taken. On the other hand, some proteins identified in the previous experiment, e.g. mitogen-activated protein kinase 1, are clearly missing from the current study. Several reasons are likely responsible for these discrepancies. First, there are major differences between ICAT- and SILAC-based quantitative proteomics. In the ICAT method, sample preparation involves extensive chromatography, and only cysteine-containing proteins are retained; this typically leads to the loss of more than 80% of peptides (
      • Turecek F.
      Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.
      ). In the SILAC method, when arginine is labeled differentially, all peptides with arginine are identified and quantified. It should be noted, however, that although the ICAT method appears to be less robust than the SILAC method, one should not come to the conclusion that ICAT is outdated because prelabeling of tissue is not always possible (e.g. in human tissue), and ICAT reduces the sample complexity and enriches low abundance proteins, which is necessary when the protein mixture is very complex. Second, current MS technologies all have limitations on reproducibility, i.e. there is low overlap when identical samples are injected multiple times. For instance, the reproducibility of our LCQ approach is about 30% when an ICAT sample with modest complexity is analyzed (
      • Zhang J.
      • Goodlett D.R.
      • Peskind E.R.
      • Quinn J.F.
      • Zhou Y.
      • Wang Q.
      • Pan C.
      • Yi E.
      • Eng J.
      • Aebersold R.H.
      • Montine T.J.
      Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid.
      ,
      • Yi E.C.
      • Marelli M.
      • Lee H.
      • Purvine S.O.
      • Aebersold R.
      • Aitchison J.D.
      • Goodlett D.R.
      Approaching complete peroxisome characterization by gas-phase fractionation.
      ). Finally the difference in database used (a more updated version 3.01 in the current study versus version 1.11 in the previous experiment) may also have a significant role in the discrepancy seen between these two studies. Our previous results related to characterization of the proteome of human cerebrospinal fluid showed that the overlap was only 66% when two different versions of databases were used (
      • Xu J.
      • Chen J.
      • Peskind E.
      • Jin J.
      • Eng J.
      • Pan C.
      • Montine T.
      • Goodlett D.
      • Zhang J.
      Characterization of proteome of human cerebrospinal fluid.
      ).
      Investigation of proteins associated with DJ-1 is not as advanced as that with α-synuclein; so far, only a handful of proteins in each of the four roughly divided categories were reported to potentially interact with DJ-1. These include: 1) nuclear proteins: p54nrb and PSF (
      • Xu J.
      • Zhong N.
      • Wang H.
      • Elias J.E.
      • Kim C.Y.
      • Woldman I.
      • Pifl C.
      • Gygi S.P.
      • Geula C.
      • Yankner B.A.
      The Parkinson’s disease-associated DJ-1 protein is a transcriptional co-activator that protects against neuronal apoptosis.
      ); homeodomain-interacting protein kinase 1 (HIPK1) (
      • Sekito A.
      • Koide-Yoshida S.
      • Niki T.
      • Taira T.
      • Iguchi-Ariga S.M.
      • Ariga H.
      DJ-1 interacts with HIPK1 and affects H2O2-induced cell death.
      ); PIASxα/ARIP3, a modulator of androgen receptor (
      • Takahashi K.
      • Taira T.
      • Niki T.
      • Seino C.
      • Iguchi-Ariga S.M.
      • Ariga H.
      DJ-1 positively regulates the androgen receptor by impairing the binding of PIASxα to the receptor.
      ); death protein Daxx (
      • Junn E.
      • Taniguchi H.
      • Jeong B.S.
      • Zhao X.
      • Ichijo H.
      • Mouradian M.M.
      Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death.
      ); and topoisomerase I and p53-binding protein Topors/p53BP3 (
      • Shinbo Y.
      • Taira T.
      • Niki T.
      • Iguchi-Ariga S.M.
      • Ariga H.
      DJ-1 restores p53 transcription activity inhibited by Topors/p53BP3.
      ); 2) chaperone proteins: hsp70, C terminus Hsp70 interacting protein, and mthsp70/grp75/mortalin (
      • Li H.M.
      • Niki T.
      • Taira T.
      • Iguchi-Ariga S.M.
      • Ariga H.
      Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress.
      ); 3) signaling protein: phosphatidylinositol 3-kinase/Akt (
      • Yang Y.
      • Gehrke S.
      • Haque M.E.
      • Imai Y.
      • Kosek J.
      • Yang L.
      • Beal M.F.
      • Nishimura I.
      • Wakamatsu K.
      • Ito S.
      • Takahashi R.
      • Lu B.
      Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling.
      ); and 4) proteins directly associated with neurodegeneration: parkin (
      • Moore D.J.
      • Zhang L.
      • Troncoso J.
      • Lee M.K.
      • Hattori N.
      • Mizuno Y.
      • Dawson T.M.
      • Dawson V.L.
      Association of DJ-1 and parkin mediated by pathogenic DJ-1 mutations and oxidative stress.
      ) and tau (
      • Rizzu P.
      • Hinkle D.A.
      • Zhukareva V.
      • Bonifati V.
      • Severijnen L.A.
      • Martinez D.
      • Ravid R.
      • Kamphorst W.
      • Eberwine J.H.
      • Lee V.M.
      • Trojanowski J.Q.
      • Heutink P.
      DJ-1 colocalizes with tau inclusions: a link between parkinsonism and dementia.
      ). Our analysis, which identified 306 candidate proteins, is the first extensive profiling attempt in elucidating proteins that may interact with DJ-1. However, all of the DJ-1-associated proteins reported in the literature mentioned above, except mortalin and PSF, were not found in our studies. Besides caveats discussed above, a few additional reasons may explain this discrepancy, including: 1) different model systems, i.e. cell types or tissues, are used between this and other studies; and 2) nuclear proteins could not be released from nuclei efficiently when the non-ionic detergent Nonidet P-40 lysis buffer was used.
      It is also noteworthy that many proteins associated with α-synuclein (Supplemental Appendix I) and DJ-1 (Supplemental Appendix II) are ribosomal proteins, translation factors, chaperones, and tubulin or tubulin-associated molecules. This finding, however, is not surprising given that protein aggregation that occurs in cells treated with rotenone involves abnormal cytoskeletal protein folding, a process capable of trapping all of these proteins.
      It is obvious that some of these candidate proteins, whether α-synuclein- or DJ-1-associated, could be contaminants acquired during affinity purification, and there is currently no good solution for this difficulty, particularly in a high throughput setting. Thus, we strongly believe that all of the candidate proteins need to be validated with additional means before their biological functions are pursued extensively. As proteins displaying quantitative changes are less likely to be nonspecific binding proteins, we deliberately chose a parkinsonian toxicant, rotenone, as a modulator, thereby adding filtering power to isolate proteins that are more likely to be important in PD pathogenesis. Using this method, we reveled that: 1) in α-synuclein-associated proteins, 82 proteins increased and 59 proteins decreased more than 20% in relative abundance after rotenone treatment; and 2) in DJ-1-associated proteins, 63 proteins increased and 56 proteins decreased more than 20% in relative abundance after rotenone treatment (see Supplemental Appendices III and IV, respectively). All of these proteins are certainly worth investigating further; however, it is not practical for us to discuss all of them here. To narrow our focus further, we paid particular attention to the 114 proteins that were observed in association with both α-synuclein and DJ-1 as one of our primary goals of this study was to identify proteins that might mediate functions between α-synuclein and DJ-1.
      As mentioned in the Introduction, it is controversial as to whether DJ-1 directly interacts with α-synuclein (
      • Zhou W.
      • Zhu M.
      • Wilson M.A.
      • Petsko G.A.
      • Fink A.L.
      The oxidation state of DJ-1 regulates its chaperone activity toward α-synuclein.
      ,
      • Meulener M.C.
      • Graves C.L.
      • Sampathu D.M.
      • Armstrong-Gold C.E.
      • Bonini N.M.
      • Giasson B.I.
      DJ-1 is present in a large molecular complex in human brain tissue and interacts with α-synuclein.
      ). The fact that we did not see direct interaction between DJ-1 and α-synuclein in two separate proteomics studies seems to suggest that they are unlikely to be directly associated. This conclusion is further supported by our IP experiments where direct interaction was not seen (Fig. 6). Interestingly a weak band was visualized when radioimmune precipitation assay buffer, a buffer containing more detergent, was used (data not shown). Because α-synuclein is a very sticky protein, these data suggest to us that the direct association between α-synuclein and DJ-1 in one earlier investigation (
      • Meulener M.C.
      • Graves C.L.
      • Sampathu D.M.
      • Armstrong-Gold C.E.
      • Bonini N.M.
      • Giasson B.I.
      DJ-1 is present in a large molecular complex in human brain tissue and interacts with α-synuclein.
      ) could be a sample preparation artifact. To state it differently, the interaction between α-synuclein and DJ-1 likely needs a docking protein(s). In fact, any of the 144 proteins associated with both DJ-1 and α-synuclein can serve this purpose. However, this is not to say that the effects of DJ-1 on α-synuclein (or vice versa) have to involve direct or indirect physical contact of these two proteins. For example, DJ-1 could influence oxidative stress (
      • Bonifati V.
      • Rizzu P.
      • Squitieri F.
      • Krieger E.
      • Vanacore N.
      • van Swieten J.C.
      • Brice A.
      • van Duijn C.M.
      • Oostra B.
      • Meco G.
      • Heutink P.
      DJ-1(PARK7), a novel gene for autosomal recessive, early onset parkinsonism.
      ,
      • Macedo M.G.
      • Anar B.
      • Bronner I.F.
      • Cannella M.
      • Squitieri F.
      • Bonifati V.
      • Hoogeveen A.
      • Heutink P.
      • Rizzu P.
      The DJ-1L166P mutant protein associated with early onset Parkinson’s disease is unstable and forms higher-order protein complexes.
      ) that subsequently modulates the function of α-synuclein. Nonetheless as one of the testable hypotheses, we have reasoned that the proteins displaying significant relative abundance changes (Table I) after rotenone treatment may also be important in mediating some of the interactions betweens DJ-1 on α-synuclein and therefore should be studied with priority in the future. But again, validation is still needed before the biological functions of these proteins are pursued. To this end, we validated five of these proteins, mortalin, nucleolin, calnexin, grp94, and clathrin, for their association with α-synuclein and DJ-1 by Western, confocal, and/or co-IP analysis.
      Mortalin is a mitochondrial stress protein and we have demonstrated in a recent study that it is decreased in PD nigral tissue as compared with controls. Furthermore mortalin likely modulates PD development via pathways involving mitochondrial and proteasomal function as well as oxidative stress (
      • Jin J.
      • Hulette C.
      • Wang Y.
      • Zhang T.
      • Pan C.
      • Wadhwa R.
      • Zhang J.
      Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: relevance to Parkinson disease.
      ). As DJ-1 has been implicated in all of these processes (
      • Taira T.
      • Saito Y.
      • Niki T.
      • Iguchi-Ariga S.M.
      • Takahashi K.
      • Ariga H.
      DJ-1 has a role in antioxidative stress to prevent cell death.
      ,
      • Yokota T.
      • Sugawara K.
      • Ito K.
      • Takahashi R.
      • Ariga H.
      • Mizusawa H.
      Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition.
      ), it is reasonable to hypothesize that mortalin may accomplish its function by interacting with DJ-1 or α-synuclein directly. The biological importance of the other four validated proteins in PD is largely unknown. Nonetheless a potential role of nucleolin, a shuttle protein in AD, has been implicated recently (
      • Dranovsky A.
      • Vincent I.
      • Gregori L.
      • Schwarzman A.
      • Colflesh D.
      • Enghild J.
      • Strittmatter W.
      • Davies P.
      • Goldgaber D.
      Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer’s disease pathology.
      ). More specifically, it appears that phosphorylation of nucleolin (
      • Reiser G.
      • Bernstein H.G.
      Altered expression of protein p42IP4/centaurin-α1 in Alzheimer’s disease brains and possible interaction of p42IP4 with nucleolin.
      ) or its interaction with p42IP4 (
      • Reiser G.
      • Bernstein H.G.
      Altered expression of protein p42IP4/centaurin-α1 in Alzheimer’s disease brains and possible interaction of p42IP4 with nucleolin.
      ) may regulate AD pathology, e.g. formation of neurofibrillary tangles. Calnexin belongs to a family called ER chaperones (
      • Bedard K.
      • Szabo E.
      • Michalak M.
      • Opas M.
      Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57.
      ), whereas grp94 has been considered as an ER stress protein (
      • Argon Y.
      • Simen B.B.
      GRP94, an ER chaperone with protein and peptide binding properties.
      ,
      • Cribb A.E.
      • Peyrou M.
      • Muruganandan S.
      • Schneider L.
      The endoplasmic reticulum in xenobiotic toxicity.
      ). Although neither calnexin nor grp94 has been linked to PD pathogenesis thus far, it has been clearly demonstrated by others that ER stress is involved in PD pathogenesis (
      • Lindholm D.
      • Wootz H.
      • Korhonen L.
      ER stress and neurodegenerative diseases.
      ). Finally clathrin is a classic protein that plays a major role in vesicular trafficking (
      • Schmid S.L.
      Clathrin-coated vesicle formation and protein sorting: an integrated process.
      ) and protein interaction with lipid raft (
      • Le Roy C.
      • Wrana J.L.
      Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling.
      ). Thus, our discovery of this protein in association with α-synuclein and DJ-1 can be significant in several ways. First, α-synuclein has been known for many years to interact with lipid and probably travels along with lipid within cells (
      • Fortin D.L.
      • Troyer M.D.
      • Nakamura K.
      • Kubo S.
      • Anthony M.D.
      • Edwards R.H.
      Lipid rafts mediate the synaptic localization of α-synuclein.
      ). With our finding, one might ask: does clathrin mediate the binding of α-synuclein with lipid? Furthermore more recent studies seem to suggest that dysfunction of vesicular trafficking comprises one of the early events in PD and α-synuclein-induced neurotoxicity. Although our Western analysis did not confirm the quantitative changes in the clathrin levels in both protein complexes after rotenone treatment, the protein could still influence α-synuclein or DJ-1 function by changing its characteristics, e.g. via oxidation or phosphorylation of the protein. In fact, post-translational modifications of clathrin could be one of the reasons for the disconcordance between our SILAC and WB results.
      In summary, we used affinity purification combined with quantitative proteomics to characterize numerous novel proteins associated with DJ-1, α-synuclein, or both, representing the most extensive profiling study of these two critical proteins to date. Furthermore we did not observe a direct interaction between α-synuclein and DJ-1, although 144 candidate proteins potentially interacting with both α-synuclein and DJ-1 were identified. Finally we validated five of these common proteins; detailed characterization of these novel proteins will likely facilitate a clearer understanding of exactly how α-synuclein and DJ-1 influence nigral neuron degeneration in PD or Lewy body formation and yield potential therapeutic targets that can prevent PD development or halt its progression clinically.

      Supplementary Material

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