Oxidative Status of DJ-1-dependent Activation of Dopamine Synthesis through Interaction of Tyrosine Hydroxylase and 4-Dihydroxy-l-phenylalanine (l-DOPA) Decarboxylase with DJ-1
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.019950
ISSN1083-351X
AutoresShizuma Ishikawa, Takahiro Taira, Takeshi Niki, Kazuko Takahashi-Niki, Chinatsu Maita, Hiroshi Maita, Hiroyoshi Ariga, Sanae M.M. Iguchi‐Ariga,
Tópico(s)Biochemical and biochemical processes
ResumoParkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-l-phenylalanine decarboxylase (DDC). DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H2O2, 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO2H and SO3H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD. Parkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-l-phenylalanine decarboxylase (DDC). DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H2O2, 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO2H and SO3H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD. Parkinson disease (PD) 2The abbreviations used are: PDParkinson diseaseH2O2hydrogen peroxideTHtyrosine hydroxylaseDDCl-DOPA decarboxylaseRT-PCRreverse transcription-PCRGSTglutathione S-transferase6-OHDA6-hydroxydopamineMPP+1-methyl-4-phenylpyridiniuml-DOPA4-dihydroxy-l-phenylalanineGPDHglycerol-3-phosphate dehydrogenaseMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMSmass spectrometryHPLChigh pressure liquid chromatography. is a neurodegenerative disease that occurs by reduction of the dopamine level through dopaminergic cell death in the substantia nigra. Genetic and environmental factors are thought to be triggers for the onset of PD, but the precise molecular mechanisms are still not known. Dopamine is synthesized by the following two steps: tyrosine is converted to l-DOPA by tyrosine hydroxylase (TH) and then l-DOPA is converted to dopamine by l-dopa decarboxylase (DDC). TH is therefore a key enzyme for dopamine synthesis and is used as a marker for dopaminergic neurons. Parkinson disease hydrogen peroxide tyrosine hydroxylase l-DOPA decarboxylase reverse transcription-PCR glutathione S-transferase 6-hydroxydopamine 1-methyl-4-phenylpyridinium 4-dihydroxy-l-phenylalanine glycerol-3-phosphate dehydrogenase matrix-assisted laser desorption ionization time-of-flight mass spectrometry high pressure liquid chromatography. DJ-1 was first identified by our group as a novel candidate of the oncogene that transformed mouse NIH3T3 cells in cooperation with activated ras (1Nagakubo D. Taira T. Kitaura H. Ikeda M. Tamai K. Iguchi-Ariga S.M. Ariga H. Biochem. Biophys. Res. Commun. 1997; 231: 509-513Crossref PubMed Scopus (667) Google Scholar). Deletion and point (L166P) mutations of DJ-1 have been shown to be responsible for onset of familial Parkinson disease, PARK7 (2Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2261) Google Scholar), and other homozygous and heterozygous mutations of DJ-1 have been identified in patients with familial or sporadic Parkinson disease (3Abou-Sleiman P.M. Healy D.G. Quinn N. Lees A.J. Wood N.W. Ann. Neurol. 2003; 54: 283-286Crossref PubMed Scopus (328) Google Scholar, 4Hague S. Rogaeva E. Hernandez D. Gulick C. Singleton A. Hanson M. Johnson J. Weiser R. Gallardo M. Ravina B. Gwinn-Hardy K. Crawley A. St. George-Hyslop P.H. Lang A.E. Heutink P. Bonifati V. Hardy J. 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DJ-1 has three cysteine residues at positions 46, 53, and 106 (Cys-46, Cys-53, and Cys-106, respectively), and these cysteine residues are oxidized after cells receive oxidative stress, resulting in scavenging of reactive oxidative species (7Taira T. Saito Y. Niki T. Iguchi-Ariga S.M. Takahashi K. Ariga H. EMBO Rep. 2004; 5: 213-218Crossref PubMed Scopus (751) Google Scholar, 8Kinumi T. Kimata J. Taira T. Ariga H. Niki E. Biochem. Biophys. Res. Commun. 2004; 317: 722-728Crossref PubMed Scopus (307) Google Scholar, 9Canet-Avilés R.M. Wilson M.A. Miller D.W. Ahmad R. McLendon C. Bandyopadhyay S. Baptista M.J. Ringe D. Petsko G.A. Cookson M.R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9103-9108Crossref PubMed Scopus (917) Google Scholar, 10Martinat C. Shendelman S. Jonason A. Leete T. Beal M.F. Yang L. Floss T. Abeliovich A. PLoS Biol. 2004; 2: e327Crossref PubMed Scopus (248) Google Scholar, 11Inden M. Taira T. Kitamura Y. Yanagida T. Tsuchiya D. Takata K. Yanagisawa D. Nishimura K. Taniguchi T. Kiso Y. Yoshimoto K. Agatsuma T. Koide-Yoshida S. Iguchi-Ariga S.M. Shimohama S. Ariga H. Neurobiol. Dis. 2006; 24: 144-158Crossref PubMed Scopus (168) Google Scholar, 12Yanagida T. Tsushima J. Kitamura Y. Yanagisawa D. Takata K. Shibaike T. Yamamoto A. Taniguchi T. Yasui H. Taira T. Morikawa S. Inubushi T. Tooyama I. Ariga H. Oxid. Med. Cell Long. 2009; 1: 36-42Crossref Scopus (69) Google Scholar). Although DJ-1 does not directly bind to DNA, DI-1 acts as a co-activator to activate various transcription factors, including the androgen receptor and p53 tumor suppressor, PSF and Nrf2, by sequestering their inhibitory factors (13Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 14Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar, 15Shinbo Y. Taira T. Niki T. Iguchi-Ariga S.M. Ariga H. Int. J. Oncol. 2005; 26: 641-648PubMed Google Scholar, 16Zhong N. Kim C.Y. Rizzu P. Geula C. Porter D.R. Pothos E.N. Squitieri F. Heutink P. Xu J. J. Biol. Chem. 2006; 281: 20940-20948Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17Clements C.M. McNally R.S. Conti B.J. Mak T.W. Ting J.P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15091-15096Crossref PubMed Scopus (655) Google Scholar, 18Tillman J.E. Yuan J. Gu G. Fazli L. Ghosh R. Flynt A.S. Gleave M. Rennie P.S. Kasper S. Cancer Res. 2007; 67: 4630-4637Crossref PubMed Scopus (84) Google Scholar). The anti-oxidative stress function of DJ-1 is therefore thought to be carried out both by self-oxidation of cysteine residues and by activation of redox-related genes. It has been reported that PSF, a transcription repressor, binds to the promoter region of the TH gene to repress its expression and that human DJ-1 binds to PSF to sequester the PSF/co-repressor complex, leading to induction of TH gene expression in cultured human cells (16Zhong N. Kim C.Y. Rizzu P. Geula C. Porter D.R. Pothos E.N. Squitieri F. Heutink P. Xu J. J. Biol. Chem. 2006; 281: 20940-20948Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). We have shown that injection of DJ-1 into the substantia nigra of PD model rats, in which dopaminergic neuronal cell death was induced by administration of 6-hydroxydopamine, prevented cell death and locomotion defect, and restored dopamine metabolism of the rats, suggesting that DJ-1 affects dopamine biosynthesis (11Inden M. Taira T. Kitamura Y. Yanagida T. Tsuchiya D. Takata K. Yanagisawa D. Nishimura K. Taniguchi T. Kiso Y. Yoshimoto K. Agatsuma T. Koide-Yoshida S. Iguchi-Ariga S.M. Shimohama S. Ariga H. Neurobiol. Dis. 2006; 24: 144-158Crossref PubMed Scopus (168) Google Scholar). However, it has not been clarified whether DJ-1 regulates the expression of genes or activity of proteins that are related to dopamine biosynthesis. In this study, we found that DJ-1 directly bonds to TH and DDC to stimulate their activities. Mutants of DJ-1 found in PD patients lost their stimulating activity to TH and DDC, and stimulation occurred in a manner dependent on the oxidative state of Cys-106 of wild-type DJ-1. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD. Establishment of SH-SY5Y cells with knocking down of the DJ-1 gene and SH-SY5Y cells harboring a vector was described previously (19Miyazaki S. Yanagida T. Nunome K. Ishikawa S. Inden M. Kitamura Y. Nakagawa S. Taira T. Hirota K. Niwa M. Iguchi-Ariga S.M. Ariga H. J. Neurochem. 2008; 105: 2418-2434Crossref PubMed Scopus (54) Google Scholar). Human SH-SY5Y cells and these cell lines were cultured in Dulbecco's modified Eagle's medium with 10% calf serum. Plasmid pcDNA3 containing FLAG-tagged wild-type or mutant DJ-1s was transfected into SH-SY5Y cells by the calcium phosphate precipitation method, and the cells were cultured in a medium in the presence of 100 μg/ml G418 for 14 days. Cells resistant to the drug were then selected, and expression of FLAG-DJ-1 was examined by Western blotting with an anti-FLAG antibody (M2, Sigma). Nucleotide sequences of the oligonucleotide used for RT-PCR and real time primers were as follows: human DJ-1 sense (RT-PCR) 5′-GGTGCAGGCTTGTAAACATATAAC-3′ and human DJ-1 antisense (RT-PCR) 5′-CTCTAAGTGATCGTCGCAGTTCGC-3′; human TH sense (RT-PCR) 5′-CGGGTCTCTAGATGGTGGATTTT-3′ and human TH antisense (RT-PCR) 5′-GCTGTGGCCTTTGAGGAGAA-3′; human DDC sense (RT-PCR) 5′-CTGGAGACTGTGATGATGGA-3 and human DDC antisense (RT-PCR) 5′-GCAAACTCCACTCCATTCA-3; human β- actin sense (RT-PCR) 5′-CCGACAGGATGCAGAAGGAG-3′ and human β-actin antisense (RT-PCR) 5′-GTGGGGTGGCTTTTAGGATG-3′; human DJ-1 sense (real time-PCR) 5′-TTGTAGGCTGAGAAATCTCTGTG-3′ and human DJ-1 antisense (real time-PCR) 5′-ATCCATTCTCACTGTGTTCGC-3′; human TH sense (real time-PCR) 5′-GCAGGCAGAGGCCATCATGT-3′ and human TH antisense (real time-PCR) 5′-GGCGATCTCAGCAATCAGCT-3′; human DDC sense (real time-PCR) 5′-GGGAAGTGCCAGTGAAGCCA-3′ and human DDC antisense (real time-PCR) 5′-GAAGTTGCCATCTGAGGGG-3′; and human β-actin sense (real time-PCR) 5′-CCCTAAGGCCAACCGRGAAA-3′ and human β-actin antisense (real time-PCR): 5′-ACGACCAGAGGCATACAGGGA-3′. Total RNAs were prepared from cells and subjected to semi-quantitative RT-PCR and real time-PCR analyses as described previously (20Yoshida T. Kitaura H. Hagio Y. Sato T. Iguchi-Ariga S.M. Ariga H. Exp. Cell Res. 2008; 314: 1217-1228Crossref PubMed Scopus (23) Google Scholar). To examine the expression levels of proteins in cells, proteins were extracted from cells with a buffer containing 150 mm NaCl, 1 mm EDTA, 20 mm Tris (pH 8.0), and 0.5% Nonidet P-40. Proteins were then separated on a 12.5% polyacrylamide gel containing SDS and subjected to Western blotting with respective antibodies. Proteins on the membrane were reacted with an IRDye800 (Rockland, Philadelphia, PA) or Alexa Fluor 680-conjugated secondary antibody (Molecular Probes, Eugene, OR) and visualized by using an infrared imaging system (Odyssey, LI-COR, Lincoln, NE). The antibodies used were anti-TH (1:1000, Chemicon, Temecula, CA), anti-DDC (1:500, Sigma), anti-actin (1:4000, Chemicon), anti-phosphorylated TH with a serine residue at amino acid number 19 (1:500, Exalpha Biologicals, Watertown, MA), and anti-DJ-1 (1:4000) antibodies. The anti-DJ-1 antibody was prepared by us as described previously (1Nagakubo D. Taira T. Kitaura H. Ikeda M. Tamai K. Iguchi-Ariga S.M. Ariga H. Biochem. Biophys. Res. Commun. 1997; 231: 509-513Crossref PubMed Scopus (667) Google Scholar). 35S-Labeled TH and DDC were synthesized in vitro using the reticulocyte lysate of the TnT transcription-translation coupled system (Promega, Madison, WI). Labeled proteins were mixed with GST or GST-DJ-1 expressed in and prepared from Escherichia coli at 4 °C for 60 min in a buffer containing 150 mm NaCl, 5 mm EDTA, 50 mm Tris (pH 7.5), 0.05% bovine serum albumin, and 0.1% Nonidet P-40. After washing with the same buffer, the bound proteins were separated in a 10% polyacrylamide gel containing SDS and visualized by fluorography. Proteins were extracted from SH-SY5Y cells by the procedure described previously (13Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Proteins were immunoprecipitated with an anti-DJ-1 antibody (1:500) or normal IgG and the precipitates were analyzed by Western blotting with an anti-TH antibody, anti-DDC antibody, or the mouse anti-DJ-1 antibody (1:2000, 3E8, MBL, Nagoya, Japan) as described above. The promoter region of the human TH gene was amplified by RT-PCR using specific primers and total RNA from human SH-SY-5Y cells as templates. Nucleotide sequences of oligonucleotides used for PCR primers are as follows: human TH sense 5′-TCAGAACCTCAGTCCTCGCATC-3′ and human TH antisense 5′-ggagatctCAACAGGGACTCAAACACCAGG-3′. Amplified cDNA containing 3416 bp of the human TH gene was digested with KpnI and BglII, and fragments obtained were inserted into KpnI and BglII sites of pGL-3 Basic (Promega). This plasmid was named pTH-Luc. SH-SY5Y cells in a 24-well dish were transfected with 0.2 μg of pTH-Luc and various amounts (0–0.2 μg) of pcDNA3-FLAG-DJ-1 together with 25 ng of pCMV-β-gal by the calcium phosphate method (21Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6495) Google Scholar). Two days after transfection, whole cell extract was prepared by addition of Triton X-100-containing solution from the Pica gene kit (Wako Pure Chemicals, Osaka, Japan) to the cells. About a one-fifth volume of the extract was used for the β-galactosidase assay to normalize the transfection efficiencies as described previously (22Mori K. Maeda Y. Kitaura H. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 1998; 273: 29794-29800Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and the luciferase activity due to the reporter plasmid was determined using a luminometer (Luminocounter Lumat LB 9507, EG & G Berthold, Bad Wildbad, Germany). Proteins in aliquots of the cell extract were analyzed by Western blotting with an anti-FLAG antibody (M2, Sigma) and visualized as described above. The same experiments were repeated three times. TH and DDC activities in cells were measured according to the published method using HPLC (16Zhong N. Kim C.Y. Rizzu P. Geula C. Porter D.R. Pothos E.N. Squitieri F. Heutink P. Xu J. J. Biol. Chem. 2006; 281: 20940-20948Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 23Tehranian R. Montoya S.E. Van Laar A.D. Hastings T.G. Perez R.G. J. Neurochem. 2006; 99: 1188-1196Crossref PubMed Scopus (79) Google Scholar). The HPLC system and column used were AKTA explorer 10 S/100 (GE Healthcare), COSMOSIL Cholester Waters (4.6 × 150 mm, Nacalai Tesque, Kyoto, Japan), and the buffer used was a buffer containing 50 mm potassium phosphate (pH 2.6), 0.1 mm EDTA, 0.2 mm heptane sulfonic acid, and 10% methanol. Proteins were extracted from SH-SY5Y cells with a buffer containing 150 mm NaCl, 1 mm EDTA, 20 mm Tris (pH 8.0), and 0.5% Nonidet P-40. Twenty μg of proteins was reacted with 100 mm l-tyrosine, 500 μm l-DOPA decarboxylase inhibitor (3-hydroxybenzylhydrazine dihydrochloride, Acros Organics) for TH, or 250 mm l-DOPA (4-dihydroxy-l-phenylalanine) for DDC, and various amounts of GST-DJ-1 at 37 °C for 15 min, and the reaction was stopped by addition of 0.1 n perchloric acid. After centrifugation of the mixture at 12,000 rpm for 30 min, the supernatant was passed through a 0.45-μm pore size filter (Cosmonice filter W, Nacalai Tesque, Japan) and applied to HPLC as described above. Cell extracts from SH-ST5Y cells were prepared as described above and immunoprecipitated with a rabbit anti-DJ-1 polyclonal antibody (final concentration of 4 μg/ml), and immunoprecipitates were separated on a 12.5% polyacrylamide gel. After the gel had been stained with Coomassie Brilliant Blue, a band corresponding to DJ-1 was cut out, reduced, alkylated with a buffer containing iodoacetamide, and digested with trypsin. The peptide solutions were desalted, mixed with α-cyano-4-hydroxycinnamic acid, and applied onto a target plate. MS/MS spectra of the Cys-106-containing peptide spanning 100–122 amino acids were obtained using an Ultraflex (Brucker Daltonics) in reflector mode and analyzed with flex analysis software (Brucker Daltonics). Protein identification was carried out with Mascot software using the National Institute of Biomedical Innovation data base. Cells were fixed with 4% paraformaldehyde and reacted with rabbit anti-DJ-1 polyclonal (1:100), mouse anti-TH monoclonal (1:100), and mouse anti-DDC monoclonal antibodies (1:100). The cells were also stained with 4′,6-diamidino-2-phenylindole. The cells were then reacted with a rhodamine-conjugated anti-rabbit IgG or an fluorescein isothiocyanate-conjugated anti-mouse IgG and observed under a Bio-Imaging system (Olympus, FSV100, Tokyo, Japan). Data are expressed as means ± S.E. Statistical analyses were performed using one-way analysis of variance followed by unpaired Student's t test. To examine the effects of DJ-1 on expression and activity of TH and DDC, human neuroblastoma SH-SY5Y cells, in which the DJ-1 gene was knocked down, were established by transfection of a vector containing short hairpin RNA targeting human DJ-1, as described previously (19Miyazaki S. Yanagida T. Nunome K. Ishikawa S. Inden M. Kitamura Y. Nakagawa S. Taira T. Hirota K. Niwa M. Iguchi-Ariga S.M. Ariga H. J. Neurochem. 2008; 105: 2418-2434Crossref PubMed Scopus (54) Google Scholar), and were named "DJ-1 knockdown cells" (19Miyazaki S. Yanagida T. Nunome K. Ishikawa S. Inden M. Kitamura Y. Nakagawa S. Taira T. Hirota K. Niwa M. Iguchi-Ariga S.M. Ariga H. J. Neurochem. 2008; 105: 2418-2434Crossref PubMed Scopus (54) Google Scholar). SH-SY5Y cells harboring the vector without short hairpin RNA were also established and named "Vector cells." Expression levels of DJ-1 and TH mRNAs in parental SH-SY5Y, DJ-1 knockdown and vector cells were examined by semi-quantitative RT-PCR (Fig. 1). β-Actin was used as a loading control. As reported previously (16Zhong N. Kim C.Y. Rizzu P. Geula C. Porter D.R. Pothos E.N. Squitieri F. Heutink P. Xu J. J. Biol. Chem. 2006; 281: 20940-20948Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), the level of TH mRNA in DJ-1 knockdown cells was significantly reduced after DJ-1 expression had been knocked down by short hairpin RNA to ∼30% to the level of DJ-1 in parental SH-SY5Y or Vector cells (Fig. 1A). To confirm this, quantitative RT-PCR analyses (real time-PCR) were carried out, and results showing 50% reduction of TH gene expression in DJ-1 knockdown cells were obtained (Fig. 1C). On the other hand, RT-PCR and real time-PCR analyses showed that expression of the DDC gene in DJ-1 knockdown cells was not reduced (Fig. 1, B and D, respectively). Corresponding to the mRNA expression levels of TH and DDC genes, the protein levels of TH and DDC were found to be reduced and not to be changed, respectively, in DJ-1 knockdown cells by Western blot analyses (Fig. 1, E and F). TH and DDC activities in DJ-1 knockdown cells were then examined. Although reduced TH activity was observed, DDC activity was also found to be reduced (Fig. 1, G and H). The same results as those for SH-SY5Y cells were obtained when human SK-N-SH cells were used (data not shown). To clarify whether inhibition of TH and DDC activities in DJ-1 knockdown cells was caused by reduced expression or by modification of enzymes, TH and DDC activities were divided by the expression levels of TH and DDC proteins (Fig. 1, I and J, respectively). The results showed that TH and DDC activities are reduced in DJ-1 knockdown cells, suggesting that a mechanism other than transcriptional regulation of the DDC gene by DJ-1 is responsible for the reduced activity of DDC in DJ-1 knockdown cells. Proteins extracted from SH-SY5Y cells were immunoprecipitated with an anti-DJ-1 antibody and nonspecific IgG, and precipitates were analyzed by Western blotting with anti-TH and anti-DDC antibodies (Fig. 2). The results showed that TH and DDC were co-immunoprecipitated with DJ-1, indicating complex formation of DJ-1 with TH and with DDC (Fig. 2, A and B). To determine whether the interaction of DJ-1 with TH and DDC is direct or indirect, pulldown assays were carried out. For these assays, purified GST-DJ-1 or GST alone was reacted with 35S-labeled TH or DDC that had been synthesized in vitro using a coupled transcription-translation system. The results clearly showed that GST-DJ-1, but not GST, bound to TH and DDC, indicating direct interaction of DJ-1 with TH and with DDC (Fig. 2, C and D). SH-SY5Y cells were then immunostained with anti-DJ-1 and anti-TH antibodies or with anti-DJ-1 and anti-DDC antibodies, and DJ-1 and TH or DDC were visualized by fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies. The results of merged images showed co-localization of DJ-1 with TH and with DDC in cells (Fig. 2, E and F). It was found that fluorescence patterns were completely or almost completely matched between DJ-1 and TH and between DJ-1 and DDC, respectively, indicating co-localization of DJ-1 with TH and with DDC. To examine a ternary complex among DJ-1, TH, and DDC, FLAG-tagged TH was transfected into SH-SY5Y cells. Forty eight h after transfection, proteins extracted from cells were immunoprecipitated with an agarose-conjugated anti-FLAG antibody or with IgG, and half of the precipitates were analyzed by Western blotting with anti-FLAG, anti-DJ-1, and anti-DDC antibodies. The other half of the precipitates was eluted with a FLAG peptide, and proteins eluted were further immunoprecipitated with an anti-DJ-1 antibody followed by Western blotting (supplemental Fig. 1A). Both DDC and DJ-1 were immunoprecipitated with the anti-FLAG antibody, indicating that FLAG-TH was associated either with DDC or with DJ-1. Double immunoprecipitation of proteins with anti-FLAG and anti-DJ-1 antibodies also precipitated DDC and DJ-1, indicating a ternary complex among DJ-1, TH, and DDC in SH-SY5Y cells. Pulldown assays in which purified GST-TH or GST alone was reacted with 35S-labeled DDC that had been synthesized in vitro were carried out to examine the direct interaction of TH with DDC. The results clearly showed that GST-TH, but not GST, bound to DDC, indicating direct interaction of TH with DDC (supplemental Fig. 1B). We then examined the effect of DJ-1 on TH and DDC activities. To do that, purified GST-DJ-1 and GST were reacted with proteins extracted from SH-SY5Y cells, which are enzyme sources for TH and DDC, in the presence of tyrosine or l-DOPA, substrates for TH and DDC, respectively, and the quantity of l-DOPA or dopamine was measured (Fig. 2, G and H). The results clearly showed that GST-DJ-1, but not GST, stimulated TH and DDC activities in a dose-dependent manner. To determine the specificity of this reaction, protein extracts were first reacted with an anti-TH antibody, anti-DDC antibody, or IgG, and TH and DDC activities were then measured in the presence of purified GST-DJ-1. The results clearly showed that the anti-TH or anti-DDC antibody, but not IgG, almost completely inhibited the enzyme activities (supplemental Fig. 2, A and B). When proteins extracted from DJ-1 knockdown SH-SY5Y cells were used as enzyme sources, TH and DDC activities lower than those using proteins from parental SH-SY5Y cells were observed in the presence of various concentrations of GST-DJ-1, suggesting that, in addition to exogenously added GST-DJ-1, DJ-1 present in protein extracts from cells also affected enzyme activities of TH and DDC (supplemental Fig. 2, C and D). To distinguish the effect of DJ-1 on activities of DJ-1-bound TH/DDC or DJ-1-unbound TH/DDC, proteins extracted from SH-SY5Y cells were mixed with GST, GST-DJ-1, or GST-L166P-DJ-1 and applied to glutathione-Sepharose columns. DJ-1-bound (pull-down) and DJ-1-unbound proteins (flow-through) were then separated (Fig. 2I). Almost all of the GST-DJ-1 and GST-L166P-DJ-1 were found to bind to glutathione-Sepharose columns under this condition, and almost equal amounts of TH or DDC were obtained as DJ-1-bound and DJ-1-unbound forms. When activities of DJ-1-bound and DJ-1-unbound forms of TH and DDC were measured, it was found that wild-type DJ-1-bound TH and DDC gave high activities but that L166P DJ-1-bound TH and DDC or DJ-1-unbound TH and DDC gave low or no activities, respectively (Fig. 2, J and K). These results clearly show that direct binding of DJ-1 to TH and DDC stimulates their activities. As it is known that the activity of TH is affected by its phosphorylation, the phosphorylation level of TH at serine 19 in the reaction mixture used in the experiment for which results are shown in Fig. 2G was examined. No change in the phosphorylation level of TH was found, suggesting that DJ-1 stimulates TH activity without modulation of TH protein (Fig. 2L). Because several mutations in the DJ-1 gene have been found in PD patients whose TH or DDC activity is supposed to be reduced, we examined the effects of mutations of DJ-1 on TH and DDC activities. M26I, E64D, and L166P DJ-1 are derived from homozygous mutations, and R98Q and D149A DJ-1 are from heterozygous mutations of the DJ-1 gene. Because oxidation of DJ-1 at Cys-106 is important for DJ-1 to exert its functions (24Takahashi-Niki K. Niki T. Taira T. Iguchi-Ariga S.M. Ariga H. Biochem. Biophys. Res. Commun. 2004; 320: 389-397Crossref PubMed Scopus (148) Google Scholar), C106S DJ-1 was also used. We first examined binding activities of these DJ-1 mutants to TH or DDC by in vitro pulldown assays. All of the mutants and wild-type DJ-1 were found to bind to TH and DDC, although there were some variations in binding activity of DJ-1 mutants to TH and DDC (Fig. 3, A and B). Effects of DJ-1 mutants on TH and DDC activities were then examined using the in vitro system. Although wild-type DJ-1 stimulated TH activity, activity to TH was not stimulated by any of the mutants (Fig. 3C, see lanes in which 20 ng of GST-mutant DJ-1 was added to mixtures). Furthermore, all of the DJ-1 mutants inhibited activity that had been obtained by wild-type DJ-1 in a dose-dependent manner. It is notable that when the same amount of mutant DJ-1 as that of wild-type DJ-1 was present in the mixture, activity of less than half of that obtained by wild-type DJ-1 alone was observed (Fig. 3C, see lanes in which 20 ng each of wild-type and mutant of DJ-1 was added), indicating that all of the mutants, even mutants from heterozygous mutations of the DJ-1 gene, acted as a dominant-negative form against wild-type DJ-1. In the case of DDC, on the other hand, all of the mutants of DJ-1 were found to have weak stimulating activity toward DDC compared with the activity of wild-type DJ-1 and to inhibit the activity of wild-type DJ-1 without a dominant-negative effect (Fig. 3D). To examine the effect of mutation of DJ-1 on TH and DDC activities in cells, SH-SY5Y cells expressing FLAG-tagged wild-type and mutants of DJ-1, M26I, L166P, and C106S were established, and expression levels of introduced DJ-1 mutants and endogenously expressing wild-type DJ-1 were analyzed by Western blotting with anti-FLAG and anti-DJ-1 antibodies, respectively (Fig. 4, C and D). M26I DJ-1 and L166P DJ-1 were found to be expressed lower than that of endogenous mouse DJ-1, and wild-type DJ-1 and C106S DJ-1 were expressed at higher levels than that of endogenous mouse DJ-1. First, the expression levels of TH and DDC mRNA in these cell lines were examined by semi-quantitative PCR (data not shown) and quantitative PCR (real time-PCR) (Fig. 4, A and B). Intensity of bands in RT-PCR was quantified (supplemental Fig. 3). Although the expression levels of DDC mRNAs in cells expressing wild-type and mutants of DJ-1 were not significantly changed, the levels of TH mRNA in cells expressing wild-type DJ-1 and mutants of DJ-1 were increased and decreased, respectively, compared with the levels in cells harboring the vector alone or parental SH-SY5Y cells (host). Expression levels of TH and DDC proteins were parallel to t
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