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

Abstract

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 H(2)O(2), 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 SO(2)H and SO(3)H 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.

FIGURE 1.

Effects of DJ-1 knockdown on expression and activity of TH and DDC. SH-SY5Y, SH-SY5Y cells with knockdown of DJ-1 expression (DJ-1 knockdown cells), and SH-SH5Y cells harboring pcDNA3 vector (Vector cells) were used. DJ-1-KD and Vector in the figures indicate DJ-1 knockdown cells and vector cells, respectively. A and B, expression levels of mRNA of TH (A), DDC (B), and DJ-1 were analyzed by RT-PCR using specific primers to respective genes and total RNAs as templates as described under “Experimental Procedures.” β-Actin was used as a loading control. C and D, expression levels of mRNA of TH (C) and DDC (D) were analyzed by real time-PCR as described under “Experimental Procedures.” Relative expression of TH and DDC to β-actin is presented. E and F, expression levels of TH (E) and DDC (F) and DJ-1 in cells were analyzed by Western blotting with anti-TH, anti-DDC, and anti-DJ-1 antibodies, respectively, as described under “Experimental Procedures.” β-Actin was used as a loading control. G and H, enzyme activity of TH (G) and DDC (H) in cells was examined as described under “Experimental Procedures.” I and J, enzyme activity of TH (I) and DDC (J) in G and H was divided by the expression level of TH and DDC described in E and F, respectively. Values are mean S.E. ± from three independent experiments. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001. N.S. means nonspecific.

FIGURE 2.

Stimulation of TH and DDC activities by DJ-1. A and B, proteins extracted from SH-SY5Y cells were immunoprecipitated (IP) with a rabbit anti-DJ-1 polyclonal antibody or IgG, and precipitates were analyzed by Western blotting with an anti-TH (A) or anti-DDC (B) antibody and a mouse anti-DJ-1 monoclonal antibody (3E8, MBL, Nagoya, Japan). C and D, GST or GST-DJ-1 was expressed in and prepared from E. coli

FIGURE 3.

Effects of mutations of DJ-1 on TH and DDC activities in vitro. A and B, GST, GST-wild-type DJ-1, or GST mutants of DJ-1 were expressed in and prepared from and reacted with ³⁵S-labeled HA-TH (A) or T7-DDC (B) that had been synthesized in vitro using a coupled transcription-translation system (Promega). After the reaction, the mixture was subjected to pulldown assays as described under “Experimental Procedures.” CBB, Coomassie Brilliant Blue. C and D, effects of wild-type (wt) DJ-1 and various mutants of DJ-1 on TH (C) and DDC (D) activities were examined in vitro as described in the legends of Fig. 1, G and H. Values are mean ± S.E. from three independent experiments. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with an amount of “no protein,” and #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with the amount of “wt:wt = 20:20.” N.S. means nonspecific.

FIGURE 4.

Effects of mutations of DJ-1 on TH and DDC activities in vivo. SH-SY5Y cells (host), SH-SY5Y cells harboring pcDNA3 vector (vector), and SH-SY5Y cells expressing wild-type (wt) or various mutants of DJ-1 were used. A and B, expression levels of mRNA of TH (A) and DDC (B) genes were analyzed by real time-PCR as described in the legends of Fig. 1, C and D. Relative expression of TH and DDC to β-actin is shown. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with an amount of host, and #, p < 0.05; ##, p < 0.01; and ###, p < 0.001 compared with the amount of vector. C and D, expression levels of TH, phosphorylated TH with a serine residue at amino acid number 19 (pTH ser 19), FLAG-DJ-1, endogenous DJ-1, and β-actin were analyzed as described in the legends of Fig. 1, E and F, and Fig. 2L. E, proteins extracted from SH-SY5Y cells expressing wild-type (wt) or various mutants of DJ-1 were immunoprecipitated (IP) with an anti-FLAG antibody or with nonspecific (N.S.) IgG, and precipitates were analyzed by Western blotting with anti-FLAG, anti-TH, and anti-DDC antibodies. F and G, TH and DDC activities in cells were measured as described in the legends of Fig. 1, G and H. Significance: *, p < 0.05; **, p < 0.01 compared with an amount of host, and #, p < 0.05; ##, p < 0.01 compared with the amount of vector. H and I, SH-SY5Y cells were transfected with various amounts of expression vectors for FLAG-tagged wild-type DJ-1 and mutants of DJ-1. Forty two h after transfection, cells were cultures in the presence of l-tyrosine and DDC inhibitor or in the presence of l-DOPA for 6 h, and then their TH and DDC activities were measured as described under “Experimental Procedures.” Values are mean ± S.E. from three independent experiments. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with an amount of substrate + lysate. J, proteins were extracted from SH-SY5Y cells described in H and J and analyzed with Western blotting with anti-FLAG and anti-DJ-1 antibodies.

FIGURE 5.

Effect of oxidation of cells on TH and DDC activities in vivo. A, SH-SY5Y cells were exposed to various concentrations of H₂O₂, 6-OHDA, and MPP⁺ for 1 h, and proteins were extracted from the cells. Proteins in cell extracts were then immunoprecipitated with an anti-DJ-1 antibody and separated on an SDS-containing polyacrylamide gel. After staining proteins in gels, a protein band corresponding to DJ-1 was subjected to TOF-MS analysis as described under “Experimental Procedures.” Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with an amount of “0 μm”. B and C, expression levels of mRNA of DJ-1 and TH in SH-SY5Y cells that had been exposed to various concentrations of H₂O₂, 6-OHDA, and MPP⁺ for 1 h were by real time-PCR (B and C) as described in the legends of Fig. 1, A–D. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with an amount of 0 μm. D, SH-SY5Y cells were transfected with a TH gene promoter-luciferase construct. At 46 h after transfection, cells were exposed to various concentrations of H₂O₂, 6-OHDA, and MPP⁺ for 1 h, and their luciferase activity was measured. Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with an amount of 0 μm. E, expression levels of TH, DDC, DJ-1, and β-actin in cells exposed to H₂O₂, 6-OHDA, and MPP⁺ were analyzed as described in the legends of Fig. 1, E and F. F, TH and DDC activities in cells exposed to H₂O₂, 6-OHDA, and MPP⁺ were measured as described in the legends of Fig. 1, G and H. Significance: *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with an amount of “0 μm”. G, GPDH activity in SH-SY5Y cells exposed to H₂O₂, 6-OHDA, and MPP⁺ for 2 h was measured according to the manufacturer's protocol (MK426, Takara Bio, Kyoto, Japan). Significance: N.S. (nonspecific) compared with an amount of DMSO or substrate + lysate. Values are mean S.E. ± from three independent experiments. H, SH-SY5Y cells were transfected with expression vectors for FLAG-tagged wild-type DJ-1, C106S DJ-1, and L166P DJ-1. Forty six h after transfection, cells were exposed to 200 μm H₂O₂ for 2 h, and their GPDH activity was measured as described in G. Significance: N.S. (nonspecific) compared with an amount of substrate + lysate. Values are mean S.E. ± from three independent experiments.

FIGURE 6.

Effect of oxidation of DJ-1 on TH and DDC activities in vitro. A, 1 μg of recombinant DJ-1 was reacted with various concentrations of H₂O₂ for 10 or 30 min, dialyzed against 1× PBS (−) containing 137 mm NaCl, 2.7 mm KCl, 8 mm Na₂HPO₄, and 1.5 mm KH₂PO₄, digested with trypsin, and subjected to TOF-MS analysis as described under “Experimental Procedures.” B, half of DJ-1 as described in A was mixed with extracts from SH-SY5Y cells, l-tyrosine, and a DDC inhibitor for TH assays or with extracts from SH-SY5Y cells and l-DOPA for DDC assays. Fifteen min after the reaction, the amount of l-DOPA or dopamine was measured using HPLC as described under “Experimental Procedures.”