Abstract
Tyrosine hydroxylase (TH), which catalyzes the conversion of L-tyrosine to L-DOPA, is the rate-limiting enzyme in the biosynthesis of catecholamines. It is well known that both α-synuclein and 14-3-3 protein family members bind to the TH molecule and regulate phosphorylation of its N-terminus by kinases to control the catalytic activity. In this present study we investigated whether other proteins aside from these 2 proteins might also bind to TH molecules. Nano-LC-MS/MS analysis revealed that 5′-nucleotidase domain-containing protein 2 (NT5DC2), belonging to a family of haloacid dehalogenase-type (HAD) phosphatases, was detected in the immunoprecipitate of PC12D cell lysates that had been reacted with Dynabeads protein G-anti-TH antibody conjugate. Surprisingly, NT5DC2 had already been revealed by Genome-Wide Association Studies (GWAS) as a gene implicated in neuropsychiatric disorders such as schizophrenia, bipolar disorder, which are diseases related to the abnormality of dopamine activity in the brain, although the role that NT5DC2 plays in these diseases remains unknown. Therefore, we investigated the effect of NT5DC2 on the TH molecule. The down-regulation of NT5DC2 by siRNA increased the synthesis of catecholamines (dopamine, noradrenaline, and adrenaline) in PC12D cells. These increases might be attributed to the catalytic activity of TH and not to the intracellular stability of TH, because the intracellular content of TH assessed by Western blotting was not changed by the down-regulation of NT5DC2. Collectively, our results indicate that NT5DC2 inhibited the synthesis of dopamine by decreasing the enzymatic activity of TH.
| Original language | English |
|---|---|
| Pages (from-to) | 1060-1065 |
| Number of pages | 6 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 516 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 03-09-2019 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
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Identification by nano-LC-MS/MS of NT5DC2 as a protein binding to tyrosine hydroxylase : Down-regulation of NT5DC2 by siRNA increases catecholamine synthesis in PC12D cells. / Nakashima, Akira; Yamaguchi, Hisateru; Kodani, Yu; Kaneko, Yoko S.; Kawata, Miho; Nagasaki, Hiroshi; Nagatsu, Toshiharu; Ota, A.
In: Biochemical and Biophysical Research Communications, Vol. 516, No. 4, 03.09.2019, p. 1060-1065.Research output: Contribution to journal › Article
TY - JOUR
T1 - Identification by nano-LC-MS/MS of NT5DC2 as a protein binding to tyrosine hydroxylase
T2 - Down-regulation of NT5DC2 by siRNA increases catecholamine synthesis in PC12D cells
AU - Nakashima, Akira
AU - Yamaguchi, Hisateru
AU - Kodani, Yu
AU - Kaneko, Yoko S.
AU - Kawata, Miho
AU - Nagasaki, Hiroshi
AU - Nagatsu, Toshiharu
AU - Ota, A.
PY - 2019/9/3
Y1 - 2019/9/3
N2 - Tyrosine hydroxylase (TH), which catalyzes the conversion of L-tyrosine to L-DOPA, is the rate-limiting enzyme in the biosynthesis of catecholamines. It is well known that both α-synuclein and 14-3-3 protein family members bind to the TH molecule and regulate phosphorylation of its N-terminus by kinases to control the catalytic activity. In this present study we investigated whether other proteins aside from these 2 proteins might also bind to TH molecules. Nano-LC-MS/MS analysis revealed that 5′-nucleotidase domain-containing protein 2 (NT5DC2), belonging to a family of haloacid dehalogenase-type (HAD) phosphatases, was detected in the immunoprecipitate of PC12D cell lysates that had been reacted with Dynabeads protein G-anti-TH antibody conjugate. Surprisingly, NT5DC2 had already been revealed by Genome-Wide Association Studies (GWAS) as a gene implicated in neuropsychiatric disorders such as schizophrenia, bipolar disorder, which are diseases related to the abnormality of dopamine activity in the brain, although the role that NT5DC2 plays in these diseases remains unknown. Therefore, we investigated the effect of NT5DC2 on the TH molecule. The down-regulation of NT5DC2 by siRNA increased the synthesis of catecholamines (dopamine, noradrenaline, and adrenaline) in PC12D cells. These increases might be attributed to the catalytic activity of TH and not to the intracellular stability of TH, because the intracellular content of TH assessed by Western blotting was not changed by the down-regulation of NT5DC2. Collectively, our results indicate that NT5DC2 inhibited the synthesis of dopamine by decreasing the enzymatic activity of TH.
AB - Tyrosine hydroxylase (TH), which catalyzes the conversion of L-tyrosine to L-DOPA, is the rate-limiting enzyme in the biosynthesis of catecholamines. It is well known that both α-synuclein and 14-3-3 protein family members bind to the TH molecule and regulate phosphorylation of its N-terminus by kinases to control the catalytic activity. In this present study we investigated whether other proteins aside from these 2 proteins might also bind to TH molecules. Nano-LC-MS/MS analysis revealed that 5′-nucleotidase domain-containing protein 2 (NT5DC2), belonging to a family of haloacid dehalogenase-type (HAD) phosphatases, was detected in the immunoprecipitate of PC12D cell lysates that had been reacted with Dynabeads protein G-anti-TH antibody conjugate. Surprisingly, NT5DC2 had already been revealed by Genome-Wide Association Studies (GWAS) as a gene implicated in neuropsychiatric disorders such as schizophrenia, bipolar disorder, which are diseases related to the abnormality of dopamine activity in the brain, although the role that NT5DC2 plays in these diseases remains unknown. Therefore, we investigated the effect of NT5DC2 on the TH molecule. The down-regulation of NT5DC2 by siRNA increased the synthesis of catecholamines (dopamine, noradrenaline, and adrenaline) in PC12D cells. These increases might be attributed to the catalytic activity of TH and not to the intracellular stability of TH, because the intracellular content of TH assessed by Western blotting was not changed by the down-regulation of NT5DC2. Collectively, our results indicate that NT5DC2 inhibited the synthesis of dopamine by decreasing the enzymatic activity of TH.
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U2 - 10.1016/j.bbrc.2019.06.156
DO - 10.1016/j.bbrc.2019.06.156
M3 - Article
VL - 516
SP - 1060
EP - 1065
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -