Department Physiological Chemistry, Fujita Health University, School of Medicine, Toyoake, Aichi, Japan.
Division of Biomedical Polymer Science, Institute of Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan.
Biochem Biophys Res Commun. 2019 Sep 3;516(4):1060-1065. doi: 10.1016/j.bbrc.2019.06.156. Epub 2019 Jul 3.
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.
酪氨酸羟化酶（TH）催化 l-酪氨酸转化为 l-DOPA，是儿茶酚胺生物合成中的限速酶。众所周知，α-突触核蛋白和 14-3-3 蛋白家族成员都与 TH 分子结合，并通过激酶调节其 N 端的磷酸化来控制催化活性。在本研究中，我们研究了除这两种蛋白之外，其他蛋白是否也可能与 TH 分子结合。纳升液相色谱-串联质谱（nano-LC-MS/MS）分析显示，5'-核苷酸酶结构域包含蛋白 2（NT5DC2）属于卤酸脱卤酶（HAD）磷酸酶家族，在与 Dynabeads 蛋白 G-抗 TH 抗体缀合物反应的 PC12D 细胞裂解物的免疫沉淀物中被检测到。令人惊讶的是，NT5DC2 已经通过全基因组关联研究（GWAS）被揭示为与神经精神疾病（如精神分裂症、双相情感障碍）相关的基因，这些疾病与大脑中多巴胺活性异常有关，尽管 NT5DC2 在这些疾病中的作用仍不清楚。因此，我们研究了 NT5DC2 对 TH 分子的影响。通过 siRNA 下调 NT5DC2 可增加 PC12D 细胞中儿茶酚胺（多巴胺、去甲肾上腺素和肾上腺素）的合成。这些增加可能归因于 TH 的催化活性，而不是 TH 的细胞内稳定性，因为 Western blot 评估的 TH 细胞内含量并未因 NT5DC2 的下调而改变。综上所述，我们的结果表明 NT5DC2 通过降低 TH 的酶活性来抑制多巴胺的合成。
