Analysis of the Tetrahydrobiopterin Biosynthesizing System in Cultured Neuron-Derived Cells

培养的神经元来源细胞中四氢生物蝶呤生物合成系统的分析

• 批准号: 10670050
• 负责人: OTA Akira
• 金额: $ 2.11万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670050/
• 关键词: NIE-115 cell line; 6R-tetrahydrobjopterin; lipopolysaccharide; GTP cyclohydrolase "I"; CD14; CD18; Toll-like receptor; tumor necrotizing factorα; リポポリサッカライド受容体; N1E-115細胞; IκB; IκBキナーゼ; 一酸化窒素; 一酸化窒素合成酵素

6R-Tetrahydrobiopterin (BH4) is a naturally occurring essential cofactor for aromatic amino acid hydroxylases and nitric oxide (NO) synthase (NOS). Lipopolysaccharide(LPS), a component of the outer membrane of gram-negative bacilli, has been shown to enhance the BH4 content of the murine neuroblastoma cell line N1E-115 as well as the release of BH4 into culture medium. Such alterations in BH4 production were at first caused by the enhancement of the expression level of RNA encoding GTP cyclohydrolase "I"(GTPCH), a rate-limiting enzyme in the BH4 de novo biosynthetic pathway. Moreover, the expression levels of mRNAs encoding 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase which place at the 2nd and the final step in BH4 biosynthesic pathway, respectively. However, we found no evidence in N1E-115 cells showing the expression of mRNA encoding CD14. CD14 is an membrane-anchoring protein and it is already recognized as the LPS receptor on macrophages. Therefore we searched the candidates for LPS receptor except for CD14 in N1E-115 cells. In a series of experiments, we found the expressions of mRNA encoding CD18, another subtype of LPS receptor on macrophages, and type 4 Toll-like receptor (T1r-4) in this cell line by way of reverse transcriptase-polymerase chain reaction. This should be the first evidence that neuron-derived cell line expresses T"I"r-4..Tumor necrotizing factor α(TNF-α) was also capable of enhancing BH4 production in N1E-115 cells. It is currently under investigation in my laboratory whether the increase in BH4 production induced by TNF-α was caused by the activation of the pathway common to the LPS stimulation.In spite of the increase in BH4 production by LPS stimulation. we could detect neither the increase in NO production nor the increase in the expression levels of mRNAs encoding neuronal and inducible NOS in N1E-115 cells. NOS enzymes in these cell lines were speculated to be saturated by BH4.

6R-四氢生物蝶呤 (BH4) 是芳香族氨基酸羟化酶和一氧化氮 (NO) 合酶 (NOS) 的天然必需辅助因子，脂多糖 (LPS) 是革兰氏阴性杆菌外膜的成分，已被证明可以增强芳香族氨基酸羟化酶和一氧化氮 (NO) 合酶 (NOS) 的功能。小鼠神经母细胞瘤细胞系 N1E-115 的 BH4 含量以及 BH4 释放到培养基中。 BH4 产生的这种变化首先是由编码 GTP 环化水解酶“I”(GTPCH) 的 RNA 表达水平的增强引起的，GTPCH 是 BH4 从头生物合成途径中的限速酶，此外，编码 6 的 mRNA 的表达水平也增强。 -丙酮酰四氢蝶呤合酶和墨蝶呤还原酶，分别位于BH4生物合成途径的第二步和最后一步。然而，我们在 N1E-115 细胞中没有发现编码 CD14 的 mRNA 表达的证据，CD14 是一种膜锚定蛋白，并且它已被识别为巨噬细胞上的 LPS 受体，因此我们在 N1E-115 细胞中寻找除 CD14 之外的候选 LPS 受体。在一系列实验中，我们发现N1E-115细胞中编码CD18（LPS受体的另一种亚型）和4型Toll样受体的mRNA表达。 (T1r-4) 在该细胞系中通过逆转录酶-聚合酶链反应的方式这应该是神经元来源的细胞系表达 T"I"r-4..肿瘤坏死因子 α(TNF-α) 的第一个证据。还能够增强 N1E-115 细胞中的 BH4 产量 目前我的实验室正在研究 TNF-α 诱导的 BH4 产量增加是否是由 LPS 常见途径的激活引起的。尽管LPS刺激增加了BH4的产生，但我们在这些细胞系中既没有检测到NO产生的增加，也没有检测到编码神经元和诱导型NOS酶的mRNA表达水平的增加。推测被 BH4 饱和。

项目成果

Ota M et al.: "Risperidone, an atypical antipsychotic, affects the mRNA expressions of monoamine oxidase type B and vesicular monoamine transporter-2 in rat brain"Biogenic Amines. 15・2. 197-216 (1999)

Ota M 等人：“利培酮，一种非典型抗精神病药，影响大鼠脑中 B 型单胺氧化酶和囊泡单胺转运蛋白 2 的 mRNA 表达”Bioogenic Amines 15·2 (1999)。

Nakashima A et al.: "Positive charge intrinsic to ArgィイD137ィエD1-ArgィイD138ィエD1 is critical for dopamine inhibition of the catalytic activity of human tyrosine hydroxylase type 1"FEBS Letters. 465-1. 59-63 (2000)

Nakashima A 等人：“ArgyD137D1-ArgyD138D1 固有的正电荷对于多巴胺抑制人酪氨酸羟化酶 1 型的催化活性至关重要”FEBS Letters 465-1 (2000)。

Akira Nakashima et al.: "Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme" Journal of Neurochemistry. in press. (1999)

Akira Nakashima 等人：“多巴胺对 1 型人酪氨酸羟化酶的抑制是由该酶 N 末端的特定部分控制的”《神经化学杂志》。

Nakashima A et al.: "Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme"Journal of Neurochemistry. 72・5. 2145-215* (1999)

Nakashima A 等：“人酪氨酸羟化酶 1 型的多巴胺抑制是由酶 N 末端的特定部分控制的”Journal of Neurochemistry 72·5* (1999)。

Nakashima A et al.: "Expression of human tyrosine hydroxylase type I in Escherichia coli as a protease-cleavable fusion protein. Short communication"Journal of Neural Transmission. 106・9/10. 819-824 (1999)

Nakashima A等人：“人酪氨酸羟化酶I型在大肠杆菌中作为蛋白酶可裂解的融合蛋白的表达。短通讯”106·9/10（1999）。