Mutant enzymes of tyrosine hydroxylase for an effective gene therapy of PD

酪氨酸羟化酶突变酶用于帕金森病的有效基因治疗

• 批准号: 14580752
• 负责人: NAKASHIMA Akira
• 金额: $ 1.34万
• 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: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580752/
• 关键词: tyrosine hydroxylase; mutant; Parkinson's disease; gene therapy; feedback inhibition

Several studies have suggested that the direct virus vector-mediated delivery of transgenes of tyrosine hydroxylase (TH) involved in atecholamine synthesis in vivo may be available for gene therapy of Parkinson's disease (PD). However, TH expressed in mammalian cells cannot produce efficiently L-dopa, because the catalytic activity of TH is inhibited by the end-products catecholamines accumulated in the cells. The purpose of our study is to produce TH with high capability of L-dopa synthesis in the mammalian cells for an effective gene therapy of PD. We produced the mutant enzymes of human TH type1 (hTH1) and then examined the characteristic of them. 1)The replacement of N-terminal residues 30-40 of hTH1 by neutral or negatively charged residues decreased the inhibitory effect of dopamine on the catalytic activity. 2)Especially, the replacement of Arg^<37> -Arg^<38> and Ser^<40> of hTH1 by negatively charged Glu or Asp gave an efficient production of DA in vitro. 3)The efficient production of DA by the mutant enzymes was detected in AtT-20 neuroendcrine cells. 4)Moreover, the mutant enzymes of hTH1 revealed a high stability in other mammalian cell lines in addition to AtT-20 cells. Collectively, our study provides useful information for the refinement of the gene-therapy approach to obtain increased production of DA in vivo.

研究表明，病毒载体介导的酪氨酸羟化酶（TH）基因的直接递送可能用于帕金森病（PD）的基因治疗。然而，在哺乳动物细胞中表达的TH不能有效地产生L-多巴，因为TH的催化活性被细胞中积累的终产物儿茶酚胺抑制。本研究的目的是在哺乳动物细胞中产生具有高合成左旋多巴能力的TH，为PD的基因治疗提供有效的载体。我们制备了人TH 1型的突变体酶（hTH 1），并对其特性进行了研究。1)用中性或带负电荷的残基取代hTH 1的N-末端残基30-40降低多巴胺对催化活性的抑制作用。（2）用带<37><38><40>负电荷的Glu或Asp取代hTH 1的Arg^ -Arg^和Ser^，能有效地在体外合成DA。3)在AtT-20神经内分泌细胞中检测到突变酶的DA的有效生产。4）此外，除了AtT-20细胞外，hTH 1的突变酶在其他哺乳动物细胞系中显示出高稳定性。总的来说，我们的研究提供了有用的信息，完善的基因治疗方法，以获得增加生产的DA在体内。

项目成果

Akira Nakashima, et al.: "Mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells"Journal of Neurochemistry. 82・1. 202-206 (2002)

Akira Nakashima 等人：“酪氨酸羟化酶 (TH) N 末端的两个氨基酸残基的突变显着增强了神经内分泌 AtT-20 细胞的催化活性”《神经化学杂志》82・1（2002 年）。 ）

Akira Nakashima, et al.: "Mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells."Journal of Neurochemistry. 82. 202-206 (2002)

Akira Nakashima 等人：“酪氨酸羟化酶 (TH) N 末端两个氨基酸残基的突变可显着增强神经内分泌 AtT-20 细胞的催化活性。”神经化学杂志。

Akira Nakashima, et al.: "Interactions between Egr1 and AP1 factors in regulation of tyrosine hydroxylase transcription."Molecular Brain Research. 112. 61-69 (2003)

Akira Nakashima 等人：“Egr1 和 AP1 因子在酪氨酸羟化酶转录调节中的相互作用。”分子脑研究。

Akira Nakashima, et al.: "Mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase(TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells."Journal of Neurochemistry. 82・1. 202-206 (2002)

Akira Nakashima 等人：“酪氨酸羟化酶 (TH) N 末端的两个氨基酸残基的突变显着增强了神经内分泌 AtT-20 细胞的催化活性。”《神经化学杂志》82・1（ 2002）

Akira Nakashima, Akira Ota, Ether L Sabban: "Interactions between Egr1 and Apt factors in regulation of tyrosine hydroxylase transcription."Molecular Brain Research. 112(1). 61-69 (2003)

Akira Nakashima、Akira Ota、Ether L Sabban：“Egr1 和 Apt 因子在酪氨酸羟化酶转录调节中的相互作用。”分子脑研究。