On the Possible Control of Allergic Inflammation based on Molecular Mechanism of Serotonin Synthesizing Enzyme

基于血清素合成酶分子机制探讨过敏性炎症的可能控制

• 批准号: 16591872
• 负责人: HASEGAWA Hiroyuki
• 金额: $ 2.34万
• 依托单位: Teikyo University of Science & Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16591872/
• 关键词: Tryptopha hydroxylase; Serotonin biosynthesis; Enzyme regulation by phosphorylation; Ubiquitination; Proteasomes; Site-directed mutagenesis; RBL2H3; トリプトファン水酸化酵素抗体; 部位特異的突然変異

Tryptophan hydroxykase, the rate limiting enzyme in serotonin biosynthesis, exists in two isoforms. In 2003, TPH2 gene was identified and the gene product was named TPH2 while the long known gene product was newly referred to TPH1. The level of tryptophan hydroxylase in cells of RBL2H3, a mast-cell line, was shown to be under very rapid molecular turnover driven by the Ubiquitin-Proteasome system in the cell. The degradation process is triggered by protein phosphorylation of the enzyme. The phosphorylation site(s) responsible for triggering is(are) not known. In this study, three major progresses were achieved: 1)Polyclonal mono-specific antibodies were raised against TPH1 and TPH2. By the use of these antibodies, dominant distribution of TPH2 in the brain and TPH1 in the peripheral tissues were confirmed. However, we found that TPH1 was also detected in the brain and TPH2 in the non-neural cells in the periphery. Further, we located TPH1 in the intestinal absorption epithelia which had been believed to lack in the hydroxylase. 2) We reported previously that in vitro immune stimulation to RBL2H3 cells with IgE-antigen complex, triggered a rapid and prominent induction of TPH1. In this study, it was demonstrated that NF-kB pathway was involved in the signal transduction of the TPH1-induction. 3) Variant TPH1 vectors were developed of which 3 serine residues (S58, S260, and S443) on the protein surface were replaced with either alanine or glutamic acid by means of site-directed mutagenesis. The variants were all active when transected to HeLa cells, a cell line lack in TPH. Currently, the variants are under examination whether they are susceptible to the molecular degradation in RBL2H3 cells which are furnished with the all the required Ubiquitin-Proteasome system specific to the wild type TPH1.

色氨酸羟化酶是5-羟色胺生物合成的限速酶，有两种亚型。2003年，TPH 2基因被鉴定，其基因产物被命名为TPH 2，而已知已久的基因产物被重新命名为TPH 1。肥大细胞系RBL 2 H3细胞中色氨酸羟化酶的水平被证明在细胞中由泛素-蛋白酶体系统驱动的非常快速的分子周转下。降解过程由酶的蛋白质磷酸化引发。负责触发的磷酸化位点未知。本研究主要取得了三个方面的进展：1）成功制备了抗TPH 1和TPH 2的多克隆抗体。通过使用这些抗体，确认了TPH 2在脑中的优势分布和TPH 1在外周组织中的优势分布。然而，我们发现TPH 1也在脑中检测到，TPH 2在外周的非神经细胞中检测到。此外，我们将TPH 1定位在肠吸收上皮细胞中，该上皮细胞被认为缺乏羟化酶。2)我们以前报道，在体外免疫刺激RBL 2 H3细胞与IgE抗原复合物，触发了快速和突出的诱导TPH 1。本研究证实NF-κ B通路参与了TPH 1诱导的信号转导过程。3)开发了变体TPH 1载体，其中蛋白质表面上的3个丝氨酸残基（S58、S260和S443）通过定点诱变被丙氨酸或谷氨酸取代。当横切到HeLa细胞（TPH中缺乏的细胞系）时，变体都是有活性的。目前，正在检查变体是否对RBL 2 H3细胞中的分子降解敏感，所述RBL 2 H3细胞配备有对野生型TPH 1特异性的所有必需的泛素-蛋白酶体系统。

项目成果

Delivery of exogenous tetrahydrobiopterin (BH4) to cells of target organs:: Role of salvage pathway and uptake of its precursor in effective elevation of tissue BH4

• DOI: 10.1016/j.ymgme.2005.09.002
• 发表时间: 2005-12-01
• 期刊: MOLECULAR GENETICS AND METABOLISM
• 影响因子: 3.8
• 作者: Hasegawa, H;Sawabe, K;Wakasugi, OK
• 通讯作者: Wakasugi, OK

Increase in tetrahydrobiopterin release from PC12 cells under hypotonic culture conditions is inhibited by HgCl_<2->

HgCl_<2-> 抑制低渗培养条件下 PC12 细胞四氢生物蝶呤释放的增加

• 发表时间: 2005
• 期刊: Pteridines 16
• 作者: Nakanishi;N.
• 通讯作者: N.

Cellular accumulation of tetrahydrobiopterin following its administration is mediated by two different processes; direct uptake and indirect uptake mediated by a methotrexate-sensitive process.

• DOI: 10.1016/j.ymgme.2005.06.020
• 发表时间: 2005-12
• 期刊: Molecular genetics and metabolism
• 影响因子: 3.8
• 作者: K. Sawabe;Yasuko Suetake;N. Nakanishi;K. Wakasugi;H. Hasegawa

Intestinal uptake of 6(R)-L-erythro-tetrahydrobioterin is distinctive from liver-type MTX-sensitive accumulation process

6(R)-L-赤型四氢生物素的肠道摄取不同于肝型 MTX 敏感的积累过程

• 发表时间: 2005
• 期刊: Pteridines 16
• 作者: Hasegawa;H.;A. Ichiyama;Hasegawa Hiroyuki;Hasegawa Hiroyuki;Nakanishi Nobuo;Sawabe Keiko;Sawabe Keiko;Keiko Sawabe
• 通讯作者: Keiko Sawabe

「研究成果報告書概要(和文)」より

摘自《研究结果报告摘要（日文）》

• 发表时间: 2005
• 作者: Kawauchi;et. al.;Nishimura et al.;Dezawa et al.;Yoshizawa et al.;星野 幹雄;星野 幹雄
• 通讯作者: 星野 幹雄