Mechanism and role of croslinking to cell membrane of cystatin A

半胱氨酸蛋白酶抑制剂A与细胞膜交联的机制和作用

• 批准号: 13670860
• 负责人: TAKAHASHI Hidetoshi
• 金额: $ 2.18万
• 依托单位: Asahikawa Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670860/
• 关键词: cystatin A; cornified envelope; 44Lys; cross-linking; keratinocyte; substitution; mutation; differentiation; GSH; GFP; 皮膚; 遺伝子調節; MAPK; ERK; JNK

We produced wild and mutant cystatin A gene, which were substituted the amino acids by in vitro mutagenesis kit. These products were cross-linked each other by the transglutaminase, then were electrohoresed by SDS-page. Only one product, which was substituted Lys at 44 amino acid to Thr, could not cross-link. Next these genes were introduced into the human keratinocytes. Then the keratinoctes were induced to form cornified cell envelope by calcium ionophore. We could observe the incorporation of cystatin A into inner surface of keratinocytes, which was induced Lys44Thr gene. The present study demonstrated that Lys at 44 amino acid is important for cross-linkinking to cell membrane.

我们获得了野生型和突变型的cystatin A基因，并用体外诱变试剂盒替代了氨基酸。这些产物通过谷氨酰胺转氨酶相互交联，然后用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离。只有一种产物不能交联，即44位氨基酸的赖氨酸被取代为苏氨酸。接下来，将这些基因导入人类角质形成细胞。然后用钙离子载体诱导角化细胞形成角化细胞被膜。可观察到Lys44Thr基因诱导的角质形成细胞内表面掺入半胱氨酸氨基转移酶A。本研究表明，44个氨基酸的赖氨酸在与细胞膜的交联中起着重要作用。

项目成果

H.Takahashi et al.: "Expression of human cystatin A by keratinocytes is positively regulated via Ras/ERK/MEKK1/MKK7/JNKsinnal transduction pathway but negatively"J.Biol Chem. 276. 36632-36638 (2001)

H.Takahashi 等人：“角质形成细胞对人胱抑素 A 的表达受到 Ras/ERK/MEKK1/MKK7/JNKsinnal 转导途径的正向调节，但受到负向调节”J.Biol Chem。

1,25-dihydroxyvitaminD3 increases human cystati A expression by in hibiting the Raf-1/MEK1/ERK signaling pathway of keratinocytes.

1,25-diHydroxyvitaminD3 通过抑制角质形成细胞的 Raf-1/MEK1/ERK 信号通路来增加人cysati A 表达。

• 发表时间: 2003
• 期刊: Arch Dermatol Res 295
• 作者: Takahashi H

H.Takahashi et al.: "Aberrant expression of apoptosis-related molecules psoriatic epidermis"J Dermatol Scie. 28. 187-197 (2002)

H.Takahashi 等：“银屑病表皮细胞凋亡相关分子的异常表达”J Dermatol Scie。

H.Takahashi et al.: "1,25-dihydroxyvitamin D(3) increases human cystatin A expression by inhibiting the Raf-1/MEK1/ERK signaling pathway of keratinocytes"Arch Dermatol Res. 295. 80-87 (2003)

H.Takahashi 等人：“1,25-二羟基维生素 D(3) 通过抑制角质形成细胞的 Raf-1/MEK1/ERK 信号通路来增加人半胱氨酸蛋白酶抑制剂 A 的表达”Arch Dermatol Res。

H.Takahashi et al.: "Similary potent action of 1,25-dihydroxyvitamin D3 and its analogues, tacalcitol, calcipotriol, and maxacalcitol on normal human keratinocyte proliferation and differentiation"J Dermatol Scie. (in press).

H.Takahashi 等人：“1,25-二羟基维生素 D3 及其类似物、他骨化醇、卡泊三醇和马沙骨化醇对正常人角质形成细胞增殖和分化的类似有效作用”J Dermatol Scie。