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.

利用体外突变试剂盒对cystatinA基因进行了氨基酸置换，获得了cystatinA基因的野生型和突变型。这些产物通过转氨酶相互交联，然后通过SDS-PAGE电泳。只有一个产物，即在44个氨基酸处由赖氨酸取代为苏氨酸，不能交联。接下来，将这些基因导入人角质形成细胞。然后用钙离子载体诱导角质形成细胞形成角质形成细胞膜。在角质形成细胞内可见Lys 44 Thr基因诱导的cystatinA掺入。本研究表明，44个氨基酸的赖氨酸是重要的交联细胞膜。

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。