Cloning of novel binding protein, which links Keratin filaments to the nuclear envelops.

克隆新型结合蛋白，将角蛋白丝与核膜连接起来。

• 批准号: 13670883
• 负责人: TAKAHASHI Kenzo
• 金额: $ 2.5万
• 依托单位: KYOTO UNIVERSITY (2002)Gunma University (2001)
• 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 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670883/
• 关键词: Keratin; unclear envelop; yeast two-hybrid; 酵母シーハイブリッド; ツーハイブリッド; ケラチン結合蛋白

In this study, we tried to clone and characterize proteins that interact with various keratins by yeast two-hybrid system. Employing the head and tail domains of epidermal type I and type II keratins as baits, we made several rounds of screening of the human skin cDNA. A novel protein termed okuribin was cloned as a type I keratin binding protein. Okuribin directly binds with the head domain of type I keratins K14, K16 and K18 but not with type II keratins. The okuribin mRNA in size of 4.1 kilo-nucleotides was expressed in the various epithelial tissues including skin as well as non-epithelial tissues like muscles. The okuribin protein predicted as a105 kDa protein encoded by 950 amino acids possessing eight nuclear localization signals as well as a signal peptides and one putative transmembrane segment. When the okuribin cDNA was fused with green fluorescence protein (GFP) or flag tag at its carboxyl terminus, and used for the forced expression into cultured keratinocytes, GFP and flag signals were observed predominantly along with the nuclear membrane. The endogenous okuribin in keratinocytes was observed mainly at the outer nuclear envelope, and occasionally in the nuclei depending on the maturation of the nuclear envelope verified by the staining intensity of lamin. The matured nuclei, where lamin and okuribin were observed strongly at the inner and outer envelop, tend to be tightly surrounded or enclosed by the keratin network in the immune-electron microscopy. From these data, okuribin is considered to act as a missing link connecting the keratin filaments and juxta-nuclear space.

在本研究中，我们试图通过酵母双杂交系统克隆和表征与多种角蛋白相互作用的蛋白。我们以表皮I型和II型角蛋白的头尾结构域为诱饵，对人皮肤cDNA进行了多轮筛选。克隆了一种名为okurbin的新型蛋白，作为I型角蛋白结合蛋白。Okuribin直接与I型角蛋白K14、K16和K18的头部结构域结合，而不与II型角蛋白结合。大小为4.1千核苷酸的okurbin mRNA在各种上皮组织（包括皮肤）和非上皮组织（如肌肉）中表达。okuribin蛋白被预测为一个105 kDa蛋白，由950个氨基酸编码，具有8个核定位信号、1个信号肽和1个推测的跨膜片段。当okurbin cDNA与绿色荧光蛋白（green fluorescence protein， GFP）或在其羧基末端的flag标签融合后，在培养的角质形成细胞中强制表达时，在核膜上主要观察到绿色荧光蛋白和flag信号。内源性okurbin在角质形成细胞中主要存在于外核膜，偶尔也存在于细胞核中，这取决于核膜的成熟程度，通过层粘胶蛋白的染色强度来验证。在免疫电镜下，成熟的细胞核被角蛋白网络紧密包围或封闭，在内外膜上可见到强烈的层粘连蛋白和库瑞宾蛋白。从这些数据来看，okurbin被认为是连接角蛋白丝和近核空间的缺失环节。

项目成果

Tanioka M, Ikoma A, Morita K, Fujii H, Toda KI, Takahashi Kenzo, Tanaka Toshihiro, Nishigori Chikako, Jin G, Higashi S, Toyokuni S, Miyachi Yoshiki: "Angiosarcoma of the scalp: absence of vascular endothelial cadherin in primary and metastatic lesions"Br

Tanioka M、Ikoma A、Morita K、Fujii H、Toda KI、Takahashi Kenzo、Tanaka Toshihiro、Nishigori Chikako、Jin G、Higashi S、Toyokuni S、Miyachi Yoshiki：“头皮血管肉瘤：原发性和中性组织中缺乏血管内皮钙粘蛋白

Eriko Torii et al.: "Expression of prostaglandin E2 receptor subtypes in the mouse hair follicles"Biochem. Biophys. Res. Commun.. 290(2). 696-700 (2002)

Eriko Torii 等：“小鼠毛囊中前列腺素 E2 受体亚型的表达”Biochem。

Olivier Bousquet, Linglei Ma, Soichiro Yamada, Changhong Gu, Toshihiro Idei, Kenzo Takahashi, Denis Wirtz and Pierre A. Coulombe: "The Non-Helical Tail Domain of Keratin 14 Promotes Filament Bundling and Enhances the Mechanical Properties of Keratin Inter

Olivier Bousquet、Linglei Ma、Soichiro Yamada、Changhong Gu、Toshihiro Idei、Kenzo Takahashi、Denis Wirtz 和 Pierre A. Coulombe：“角蛋白 14 的非螺旋尾部结构域促进细丝集束并增强角蛋白间的机械性能

Eriko Trii et al.: "Expression of prostaglandin E2 receptor subtype・・・"Biochem Biophys Res Commun. 290(2). 696-700 (2002)

Eriko Trii 等人：“前列腺素 E2 受体亚型的表达……”Biochem Biophys Res Commun. 290(2) 696-700 (2002)。

高橋 健造 ほか: "創傷治癒のメカニズム"Progress in Medicine. 21(8). 1809-1813 (2001)

高桥贤三 (Kenzo Takahashi) 等人：“伤口愈合机制”，医学进展 21(8) (2001)。