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

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。

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.