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Studies on the structure and biological function of human hair follicle peptidylarginine deiminase

人毛囊肽基精氨酸脱亚胺酶的结构及生物学功能研究

基本信息

批准号：
13670904
负责人：
KAWADA Aklira
金额：
$ 2.3万
依托单位：
Kinki University
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

项目摘要

The hair is composed of inner and root sheath, cuticle, and medulla cells which are differentiated from hair mother cell. The terminal differentiation of hair mother cell is common in tracing epidermal basal cells, but the biochemical study regarding the process of hair differentiation is very few.Peptidylarginine deiminase(PAD) is a post-translational modification enzyme which gives the electrostatic change by converting basic region of the target protein into the radio valence, causing the different interaction between target proteins and another protein. Since the first finding of G.E Rogers in 1977, that enzymatic activity converts arginine residue of the protein to the citrulline residue in extract of guinea pig hair follicle, biochemical studies of PADs were advanced in the various tissues. However the research on the PAD of the hair follicle is seldom carried out. In this study, we prepared the human hair cells by the primary culture method, and the RNA, and then cloned the enti … More re cDNA of the human hairy cell PAD(PAD T4) by homologue polymerase chain reaction. The complete amino acid sequence was clarified by the nucleotide sequence analysis. We constructed the human PAD T4 expression plasmid in an Echerichia coli and purification and enzymatic properties of the recombinant enzyme were clarified. Especially, the role of which the hairy cell peculiar protein(trichohyalin) is important for the regulation of the hair construct and the citrulline residues were found in the trichohyalin, that the trichohyalin is a candidate target for PAD T4. In this study we proved that PAD T4 modified the trichohyalin effectively. Next, we made a monoclonal antibody against specific for PAD T4 is and we examined the cellular distribution of PAD T4 in the hair follicle. The immunohistochemisty showed that the PAD T4 specifically localized in the inner root sheath in the hairy cells. The knowledge which proved that the above-mentioned trichohyalin also exists in this organization locally and that this enzyme has made the trichohyalin to be a target. Less

毛发由毛母细胞分化而来的内、根鞘细胞、角质层细胞和髓质细胞组成。毛母细胞终末分化在表皮基底细胞中很常见，但关于毛分化过程的生化研究却很少。肽精氨酸脱亚胺酶（PAD）是一种翻译后修饰酶，它通过将靶蛋白的碱基区转化为放射价区，使靶蛋白与另一种蛋白发生不同的相互作用，从而产生静电变化。自1977年G.E Rogers首次发现在豚鼠毛囊提取物中酶活性将蛋白的精氨酸残基转化为瓜氨酸残基以来，在各种组织中开展了PADs的生化研究。然而，对毛囊外周动脉病变的研究却很少。本研究采用原代培养的方法制备人毛细胞，提取RNA，并通过同源聚合酶链反应克隆人毛细胞PAD（PAD T4）的完整cDNA。通过核苷酸序列分析确定了完整的氨基酸序列。我们在大肠杆菌中构建了人PAD T4表达质粒，并对重组酶的纯化和酶学性质进行了澄清。特别是毛细胞特有蛋白（trichohyalin）在毛发结构调控中的重要作用，并且在trichohyalin中发现瓜氨酸残基，表明trichohyalin是PAD T4的候选靶点。在本研究中，我们证明了PAD - T4对毛透明蛋白的有效修饰。接下来，我们制备了一种针对PAD T4特异性的单克隆抗体，并检测了PAD T4在毛囊中的细胞分布。免疫组化结果显示，PAD T4特异性定位于毛细胞内根鞘。这些知识证明了上述trichohyalin在该组织局部也存在，并且该酶使trichohyalin成为靶标。少