S100A7 AND KERATINOCYTE DIFFERENTIATION IN PSORIASIS

银屑病中的 S100A7 和角质形成细胞分化

• 批准号: 6100498
• 负责人: NANCY A ROBINSON
• 金额: $ 4.59万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6100498
• 关键词: biological signal transduction; calcium binding protein; cell differentiation; crosslink; enzyme mechanism; enzyme substrate; gel electrophoresis; human tissue; immunologic assay /test; immunoprecipitation; intermolecular interaction; keratinocyte; protein glutamine gamma glutamyltransferase; protein localization; protein sequence; psoriasis; tissue /cell culture; western blottings

Calcium is a key regulator in the normal differentiation of epidermal keratinocytes. The calcium signal is transduced via calcium-binding proteins. The S100 family of calcium-binding proteins are strongly implicated in tissue-specific mediation of the calcium signal. Given that calcium is an important regulator of differentiation and that keratinocytes synthesize several S100 proteins, aberrant differentiation of keratinocytes may, in part, be due to altered expression of this important class of calcium-binding proteins. In support of this idea, psoriatic keratinocytes display an altered expression of this important expression with S100A8, S100A9 and S100A7 being upregulated. In this proposal, the role of one of these proteins, S100A7, or psoriasin, in psoriasis will be examined. S100A7 has a limited tissue distribution, being present in fetal skin, ear, and tongue. This S100 protein is not synthesized in normal human interfollicular skin, however it is highly expressed in psoriatic lesional plaques. The mechanism by which S100A7 influences abnormal keratinocyte differentiation is not known. In general, S100 proteins mediate calcium signal through a calcium-dependent conformational change. The calcium-activated S100 protein binds to, and modifies the activity of, specific target proteins. While many targets of S100 proteins have been identified, the target protein(s) of S100A7 is not known. In addition, some of the S100 proteins are post-translationally modified, which potentially affects their physiological function. For example, S100A11 and S100A10 are covalently cross-linked by epidermal transglutaminases (TGs) in vivo. Based on the shared sequence and structural homology within the S100 protein family, it is likely that S100A7 is similarly modified by TG. It is suspected that this modification could prove to be a critical step in regulation of differentiation since it links together calcium (a key regulator of the process), S100 protein (a component of the transduction pathway) and TG (the enzyme responsible for the formation of the cornified envelope- a terminal differentiation product). The overall goals of this study are to (i) characterize, in vivo and in vitro, the ability of TGs to covalently modify S100A7 and (ii) to identify the target protein(s) of S100A7. In preparation for these studies, the coding region of S100A7 has been cloned into a prokaryotic expression vector. The antibody elicited against rhS100A7, however, cross-reacts with other S100 proteins. In specific aim 1 a antibody directed against the V8 protease released carboxyl-terminal peptide of S100A7 will be produced. This peptide is from an area of divergence within the S100 family. In specific aim 2 the ability of TGs to cross-link S100A7 in vitro and in vivo will be investigated. rhS100A7 will be incubated in vitro with TG and the products analyzed by polyacrylamide gel. Specific sites of cross-link formation will be identified after trypsin digestion of S100A7, peptide purification and sequence analysis. Keratinocyte extracts, from cell culture and from normal and psoriatic epidermis, will be analyzed by western blot to determine if S100A7 is modified in vivo by TG. The goal of Specific Aim 4 is to identify S100A7 target protein(s). This fundamentally important question will be answered using ligand blots, co-immunoprecipitation assays, affinity chromatography and protein microsequencing.

钙是表皮正常分化的关键调节剂 角质形成细胞。 钙信号通过钙结合转导 蛋白质。 钙结合蛋白 S100 家族具有很强的 涉及钙信号的组织特异性介导。 给定 钙是分化的重要调节剂 角质形成细胞合成多种 S100 蛋白，异常 角质形成细胞的分化可能部分是由于改变 这一类重要的钙结合蛋白的表达。 在 支持这一观点的是，银屑病角质形成细胞表现出改变 这一重要表达与 S100A8、S100A9 和 S100A7 的表达 被上调。 在此提案中，其中之一的作用 将检查牛皮癣中的蛋白质、S100A7 或牛皮癣素。 S100A7 组织分布有限，存在于胎儿皮肤、耳朵、 和舌头。 这种 S100 蛋白在正常人体内不合成 毛囊间皮肤，但在银屑病中高度表达 病变斑块。 S100A7影响异常的机制 角质细胞分化尚不清楚。 一般来说，S100 蛋白 通过钙依赖性构象介导钙信号 改变。 钙激活的 S100 蛋白结合并修饰 特定靶蛋白的活性。虽然S100的目标很多 蛋白质已被鉴定，S100A7 的目标蛋白质尚未确定 已知。此外，一些 S100 蛋白是翻译后蛋白 修改，这可能会影响其生理功能。为了 例如，S100A11和S100A10通过表皮共价交联 体内转谷氨酰胺酶（TG）。基于共享序列和 S100 蛋白家族内的结构同源性，很可能 S100A7同样由TG改装。疑似这 修改可能被证明是监管的关键一步 差异化，因为它将钙（钙的关键调节剂）连接在一起 过程）、S100 蛋白（转导途径的一个组成部分）和 TG（负责形成角质化包膜的酶 - 终端差异化产品）。本研究的总体目标 (i) 在体内和体外表征 TG 的能力 共价修饰 S100A7 并 (ii) 鉴定 S100A7 的靶蛋白 S100A7。 为了准备这些研究，S100A7 的编码区 已被克隆到原核表达载体中。 抗体 针对 rhS100A7 引发，但与其他 S100 发生交叉反应 蛋白质。 在具体目标 1 中，针对 V8 的抗体 将产生S100A7的蛋白酶释放的羧基端肽。 该肽来自 S100 家族的一个分歧领域。 在 具体目标 2 TG 在体外和体内交联 S100A7 的能力 体内将被调查。 rhS100A7 将在体外与 TG和聚丙烯酰胺凝胶分析产物。 具体站点 胰蛋白酶消化后可鉴定交联形成 S100A7，肽纯化和序列分析。 角质形成细胞 从细胞培养物以及正常和牛皮癣表皮中提取， 将通过蛋白质印迹分析以确定 S100A7 是否被修饰 TG 的vivo。 具体目标4的目标是确定S100A7目标 蛋白质。 这个至关重要的问题将得到解答 使用配体印迹、免疫共沉淀、亲和力 色谱法和蛋白质微测序。