HEMIDESMOSOMAL PROTEIN LINKAGE MAP BY TWO-HYBRID SYSTEM

目前的建议是基于利用酵母双杂交系统揭示以180-kD大疱性类天疱疮抗原（BPAG2/BP180）为中心的半粒染色体蛋白网络的发现，这是一种跨膜分子，也称为XVI型胶原。BP180的胞内结构域作为诱饵，与β 4整合素胞内结构域、与细胞角蛋白18、与p120分子，以及与plectin和periplakin的羧基端球状结构域内的特异性结构域（linker-region）相互作用。通过对正常蛋白的功能、转基因小鼠的表型、基因突变或自身免疫性疾病患者的研究，揭示了plakin蛋白家族的完整性，目前由5个成员（plectin, desmoplakin, BP230， envoplakin和periplakin）组成。本研究的总体目标是利用酵母双杂交系统揭示半染色体蛋白连锁图谱。本研究的重点是揭示XVII型胶原/BPAG2/BP180分子的氨基端球状结构域与periplakin的羧基端球状尾部结构域“连接子”之间的双杂交相互作用的生物学意义。为了实现这一目标，将开发识别periplakin和BP180感兴趣区域内表位的特异性抗体，作为1)在细胞和组织中检测和定位这些蛋白质的特定工具，并通过免疫荧光研究它们的共定位，特别强调由初步结果识别的组织；2)通过体外结合实验确认酵母双杂交相互作用；3)最相关的生物学数据将从“建立连接/断开连接”型实验中获得。转染细胞中选定的蛋白质和蛋白质结构域的表达将用于揭示体内相互作用或这种相互作用的丧失；4)利用酵母双杂交系统进一步表征BP180与plakin家族蛋白的相互作用。预计这些研究将提供BP180/periplakin相互作用的关键信息，以实现理解为皮肤基底膜区提供稳定性的蛋白质-蛋白质网络的总体目标。

The present proposal is based on the discoveries which were made while utilizing the yeast two-hybrid system to reveal the hemidesmosomal network of proteins Centering on the 180-kD bullous pemphigoid antigen (BPAG2/BP180), a transmembrane molecule also known as a type XVI Collagen. An intracellular domain of BP180, when used as a bait, showed interaction with beta4 integrin intracellular domain, with cytokeratin 18, with p120 molecule, and with the specific domain, called linker-region within the carboxy-terminal globular domains of plectin and periplakin. The plakin family of proteins, currently consisting of five members (plectin, desmoplakin, BP230, envoplakin and periplakin) gives integrity to cells and tissues as revealed through the studies on the function of normal proteins, phenotype of transgenic mice, or patients with gene mutations or autoimmune diseases. The overall goal of the current study is to disclose the hemidesmosomal protein linkage map using yeast two- hybrid system. The main focus of the proposed work is to reveal the biological significance of the two-hybrid interaction between the amino- terminal globular domain of type XVII collagen/BPAG2/BP180 molecule and the carboxy-terminal globular tail domain "linker" of periplakin. Towards this goal, specific antibodies recognizing epitopes within the domains of interest in periplakin and BP180 will be developed, to be used as specific tools for 1) the detection and localization of these proteins in cells and tissues and to study their co-localization by immunofluorescence with specific emphasis on tissues identified by the preliminary results; 2) In vitro binding assays will be conducted to confirm the yeast two-hybrid interactions; 3) The most relevant biological data will be obtained from the "making a connection/breaking the connection" -type experiments. The expression of selected proteins and protein domains in the transfected cells will be used to reveal the in vivo interaction or the loss of such interaction; 4) The yeast two-hybrid system will be used to further characterize the interaction between BP180 and the plakin family of proteins. It is expected that these studies will provide critical information on BP180/periplakin interactions towards the overall goal of understanding the protein-protein network providing stability to the cutaneous basement membrane zone.