HEMIDESMOSOMAL PROTEIN LINKAGE MAP BY TWO-HYBRID SYSTEM

本提案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)为中心的蛋白质半桥粒网络的发现，BPAG2/BP180是一种跨膜分子，也被称为XVI型胶原。BP180的胞内区作为诱饵，与β4整合素胞内区、细胞角蛋白18、p120分子相互作用，以及与plectin和周斑蛋白羧基末端球状结构域内的连接区相互作用。Plakin家族目前由5个成员(plectin、desmoplakin、BP230、envoplakin和periplakin)组成，通过对正常蛋白质的功能、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发现，plakin家族为细胞和组织提供了完整性。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质连锁图谱。本研究的主要目的是揭示XVI型胶原/BPAG2/BP180分子的氨基末端球状结构域与周斑蛋白的羧基末端球状结构域“连接体”之间的双杂交作用的生物学意义。为此，将开发识别周斑蛋白和BP180感兴趣区域内的表位的特定抗体，用作以下特定工具：1)检测和定位这些蛋白质在细胞和组织中的位置，并通过免疫荧光研究它们的共存，重点是初步结果确定的组织；2)将进行体外结合试验，以确认酵母双杂交作用；3)最相关的生物学数据将从“建立连接/断开连接”类型的实验中获得。4)酵母双杂交系统将用于进一步研究BP180与plakin家族蛋白家族之间的相互作用。预计这些研究将提供关于BP180/周膜蛋白相互作用的关键信息，从而了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.