Molecular Analysis of Tight Junctions in Liver and Gut

描述（由申请人提供）：本项目的长期目标是了解调节溶质通过胃肠道和肝脏上皮细胞紧密连接的细胞旁转运的分子基础。在电阻、溶质通量和离子电荷选择性方面，紧密连接的屏障特性在细胞类型之间变化很大。当屏障被不同组织中的致病因素（炎症、特定细菌毒素、药物等）破坏时，转运被阻止，导致腹泻、胆汁淤积或抗原和微生物的进入增强。目前，对屏障的分子基础、其可变特性和调节知之甚少。在拟议的研究中，我们将追求的假设，一个新描述的跨膜蛋白家族称为claudins负责形成屏障及其选择性特性。首先，我们将研究是否选定的成员的20个claudins显示不同的免疫组织化学位置之间的不同类型的胃肠道细胞。我们将在人体组织中确定结肠和小肠中的表达水平和模式是否会因癌症和炎症而发生变化。差异表达模式和反应将被认为与提供连接的可变特性的作用一致。第二，我们将直接测试单个claudin在培养的上皮细胞中表达时改变屏障性质如电阻、溶质通量和离子选择性的能力。我们将尝试通过细胞外序列的定点诱变来确定参与创造屏障可变特性的蛋白质序列。第三，我们将确定的蛋白质结构的基础上的障碍，通过确定的寡聚状态的claudins溶解从膜到非离子洗涤剂，使用生物化学和生物物理方法和化学交联。我们将确定屏障的基本蛋白单元是同源还是异源的，并尝试使用冷冻电子显微镜进行直接结构分析。这将使我们能够看到蛋白质如何折叠和接触，以创建一个选择性屏障。结构信息将与生理学和诱变研究相关。第二紧密连接跨膜蛋白occludin的生理和结构特性将与claudins进行比较。这些研究将为我们理解疾病中细胞旁转运是如何调节和改变的提供重要和新颖的进展

DESCRIPTION (provided by applicant): The long-range goal of this project is to understand the molecular basis for regulating paracellular transport of solutes across the tight junctions of epithelia in the gastrointestinal tract and liver. Barrier characteristics of tight junctions vary widely among cell types in terms of electrical resistance, solute flux and ionic charge selectivity. When the barrier is disrupted by pathogenic factors in different tissues (inflammation, specific bacterial toxins, drugs, etc.) transport is arrested leading to diarrhea, cholestasis, or enhanced entry of antigens and microbes. Presently the molecular basis for the barrier, its variable properties and regulation are poorly understood. In the proposed studies we will pursue the hypothesis that a newly described family of transmembrane proteins called the claudins are responsible for forming the barrier and its selectivity properties. First, we will examine whether selected members of the 20 claudins show different immunohistochemical location among different cell types of the GI tract. We will determine in human tissues whether the expression levels and patterns change in the colon and small bowel in response to cancer and inflammation. Differential expression patterns and responses will be considered consistent with a role in providing the junction's variable properties. Second, we will directly test the ability of individual claudins to alter barrier properties such as electrical resistance, solute flux and ion selectivity when expressed in cultured epithelial cells. We will attempt to define the protein sequences involving in creating the barrier's variable properties through site-directed mutageneis of the extracellular sequences. Third, we will define the protein structural basis for the barrier by determining the oligomeric state of claudins solubilized from membranes into non-ionic detergents using biochemical and biophysical methods and chemical cross linking. We will determine whether the basic protein unit of the barrier is homo- or heteromeric and attempt direct structural analysis using cryo-electron microscopy. This will allow us to see how the proteins fold and contact to create a selective barrier. Structural information will be correlated with the physiologic and mutagenesis studies. The physiologic and structural properties of a second Tight Junction transmembrane protein occludin, will be compared with the claudins. Together these studies will provide significant and novel advancements in our understanding of how paracellular transport is regulated and is altered in disease