Molecular Analysis of Tight Junctions in Liver and Gut

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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