REGULATION OF TIGHT JUNCTIONS AND ROLE IN DIARRHEA OF ZO

细胞旁途径是被动溶质流过肠上皮屏障的主要途径，其渗透性取决于细胞间紧密连接（tj）（也称为闭合小带（ZO））的调节。 一个世纪前，这些结构被认为是一种分泌的细胞外粘固剂，在细胞旁空间内形成绝对和不受调节的屏障。 过去几十年的生物学研究表明，tj是动态结构，其生理调节在很大程度上仍不明确。在我们最初的资助中，我们使用了封闭小带毒素（Zot），一种由霍乱弧菌精心制作的蛋白质，作为研究tj功能调节的工具。 在3年的资助期间获得的结果表明，Zot通过与特异性表面受体相互作用可逆地打开tj，所述特异性表面受体与复杂的细胞内信号传导途径偶联，所述细胞内信号传导途径涉及肌动蛋白单体的PKCa依赖性聚合成肌动蛋白微丝，所述肌动蛋白微丝策略性地定位以调节细胞旁途径。 此外，我们能够纯化和部分表征的人真核Zot类似物，我们命名zonulin，这是一种新的，内源性调节剂的tj功能。本申请的长期目标是进一步确定Zot和zonulin的作用机制，并纯化和克隆Zot表面受体以获得对tj调节的了解，特别是在细胞和分子水平。

The paracellular route is the dominant pathway for passive solute flow across the intestinal epithelial barrier, and its permeability depends on the regulation of intercellular tight junctions (tj), also known as the zonula occludens (ZO). A century ago, these structures were thought to be a secreted extracellular cement forming an absolute and unregulated barrier within the paracellular space. Biological studies of the past several decades have shown that tj are dynamic structures whose physiological regulation remains largely undefined. In our original grant, we used zonula occludens toxin (Zot), a protein elaborated by Vibrio cholerae, as a tool to study the regulation of tj function. The results obtained during the 3 years of funding demonstrated that Zot reversibly opens tj by interacting with a specific surface receptor(s) coupled to a complex intracellular signaling pathway involving PKCalpha-dependent polymerization of actin monomers into actin microfilaments strategically localized to regulate the paracellular pathway. Furthermore, we were able to purify and partially characterize a human eukaryotic Zot analogue, that we named zonulin, that represents a novel, endogenous modulator of tj function. The long term objectives of the proposed application are to further define the mechanism(s) of action of both Zot and zonulin and to purify and clone the Zot surface receptor to gain insights into tj regulation, particularly at the cellular and molecular levels.