REGULATION OF TIGHT JUNCTIONS AND ROLE IN DIARRHEA OF ZO

细胞旁途径是被动溶质通过肠上皮屏障的主要途径，其通透性依赖于细胞间紧密连接(TJ)的调节，也称为闭锁带(ZO)。一个世纪前，这些结构被认为是一种分泌性的细胞外粘合剂，在细胞旁空间内形成了一种绝对的、不受监管的屏障。过去几十年的生物学研究表明，TJ是一种动态结构，其生理调节在很大程度上仍未明确。在我们最初的资助中，我们使用了由霍乱弧菌阐述的一种蛋白质--闭锁小带毒素(Zot)作为研究TJ功能调节的工具。三年的研究结果表明，Zot通过与一种特殊的表面受体(S)相互作用而可逆地开放TJ，该受体连接到一个复杂的细胞内信号通路，该通路涉及依赖于PKCalpha的肌动蛋白单体聚合成具有战略定位的肌动蛋白微丝，以调节细胞外途径。此外，我们能够提纯并部分鉴定一种人类真核Zot类似物，我们将其命名为zonlin，它代表了一种新的内源性TJ功能调节剂。拟议应用的长期目标是进一步确定Zot和Zonlin的作用机制(S)，并纯化和克隆Zot表面受体，以深入了解TJ的调节，特别是在细胞和分子水平上。

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.