MECHANISTIC UNDERSTANDING OF PROTEIN INTERACTIONS AT THE TIGHT JUNCTION: STRUCTURAL REGULATION OF CANONICAL AND NONCANONICAL FUNCTIONS

总结 屏障功能在感染性和免疫介导的肠道和全身性疾病中受损。这 该计划现已完成第四个资助周期，我们的长期目标是了解 肠上皮屏障调节在一个基本的，分子水平和定义如何调节， 失调影响疾病。这些知识是发展理性的、基于机制的 治疗方法。在之前的周期中，我们已经做出了范式转变的发现，包括识别 在紧密连接中发生连续的分子重塑。这一点和其他新的见解提供了 我们先前的工作使我们探索了蛋白质的分子相互作用和功能后果， 紧密连接处的相互作用。我们的初步数据证明了claudin-4、occludin和ZO-4的新活性。 1与它们形成紧密连接的能力无关。使用结构方法，我们发现， occludin尾部磷酸化掩盖了ZO-1结合位点，而去磷酸化则触发了构象变化。 增强与ZO-1结合的变化。所得的闭合蛋白/ZO-1复合物然后形成稳定的相互作用 与claudin-2一起破坏通道功能。在体内，我们发现免疫介导的结肠炎的严重程度 分别在claudin-2敲除或转基因小鼠中显著降低或增加。我们组合 体外结构和体内功能数据，以抑制闭合蛋白磷酸化，阻断闭合蛋白-2通道， 在体内减弱免疫介导的结肠炎。在探索claudin-4过表达作为一种免疫调节剂的潜力时， 我们发现，敲除或过表达 claudin-4影响紧密连接通透性。然而，Claudin-4能够增强屏障功能， 与紧密连接蛋白-2一起沿着表达。这些和其他初步数据表明，与传统的 智慧，claudin-4不形成障碍，而是通过直接破坏claudin-2来降低渗透性 聚合物我们的膜片钳研究表明，紧密连接通道是主动门控的，这表明， 改变渗透性的极端方法，例如稳定通道的闭合状态， 可能更细致的方法可能在治疗上很重要，因为我们的体内研究表明， 完全的紧密连接蛋白-2通道抑制可能是有害的。例如，claudin-2介导的细胞旁 水和Na+流出导致紧密连接蛋白-2敲除小鼠的病原体清除缺陷。单独研究 occludin使我们意外地观察到，通过与屏障功能无关的机制，occludin是一个关键， 上皮存活的调节因子。同时，我们发现ZO-1是上皮定向所必需的， 增殖和顶端结构。这些数据清楚地表明，单独研究这些蛋白质将导致 不完整的理解，将限制调查结果的效用。因此，本建议旨在了解 不同紧密连接蛋白功能之间的关系，指导这些功能的分子相互作用， 活动，以及这种知识的潜力，以指导发展的结构为基础的，合理的治疗。

SUMMARY Barrier function is compromised in infectious and immune-mediated intestinal and systemic diseases. This program, now completing its fourth funding cycle, has been guided by our long-term goal of understanding intestinal epithelial barrier regulation at a fundamental, molecular level and defining how regulation and dysregulation impact disease. This knowledge is required for development of rational, mechanism-based therapeutic approaches. In previous cycles, we have made paradigm-shifting discoveries including recognition that continuous molecular remodeling occurs within the tight junction. This and other new insight provided by our previous work led us to explore the molecular interactions and functional consequences of protein interactions at tight junctions. Our preliminary data demonstrate novel activities of claudin-4, occludin, and ZO- 1 that are unrelated to their ability to form tight junctions. Using a structural approach, we have discovered that occludin tail phosphorylation masks the ZO-1 binding site, while dephosphorylation triggers conformational change that enhances binding to ZO-1. The resulting occludin/ZO-1 complexed then form stable interactions with claudin-2, which disrupt channel function. In vivo, we found that the severity of immune-mediated colitis was markedly reduced or increased in claudin-2 knockout or transgenic mice, respectively. We combined the in vitro structural and in vivo functional data to inhibit occludin phosphorylation, block claudin-2 channels, and attenuate immune-mediated colitis in vivo. While exploring the potential of claudin-4 overexpression as a therapeutic intervention to enhance barrier function we found that neither knockout nor overexpression of claudin-4 affected tight junction permeability. Claudin-4 was, however, able to enhance barrier function when expressed along with claudin-2. These and other preliminary data indicate that, contrary to conventional wisdom, claudin-4 does not form barriers, but rather reduces permeability by directly disrupting claudin-2 polymers. Our patch-clamp studies showing that tight junction channels are actively gated suggest that less extreme approaches to modifying permeability, such as stabilizing the closed state of the channel, are possible. More nuanced approaches could be therapeutically important, as our in vivo studies indicate that complete claudin-2 channel inhibition can be detrimental. For example, loss of claudin-2-mediated paracellular water and Na+ efflux resulted in defective pathogen clearance by claudin-2 knockout mice. Separate studies of occludin led to our unexpected observation that, by mechanisms unrelated to barrier function, occludin is a key regulator of epithelial survival. In parallel we discovered that ZO-1 is required for epithelial orientation, proliferation, and apical structure. These data make it clear that studying these proteins individually will lead to incomplete understanding and will limit utility of the findings. This proposal therefore seeks to understand the relationships between diverse tight junction protein functions, the molecular interactions that direct these activities, and the potential of that knowledge to guide development of structure-based, rational therapies.