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

摘要 在传染性和免疫介导的肠道和全身疾病中，屏障功能受到损害。这 该计划现已完成其第四个资金周期，一直以我们理解的长期目标为指导 在基础、分子水平上的肠上皮屏障调节，以及如何定义调节和 调节失调会影响疾病。这些知识是开发合理的、基于机制的 治疗方法。在之前的周期中，我们已经做出了改变范式的发现，包括认识 这种持续的分子重塑发生在紧密连接中。这一点和其他由 我们之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 紧密交汇处的相互作用。我们的初步数据显示了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 阻滞素尾部磷酸化掩盖ZO-1结合位点，而去磷酸化触发构象 增强与ZO-1结合的变化。由此产生的闭塞素/ZO-1络合，然后形成稳定的相互作用 与claudin-2有关，它扰乱了通道功能。在体内，我们发现免疫介导的结肠炎的严重程度 在claudin-2基因敲除小鼠和转基因小鼠中，分别显著减少或增加。我们结合了 体外结构和体内功能数据，以抑制阻滞素磷酸化，阻断claudin-2通道，以及 在体内减轻免疫介导的结肠炎。在探索claudin-4过度表达作为一种 增强屏障功能的治疗干预我们发现，无论是基因敲除还是过表达 Claudin-4影响紧密连接通透性。然而，claudin-4在以下情况下能够增强屏障功能 与claudin-2一起表达。这些和其他初步数据表明，与常规数据相反 明智的是，claudin-4不会形成屏障，而是通过直接破坏claudin-2来降低通透性。 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 改变渗透率的极端方法，如稳定通道的关闭状态，有 有可能。更细微的方法可能在治疗上很重要，因为我们的体内研究表明 完全抑制claudin-2通道可能是有害的。例如，Claudin-2介导的细胞旁的丢失 水和Na+外流导致Claudin-2基因敲除小鼠病原体清除障碍。单独研究 阻塞素使我们意外地观察到，通过与屏障功能无关的机制，阻塞素是一个关键 上皮细胞存活的调节剂。与此同时，我们发现ZO-1是上皮定位所必需的， 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 不完全的理解，并将限制调查结果的实用性。因此，这项提议试图理解 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，以及这些知识指导以结构为基础的合理疗法发展的潜力。

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.