Perijunctional myosin light chain kinase recruitment: A novel, non-enzymatic target for therapeutic intestinal barrier restoration

摘要： 在感染性和免疫介导性肠道疾病中，肠道屏障功能受损。这 该计划现已完成其第三个供资周期，重点是肠道疾病的机制和影响 上皮屏障功能障碍。在以前的周期中，我们将上皮肌球蛋白轻链激酶(MLCK)定义为 致密连接屏障的关键调节因子对炎症刺激的反应。这项工作已经完成了 被广泛复制并扩展到其他系统。我们进一步证明了MLCK依赖增加了 通透性促进炎症性肠病和移植物抗宿主病的进展。我们也 定义了MLCK激活下游的信号事件，并发现虽然屏障由 免疫系统中，MLCK依赖的紧密连接通透性增加也能够调节粘膜 引发claudin-2表达增加的免疫力。这导致我们共同发现了毛孔和渗漏 跨紧连接磁通的路径；一个现在被广泛接受的模型。在当前的周期中，我们专注于 对毛孔和渗漏途径的研究发现，在感染疾病的背景下，紧密连接增加 Claudin-2表达导致的通透性是有益的，并促进病原体清除。此外，我们 发现了一个特定的MLCK剪接变异体MLCK1，作为紧密连接通透性的关键调节因子。在……里面 除了激活MLCK转录和酶活性外，我们还发现炎症刺激导致 MLCK1被招募到连接周围的肌动球蛋白环。我们确定了一个特定的区域，免疫球蛋白- 细胞黏附分子结构域3(IgCAM3)是MLCK1募集所必需的，并足以作为 显性否定以阻止招聘。我们继续解决了IgCAM3的晶体结构，确定了一个潜在的 在人和小鼠IgCAM3之间保守但在其他MLCK中不存在的药物结合口袋 并对约140,000个类药物分子的NCI文库进行电子筛选。我们确认了一个， 称为Divertin，在不抑制上皮或平滑肌MLCK的情况下阻止MLCK1的募集 酶功能。通过阻止MLCK1募集到连接周围的肌动球蛋白环，Divertin阻止 MLCK1在该位点磷酸化肌球蛋白II调节轻链。这反过来又能阻止炎症-- 在体外、体内(小鼠)和体外(人体肠道活检)诱导屏障丧失。Divertin使所有 实验性免疫介导性炎症性肠病(小鼠)的特点，包括屏障丧失，免疫 激活和死亡率。这项提议的目的是确定由以下因素改变的细胞内蛋白质相互作用 Divertin并确定MLCK1募集的分子机制。所描述的研究将表征 我们已经发现的MLCK1结合伙伴以及通过 前沿的蛋白质组学方法。完成后，这些研究将定义MLCK1相互作用体 以及管理招募的机制以及限制招募的潜在好处和风险 MLCK1招募。这些进展将使人类使用的Divertin类药物的未来发展成为可能。

SUMMARY: Intestinal barrier function is compromised in infectious and immune-mediated intestinal diseases. This program, now completing its third funding cycle, has focused on the mechanisms and impact of intestinal epithelial barrier dysfunction. In previous cycles we defined epithelial myosin light chain kinase (MLCK) as a critical regulator of tight junction barrier function in response to inflammatory stimuli. This work has been replicated widely and extended to other systems. We went on to show that MLCK-dependent increases in permeability promote progression of inflammatory bowel disease and graft-versus-host disease. We also defined signaling events downstream of MLCK activation and found that while the barrier is regulated by the immune system, MLCK-dependent increases in tight junction permeability are also able to regulate mucosal immunity that triggers increases in claudin-2 expression. This led to our co-discovery of the pore and leak pathways of trans-tight junction flux; a model that is now widely accepted. In the current cycle we focused on the pore and leak pathways and found that, in the context of infectious disease, increased tight junction permeability as a result of claudin-2 expression is beneficial and promotes pathogen clearance. In addition, we identified a specific MLCK splice variant, MLCK1, as a critical regulator of tight junction permeability. In addition to activating MLCK transcription and enzymatic activity, we found that inflammatory stimuli cause MLCK1 to be recruited to the perijunctional actomyosin ring. We identified a specific domain, immunoglobulin- cell adhesion molecule domain 3 (IgCAM3) as being required for MLCK1 recruitment and sufficient to act as a dominant negative to block recruitment. We went on to solve the IgCAM3 crystal structure, identify a potential drug-binding pocket that was conserved between human and mouse IgCAM3 but absent in other MLCK IgCAMs, and perform an in silico screen of a NCI library of ~140,000 drug-like molecules. We identified one, termed Divertin, that prevents MLCK1 recruitment without inhibiting epithelial or smooth muscle MLCK enzymatic function. By blocking MLCK1 recruitment to the perijunctional actomyosin ring, Divertin prevents MLCK1 from phosphorylating myosin II regulatory light chain at that site. This, in turn, blocks inflammation- induced barrier loss in vitro, in vivo (mice), and ex vivo (human intestinal biopsies). Divertin attenuated all features of experimental immune-mediated inflammatory bowel disease (mice), including barrier loss, immune activation, and mortality. The aim of this proposal is to identify the intracellular protein interactions modified by Divertin and define the molecular mechanisms of MLCK1 recruitment. The studies described will characterize MLCK1 binding partners we have already discovered as well as new binding partners identified through cutting-edge proteomic approaches. At completion, these studies will have defined the MLCK1 interactome and mechanisms by which recruitment is regulated as well as the potential benefits and risks of inhibiting MLCK1 recruitment. These advances will enable future development of Divertin-like agents for human use.