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

总结： 肠道屏障功能在感染性和免疫介导的肠道疾病中受损。这 该计划目前正在完成第三个资助周期，重点关注肠道疾病的机制和影响。 上皮屏障功能障碍在以前的研究中，我们将上皮肌球蛋白轻链激酶（MLCK）定义为一种 对炎症刺激作出反应的紧密连接屏障功能的关键调节剂。本作品已 广泛复制并推广到其他系统。我们继续表明，MLCK依赖性增加， 通透性促进炎性肠病和移植物抗宿主病进展。我们也 定义了MLCK激活下游的信号传导事件，并发现虽然屏障由 在免疫系统中，紧密连接通透性MLCK依赖性增加也能够调节粘膜 引发紧密连接蛋白-2表达增加的免疫力。这导致了我们共同发现的孔隙和泄漏 trans-tight junction通量的途径;现在被广泛接受的模型。在当前的周期中，我们专注于 孔和泄漏途径，并发现，在感染性疾病的情况下，增加紧密连接 由于紧密连接蛋白-2表达而导致的渗透性是有益的，并促进病原体清除。另外我们 确定了一个特定的MLCK剪接变异体，MLCK 1作为紧密连接渗透性的关键调节因子。在 除了激活MLCK转录和酶活性外，我们发现炎症刺激引起 MLCK 1被募集到连接周围肌动球蛋白环。我们发现了一个特殊的区域，免疫球蛋白- 细胞粘附分子结构域3（IgCAM 3）是MLCK 1募集所需的，并且足以作为MLCK 1的一部分。 显性阴性以阻止募集。我们继续解析IgCAM 3的晶体结构， 在人和小鼠IgCAM 3之间保守但在其他MLCK中不存在的药物结合口袋 IgCAM，并对约140，000种药物样分子的NCI文库进行计算机筛选。我们找到了一个， 称为Divertin，其阻止MLCK 1募集而不抑制上皮或平滑肌MLCK 酶的功能通过阻断MLCK 1募集到连接周围肌动球蛋白环， MLCK 1在该位点磷酸化肌球蛋白II调节轻链。这反过来又阻止了炎症- 在体外、体内（小鼠）和离体（人肠活检）中诱导屏障丧失。变应素减弱了所有 实验性免疫介导的炎症性肠病（小鼠）的特征，包括屏障丧失、免疫抑制、免疫抑制和免疫抑制。 激活和死亡。该建议的目的是确定细胞内蛋白质相互作用的修饰， Divertin和定义MLCK 1募集的分子机制。所描述的研究将描述 我们已经发现的MLCK 1结合伴侣以及通过以下方法鉴定的新结合伴侣： 尖端的蛋白质组学方法完成时，这些研究将确定MLCK 1相互作用组 和机制，以及禁止招募的潜在好处和风险。 MLCK 1招募。这些进展将使未来的开发Divertin样药物用于人类使用。

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.