抑制FKBP8介导的MLCK1转运对TNF诱导肠道粘膜损伤修复的影响及机制研究

Intestinal barrier dysfunction contributes to the progression of inflammatory bowel and systemic disease. However, there is no specific treatment due to the unclear mechanisms. It has been proved that myosin light chain kinase (MLCK)activation and trafficking resulting in tight junction destruction is the primary cause of TNF-induced small intestinal barrier loss and experimental colitis. Therefore,inhibition of MLCK activity and trafficking is a therapeutic target for intestinal mucosal injury. MLCKs include smooth muscle MLCK and non-smooth muscle MLCK. Unfortunately, the inhibitors of MLCK cannot make a distinction between intestinal epithelial MLCK and smooth muscular MLCK, which results in severe illnesses, toxicities, and fatalities.Thus, there is a fundamental gap that separates our previous elucidation of mechanisms and clinicopathologic significance of barrier regulation in disease from the development of strategies that can be used to modulate intestinal epithelial tight junction function for therapeutic purposes. This proposal seeks to bridge that gap by building on our recent observations regarding regulation of the MLCK-myosin phosphatase axis in disease. Specifically, we will focus on understanding trafficking of the non-smooth muscle MLCK splice variant, MLCK1 and MLCK2. Recent studies indicate that tumor necrosis factor (TNF) or chronic disease cause MLCK1 recruitment to the perijunctional actomyosin ring, to regulate tight junction permeability.Our previous study demonstrated that FKBP8 interact with the IgCAM3 domain of MLCK1, the inhibition of FKBP8 can prevent MLCK1 trafficking. Here we propose to define the molecular mechanisms of basal and TNF-induced MLCK1 trafficking and to characterize the therapeutic potential of newly-discovered trafficking inhibitors(FKBP8 inhibitors) in barrier dysfunction. We will define the interaction of MLCK1 and FKBP8 by the FRET, MST. Cells and mice will be used to detect the function of FKBP8 inhibitors in MLCK trafficking and barrier dysfunction. The proposal is innovative because it will define novel regulatory mechanisms and will result in a significant shift in our understanding of means to correct barrier function and actomyosin contractile status for therapeutic benefit. The proposed research is significant because it will link specific mechanisms of barrier loss to disease and identify novel therapeutic approaches.

炎症性肠病伴随肠粘膜损伤，但缺乏特异性治疗。研究证实肌球蛋白轻链激酶(MLCK)激活和转运破坏紧密连接是TNF诱导肠屏障丧失和实验性肠炎主要原因，因此抑制MLCK活性和转运是肠粘膜损伤治疗靶点。MLCK分平滑肌和非平滑肌两种，目前所有MLCK抑制剂会降低两者活性导致低血压、蠕动不足、伤口愈合慢等毒副作用。若能特异性抑制肠上皮非平滑肌MLCK可避免副作用。非平滑肌MLCK分MLCK1和2，TNF仅使MLCK1向肌动球蛋白环转运，与其特有IgCAM3结构域密切相关。经筛选发现FKBP8可直接结合MLCK1的IgCAM3但机制不明。本研究旨在探讨是否抑制FKBP8可阻止MLCK1转运，延缓或恢复TNF诱导肠粘膜损伤。课题在前期基础上，明确FKBP8与MLCK1结合及影响因素；并在MLCK过表达细胞和实验鼠验证FKBP8抑制剂对MLCK1转运及肠黏膜损伤影响。项目实施为治疗肠炎提供新思路及理论依据

目的：肠屏障丧失是克罗恩病 (CD) 的危险因素。肌球蛋白轻链激酶 1 (MLCK1) 的表达和活性增加诱导肠粘膜通透性增加,与 CD 严重程度相关。此外，临床前研究表明，MLCK1肌动球蛋白环募集是屏障丧失和实验性 IBD 进展所必需的。本课题探讨 MLCK1 募集的机制，并在药理学上针对这一过程寻找潜在的药物靶点.方法：在体外评估了 FK506 结合蛋白 8 (FKBP8) 和 MLCK1 之间的蛋白质相互作用。转基因和敲除上皮细胞和小鼠被用作临床前模型。最终这些现象在 CD 患者和对照受试者的活检组织中得到进一步验证。.结果：MLCK1 与FKBP8 PPI 结构域特异性相互作用。敲除或FKBP8突变体表达可防止 TNF 诱导的 MLCK1 募集和体外屏障丧失。 MLCK1-FKBP8 结合被FK506阻断，这在体外和体内逆转了 TNF 诱导的 MLCK1-FKBP8 相互作用、MLCK1 募集和屏障丧失。 CD 患者活检显示，与对照受试者相比，细胞间连接处的 MLCK1-FKBP8 相互作用数量增加。.结论：与可被FK506阻断的 FKBP8 结合是 MLCK1 募集到肌动球蛋白环和导致免疫介导屏障丧失的下游事件所必需的。结果证实 在CD病人中, MLCK1 活性和募集增加、 MLCK1-FKBP8 相互作用增加，提示靶向该过程可能对人类疾病具有治疗作用。这些对疾病相关屏障丧失机制的新见解为 FKBP8-MLCK1 相互作用的治疗开发提供了重要基础。

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