Finding a mate for MLCK: improving the potential for therapeutic targeting of gut permeability.
Finding a mate for MLCK: improving the potential for therapeutic targeting of gut permeability.
复制标题
寻找 MLCK 的伴侣：提高肠道通透性治疗靶向的潜力。
DOI：
10.1136/gutjnl-2022-327599
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发表时间：
2022
期刊：
影响因子：
24.5
通讯作者：
McCole,DeclanF
中科院分区：
文献类型：
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作者：
McCole,DeclanF
肠通透性升高长期以来被认为是肠道炎性疾病，特别是炎性肠病（IBD）的病理生理学标志物。除了IBD风险遗传变异与肠道通透性和屏障功能障碍的多个方面的关联之外，通透性增加已被确定为克罗恩病患者的健康一级亲属中IBD发作和克罗恩病患者复发的预测因子。1-4然而，尽管对炎症在疾病相关渗透性中的作用进行了广泛的研究，但除广泛作用的抗炎剂外，屏障恢复剂的开发已被证明极具挑战性。在GUT中，Zuo等人提出了一项创新性研究，确定他克莫司结合蛋白FKBP 8是紧密连接调节介质肌球蛋白轻链激酶1（MLCK 1）的特异性结合伴侣。MLCK 1是典型紧密连接调节蛋白MLCK的两种剪接变体之一。MLCK磷酸化连接周肌动球蛋白环内的肌球蛋白II调节轻链（MLC），以增加紧密连接对直径高达~ 100 μ m分子的渗透性。6这种细胞旁渗透途径通常被称为“渗漏”途径。7在使用转基因小鼠和酶抑制剂的研究中，MLCK已被确定为急性麻黄碱诱导的腹泻以及慢性免疫介导的实验性IBD的驱动因素。6此外，MLCK作为炎性细胞因子肿瘤坏死因子（TNF）-α和干扰素（IFN）-γ的关键下游介质，最终导致上皮紧密连接（调节上皮通透性的关键蛋白质聚集体结构）的改变。然而，靶向MLCK用于治疗性干预以恢复屏障功能已被难以避免对在心肌、平滑肌和骨骼肌中表达的MLCK的不希望的副作用所极大地阻碍。本文通过确定一种途径来规避广泛MLCK抑制的非特异性效应，通过将FKBP 8确定为可靶向减少MLCK 1在紧密连接处积累的额外因子，来解决一个重要问题。在他们之前的工作中，作者阐明了TNF诱导的MLCK 1（而不是MLCK 2剪接变体）向连接周围肌动球蛋白环的募集对MLCK 1特有的氨基末端免疫球蛋白样结构域IgCAM 3的小分子阻断敏感。8他们假设，阻断MLCK 1募集的小分子通过干扰IgCAM 3与另一种尚未鉴定的蛋白质的结合而起作用。为了鉴定这种假定的介质，他们使用酵母双杂交筛选来探测MLCK 1特异性结合蛋白的人肠上皮互补DNA文库。他们鉴定了FK 506结合蛋白FKBP 8，也称为FKBP 38，这是一种与自噬，线粒体自噬和未折叠蛋白反应有关的蛋白质，但以前在肠上皮细胞中没有功能特征。10使用蛋白结合和邻近连接方法，他们表明MLCK 1直接结合FKBP 8，并且这些相互作用对于肠上皮细胞系和人肠类器官培养物中MLCK 1募集、MLC磷酸化和TNF诱导的屏障丧失是必不可少的（参见图1）。值得注意的是，虽然TNF增加MLCK 1表达，但TNF对FKBP 8的作用在促进MLCK-FKBP 8相互作用方面是特异性的，
Elevated intestinal permeability has long been appreciated as a pathophysiological marker of gut inflammatory disease, in particular in inflammatory bowel disease (IBD). In addition to the association of IBD risk genetic variants with multiple aspects of intestinal permeability and barrier dysfunction, increased permeability has been identified as a predictor of IBD onset in healthy first-degree relatives of patients with Crohn’s disease, and of relapse in patients with Crohn’s disease. 1–4 However, despite extensive investigation of the role of inflammation in diseaseassociated permeability, development of barrier-restoring agents beyond broadacting anti-inflammatory agents has proven extremely challenging. In GUT, Zuo et al present an innovative study identifying that the tacrolimusbinding protein FKBP8 is a specific binding partner for the tight junction regulatory mediator, myosin light-chain kinase 1 (MLCK1). 5 MLCK1 is one of two splice variants of the canonical tight junction regulatory protein, MLCK. MLCK phosphorylates myosin II regulatory light chain (MLC) within the perijunctional actomyosin ring to increase tight junction permeability to molecules with diameters up to~ 100 Å. 6 This route of paracellular permeability is often referred to as the ‘leak’pathway. 7 In studies using genetically-modified mice and enzymatic inhibitors, MLCK has been identified as a driver of acute cytokine-induced diarrhoea as well as chronic immune-mediated experimental IBD. 6 Moreover, MLCK acts as a critical downstream mediator of the inflammatory cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ, culminating in alterations of epithelial tight junctions the key protein aggregate structures that regulate epithelial permeability. 6 8 9 However, targeting of MLCK for therapeutic intervention to restore barrier function has been greatly hampered by the difficulty in avoiding unwanted side effects on MLCK expressed in cardiac, smooth and skeletal muscle. This paper addresses a significant problem by determining a route to circumvent the non-specific effects of broad MLCK inhibition, by identifying FKBP8 as an additional factor that can be targeted to reduce accumulation of MLCK1 at tight junctions. In their prior work, the authors elucidated that TNF-induced recruitment of MLCK1—but not the MLCK2 splice variant—to the perijunctional actomyosin ring was sensitive to small molecule blockade of an amino-terminal immunoglobulin-like domain, IgCAM3, that is unique to MLCK1. 8 They hypothesised that the small molecule that blocked MLCK1 recruitment acted by interfering with IgCAM3 binding to another—as yet unidentified—protein. To identify this putative mediator, they used a yeast twohybrid screen to probe a human intestinal epithelial complementary DNA library for MLCK1-specific binding proteins. They identified FK506-binding protein FKBP8, also known as FKBP38, a protein that has been linked to autophagy, mitophagy and the unfolded protein response but had not previously been functionally characterised in intestinal epithelial cells. 10 Using protein binding and proximity ligation approaches, they showed that MLCK1 binds directly to FKBP8 and that these interactions are essential for MLCK1 recruitment, MLC phosphorylation and TNF-induced barrier loss in intestinal epithelial cell lines and human intestinal organoid cultures (see figure 1). Notably, while TNF increases MLCK1 expression, the effect of TNF on FKBP8 was specific in promoting MLCK-FKBP8 interactions as