PDF(Pubmed)
Abstract：
Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn\'s disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.
摘要：
肠上皮表达两个长肌球蛋白轻链激酶（MLCK）剪接变体，MLCK1和MLCK2的不同之处在于MLCK2内不存在完整的免疫球蛋白样(Ig)结构域3。只有MLCK1与稳态时的结周肌动球蛋白环相关，并且这种定位被包括肿瘤坏死因子(TNF)的炎症刺激增强。在这里，我们试图鉴定指导结周MLCK1定位的MLCK1结构域及其与疾病的相关性。克罗恩病患者回肠活检显示，相对于健康对照，MLCK1表达和结周定位优先增加。与MLCK1相反，在肠上皮细胞中表达的MLCK2主要与基础应力纤维有关，两种亚型对上皮迁移和屏障调节有不同的影响。MLCK1（Ig1-4）和MLCK（Ig1-3），但不是MLCK2（Ig1-4）或MLCK1（Ig3），在体外直接与F-肌动蛋白结合，并在肠上皮细胞中直接进行结周募集。进一步的研究表明，Ig1是不必要的，但是，与Ig3一样，Ig1和Ig2之间的非结构化接头（Ig1/2us）对于募集至关重要。尽管无法独立结合F-肌动蛋白或直接招募，Ig3确实具有显性负功能，使其能够取代结周MLCK1，增加稳态屏障功能，防止TNF诱导的MLCK1募集，并减弱TNF诱导的屏障丧失。这些数据定义了MLCK1定位所需的最小域，并提供了对MLCK1募集过程的机械洞察。总的来说,这些结果为分子靶向疗法的开发奠定了基础，该疗法靶向关键的MLCK1域以防止招募，恢复屏障功能，并限制炎症性肠病的进展。
