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

接合周围肌球蛋白轻链激酶募集：用于治疗性肠屏障恢复的新型非酶靶点

基本信息

批准号：
10441427
负责人：
JERROLD R. TURNER
金额：
$ 68.82万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2024-06-30
项目状态：
已结题

项目摘要

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.

概括：肠道屏障功能在感染性和免疫介导的肠道疾病中受到损害。这该计划现已完成第三个资助周期，重点关注肠道的机制和影响上皮屏障功能障碍。在之前的周期中，我们将上皮肌球蛋白轻链激酶 (MLCK) 定义为响应炎症刺激的紧密连接屏障功能的关键调节剂。这项工作已广泛复制并扩展到其他系统。我们继续证明 MLCK 依赖性增加渗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，发现虽然屏障受免疫系统中，MLCK 依赖性的紧密连接通透性增加也能够调节粘膜触发claudin-2表达增加的免疫。这导致我们共同发现了孔隙和泄漏跨密封连接通量的路径；现在已被广泛接受的模型。在当前周期中，我们重点关注孔隙和泄漏途径，发现在传染病的背景下，紧密连接增加 Claudin-2 表达带来的渗透性是有益的，可促进病原体清除。此外，我们确定了一种特定的 MLCK 剪接变体 MLCK1，作为紧密连接通透性的关键调节因子。在除了激活 MLCK 转录和酶活性外，我们发现炎症刺激会导致 MLCK1 被招募到连接周围肌动球蛋白环。我们确定了一个特定的域，免疫球蛋白- 细胞粘附分子结构域 3 (IgCAM3) 是 MLCK1 招募所必需的，并且足以充当显性阴性阻止招募。我们继续解决了 IgCAM3 晶体结构，确定了潜在的药物结合袋在人和小鼠 IgCAM3 之间保守，但在其他 MLCK 中不存在 IgCAM，并对约 140,000 个药物样分子的 NCI 文库进行计算机筛选。我们确定了一个，称为 Divertin，可防止 MLCK1 募集而不抑制上皮或平滑肌 MLCK 酶的功能。通过阻断 MLCK1 募集到连接周围肌动球蛋白环，Divertin 可防止 MLCK1 来自磷酸化肌球蛋白 II 调节轻链的该位点。这反过来又可以阻止炎症—— 在体外、体内（小鼠）和离体（人肠道活检）中诱导屏障丧失。 Divertin 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、免疫激活和死亡率。该提案的目的是确定通过修饰修饰的细胞内蛋白质相互作用 Divertin 并定义了 MLCK1 募集的分子机制。所描述的研究将表征我们已经发现了 MLCK1 结合伙伴以及通过以下方式确定的新结合伙伴尖端蛋白质组学方法。完成后，这些研究将定义 MLCK1 相互作用组招募的监管机制以及抑制的潜在好处和风险 MLCK1 招募。这些进步将使未来开发人类使用的类地弗汀制剂成为可能。

项目成果

期刊论文数量（64）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inhibiting myosin light chain kinase retards the growth of mammary and prostate cancer cells.

抑制肌球蛋白轻链激酶可延缓乳腺癌和前列腺癌细胞的生长。

DOI：
发表时间：
2006-05
期刊：
影响因子：
0
作者：
Gu, Lian;Hu, Wen;Antic, Nenad;Mehta, Rajendra;Turner, Jerrold R;de Lanerolle, Primal
通讯作者：
de Lanerolle, Primal

Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.

活细胞中蛋白质-蛋白质相互作用的时间分辨发光共振能量转移成像。

DOI：
发表时间：
2010-08-03
期刊：
影响因子：
11.1
作者：
Rajapakse, Harsha E;Gahlaut, Nivriti;Mohandessi, Shabnam;Yu, Dan;Turner, Jerrold R;Miller, Lawrence W
通讯作者：
Miller, Lawrence W

Good fences make good neighbors: Gastrointestinal mucosal structure.

好围栏造就好邻居：胃肠粘膜结构。