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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10441427
• 关键词: Acquired Immunodeficiency Syndrome; Actomyosin; Affinity; Apical; Attenuated; Benefits and Risks; Binding; Biopsy; Catalytic Domain; Celiac Disease; Cell Adhesion Molecules; Cessation of life; Citrobacter rodentium; Colitis; Communicable Diseases; Complement; Crystallization; Cytoplasm; Data; Development; Diabetes Mellitus; Disease; Dominant-Negative Mutation; Enzymes; Epithelial; Event; FK506; Foundations; Functional disorder; Funding; Future; Gastrointestinal Diseases; Genetic Transcription; Goals; Grant; Gut Mucosa; Health; Human; Immune; Immune system; Immunoglobulins; In Vitro; Infectious colitis; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Intercellular Junctions; Intestinal Content; Intestinal Diseases; Intestines; Knock-out; Knowledge; Lead; Learning; Left; Libraries; Light; Mediating; Modeling; Molecular; Mucosal Immunity; Mucositis; Mucous Membrane; Mucous body substance; Mus; Myosin Alkali Light Chains; Myosin Light Chain Kinase; Myosin Type II; Outcome; Pathologic; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacology; Physiological; Protein Isoforms; Proteins; Proteomics; RNA Splicing; Regulation; Rheumatoid Arthritis; Role; Signal Transduction; Site; Stimulus; Structure; System; Systemic disease; TNF gene; Therapeutic; Therapeutic Agents; Tight Junctions; Tissues; Toxic effect; Variant; Work; druggable target; effective therapy; epithelial injury; graft vs host disease; immune activation; immunoregulation; improved; in silico; in vivo; intestinal barrier; intestinal epithelium; microbiome; mortality; novel; novel strategies; novel therapeutic intervention; pathogen; preservation; prevent; programs; protein protein interaction; recruit; repaired; response; restoration; small molecule; targeted treatment; Acquired Immunodeficiency Syndrome; Actomyosin; Affinity; Apical; Attenuated; Benefits and Risks; Binding; Biopsy; Catalytic Domain; Celiac Disease; Cell Adhesion Molecules; Cessation of life; Citrobacter rodentium; Colitis; Communicable Diseases; Complement; Crystallization; Cytoplasm; Data; Development; Diabetes Mellitus; Disease; Dominant-Negative Mutation; Enzymes; Epithelial; Event; FK506; Foundations; Functional disorder; Funding; Future; Gastrointestinal Diseases; Genetic Transcription; Goals; Grant; Gut Mucosa; Health; Human; Immune; Immune system; Immunoglobulins; In Vitro; Infectious colitis; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Intercellular Junctions; Intestinal Content; Intestinal Diseases; Intestines; Knock-out; Knowledge; Lead; Learning; Left; Libraries; Light; Mediating; Modeling; Molecular; Mucosal Immunity; Mucositis; Mucous Membrane; Mucous body substance; Mus; Myosin Alkali Light Chains; Myosin Light Chain Kinase; Myosin Type II; Outcome; Pathologic; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacology; Physiological; Protein Isoforms; Proteins; Proteomics; RNA Splicing; Regulation; Rheumatoid Arthritis; Role; Signal Transduction; Site; Stimulus; Structure; System; Systemic disease; TNF gene; Therapeutic; Therapeutic Agents; Tight Junctions; Tissues; Toxic effect; Variant; Work; druggable target; effective therapy; epithelial injury; graft vs host disease; immune activation; immunoregulation; improved; in silico; in vivo; intestinal barrier; intestinal epithelium; microbiome; mortality; novel; novel strategies; novel therapeutic intervention; pathogen; preservation; prevent; programs; protein protein interaction; recruit; repaired; response; restoration; small molecule; targeted treatment

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： 肠道屏障功能在传染性和免疫介导的肠道疾病中受到损害。这 计划现在完成其第三个融资周期，重点关注肠的机制和影响 上皮屏障功能障碍。在以前的周期中，我们将上皮肌球蛋白轻链激酶（MLCK）定义为 紧密连接屏障功能的关键调节因子响应炎症刺激。这项工作已经 被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。我们也是 定义MLCK激活下游的信号事件，发现屏障受到调节 免疫系统，紧密连接渗透性的MLCK依赖性增加也能够调节粘膜 触发Claudin-2表达增加的免疫力。这导致了我们对毛孔的共发现和泄漏 跨距连接通量的途径；现在被广泛接受的模型。在当前周期中，我们专注于 孔隙和泄漏途径，发现在传染病的背景下，紧密连接增加 由于Claudin-2表达而导致的渗透性是有益的，并促进了病原体清除率。另外，我们 将特定的MLCK剪接变体MLCK1确定为紧密连接渗透性的关键调节剂。在 除了激活MLCK转录和酶活性，我们发现炎症刺激引起 MLCK1将被招募到肌周围的肌球蛋白环。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募集所必需的，足以充当A 主要负面对阻止招聘。我们继续解决IGCAM3晶体结构，确定潜力 人类和小鼠IgCAM3之间保守但在其他MLCK中保守的药物结合口袋 IgCAM，并在〜140,000种药物样分子的NCI库中执行一个硅筛。我们确定了一个 称为脱水素，可防止MLCK1募集而不抑制上皮或平滑肌MLCK 酶促功能。通过将MLCK1募集阻塞到肌膜外肌球蛋白环上，Everertin可防止 MLCK1来自该部位磷酸化肌球蛋白II调节轻链。反过来，这会阻止炎症 - 在体外，体内（小鼠）和体内（人肠活检）诱导屏障丧失。分离蛋白减弱了所有 实验性免疫介导的炎症性肠病（小鼠）的特征，包括屏障损失，免疫 激活和死亡率。该建议的目的是确定由 分离并定义MLCK1募集的分子机制。描述的研究将表征 MLCK1约束伙伴我们已经发现了以及通过 尖端的蛋白质组学方法。完成后，这些研究将定义MLCK1 Interactome 以及监管招聘的机制以及抑制的潜在益处和风险 MLCK1招聘。这些进步将使未来的类似神经蛋白的药物用于人类使用。