Cytoskeletal rescue of polarized atypical PKC in intestinal junctions under infla

炎症条件下肠连接处极化非典型 PKC 的细胞骨架拯救

• 批准号: 8053457
• 负责人: Pedro Salas
• 金额: $ 31.43万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8053457
• 关键词: Affect; American; Animal Model; Animals; Antigens; Apical; Bacteria; Binding; Biological Assay; Blood; Brain; Catalytic Domain; Cell Fractionation; Cells; Chronic; Colitis; Competence; Complex; Connective Tissue; Cytoskeleton; Data; Degradation Pathway; Development; Diffusion; Disease; Epithelial; Epithelial Cells; Epithelium; Excision; Family member; Health; Heat-Shock Proteins 70; Human; In Vitro; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Intermediate Filaments; Intestines; Keratin; Laboratories; Lead; Lung; Maintenance; Malignant Neoplasms; Measures; Mediating; Molecular; Molecular Chaperones; Molecular Conformation; Oncogenes; Organ; Pathogenesis; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiologic pulse; Physiological; Principal Investigator; Proteins; Reaction; Reactive Oxygen Species; Recruitment Activity; Research; Role; Scientist; Signal Transduction; Site; Sodium Dextran Sulfate; Specificity; System; TNF gene; Testing; Tight Junctions; Tissues; Toxin; Transgenic Mice; atypical protein kinase C; base; cytokine; deletion analysis; in vivo; intestinal crypt; intestinal epithelium; knock-down; mutant; novel; overexpression; reconstitution; research study; scaffold; therapy design; tissue culture

DESCRIPTION (provided by applicant): Opening of tight junctions is involved in the pathogenesis of inflammatory bowel disease (IBD). Many studies have focused on the role of Tyr-kinases in the removal of tight junction components. However, no one has yet studied the role of atypical Protein Kinase C (aPKC), the evolutionarily conserved organizer of tight junctions, in inflammation. Our preliminary data shows a steep decrease of aPKC in intestinal epithelia exposed to pro- inflammatory signals. We have also shown that this effect is post-trancriptional and related to an Hsp-70- intermediate filament- dependent mechansim of rescue of aPKC. We hypothesize that post-translational mechanisms that recruit and maintain atypical PKC at the apical domain are impaired by inflammatory signals, resulting in increased tight junction permeability in the intestinal epithelium. We propose to: (1) Determine the molecular mechanisms by which Hsp70 chaperones rescue misfolded aPKC otherwise targeted for degradation. A combination of tissue culture of human intestinal cells, subcellular fractionation, and an in vitro reconstitution assay developed in our laboratory will be used to analyze the components of the aPKC rescue mechanisms. (2) Characterize molecular mechanisms that can antagonize loss of aPKC active conformation or degradation. Specific knock-down of Thr/Ser phosphatase catalytic subunits and expression of keratin intermediate filaments in human cells that normally lack them will be used to assess possible mechanisms to protect aPKC from losing its active conformation or to enhance the rescue mechanism. These observations will be validated in transgenic mice overexpressing keratin intermediate filaments. (3) Identify pro-inflammatory signals that downregulate aPKC and their targets in the degradation and rescue machinery that maintains aPKC physiologic levels. We will screen for pro-inflammatory cytokines that decrease aPKC levels, aside of TNF-, and analyze the step of the activation and degradation pathways of aPKC that are affected by pro-inflammatory signaling. Again, the results will be validated in an animal model PUBLIC HEALTH RELEVANCE: IBD is a chronic invalidating disease that affects around 3 million mericans. Increased permeability across the epithelial intestinal barrier is not the cause, but it is widely accepted as an important factor to perpetuate the inflammation. The studies in this project are intended to understand the molecular basis of a novel, as yet non studied mechanism to organize and maintain the "tightness" (tight junction competence) of the epithelial barrier.

描述（由申请人提供）：紧密连接的开放与炎症性肠病（IBD）的发病机理有关。许多研究集中在Tyr-激酶在去除紧密连接组件中的作用。然而，尚无人研究非典型蛋白激酶C（APKC）在炎症中的进化保守的紧密连接的组织者。我们的初步数据显示，暴露于炎症信号的肠上皮中的APKC急剧下降。我们还表明，这种作用是终文后的，并且与HSP-70-依赖于APKC的HSP-70中间丝丝相关。我们假设炎症信号会损害募集和维持非典型PKC的翻译后机制，从而导致肠上皮上皮的紧密连接性渗透率增加。我们建议：（1）确定HSP70伴侣挽救异折叠APKC的分子机制，另有针对降解。在我们实验室开发的人类肠细胞的组织培养物，亚细胞分馏和体外重构测定法的结合将用于分析APKC救援机制的组成部分。 （2）表征可以拮抗APKC主动构象或降解的分子机制。 THR/SER磷酸酶催化亚基的特定敲低以及通常缺乏它们的角蛋白中间丝的表达，将用于评估保护APKC的可能机制，以防止APKC失去其活性构型或增强救援机制。这些观察结果将在过表达角蛋白中间细丝的转基因小鼠中得到验证。 （3）确定在维持APKC生理水平的降解和救援机制中下调APKC及其目标的促炎信号。我们将筛选出降低TNF-降低APKC水平的促炎性细胞因子，并分析受促炎性信号传导影响的APKC的激活和降解途径的步骤。同样，结果将在动物模型中得到验证 公共卫生相关性：IBD是一种慢性无效疾病，影响了约300万梅美洲人。在上皮肠壁上的渗透性提高并不是原因，而是被广泛认为是使炎症永存的重要因素。该项目中的研究旨在理解新颖的分子基础，但尚未研究的机制，以组织和维持上皮屏障的“紧密连接能力”（紧密连接能力）。