Stretch and Shear Stress Induced Degradation of Keratin

描述(由申请人提供)： 急性肺损伤患者接受正压机械通气，以改善气体交换。然而，机械通气可能会在充满液体的肺泡的重复再开放过程中产生巨大的剪切力，并使备用的充气肺泡过度膨胀，从而导致患者预后不良和肺损伤恶化。角蛋白中间丝是肺泡上皮细胞中维持细胞机械完整性的主要结构蛋白。角蛋白在剪切力作用下会发生适应性变化。在初步研究中，我们观察到在切应力作用下，肺泡上皮细胞角蛋白IF网络解体，这与肺泡上皮细胞损伤有关。本应用拟通过三个相互关联的特定目标，利用细胞和分子生物学技术研究肺泡上皮细胞角蛋白IF对周期性剪应力和拉伸的反应：具体目标1：确定周期性剪应力和/或拉伸是否导致肺泡上皮细胞分解和降解角蛋白；具体目标2：确定周期性剪应力和/或拉伸是否通过泛素-蛋白酶体途径导致角蛋白降解；具体目标3：确定机械应变下泛素介导的肺泡上皮细胞角蛋白降解所涉及的E2和E3酶。这些研究的完成将对机械通气引起肺泡上皮细胞损伤的机制提供新的见解。

DESCRIPTION (provided by applicant): Patients with acute lung injury are placed on positive-pressure mechanical ventilation to improve gas exchange. However, mechanical ventilation may contribute to poor patient outcomes and worsening lung injury by generating large shear forces during repetitive reopening of fluid-filled alveoli and over-distending spared air-filled alveoli. Keratin intermediate filaments (IF) are the major structural proteins in alveolar epithelial cells that maintain the cell's mechanical integrity. Keratin IF undergo adaptive changes in response to shear stress. In preliminary studies we observed that the keratin IF network was disassembled in alveolar epithelial cells exposed to shear stress and that this was associated with alveolar epithelial cell injury. This application proposes to study the response of keratin IF to cyclic shear stress and stretch in alveolar epithelial cells utilizing cellular and molecular biology techniques via three interrelated specific aims: Specific Aim 1: To determine whether cyclic shear stress and/or stretch of alveolar epithelial cells causes keratin IF disassembly and degradation; Specific Aim 2: To determine whether cyclic shear stress and/or stretch causes degradation of keratin proteins occurs via the ubiquitin-proteasome pathway; Specific Aim 3: To identify the E2 and E3 enzymes involved in the ubiquitin-mediated degradation of alveolar epithelial cell keratin IF exposed to mechanical strain. Completion of these studies will provide new insights into the mechanisms responsible for ventilation induced mechanical injury to alveolar epithelial cells.