TRAUMA-INDUCED REPROGRAMMING: CHANGES IN LIPID RAFT PROTEIN CONTENT

创伤引起的重编程：脂筏蛋白含量的变化

• 批准号: 7359117
• 负责人: Joseph Cuschieri
• 金额: $ 2.1万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7359117
• 关键词: TRAUMA; INDUCED; REPROGRAMMING; CHANGES; LIPID

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Following severe trauma mononuclear cells are reprogrammed leading to alterations in innate immunity. These phenotypes are responsible for increased susceptibility to invading organisms leading to the development of organ failure. This state has been recreated in vitro by subjecting mononuclear cells to factors induced by trauma, including platelet activating factor (PAF), oxidant stress and complement 5a (C5a). Although the mechanism(s) responsible for this reprogramming remain unknown, previous work has demonstrated that this process may be associated with alterations in the protein content within specific plasma membrane microdomains that are rich in cholesterol and sphingolipids termed lipid rafts. Following injury, we hypothesize that factors induced by trauma result in the production of the lipid mediator ceramide from lipid rafts. Ceramide once produced fuses within rafts leading to the formation of macrodomains resulting in changes in membrane fluidity. Due to these changes, various proteins are recruited to the lipid raft resulting in the formation of focal adhesion-like complexes that contain some but not all of the Toll-like receptor (TLR) components. The following experimental approach will be followed: Differentiated THP-1 cells will be subjected to lipopolysaccharide (LPS) stimulation for various periods of time up to 60 min. Selected cells will be pre-treated with PAF, hydrogen peroxide or C5a for periods of time up to 30-60 min. Lipid raft protein extraction will be performed using sucrose gradient centrifugation. Harvested proteins will then be used for analysis using the LC-ESI-MS system. It is our hypothesis that assembly of these complexes and changes in lipid raft content are responsible for subsequent reprogramming that induces enhanced activation in response to subsequent infection. Based on these in vitro observations, it is our hope to then explore potential changes that occur in severely injured trauma patients in order to determine potential prognostic and therapeutic targets.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。严重创伤后，单核细胞被重新编程，导致先天免疫的改变。这些表型导致对入侵生物体的易感性增加，从而导致器官衰竭的发展。这种状态已经在体外通过使单核细胞经受由创伤诱导的因子（包括血小板活化因子（PAF）、氧化应激和补体5a（C5 a））来重建。虽然负责这种重编程的机制仍然未知，但以前的工作已经证明，这一过程可能与特定质膜微区中蛋白质含量的改变有关，这些微区富含胆固醇和鞘脂，称为脂筏。损伤后，我们假设创伤引起的因素导致脂质介体神经酰胺从脂筏的生产。神经酰胺一旦产生就在筏内融合，导致大域的形成，从而导致膜流动性的变化。由于这些变化，各种蛋白质被募集到脂筏，导致形成粘着斑样复合物，其含有一些但不是全部的Toll样受体（TLR）组分。 将遵循以下实验方法：分化的THP-1细胞将接受脂多糖（LPS）刺激不同时间段，最长60 min。选择的细胞将用PAF、过氧化氢或C5 a预处理30-60 min。将使用蔗糖梯度离心进行脂筏蛋白提取。然后将收获的蛋白质用于使用LC-ESI-MS系统进行分析。我们的假设是，这些复合物的组装和脂筏含量的变化是负责随后的重编程，诱导增强激活响应随后的感染。基于这些体外观察，我们希望探索严重创伤患者发生的潜在变化，以确定潜在的预后和治疗靶点。