小窝蛋白脚手架区变异多肽解除HO-1活性内源性抑制的实验研究

HO-1 can protect against acute lung injury (ALI) due to its anti-oxidant, anti-inflammatory and anti-apoptotic properties. Heme, HO-1 specific inducer, administration 12h before LPS challenge effectively protected against ALI. However, heme administration after LPS challenge failed to attenuate LPS-induced ALI because of lower HO-1 bioactivity. Inhibition of HO-1 bioactivity by endogenous caveolin-1 (Cav-1) has been well characterized in vitro and in vivo. Cav-1 inhibits HO-1 bioactivity via the five-amino acid sequence (residues 97?101) between the caveolin scaffolding domain (CSD), but which amino acid residue(s) is(are) critical to inhibit HO-1 activity remains to determine. Cav-1 CSD also contacts and suppresses eNOS activity. Phe-92 (F92) amino acids residue is crucial for Cav-1 mediated inhibition of eNOS. Mutation of F92 to A92 in the Cav-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cav-1. AP-F92A-Cav-1CSD, a cell permeable peptide (AP) harboring a mutant form of the Cav-1 CSD, competitively interacted with eNOS and specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release in mice. These observations have led us to propose the overall hypothesis that AP- mutation amino acids residue -Cav-1CSD can competitively interact with HO-1 and specifically disrupted the inhibitory actions of endogenous Cav-1 toward HO-1 and thereby enhanced HO-1 bioactivity in vivo. In order to test this hypothesis, firstly, we will explore the amino acid residue(s), which is(are) critical to inhibit HO-1 activity via surface plasmon resonance measurement, in vitro binding assay and heme titration experiment. Secondly, we will design and synthesis AP- mutation amino acids residue -Cav-1CSD peptide, theoretically, which interact with HO-1 and do not inhibit HO-1activity. Next, we determine the protective role of AP- mutation amino acids residue -Cav-1CSD peptide on A549 alveolar epithelial cell, alveolar macrophage and rat lung after LPS challenge. Finally, we employ HO-1siRNA mice to test the beneficial function of this mutational Cav-1 CSD peptide on ALI is HO-1 dependant. Upon completion of the studies, we will gain important insights into the modulating mechanisms of HO-1 activity by caveolin-1, may get a new way to increase HO-1 activity, and thereby identify novel therapeutic method for ALI.

血红素加氧酶-1（HO-1）对急性肺损伤（ALI）有重要保护作用，但由于小窝蛋白-1（Cav-1）能内源性抑制HO-1的活性，使HO-1不能有效发挥对ALI的治疗功效。已知抑制HO-1活性的结构是Cav-1脚手架区（CSD）第97-101位5个氨基酸残基。但哪一个或哪几个是抑制HO-1活性的关键残基尚未清楚；能否合成活性抑制区变异的CSD多肽来干预体内野生型CAV-1与HO-1间的结合，使HO-1活性免于抑制是值得探讨的问题。本研究拟寻找抑制HO-1活性的关键氨基酸残基，创建能与体内野生型Cav-1竞争性结合HO-1而不抑制HO-1活性的Cav-1CSD变异多肽；观察Cav-1CSD变异肽联合血晶素对ALI的保护作用和机制；使用HO-1活性阻断剂锌原卟啉确认该多肽对ALI的治疗作用是HO-1依赖性的。本研究旨在探索解除内源性抑制HO-1活性的新方法，为HO-1临床应用构建理论基础。

血红素加氧酶-1（HO-1）对LPS诱导的急性肺损伤具有重要保护作用，然而其生物催化活性被小窝蛋白-1脚手架区（caveolin-1 scaffolding domain，CSD）内源性抑制，制约其保护效果；本研究利用生物信息学技术、激光共聚焦显微镜观察实时荧光定量PCR和Western-blot等手段，重点分析了与HO-1结合的CSD第97-101位5个氨基酸残基，设计了设计F99A突变型和101位氨基酸截断型CSD多肽，在细胞水平和动物水平比较了2种突变型CSD多肽和野生型全长CSD多肽对HO-1活性的影响及其对LPS损伤模型的保护作用。研究发现：与预期的结果不一致，野生型CSD多肽可使HO-1与肺泡巨噬细胞细胞膜Cav-1的结合减少，HO-1的活性增高2倍；野生型CSD多肽可降低TNF-α和iNOS IL-1β、MCP-1等炎症因子mRNA表达水平和NO含量明显下降；同时提高IL-10的表达水平; 野生生型CSD多肽也可使肺组织中HO-1与Cav-1的结合减少，HO-1的活性增高；促炎因子表达明显降低，从而肺湿/干比、肺泡灌洗液细胞数、血清中乳酸脱氢酶活性和肺组织病理形态变化明显减轻。显著降低。F99A突变型CSD多肽不能减少HO-1与肺泡巨噬细胞细胞膜Cav-1结合，抗炎效果不明显；截断型CSD多肽与HO-1的结合能为-69.72732，细胞实验也证实其能减少HO-1与肺泡巨噬细胞细胞膜Cav-1结合；但其对HO-1的活性影响不大；仅能使MCP-1 和NO mRNA等部分促炎因子表达水平下降。研究结果阐明野生型CSD能够明显增高HO-1活性， 有效保护LPS诱导的急性肺损伤。本项目有望提高一种治疗LPS诱导的急性肺损伤的新方法。从药物研发的角度考虑，突变的多肽具有潜在的毒性作用，野生型多肽更具有药物开发价值。我们正在申请专利。