Lipid-Modification of Proteins in Alzheimer's Disease

大量证据表明，淀粉样β蛋白（A β）寡聚化和环氧合酶（考克斯）活性参与阿尔茨海默病（AD）的发病机制。这一提议旨在确定这两个过程之间相互作用的分子基础。环加氧酶的直接产物是前列腺素H2（PGH 2），其在水溶液中快速重排，其中约20%转化为左旋花素E2（LGE 2）和LGD 2。这些高度反应性的γ-酮醛迅速加合到胺基，特别是蛋白质的赖氨酸残基的ε-胺，并且还可以交联蛋白质。我们已经发现，PGH 2，通过左旋兰途径，加速形成 具有Abeta衍生的可扩散配体的免疫化学、电泳和超微结构特征的Abeta可溶性寡聚体，并且这些寡聚体具有神经毒性。为了分析蛋白质的左甘氨酰加合物的形成，我们已经表征了左甘氨酰-赖氨酸内酰胺加合物的结构，并开发了一种用串联质谱法对其进行分析的方法。利用这种敏感而特异的方法， 在AD患者的大脑海马体中，左旋丙氨酰蛋白加合物的水平比在年龄匹配的对照大脑中发现的水平高12倍。脑蛋白的这种COX衍生的脂质修饰的水平与阿尔茨海默病的Braak阶段的严重程度高度相关，并为考克斯活性参与该疾病提供了切实的新证据。该提案解决了AD中由左甘肽加合的蛋白质的表征。拟议研究的最初目的是确定AD大脑中特异性Abeta上的左旋甘氨酰加合物的存在。这将通过利用LC-串联质谱分析免疫沉淀的Abeta蛋白水解衍生的加合氨基酸残基和肽来解决。此外，本发明还提供了一种方法， 将检查参与A β生物合成的蛋白质的左甘氨酰加合物的存在，并利用无偏蛋白质组学分析，确定AD中其它蛋白质的左甘氨酰加合物的存在。总之，脂质修饰的蛋白质提供了一个新的范例，了解考克斯活动的后果，这一建议将表征蛋白质修饰的COX衍生的脂质加合物在AD的大脑。

Substantial evidence indicates that both amyloid beta (Abeta) oligomerization and cyclooxygenase (COX) activity contribute to the pathogenesis of Alzheimer's disease (AD). This proposal seeks to determine the molecular basis of the interaction between these two processes. The immediate product of the cyclooxygenases is prostaglandin H2(PGH2), which rapidly rearranges in aqueous solution with approximately 20% being converted to levuglandin E2(LGE2) and LGD2. These highly reactive gamma-ketoaldehydes rapidly adduct to amine groups, notably the epsilon-amine of the lysine residues of proteins, and also can crosslink proteins. We have discovered that PGH2, via the levuglandin pathway, accelerates formation of soluble oligomers of Abeta that have the immunochemical, electrophoretic, and ultrastructural characteristics of Abeta-derived diffusible ligands, and these oligomers are neurotoxic. In order to analyze the formation of levuglandinyl adducts of proteins, we have characterized the structure of the levuglandinyl-lysine lactam adduct and developed a method for its analysis with tandem mass spectrometry. Utilizing this sensitive and specific method we have now identified levuglandinyl adducts of protein in hippocampus of brains from patients who had AD, at levels that are increased 12 fold above those found in age-matched control brains. The level of this COX-derived lipid modification of brain proteins is highly correlated with the Braak stage of severity of the Alzheimer's disease and provides tangible new evidence for participation of COX activity in the disease. This proposal addresses the characterization of proteins that are adducted by levuglandins in AD. An initial aim of the proposed research is to determine the presence of levuglandinyl adducts specifically on Abeta in AD brains. This will be addressed by the analysis of adducted amino acid residues and peptides proteolytically derived from immunoprecipitated Abeta, utilizing LC-tandem mass spectrometry for analysis. In addition, the presence of levuglandinyl adducts of proteins involved in biosynthesis of Abeta will be examined, and utilizing an unbiased proteomic analysis, the presence of levuglandinyl adducts of other proteins in AD will be determined. In summary, lipid modification of proteins provides a new paradigm for understanding the consequences of COX activity, and this proposal will characterize proteins that are modified by COX-derived lipid adducts in the brains of AD.