Lipid-Modification of Proteins in Alzheimer's Disease

大量证据表明，淀粉样β蛋白(Abeta)寡聚和环氧合酶(COX)活性均参与了阿尔茨海默病(AD)的发病机制。这一提议试图确定这两个过程之间相互作用的分子基础。环氧合酶的直接产物是前列腺素H2(PGH2)，它在水溶液中迅速重排，大约20%转化为左旋糖苷E2(LGE2)和LGD2。这些高活性的伽马酮醛能迅速地加成胺基，特别是蛋白质赖氨酸残基的epsilon-胺，也能使蛋白质交联。我们发现，PGH2通过左旋糖苷途径，加速血管形成。 Abeta的可溶性低聚体，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，这些低聚体具有神经毒性。为了分析蛋白质的左旋糖基加合物的形成，我们对左旋糖基赖氨酸内酰胺加合物的结构进行了表征，并建立了串联质谱仪分析它的方法。利用这一敏感而具体的方法，我们现在已经确定 阿尔茨海默病患者大脑海马区蛋白质的左旋糖基加合物的水平比年龄匹配的对照组大脑中的水平高12倍。这种COX衍生的脑蛋白脂质修饰水平与阿尔茨海默病的Braak分期高度相关，并为COX活性参与阿尔茨海默病提供了切实的新证据。这项建议涉及AD中左旋糖苷加成的蛋白质的特征。这项拟议研究的最初目的是确定AD大脑中是否存在专门针对Abeta的左旋糖基加合物。这将通过分析来自免疫沉淀Abeta的加合氨基酸残基和多肽来解决，利用LC-串联质谱仪进行分析。此外, 将检查参与Abeta生物合成的蛋白质的左旋糖基加合物的存在，并利用无偏倚的蛋白质组学分析，将确定AD中其他蛋白质的左旋糖基加合物的存在。总之，蛋白质的脂质修饰为理解COX活性的后果提供了一个新的范式，这一提议将表征AD大脑中被COX衍生的脂质加合物修饰的蛋白质。

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.