Development of chain selective polyubiquitin markers as early detectors of Parkinson’s and Alzheimer’s Disease

哺乳动物细胞通过泛素(Ub)蛋白酶体途径降解错误折叠的蛋白质 和自噬。神经元功能障碍或神经元死亡的发展的第一步是缺乏 对错误折叠的蛋白质的反应。神经元中Ub蛋白酶体途径的功能障碍导致蓄积 神经元中的聚集体和斑块。蛋白质的泛素化不仅控制蛋白质的半衰期，而且 也起到调节修饰的作用，例如，组蛋白泛素化。由于Ub共轭的动态性质 和去结合，泛素化的蛋白质是非常困难的分离和研究。用来识别蛋白质 在修饰的蛋白质池中，几乎不可能有一个特定的Ub翻译后修饰 没有选择性的工具。更复杂的是在多泛素中存在多种类型的Ub-Ub连接 锁链。泛素通过异肽键与目标蛋白中的赖氨酸残基相连。这些Ub-Moiets 然后可以作为其他瑞银结合的底物，也是通过形成异肽 一个Ub的C-末端与目标Ub的七(7)个赖氨酸中的任何一个或目标Ub的N-末端之间的键。UB链 不同的连接向细胞传递不同的信息，从而决定蛋白质的最终命运 --降解、移位和氧化应激反应，仅举几例。编码的精确信息 由于缺乏选择性识别这些的试剂，在各种链上的连接在很大程度上是未知的 联系。这项提议的目标是开发工具，允许选择性地识别、量化和 分离含有难以研究的连接的单泛素链和多泛素链修饰的蛋白质。这将是 使用包含来自不同种类的所有已知Ub结合基序的新型Ub结合微阵列来完成 蛋白质。在第一阶段，我们将确定和表征新的Ub结合结构域(UBD)，该结构域具有选择性 单泛素、单泛素化底物和特定的多泛素连接。考虑到事实是 Ub蛋白酶体途径的失调和自噬被强烈地认为是 神经退行性疾病，敏感而新颖的链选择工具的开发将导致早期 阿尔茨海默氏症和帕金森氏病的诊断(第二阶段)。

Mammalian cells remove misfolded proteins by degradation mediated by the ubiquitin (Ub) proteasome pathway and autophagy. The first step in the development of neuronal dysfunction or neuronal death is the lack of a response to misfolded proteins. Dysfunction in the Ub proteasome pathway in neurons leads to the accumulation of aggregates and plaques in neurons. Ubiquitylation of proteins not only controls the half-lives of proteins but also acts as a regulatory modification, e.g., histone ubiquitylation. Due to the dynamic nature of Ub conjugation and deconjugation, ubiquitylated proteins are extremely difficult to isolate and study. Identifying a protein with one particular Ub post translational modification out of the pool of modified proteins becomes nearly impossible without selective tools. A further complication is the presence of multiple types of Ub-Ub linkages in polyubiquitin chains. Ubiquitin is attached, via isopeptide bonds, to lysine residues in the target protein. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, again through the formation of isopeptide bonds between the C-terminus of one Ub and any of seven (7) lysines, or N-terminus of the target Ub. Ub chains with different linkages convey different messages to cells and, hence, determine the ultimate fate of the protein -- degradation, translocation, and oxidative stress response, to name a few. The precise information encoded in the various chain linkages is largely unknown due to a lack of reagents that selectively recognize these linkages. The goal of this proposal is to develop tools that allow the selective identification, quantification, and isolation of proteins modified by mono and polyubiquitin chains containing difficult to study linkages. This will be accomplished using a novel Ub binding microarray that contains all of the known Ub binding motifs from various proteins. In Phase I, we will identify and characterize novel Ub binding domains (UbDs) exhibiting selectivity for monoubiquitin, monoubiquitylated substrates and specific polyubiquitin linkages. Given the fact that dysregulation of the Ub proteasome pathway and autophagy have been strongly implicated as a first step in neurodegenerative diseases, the development of sensitive and novel chain selective tools will lead to early diagnosis of Alzheimer’s and Parkinson’s diseases (phase II).