NQ01, Oxidative Stress and Proteasomal Inhibition

描述(由申请人提供)：多巴胺能神经元氧化应激增加被认为是帕金森氏病(PD)的一个致病因素。在这些细胞中产生的儿茶酚、DOPA、多巴胺和DOPAC可以被氧化，产生具有侵略性的氧物种和活性芳基化苯二酚，这些都能够对细胞造成损害。我们已经确定了NQO1基因(NQO1*2)的缺失多态，并发现携带NQO1*2变异等位基因的个体与强调NQO1作为帕金森病危险因素的对照个体相比，患帕金森病的风险显著增加。PD还与泛素-蛋白酶体系统(UPS)的抑制有关。在神经退行性疾病中，聚集蛋白抑制UPS会导致氧化应激增加。由于氧化应激可导致蛋白质的错误折叠和聚集，从而导致UPS进一步受挫，因此开始出现一幅图景，其中氧化应激和UPS抑制都是恶性循环的一部分，并且是PD病因的重要因素。在这项提议中，我们将检验以下假设：1)NQO1保护细胞免受DOPA、多巴胺和DOPAC代谢过程中产生的反应性芳基苯醌和氧化物种的影响。我们将使用多巴胺能细胞、基于机制的NQO1抑制剂和我们实验室开发的等基因细胞模型来探索NQO1在这些研究中的作用；2)DOPA、多巴胺或DOPAC衍生的邻苯二酚有助于抑制UPS，而NQO1可对抗这种抑制作用；3)NQO1可防止因抑制UPS而产生的氧化应激。UPS在细胞中的抑制将通过使用化学蛋白酶体抑制剂和转导突变形式的α-突触核蛋白来实现，这些突变形式与PD相关，并通过涉及蛋白质聚集的机制抑制UPS；4)突变的NQO1*2蛋白通常被UPS快速降解，产生氧自由基，导致氧化应激增加。突变的NQO1*2蛋白产生氧自由基的能力在UPS被抑制和蛋白质积累的条件下变得特别重要。

DESCRIPTION (provided by applicant): Increased oxidative stress in dopaminergic neurons has been implicated as a causative factor in Parkinson's disease (PD). The catechols DOPA dopamine and DOPAC generated in these cells can undergo oxidation to produce aggressive oxygen species and reactive arylating quinones which are capable of cellular damage. We have characterized a null polymorphism in the NQO1 gene (NQO1 *2) and individuals carrying the variant NQO1 *2 allele have been found to be at a markedly increased risk of PD relative to control individuals emphasizing the role of NQO1 as a risk factor for PD. PD has also been associated with inhibition of the ubiquitin-proteasomal system (UPS). Inhibition of the UPS by aggregated proteins in neurodegenerative disease leads to increased oxidative stress. Since oxidative stress can result in the misfolding and aggregation of proteins resulting in further frustration of the UPS, a picture is beginning to emerge where both oxidative stress and UPS inhibition are part of a vicious cycle and are important factors in the etiology of PD.In this proposal, we will test the following hypotheses; 1) that NQO1 protects cells against reactive arylating quinones and oxidizing species generated during metabolism of DOPA, dopamine and DOPAC. We will use dopaminergic cells, mechanism based inhibitors of NQO1 and an isogenic cellular model developed in our lab to explore the role of NQOl in these studies; 2) that DOPA, dopamine or DOPAC derived o-quinones contribute to UPS inhibition and that NQO1 protects against this inhibitory effect; 3) that NQO1 protects against oxidative stress generated as a result of inhibition of the UPS. UPS inhibition in cells will be achieved by the use of both chemical proteasome inhibitors and by transfection of mutant forms of alpha-synuclein which have been associated with PD and inhibit the UPS by mechanisms involving protein aggregation and 4) that mutant NQO1*2 protein, which is normally rapidly degraded by the UPS, generates oxygen radicals leading to increased oxidative stress. The ability of mutant NQO1 *2 protein to generate oxygen radicals becomes particularly important under conditions where the UPS is inhibited and the protein accumulates.