Mechanisms of LRRK2 Mediated Neurotoxicity

描述(由申请人提供)：帕金森氏病(PD)是美国发病率和死亡率的主要原因。病因在很大程度上还不清楚，减缓或阻止疾病持续发展的疗法还不存在。富含亮氨酸的重复蛋白激酶2(LRRK2)基因的显性错义突变是帕金森病最常见的特定病因，LRRK2突变与模仿典型晚发性疾病的疾病表型有关。LRRK2编码一个异常大的蛋白激酶，而导致帕金森病的最常见突变可能上调LRRK2激酶的活性。由于小分子蛋白激酶抑制剂对多种类型癌症的治疗有影响，LRRK2是一种潜在的治疗帕金森病的靶点，因为在模型系统中，蛋白激酶活性与神经退行性变相关。通过设计编码LRRK2开放阅读框架的新的高容量病毒载体，无论是引起PD的突变上调激酶活性还是突变失活激酶活性，LRRK2激酶活性在体内引导多巴胺能神经变性的重要性将被确定。致病突变对LRRK2功能和活性的具体影响尚不完全清楚，但可能允许一种直接的途径来破译与疾病相关的LRRK2功能。LRRK2编码活性激酶和GTPase蛋白，这是人类蛋白质组中几乎唯一的排列，并且这两个酶结构域都可能发生致病突变。寡聚和二聚化是许多蛋白激酶和GTP酶蛋白的内在调节机制。我们将对寡聚和二聚体LRRK2蛋白进行生化表征，并确定致病LRRK2突变对LRRK2构象组成和相关活性的影响。此外，我们开发了一种技术，可以直接显示活细胞中LRRK2酶的活性，并探索对激酶活性的抑制和激活。通过对LRRK2自动磷酸化位点的解析，我们发现了在可能的相互调节的激酶和GTPase活性中的一种意想不到的复杂性，其中激酶活性可能在介导GTPase依赖的LRRK2二聚化和激酶介导的底物的磷酸化中发挥双重作用。我们将表征LRRK2自动磷酸化在酶调节中的作用，以及PD相关突变如何扰乱正常活动。 公共卫生相关性：探索LRRK2基因突变致病的生化机制，以确定治疗开发的潜在靶点。LRRK2基因目前是帕金森病最常见的已知原因。识别与LRRK2介导的神经退行性变相关的细胞通路将进一步增强我们对帕金森氏病的了解，并刺激合理设计的药物的开发，以减缓或阻止疾病。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a major cause of morbidity and mortality in the United States. The etiology is largely unknown and therapies that slow or halt the relentless progression of disease do not yet exist. Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known specific cause of PD, and LRRK2 mutations associate with disease phenotypes that mimic typical late-onset disease. LRRK2 encodes an unusually large protein kinase, and the most common mutations that cause PD may up-regulate LRRK2 kinase activity. Since small molecule protein kinase inhibitors have made an impact on the treatment of multiple types of cancer, LRRK2 represents a potential therapeutic target for the treatment of PD should kinase activity correlate with neurodegeneration in model systems. Through the design of novel high capacity viral vectors encoding the LRRK2 open-reading frame modified with either PD-causing mutations that up-regulate kinase activity or mutations that inactivate kinase activity, the importance of LRRK2 kinase activity in directing dopaminergic neurodegeneration in vivo will be determined. The specific effects of pathogenic mutations on LRRK2 function and activity are not fully understood, but may allow for a straightforward route to decipher disease-related LRRK2 functions. LRRK2 encodes an active kinase and GTPase protein, a near unique arrangement in the human proteome, and pathogenic mutations can occur in both enzymatic domains. Oligomerization and dimerization are intrinsic mechanisms of regulation for many protein kinases and GTPase proteins. We will biochemically characterize oligomeric and dimeric LRRK2 protein and determine the effect of pathogenic LRRK2 mutations on the composition of LRRK2 conformations and associated activities. Further, we develop technology that may allow direct visualization of LRRK2 enzyme activity in living cells and explore both inhibition and activation of kinase activity. Through the resolution of LRRK2 autophosphorylation sites, we have uncovered an unexpected complexity in the probable reciprocal regulation of kinase and GTPase activities, where kinase activity may play a dual role in mediating GTPase-dependent LRRK2 dimerization and kinase-mediated phosphorylation of substrates. We will characterize the role of LRRK2 autophosphorylation on enzymatic regulation and how PD-associated mutations perturb normal activities. PUBLIC HEALTH RELEVANCE: The biochemical mechanisms underlying disease-causing mutations in the LRRK2 gene, currently the most common known cause of Parkinson's disease, are explored in order to determine potential targets for therapeutics development. Identification of cellular pathways linked with LRRK2-mediated neurodegeneration will further enhance our understanding of Parkinson's disease and spur the development of rationally designed drugs that slow or halt the disease.