Mechanisms of LRRK2 Mediated Neurotoxicity

描述（由申请人提供）：帕金森病（PD）是美国发病率和死亡率的主要原因。病因在很大程度上是未知的，减缓或阻止疾病无情进展的疗法还不存在。富含亮氨酸重复序列激酶2（LRRK2）基因中的显性错义突变是PD最常见的已知特异性病因，LRRK2突变与模拟典型迟发性疾病的疾病表型相关。LRRK2编码一种异常大的蛋白激酶，导致PD的最常见突变可能上调LRRK2激酶活性。由于小分子蛋白激酶抑制剂对多种类型癌症的治疗产生了影响，因此LRRK2代表了治疗PD的潜在治疗靶点，如果激酶活性与模型系统中的神经变性相关。通过设计编码LRRK2开放阅读框的新型高容量病毒载体，所述LRRK2开放阅读框用上调激酶活性的引起PD的突变或抑制激酶活性的突变修饰，将确定LRRK2激酶活性在体内指导多巴胺能神经变性中的重要性。致病突变对LRRK2功能和活性的具体影响尚未完全了解，但可能允许一种直接的途径来破译疾病相关的LRRK2功能。LRRK2编码一种活性激酶和GTP酶蛋白，这是人类蛋白质组中一种近乎独特的排列，致病性突变可发生在两个酶结构域中。寡聚化和二聚化是许多蛋白激酶和GTP酶蛋白的内在调节机制。我们将生物化学表征寡聚体和二聚体LRRK2蛋白，并确定致病性LRRK2突变对LRRK2构象组成和相关活性的影响。此外，我们开发的技术，可以允许直接可视化的LRRK2酶活性在活细胞中，并探索抑制和激活激酶活性。通过解析LRRK2自磷酸化位点，我们发现了激酶和GTP酶活性可能相互调节的意外复杂性，其中激酶活性可能在介导GTP酶依赖性LRRK2二聚化和激酶介导的底物磷酸化中发挥双重作用。我们将描述LRRK2自身磷酸化对酶调节的作用以及PD相关突变如何干扰正常活动。

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.