Biophysical analysis of LRRK2

描述（由申请方提供）：富含亮氨酸重复激酶2（LRRK 2）基因突变导致帕金森病（PD）的常染色体显性形式。细胞裂解物中LRRK 2的生化分析表明，蛋白质二聚化，并且激酶活性的损伤导致二聚体的消耗和高阶寡聚体的形成。有趣的是，PD相关突变将平衡向LRRK 2的二聚体（活性）状态转移。由于LRRK 2激酶活性明显依赖于其寡聚化状态，因此我们在目标1中提出使用荧光波动光谱（FFS）方法阐明活细胞中LRRK 2自缔合的过程。将采用三种FFS模式：1。数量和亮度（N&B）模式将揭示寡聚状态并估计整个细胞中LRRK 2和LRRK 2突变体的亲和力; 2.双色交叉相关模式将用于检测LRRK 2的野生型和突变形式之间的异源寡聚化，作为验证这种方法的手段，用于研究LRRK 2及其结合伴侣之间的异源相互作用; 3.全内反射荧光（TIRF）模式下的FFS将使我们能够特别关注质膜上的LRRK 2寡聚化。这些方法将用于确定LRRK 2自缔合如何受到影响其磷酸化状态，其激酶和GTdR活性及其棕榈酰化状态的干预的影响，棕榈酰化是我们实验室最近发现的LRRK 2的修饰。将平行进行密度梯度离心、激酶和GT3测定以及细胞毒性分析，以表征这些干预对LRRK 2和LRRK 2表达细胞特性的影响。目的2将致力于阐明LRRK 2和α-突触核蛋白之间的潜在相互作用，α-突触核蛋白是PD（路易体）的特异性神经元包涵体的主要成分。我们最近观察到LRRK 2增加丝氨酸129处磷酸化的α-突触核蛋白的水平，其是人类PD脑中病理学的选择性标志物。最引人注目的是，我们发现磷酸化的α-突触核蛋白显著增加了转染细胞中LRRK 2的丰度和毒性。因此，我们建议使用FFS方法来定义LRRK 2和α-突触核蛋白之间形成的复合物的性质，无论是在胞质溶胶中还是在膜上。综上所述，我们的研究将定义与细胞毒性以及与高水平和低水平的激酶和GTP酶活性相关的LRRK 2的生物物理状态，并将阐明LRRK 2和α-突触核蛋白突变的致病机制之间的相互作用。

DESCRIPTION (provided by applicant): Mutations in the gene for Leucine-Rich Repeat Kinase 2 (LRRK2) are responsible for an autosomal dominant form of Parkinson's Disease (PD). Biochemical analyses of LRRK2 in cell lysates indicate that the protein dimerizes, and that impairment of kinase activity results in depletion of dimers and formation of higher-order oligomers. Interestingly, PD-associated mutations shift the balance toward the dimeric (active) state of LRRK2. Because LRRK2 kinase activity is apparently dependent on its oligomerization state, we propose in Aim 1 to elucidate the process of LRRK2 self-association in living cells using Fluorescence Fluctuation Spectroscopy (FFS) approaches. Three modes of FFS will be employed: 1. The Number and Brightness (N&B) mode will reveal the oligomeric states and estimate the affinities of LRRK2 and LRRK2 mutants throughout the cell; 2. The two-color cross-correlation mode will be used to detect hetero-oligomerization between wild-type and mutant forms of LRRK2 as a means of validating this approach for investigations of hetero-interactions between LRRK2 and its binding partners; 3. FFS in the Total Internal Reflection Fluorescence (TIRF) mode will allow us to focus specifically on LRRK2 oligomerization on the plasma membrane. These methods will be used to determine how LRRK2 self-association is affected by interventions that affect its phosphorylation state, its kinase and GTPase activities, and its state of palmitoylation, a modification of LRRK2 recently identified in our laboratory. Density gradient centrifugation, kinase and GTPase assays, and cell toxicity analyses will be performed in parallel to characterize the effects of these interventions on the properties of LRRK2 and LRRK2- expressing cells. Aim 2 will be directed at elucidating potential interactions between LRRK2 and a-synuclein, the major component of the pathognomonic neuronal inclusions of PD (Lewy bodies). We recently observed that LRRK2 increases the level of a-synuclein phosphorylated at serine 129, which is a selective marker of pathology in human PD brains. Most strikingly, we found that phosphorylated a-synuclein significantly increases LRRK2 abundance and toxicity in transfected cells. Therefore, we propose to use FFS approaches to define the nature of complexes formed between LRRK2 and a-synuclein, either in the cytosol or on membranes. Taken together, our studies will define the biophysical states of LRRK2 that are associated with cell toxicity and with high and low levels of kinase and GTPase activity, and will elucidate the interplay between pathogenic mechanisms of LRRK2 and a-synuclein mutations.