Biophysical analysis of LRRK2

描述（由申请人提供）：富含亮氨酸重复激酶2 （LRRK2）基因突变导致常染色体显性帕金森病（PD）。细胞裂解物中LRRK2的生化分析表明，该蛋白二聚体，激酶活性的损害导致二聚体的消耗和高阶低聚物的形成。有趣的是，pd相关突变将平衡转向LRRK2的二聚体（活性）状态。由于LRRK2激酶活性明显依赖于其寡聚化状态，我们在Aim 1中提出使用荧光波动光谱（FFS）方法来阐明活细胞中LRRK2自结合的过程。FFS将采用三种模式：Number and Brightness （N&B）模式将揭示LRRK2和LRRK2突变体在整个细胞中的寡聚状态并估计其亲和性；2. 双色互相关模式将用于检测野生型和突变型LRRK2之间的异齐聚化，作为验证LRRK2与其结合伙伴之间异相互作用的研究方法的一种手段；3. 全内反射荧光（TIRF）模式下的FFS将使我们能够专门关注质膜上的LRRK2寡聚化。这些方法将用于确定影响LRRK2磷酸化状态、激酶和GTPase活性以及棕榈酰化状态（我们实验室最近发现的LRRK2的一种修饰）的干预措施如何影响LRRK2的自我关联。密度梯度离心、激酶和GTPase测定以及细胞毒性分析将同时进行，以表征这些干预对LRRK2和表达LRRK2的细胞特性的影响。目的2将旨在阐明LRRK2和a-突触核蛋白之间的潜在相互作用，a-突触核蛋白是PD（路易小体）的病理神经元包涵体的主要成分。我们最近观察到LRRK2增加了丝氨酸129磷酸化的a-synuclein水平，这是人类PD大脑病理的选择性标记。最引人注目的是，我们发现磷酸化的a-synuclein显著增加了转染细胞中LRRK2的丰度和毒性。因此，我们建议使用FFS方法来定义LRRK2和a-synuclein之间形成的复合物的性质，无论是在细胞质中还是在膜上。总之，我们的研究将确定LRRK2的生物物理状态，这些状态与细胞毒性以及激酶和GTPase活性的高低水平有关，并将阐明LRRK2的致病机制与a-synuclein突变之间的相互作用。

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. PUBLIC HEALTH RELEVANCE: Dominantly inherited mutations in LRRK2 are the most common cause of familial Parkinson's disease. These studies will establish methods to measure the oligomerization state and membrane association of LRRK2 in cells. The results will help determine the normal biophysical properties of LRRK2 and the potential mechanisms by which mutations result in altered LRRK2 functions or altered interactions with other proteins, which may cause cell toxicity and neurodegeneration.