Biophysical analysis of LRRK2

LRRK2 的生物物理分析

• 批准号: 8249312
• 负责人: Matthew S Goldberg
• 金额: $ 24.15万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8249312
• 关键词: 2-bromopalmitate; Affect; Affinity; Binding; Biochemical; Biological Assay; Brain; Cell Extracts; Cell Fraction; Cell Survival; Cell membrane; Cells; Centrifugation; Chemicals; Color; Complex; Cytoplasm; Cytosol; Data; Density Gradient Centrifugation; Dependence; Dimerization; Disease; Engineering; Equilibrium; Figs - dietary; Fluorescence; Fractionation; GTP Binding; Gel Chromatography; Genes; Glycerol; Guanosine Triphosphate Phosphohydrolases; Homo; Human; Impairment; In Vitro; Inherited; Intervention; Investigation; Laboratories; Length; Life; Link; Measures; Mediating; Membrane; Methods; Modification; Molecular; Mutation; Nature; Nerve Degeneration; Neurons; Parkinson Disease; Pathology; Phosphorylation; Phosphorylation Site; Phosphotransferases; Point Mutation; Post-Translational Protein Processing; Process; Property; Proteins; Serine; Spectrum Analysis; Testing; Toxic effect; Variant; alpha synuclein; crosslink; dimer; enzyme activity; innovation; intervention effect; leucine-rich repeat kinase 2; monomer; mutant; palmitoylation; synuclein

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.

描述(由申请人提供)：富亮氨酸重复蛋白激酶2(LRRK2)基因突变是一种常染色体显性帕金森氏病(PD)的原因。对细胞裂解物中LRRK2的生化分析表明，该蛋白发生二聚体，而激酶活性的受损导致二聚体的耗尽和更高阶低聚物的形成。有趣的是，PD相关突变使LRRK2的平衡向二聚体(活性)状态移动。由于LRRK2的活性明显依赖于其寡聚状态，因此我们在目标1中建议使用荧光涨落光谱(FFS)方法来阐明活细胞中LRRK2的自结合过程。FFS的三种模式将被使用：1.数量和亮度(N&B)模式将揭示LRRK2和LRRK2突变体在整个细胞中的低聚状态并估计LRRK2突变体的亲和力；2.双色互相关模式将被用于检测LRRK2野生型和突变型LRRK2之间的异源齐聚，作为验证该方法的一种手段，用于研究LRRK2与其结合伙伴之间的异源相互作用；3.全内反射荧光(TIRF)模式中的FFS将使我们能够专门关注质膜上的LRRK2齐聚。这些方法将被用来确定干预措施如何影响LRRK2的自结合，从而影响其磷酸化状态、其激酶和GTPase活性以及其棕榈酰化状态，这是我们实验室最近发现的LRRK2的一种修饰。将同时进行密度梯度离心法、激酶和GTPase分析以及细胞毒性分析，以表征这些干预措施对LRRK2和LRRK2表达细胞特性的影响。目的2旨在阐明LRRK2与α-突触核蛋白之间的潜在相互作用，α-突触核蛋白是帕金森病原性神经元包涵体(路易小体)的主要成分。我们最近观察到，LRRK2增加了丝氨酸129处的α-突触核蛋白的磷酸化水平，丝氨酸129是帕金森病脑内一种选择性的病理标志。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LRRK2在转基因细胞中的丰度和毒性。因此，我们建议使用FFS方法来定义LRRK2与a-突触核蛋白之间形成的复合体的性质，无论是在胞浆中还是在膜上。综上所述，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高水平和低水平的激酶和GTP酶活性有关，并将阐明LRRK2与α-突触核蛋白突变之间的致病机制之间的相互作用。 公共卫生相关性：LRRK2的主要遗传突变是家族性帕金森病的最常见原因。这些研究将建立测量LRRK2在细胞中的寡聚状态和膜结合的方法。这些结果将有助于确定LRRK2的正常生物物理性质，以及突变导致LRRK2功能改变或与其他蛋白质相互作用改变的潜在机制，这可能导致细胞毒性和神经退化。