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）：富含亮氨酸的重复激酶2（LRRK2）基因中的突变是帕金森氏病（PD）的常染色体显性形式。 LRRK2在细胞裂解物中的生化分析表明蛋白质二聚体二聚体，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，与PD相关的突变将平衡转移到LRRK2的二聚体（活性）状态。由于LRRK2激酶的活性显然取决于其寡聚态，因此我们在AIM 1中提出，使用荧光波动光谱（FFS）方法阐明活细胞中LRRK2自我关联的过程。将采用三种FF的模式：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LRRK2和LRRK2突变体的亲和力； 2。两种颜色的互相关模式将用于检测LRRK2的野生型和突变形式之间的异质 - 异构化，以此作为验证这种方法以研究LRRK2及其结合伙伴之间异质互动的方法； 3。在总内反射荧光（TIRF）模式下的FFS将使我们能够专门针对质膜上的LRRK2低聚。这些方法将用于确定LRRK2自我关联如何受到影响其磷酸化状态，激酶和GTPase活性及其棕榈酰化状态的干预措施的影响，这是对我们实验室中最近确定的LRRK2的修改。密度梯度离心，激酶和GTPase分析以及细胞毒性分析将平行进行，以表征这些干预措施对LRRK2和LRRRK2-表达细胞的特性的影响。 AIM 2将针对LRRK2和A-突触核蛋白之间的潜在相互作用，这是PD（Lewy Bodies）病理学神经元内夹杂物的主要成分。我们最近观察到，LRRK2增加了在丝氨酸129处磷酸化的A-核蛋白的水平，这是人类PD大脑中病理的选择性标记。最引人注目的是，我们发现磷酸化的A-核蛋白显着增加了转染细胞中的LRRK2丰度和毒性。因此，我们建议使用FFS方法来定义在细胞质或膜上在LRRK2和A-核蛋白之间形成的复合物的性质。综上所述，我们的研究将定义与细胞毒性以及高水平和低水平的激酶和GTPase活性相关的LRRK2的生物物理状态，并将阐明LRRK2的致病机制和A-核蛋白突变之间的相互作用。 公共卫生相关性：LRRK2中主要遗传的突变是家族性帕金森氏病的最常见原因。这些研究将建立测量细胞中LRRK2的寡聚状态和膜关联的方法。结果将有助于确定LRRK2的正常生物物理特性以及突变导致LRRK2功能改变或与其他蛋白质的相互作用改变的潜在机制，这可能导致细胞毒性和神经变性。