Biochemical and structural studies on LRRK2 regulation.

LRRK2 调控的生化和结构研究。

• 批准号: 407661177
• 负责人: Professor Dr. Stefan Knapp
• 金额: --
• 依托单位: Institut für Pharmazeutische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/407661177?language=en
• 关键词: Biochemical; structural; studies; LRRK2; regulation.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. In the last decade several gene loci (PARK 1-16) have been linked to the pathogenesis of PD, among them the PARK8 locus that encodes for the Leucine-rich repeat kinase 2 (LRRK2). LRRK2 has been associated with both, sporadic as well as inherited PD suggesting a central role in disease pathology. LRRK2 has kinase as well as GTPase activity and acts also as a scaffold. Several mutations in LRRK2 have been correlated to familial PD, but despite significant research efforts, little is known about the regulation of this 285 kDa multi-domain phosphoprotein. In this application we want to study the molecular basis of LRRK2 regulation addressing both, enzymatic and scaffolding functions. In particular, the proposed structure function approach will allow us to achieve a better mechanistic understanding of the functional consequences of the PD-relevant mutations. We will aim to solve first high-resolution crystal structures of domains of the human LRRK2 protein. Therefore, we have formed an interdisciplinary team between Kassel University (protein biochemistry, biophysics, cell culture) and the Goethe University Frankfurt (Xray-crytallography, structure determination). Applying innovative concepts like “kinase spines” and employing novel tools, i.e. nanobodies and DARPins, we want to investigate the interplay of the GTPase domain with the protein kinase domain. In addition, we want to study the binding of known interaction partners such as 14-3-3-isoforms and their influence on conformation and activity of LRRK2. We already described an essential function for cAMP-dependent protein kinase (PKA) and 14-3-3 interaction in the negative regulation of LRRK2 kinase activity. We want to investigate the temporal and spatial regulation of the LRRK2/PKA signaling module via A-kinase anchoring proteins (AKAPs). Employing a combination of structural, molecular, biochemical, cellular and biophysical techniques will help us to describe the modulation of LRRK2 function in healthy and diseased states. Understanding LRRK2 regulation on a molecular level will not only provide insights into mechanisms of PD neurodegeneration, but may also facilitate the development of therapeutics.

帕金森病（PD）是最常见的神经退行性运动障碍。在过去的十年中，几个基因位点（PARK 1-16）已被链接到PD的发病机制，其中PARK 8基因座编码的富含亮氨酸的重复激酶2（LRRK 2）。LRRK 2与散发性和遗传性PD相关，表明在疾病病理学中起着重要作用。LRRK 2具有激酶以及GT3活性，并且还充当支架。LRRK 2中的几个突变与家族性PD相关，但尽管进行了大量研究，但对这种285 kDa多结构域磷蛋白的调控知之甚少。在本申请中，我们希望研究LRRK 2调节的分子基础，解决酶和支架功能。特别是，建议的结构功能的方法将使我们能够实现更好的机制理解的功能后果的PD相关的突变。我们的目标是解决人类LRRK 2蛋白结构域的第一个高分辨率晶体结构。因此，我们在卡塞尔大学（蛋白质生物化学、生物物理学、细胞培养）和歌德大学（法兰克福）（X射线晶体学、结构测定）之间组建了一个跨学科团队。应用“激酶棘”等创新概念并采用新型工具，即纳米抗体和DARPins，我们希望研究GTPase结构域与蛋白激酶结构域的相互作用。此外，我们希望研究已知的相互作用伙伴，如14-3-3-异构体的结合及其对LRRK 2构象和活性的影响。我们已经描述了cAMP依赖性蛋白激酶（PKA）和14-3-3相互作用在LRRK 2激酶活性的负调节中的基本功能。我们希望通过A激酶锚定蛋白（AKAP）研究LRRK 2/PKA信号模块的时空调控。采用结构，分子，生物化学，细胞和生物物理技术的组合将有助于我们描述在健康和疾病状态下LRRK 2功能的调节。了解LRRK 2在分子水平上的调控不仅可以深入了解PD神经退行性变的机制，还可以促进治疗方法的开发。