Biochemical and structural studies on LRRK2 regulation.

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，一些基因(Park 1-16)与帕金森病的发病机制有关，其中Park8基因座编码富含亮氨酸的重复蛋白激酶2(LRRK2)。LRRK2与散发性和遗传性帕金森病都有关联，这表明在疾病病理中发挥了核心作用。LRRK2除了具有GTP酶活性外，还具有激酶活性，并作为支架发挥作用。LRRK2的几个突变与家族性帕金森病有关，但尽管进行了大量的研究，但对这个285 kDa的多结构域磷酸蛋白的调节知之甚少。在这个应用中，我们想要研究LRRK2调节的分子基础，涉及酶和支架功能。特别是，所提出的结构功能方法将使我们能够更好地从机制上理解PD相关突变的功能后果。我们的目标是首先解决人类LRRK2蛋白结构域的高分辨率晶体结构。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)和法兰克福歌德大学(X射线结晶术、结构测定)之间组成了一个跨学科的团队。应用创新的概念，如“激酶脊椎”，并使用新的工具，即纳米机构和DARPins，我们想要研究的相互作用的GTPase结构域与蛋白激酶结构域。此外，我们还想研究14-3-3-亚型等已知相互作用伙伴的结合及其对LRRK2构象和活性的影响。我们已经描述了cAMP依赖的蛋白激酶(PKA)和14-3-3相互作用在LRRK2活性负调控中的重要作用。我们希望通过A-激酶锚定蛋白(AKAP)来研究LRRK2/PKA信号模块的时间和空间调节。利用结构、分子、生化、细胞和生物物理技术的组合将有助于我们描述LRRK2在健康和疾病状态下的功能调节。在分子水平上了解LRRK2的调控，不仅有助于深入了解帕金森病神经退行性变的机制，而且可能有助于治疗学的发展。

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.