Structure and function of DISC1 in the cAMP pathway

DISC1(精神分裂症中的精神障碍-1)已经成为一种重要的遗传风险因素 一系列精神疾病，如精神分裂症、躁郁症和抑郁症。DISC1的产品是 一种在几个神经元信号通路中起关键作用的长支架蛋白。尽管不断增长 关于它在精神障碍病因学中的作用，人们几乎对其结构一无所知 DISC1及其相互作用和调控机制。这项研究项目将专门关注 DISC1在cAMP途径中的作用在患有重大疾病的患者中发现cAMP水平降低 抑郁症，并与神经退行性疾病有关。控制细胞内水平的酶 CAMP的活性，如磷酸二酯酶家族PDE4，对 抗抑郁和增强记忆药物的发展。这个项目的总体目标是 提供一个结构框架，描述DISC1在cAMP途径中的作用，并了解 这一通路的改变有助于精神障碍的病因学。AIM 1将提供完整的 DISC1的结构模型，包括其寡聚和磷酸化调控的机制。 我们还将调查疾病相关突变的结构后果。我们正在提议 结合氢-氢交换质谱学的综合结构方法 (HDX-MS)、溶液核磁共振光谱、X射线结晶学、小角X射线散射(SAXS)和 分子模拟重建DISC1的完整结构和动力学特征。我们的 初步数据表明，我们可以用溶液核磁共振表征DISC1N-末端结构 光谱，而包含中心和/或C-末端结构域的结构很适合X- 射线结晶学研究。目标2将结合生化和结构方法来表征 DISC1与主要转录因子ATF4的相互作用cAMP途径控制 Cre元件和磷酸二酯酶PDE4D9的表达。我们的初步数据显示， DISC1-ATF4复合体可以在体外用这两种蛋白质的截短结构进行重组。这一目标将 提供一个机制框架来理解DISC1作为转录共抑制因子和 DISC1-ATF4复合体在cAMP途径中的作用。目标3将解开变构的机制 DISC1对长和短PDE4异构体的抑制，使用生化、结构和 计算技术。总体而言，这一项目的完成将提供一个全面、统一的 了解DISC1在cAMP途径中作用的框架。我们的工作也将提供新的 开发调节cAMP水平的分子的结构靶点。

DISC1 (Disrupted-In-Schizophrenia-1) has emerged as a significant genetic risk factor for a wide range of mental illness such as schizophrenia, bipolar disorders and depression. The product of DISC1 is a long scaffold protein that plays a critical role in several neuronal signaling pathways. Despite growing appreciation of its role in the etiology of mental disorders, almost nothing is known about the structure of DISC1 and its mechanisms of interaction and regulation. This research project will specifically focus on the role of DISC1 in the cAMP pathway. Reduced cAMP levels are found in patients suffering from major depression and are associated with neurodegenerative diseases. Enzymes controlling intracellular levels of cAMP, such as the phosphodiesterase family PDE4, have considerable pharmaceutical importance for the development of antidepressant and memory enhancing drugs. The overall goal of this project is to provide a structural framework describing the role of DISC1 in the cAMP pathway and to understand how alterations in this pathway contribute to the etiology of psychiatric disorders. Aim 1 will provide a complete structural model of DISC1, including its mechanism of oligomerization and modulation by phosphorylation. We will also investigate the structural consequences of disease-associated mutations. We are proposing here an integrative structural approach combining Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS), solution NMR spectroscopy, X-ray crystallography, Small-angle X-ray Scattering (SAXS) and molecular simulations to reconstitute the complete structural and dynamical features of DISC1. Our preliminary data demonstrate that we can characterize DISC1 N-terminal constructs by solution NMR spectroscopy while constructs encompassing the central and/or C-terminal domains are well suited for X- ray crystallography study. Aim 2 will combine biochemical and structural approaches to characterize the interaction between DISC1 and ATF4, a major transcription factor the cAMP pathway controlling the expression of the CRE-elements and of the phosphodiesterase PDE4D9. Our preliminary data show that DISC1-ATF4 complex can be reconstituted in vitro with truncated constructs of both proteins. This aim will provide a mechanistic framework for understanding the role of DISC1 as transcriptional co-repressor and the function of DISC1-ATF4 complex in the cAMP pathway. Aim 3 will unravel the mechanisms of allosteric inhibition of long and short PDE4 isoforms by DISC1, using a combination of biochemical, structural and computational techniques. Overall, completion of this project will provide a comprehensive, unifying framework for understanding the role of DISC1 in the cAMP pathway. Our work will also provide new structural targets for the development of molecules regulating cAMP levels.