Interactions of intracellular calcium sensors with the pre-mRNA splicing factor Prp40

项目摘要 亨廷顿病(HD)是一种以功能障碍和死亡为特征的严重神经系统疾病。 特定的大脑神经元。HD与Htt蛋白功能异常直接相关。 N-末端有多聚谷氨酰胺扩张的个体(Htt-e)。HTT作为一种蛋白质支架和一种 Htt-e功能障碍的一个方面是它与其结合伙伴的相互作用的改变，包括Pre-mRNA 加工蛋白40同系物A(Prp40A)。有趣的是，之前的研究表明，两者的活动 HTT和Prp40A是由细胞内的钙离子传感器以钙依赖的方式调节的。一种Htt结合的钙离子 传感器是普遍存在的钙调蛋白(CaM)，其相互作用调节转谷氨酰胺酶2的激活 Htt-e的交联导致HD脑中的聚集。Centrin，另一个与之密切相关的EF-Hand钙传感器 已发现在中心体和光感受器纤毛上与Htt共定位于CaM，两者 蛋白质在纤毛发生中起着调节作用。发现酵母Prp40与酵母CaM在一种蛋白质中相互作用 基因芯片实验。我们的研究小组最近报道了一个hPrp40A肽基序与人类相互作用 Centin 2(Cen2)以钙依赖的方式被激活。综上所述，这里总结的结果表明Htt，hPrp40A， CaM和Cen2形成一个受细胞内钙信号调控的网络，可能参与HD 发病机制。为了探索这一有趣的可能性并最终确定该网络是否可行 作为发展HD疗法的目标，有必要更好地了解其分子基础和功能 这些互动的结果。在这里，我建议迈出第一步，通过实施以下措施来填补这一关键的知识空白 HPrp40A与EF-Hand钙离子传感器相互作用的生物物理和结构分析 HCen2。在目标1中，我将完成hPrp40A和hCen2相互作用的分析，通过获得高- 用X-射线单晶衍射法测定了该配合物的分辨结构。目标2将验证物理相互作用 HPrp40A和hCaM，用等温滴定量热法测定其结合亲和力，并测定其高亲和力。 分辨X射线晶体结构的它们的络合物。在目标3中，我将生成hCAM的结构模型- 结合小角X射线散射、负染电子的hPrp40A-hCen2三元络合物 显微镜和计算建模。这些结果将对随后分析这一现象是如何发生的 蛋白质网络功能，评价其在HD中的作用，并为我的长期研究目标做好铺垫 确定Htt-Prp40A-Cen2-CaM网络是否是开发HD疗法的可行靶点。

Project Summary Huntington’s disease (HD) is a severe neurological disorder that is characterized by the dysfunction and death of specific brain neurons. HD is directly associated with abnormal function of the protein huntingtin (Htt) in individuals with a polyglutamine expansion at the N-terminus (Htt-e). Htt functions as a protein scaffold and one aspect of Htt-e malfunction is alterations in its interactions with its binding partners, including the pre-mRNA processing protein 40 homolog A (Prp40A). Interestingly, previous studies have shown that the activities of both Htt and Prp40A are modulated by intracellular Ca2+ sensors in a Ca2+-dependent manner. One Htt-binding Ca2+ sensor is the ubiquitous calmodulin (CaM), whose interaction modulates the activation of transglutaminase 2 cross-linking of Htt-e that leads to aggregation in HD brain. Centrin, another EF-hand Ca2+ sensor closely related to CaM, has been found to colocalize with Htt at the centrosome and in the photoreceptor cilium, and both proteins have a regulatory role in ciliogenesis. Yeast Prp40 was found to interact with yeast CaM in a protein microarray experiment. Our research group recently reported that a hPrp40A peptide motif interacts with human centrin 2 (Cen2) in a Ca2+-dependent manner. Together, the results summarized here suggest that Htt, hPrp40A, CaM, and Cen2 form a network that is modulated by intracellular Ca2+ signals and may be involved in HD pathogenesis. In order to explore this intriguing possibility and ultimately determine if this network is a viable target for developing HD therapeutics, it will be necessary to better understand the molecular basis and functional outcomes of these interactions. Here, I propose a first step to fill this critical gap in knowledge by carrying out biophysical and structural analysis of the interaction between hPrp40A and the EF-hand Ca2+ sensors hCaM and hCen2. In Aim 1 I will complete the analysis of the interaction of hPrp40A and hCen2 by obtaining a high- resolution structure of the complex by X-ray crystallography. Aim 2 will validate the physical interaction of hPrp40A and hCaM, measure the binding affinity by isothermal titration calorimetry and determine the high- resolution X-ray crystal structure of their complex. In Aim 3, I will generate a structural model of the hCaM- hPrp40A-hCen2 ternary complex using a combination of small angle X-ray scattering, negative-stain electron microscopy, and computational modeling. These results will be valuable for subsequent analyses of how this protein network functions, evaluation of its role in HD, and set the stage for my long-term research goal of determining if the Htt-Prp40A-Cen2-CaM network is a viable target for developing an HD therapy.