Identifying The Human Calcineurin Signaling Network

 描述(申请人提供)：缺乏用于全球鉴定磷酸酶底物的实验和计算工具，在我们对细胞调控网络的理解中留下了重大空白，并阻碍了对磷酸酶信号的系统水平的分析。该提案通过系统地确定钙调神经磷酸酶(CN)的靶标、免疫抑制剂FK506和环孢菌素A的靶标来解决这一知识鸿沟，CN是普遍存在的钙/钙调蛋白依赖的蛋白磷酸酶，目前在哺乳动物中只有25种底物被归因于CN--与酵母的底物相同，其蛋白质组小约十倍，并通过干扰未知的调节事件在患者病因的副作用中长期抑制CN。这表明，在人类中，大多数CN底物及其调节功能仍有待确定。最近对CN底物识别的见解推动了我们的方法：CN与短线性基序(SLIMs)结合，称为PxIxIT和LxVP，可以在数百个残基以外的底物去磷酸化位点发生。突变这些基序或阻止它们与CN上保守的表面结合，即用FK506、CySA或病毒抑制剂A238L，可以阻止去磷酸化。在我们之前成功地在酵母中建立CN信号网络的基础上，我们正在通过系统地鉴定蛋白质组中的CN结合多肽来确定人的CN底物，由于它们的低亲和力和简并序列，这是具有挑战性的。本工作旨在1)通过实验和计算方法的协同作用，系统地发现人类PxIxIT和LxVP型序列。CN结合序列直接选自噬菌体展示文库，该文库包含人类蛋白质组的所有无序区域，其中存在苗条。PxIxIT也是通过利用它们特有的结构特征并使用一种新的方法预测与保守的PxIxIT在CN上的结合而在Silico中被识别的。候选序列在体外被验证为CN结合，在Aim 2中，在培养的动物细胞中测试它们的亲本蛋白与CN的相互作用和/或被CN去磷酸化。在目标3中，我们表征了一个新发现的CN结合基序YLxxLF的功能和结合模式，它可能识别一类不同的CN相互作用蛋白，并为选择性干扰CN信号事件提供途径。这种对人类CN信号的系统水平分析最终将确定与其他调控网络的交叉点，这些网络可以用于治疗，例如提供策略来改善免疫抑制剂的副作用。此外，这项工作将为磷酸酶如何实现特异性提供基本的见解，并将为研究钙离子或磷酸化依赖的蛋白质功能调节的研究人员创造一个关键的新资源。

 DESCRIPTION (provided by applicant): The absence of experimental and computational tools for global identification of phosphatase substrates leaves a major gap in our understanding of cellular regulatory networks and prevents systems-level analyses of phosphatase signaling. The proposal addresses this knowledge gap by systematically identifying targets of calcineurin (CN), the ubiquitous Ca2+/calmodulin-dependent protein phosphatase and target of immunosuppressants, FK506 and cyclosporin A. Only 25 substrates are currently attributed to CN in mammals--the same as in yeast, whose proteome is ~tenfold smaller, and chronic inhibition of CN in patient's cause's side-effects by disrupting unidentified regulatory events. This suggests that in humans, the majority of CN substrates, and thus its regulatory functions, remain to be identified. Recent insights into CN substrate recognition drive our approach: CN binds to short linear motifs (SLiMs), termed PxIxIT and LxVP, which can occur hundreds of residues away substrate dephosphorylation sites. Mutating these motifs or preventing their binding to conserved surfaces on CN, i.e. with FK506, CysA, or viral inhibitor A238L, blocks dephosphorylation. Building upon our previous success establishing the CN signaling network in yeast, we are determining human CN substrates by systematically identifying CN- binding peptides in the proteome, which is challenging due to their low affinities and degenerate sequences. This work aims to 1) systematically discover human PxIxIT and LxVP-type sequences by synergizing experimental and computational methods. CN-binding sequences are directly selected from phage display libraries that contain all disordered regions of the human proteome, where SLiMs reside. PxIxITs are also identified in silico by leveraging their characteristic structural features and using a novel method to predict binding to the conserved PxIxIT-docking surface on CN. Candidate sequences are validated for CN-binding in vitro, and in Aim 2 their parent proteins are tested for interaction with and/or dephosphorylation by CN in cultured animal cells. In Aim 3 we characterize the functions and binding mode of a newly discovered CN-binding motif, YLxxLF, which may identify a distinct class of CN- interacting proteins and provide avenues for selective disruption of CN signaling events. This systems- level analysis of CN signaling in humans will ultimately identify intersections with other regulatory networks that can be exploited therapeutically, such as providing strategies to ameliorate immunosuppressant side effects. Furthermore, this work will provide fundamental insights into how phosphatases achieve specificity and will create a critical new resource for researchers studying Ca2+ or phosphorylation-dependent regulation of protein function.