Identifying The Human Calcineurin Signaling Network

识别人类钙调神经磷酸酶信号网络

• 批准号: 9276716
• 负责人: Martha S. Cyert
• 金额: $ 48.31万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276716
• 关键词: Address; Adverse effects; Affinity; Animals; Binding; Binding Proteins; Binding Sites; Biochemical; Bioinformatics; Calcineurin; Calcineurin inhibitor; Calmodulin; Catalytic Domain; Cells; Characteristics; Chronic; Clinical; Complement; Complex; Computer Simulation; Computing Methodologies; Coronary Arteriosclerosis; Cyclosporine; Diabetes Mellitus; Diagnosis; Disease; Docking; Etiology; Event; FK506; Heart Diseases; Human; Immune System Diseases; Immunosuppressive Agents; In Vitro; Ion Channel; Kidney Failure; Knowledge; Libraries; Malignant Neoplasms; Mammals; Mediating; Methods; Mutate; Organism; Parents; Patients; Peptides; Phage Display; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Process; Protein Dephosphorylation; Protein Region; Protein phosphatase; Proteins; Proteome; Proteomics; Regulation; Renal function; Research Personnel; Resources; Schizophrenia; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Structure; Surface; System; Techniques; Testing; Therapeutic; Viral Proteins; Virus Diseases; Virus Inhibitors; Work; Yeasts; base; computerized tools; drug discovery; experience; flexibility; in vivo; insight; novel; novel strategies; novel therapeutic intervention; prevent; protein function; protein protein interaction; public health relevance; receptor; research study; success; transcription factor; yeast protein

 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.

 描述（由申请人提供）：缺乏用于磷酸酶底物全局鉴定的实验和计算工具，这在我们对细胞调控网络的理解中留下了重大空白，并阻止了磷酸酶信号传导的系统水平分析。该提案通过系统地鉴定钙调磷酸酶（CN）的靶点和免疫抑制剂FK 506和环孢菌素A的靶点来解决这一知识缺口。CN是普遍存在的Ca 2 +/钙调蛋白依赖性蛋白磷酸酶。目前在哺乳动物中只有25种底物归因于氯化萘-与酵母中的底物相同，酵母的蛋白质组比氯化萘小约10倍，氯化萘通过破坏未鉴定的调节事件在患者病因的副作用中受到慢性抑制。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（SLiM）结合，称为PxIxIT和LxVP，这可能发生在距离底物去磷酸化位点数百个残基处。突变这些基序或阻止它们与CN上的保守表面结合，即用FK 506、CysA或病毒抑制剂A238 L，阻断去磷酸化。基于我们先前在酵母中成功建立CN信号网络的基础上，我们通过系统地鉴定蛋白质组中的CN结合肽来确定人CN底物，这是具有挑战性的，因为它们的低亲和力和简并序列。本工作旨在1）通过协同实验和计算方法系统地发现人类PxIxIT和LxVP型序列。CN结合序列直接选自噬菌体展示文库，所述噬菌体展示文库含有SLiM所在的人蛋白质组的所有无序区域。PxIxIT还通过利用其特征性结构特征并使用新方法预测与CN上保守的PxIxIT对接表面的结合来在计算机上鉴定。候选序列在体外验证CN结合，并且在Aim 2中，在培养的动物细胞中测试其亲本蛋白与CN的相互作用和/或通过CN的去磷酸化。在目标3中，我们表征了新发现的CN结合基序YLxxLF的功能和结合模式，其可以识别不同类别的CN相互作用蛋白，并提供选择性破坏CN信号传导事件的途径。这种对人类CN信号传导的系统级分析将最终确定与其他可用于治疗的调控网络的交叉点，例如提供改善免疫抑制剂副作用的策略。此外，这项工作将为磷酸酶如何实现特异性提供基本见解，并为研究Ca 2+或磷酸化依赖的蛋白质功能调节的研究人员创造一个重要的新资源。