Determining the Substrate Specificity of Wild-Type and Encephalopathy-Associated Mutants of Protein Kinase CDKL5

确定蛋白激酶 CDKL5 野生型和脑病相关突变体的底物特异性

• 批准号: 9354519
• 负责人: Daniel Schwartz
• 金额: $ 19.24万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9354519
• 关键词: Address; Antiepileptic Agents; Bacterial Model; Bioinformatics; Brain; Child; Communities; Companions; Computer software; Custom; Cyclin-Dependent Kinases; Data; Development; Disease; Drug resistance; Encephalopathies; Epilepsy; Etiology; Exhibits; Gene Expression; Generations; Goals; Individual; Infantile spasms; Knowledge; Laboratories; Link; Manuscripts; Mental Retardation; Missense Mutation; Modeling; Morphogenesis; Mutation; Nature; Neurons; Pathway interactions; Patients; Phenotype; Phosphotransferases; Probability; Protein Kinase; Protein-Serine-Threonine Kinases; Public Health; Research; Research Personnel; Rett Syndrome; Role; Scanning; Seizures; Specificity; Substrate Specificity; Testing; Therapeutic; Treatment Efficacy; Variant; Work; base; computerized tools; early onset; epileptic encephalopathies; infancy; innovation; insight; loss of function; mutant; novel; novel therapeutics; premature; prevent; tool; user-friendly

PROJECT SUMMARY Mutations in the serine/threonine kinase Cyclin-Dependent Kinase-Like 5 (CDKL5) are strongly implicated in infantile epileptic encephalopathy (IEE), yet at present only a few substrates have been identified, and the mechanisms behind CDKL5 mutation in disease etiology remains largely unknown. Until these knowledge gaps are addressed, it is likely that the field will struggle to understand, and have difficulty treating, CDKL5- associated IEE. The long-term goal is to understand the fundamental mechanisms behind infantile epileptic encephalopathy, in order to develop new, effective therapeutic strategies. This requires a precise understanding of the role(s) of CDKL5 in both normal brain development and IEE. The objective in this application, therefore, is to identify the substrate specificity of wild-type and mutant CDKL5, and to create publically accessible CDKL5 substrate predictors. The central hypothesis is that IEE-associated CDKL5 mutations will rewire kinase substrate specificity, resulting in distinct substrate pools for individual CDKL5 variants. This hypothesis has been formulated based on preliminary data from the applicant's laboratory. The rationale underlying the proposed research is that characterizing the substrate specificity of wild-type and IEE- associated CDKL5 mutants, and creating freely available tools that use this data to identify high-confidence putative substrates, will allow the field to advance the understanding of CDKL5-associated encephalopathy. Guided by strong preliminary data, the central hypothesis will be tested through the first of two specific aims: 1) Identify the wild-type and encephalopathy-associated mutant substrate specificities of the CDKL5 kinase; the critical need for accessible prediction tools will be addressed in the second specific aim: 2) Create freely accessible, custom substrate predictors for each CDKL5 variant. The first aim will be conducted using a novel bacterial approach for determining protein kinase specificity, while the second aim will be accomplished by a companion computational tool for substrate prediction; both the bacterial approach and computational tool were pioneered by the applicant's group. The proposed research is innovative, in the applicant's opinion, because it combines their lab's approach for specificity determination with powerful computational tools, and represents a significant departure from the status quo by shifting the focus of CDKL5 disease etiology to substrate specificity rewiring. The research will be significant because it is expected to offer critical insight into the etiology of CDKL5-associated IEE. Ultimately, this insight has the potential to guide the treatment of IEE, and inform the development of novel therapeutic strategies.

项目总结 丝氨酸/苏氨酸激酶细胞周期蛋白依赖性蛋白激酶样蛋白5(CDKL5)的突变与 婴儿癫痫脑病(IEE)，但目前只发现了几种底物，而 CDKL5突变在疾病病因学中的机制在很大程度上仍不清楚。直到这些知识 如果差距得到解决，很可能该领域将难以理解并难以处理CDKL5- 关联的IEE。长期目标是了解婴儿癫痫背后的基本机制。 脑病，以开发新的、有效的治疗策略。这需要一个精确的 理解CDKL5在正常脑发育和IEE中的作用(S)。这件事的目的是 因此，应用于鉴定野生型和突变型CDKL5的底物特异性，并创建 公众可访问的CDKL5底物预报器。中心假设是IEE相关的CDKL5 突变将重新连接激酶底物专一性，导致单个CDKL5的不同底物池 变种。这一假设是基于申请人实验室的初步数据提出的。这个 这项拟议研究的基本原理是，表征野生型和IEE的底物专一性- 相关的CDKL5突变体，并创建免费可用的工具，使用这些数据来识别高置信度 可能的底物，将允许该领域推进对CDKL5相关脑病的理解。 在强劲的初步数据的指导下，中心假设将通过两个具体目标中的第一个进行检验：1) 鉴定CDKL5激酶的野生型和脑病相关突变底物的特异性； 对可用的预测工具的迫切需求将在第二个具体目标中得到解决：2)自由创建 适用于每个CDKL5变种的可访问、定制的底物预测器。第一个目标将通过一部小说来实现 细菌方法来确定蛋白激酶的特异性，而第二个目的将通过一种 用于底物预测的配套计算工具；细菌方法和计算工具 都是由申请者所在的团体首创的。申请人认为，拟议的研究是创新的， 因为它结合了他们实验室确定特异性的方法和强大的计算工具，并且 将CDKL5疾病病因学的重点转移到 底物特异性重新布线。这项研究将具有重要意义，因为它有望为 CDKL5相关IEE的病因学研究。最终，这一见解有可能指导IEE的治疗， 并为新的治疗策略的开发提供信息。

项目成果

Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach.

使用蛋白质组肽库 (ProPeL) 方法表征蛋白激酶底物特异性。

• DOI: 10.1002/cpch.38
• 发表时间: 2018
• 期刊: Current protocols in chemical biology
• 作者: Lubner,JoshuaM;Balsbaugh,JeremyL;Church,GeorgeM;Chou,MichaelF;Schwartz,Daniel
• 通讯作者: Schwartz,Daniel