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: 9245394
• 负责人: Daniel Schwartz
• 金额: $ 23.22万
• 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 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245394
• 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; Housing; 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; Variant; Work; base; computerized tools; early onset; 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.

项目摘要 丝氨酸/苏氨酸激酶细胞周期蛋白依赖性激酶样5（CDKL 5）的突变强烈地涉及 婴儿癫痫性脑病（IEE），但目前只有少数底物已被确定， 疾病病因学中CDKL 5突变背后的机制仍在很大程度上未知。直到这些知识 如果这些差距得到解决，该领域很可能难以理解CDKL 5，并且难以治疗CDKL 5- 相关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 了解CDKL 5在正常脑发育和IEE中的作用。在这方面的目标 因此，本发明的应用是鉴定野生型和突变型CDKL 5的底物特异性，并产生 免疫学可及的CDKL 5底物预测因子。中心假设是IEE相关的CDKL 5 突变将重新连接激酶底物特异性，导致单个CDKL 5的不同底物库 变体。该假设是根据申请人实验室的初步数据制定的。的 所提出的研究的基本原理是，表征野生型和IEE的底物特异性， 相关的CDKL 5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 假定的底物，将允许该领域推进对CDKL 5相关脑病的理解。 在强有力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来检验： 鉴定CDKL 5激酶的野生型和脑病相关突变底物特异性; 第二个具体目标将解决对可访问的预测工具的关键需求：2）自由创建 每个CDKL 5变体的可访问的定制底物预测因子。第一个目标将使用小说进行 细菌的方法来确定蛋白激酶的特异性，而第二个目标将通过 用于底物预测的伴随计算工具;细菌方法和计算工具 都是由申请人所在的组织开创的。申请人认为，拟议的研究具有创新性， 因为它结合了他们实验室的特异性测定方法和强大的计算工具， 通过将CDKL 5疾病病因学的焦点转移到 底物特异性重新布线。这项研究将是重要的，因为它预计将提供关键的洞察力， CDKL 5相关IEE的病因。最终，这种见解有可能指导IEE的治疗， 并为新的治疗策略的发展提供信息。