Functional analysis of an essential Trypanosoma brucei protein kinase

布氏锥虫必需蛋白激酶的功能分析

基本信息

批准号：
9316312
负责人：
Marilyn Parsons
金额：
$ 24.5万
依托单位：
SEATTLE BIOMEDICAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-01-16 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9316312
关键词：
Acute Address Affect African Trypanosomiasis Allosteric Site Alpha Cell Binding Binding Sites Biological Blood Circulation C-terminal Cell Cycle Cell Death Cell physiology Cells Cessation of life Chagas Disease Chemicals Consensus Coupled Cutaneous Cytokinesis Cytology Cytolysis Data Defect Development Disease Drug Targeting Drug resistance Event Foundations Future G1 Phase Genetic Growth Human Hydrophobicity Immune Evasion Immunofluorescence Immunologic In Vitro Individual Infection Knock-out Knowledge Learning Leishmania Lesion Lytic Mammalian Cell Mass Spectrum Analysis Mediating Modeling PDPK1 gene Parasites Pathogenicity Pathway interactions Peptides Pharmaceutical Preparations Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Process Protein Kinase Proteins Proteomics RNA Interference Regulation Resistance Role Scheme Sequence Analysis Signal Transduction Site Testing Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Vaccines Validation Visceral analog base design environmental change fighting human disease inhibitor/antagonist kinase inhibitor knock-down mutant neglect novel novel therapeutics pathogen response thiophosphate tool vaccine discovery

项目摘要

Trypanosomatid parasites cause some of the most challenging neglected diseases worldwide. The human- pathogenic triumvirate of Trypanosoma brucei spp., Trypanosoma cruzi, and Leishmania spp. affect hundreds of thousands of individuals, causing human African trypanosomiasis, Chagas’ disease, and a range of visceral to cutaneous lesions, respectively. With no approved vaccines and treatments challenged by resistance, delivery, and efficacy concerns, discovery of new drugs is critical for therapy, control, and elimination of these diseases. Hence, new, validated targets are needed to enter the discovery pipeline. We have recently genetically and chemically validated the T. brucei protein kinase, AEK1, which is both highly conserved in these agents yet different from human protein kinases. The lytic phenotype following knockdown argues that important pathways are controlled by AEK1, yet nothing is known about its regulation or downstream effectors. We propose to fill this knowledge gap. We will test specific cis and trans interactions that we postulate regulate AEK1 activity, including probing a predicted allosteric site and assessing a candidate activating kinase. Our data shows that inhibition of AEK1 leads to inhibition of one or more events required for cytokinesis. To place its action within the cellular context, we will pinpoint the cytologic defect that occurs when AEK1 is inhibited. Because protein kinases exert their functions via modulating downstream effector molecules, the essentiality of AEK1 must reflect essentiality of one or more of its substrates. To identify substrates, we will use a genetically modified AEK1 that specifically accepts large thio-ATP analogs to enable specific thiophosphorylation of its substrates. Substrates will then be captured by virtue of the transferred thiophosphate. Identification of the thiophosphorylated peptides by mass spectrometry will allow development of a list of candidate substrates and a consensus phosphorylation site sequence that will enable identification of additional candidates. The identified molecules will be reviewed for their relationship to the phenotypic processes identified to aid in prioritization for future studies. By revealing mechanisms of regulation and identifying candidate substrates, this project will inform us about the pathway in which AEK1 functions. Given the high similarity of AEK1 across trypanosomatids, it is likely that many of our findings will be applicable to other trypanosomatid pathogens.

锥虫寄生虫会导致世界范围内一些最具挑战性的被忽视的疾病。布氏锥虫、克氏锥虫和利什曼原虫的致病性三巨头影响数百种。成千上万的个体，导致人类非洲锥虫病、恰加斯病和一系列内脏疾病分别针对皮肤病变，没有经过批准的疫苗和治疗方法受到耐药性的挑战，递送和疗效问题，新药的发现对于治疗、控制和消除这些问题至关重要因此，我们最近需要新的、经过验证的靶点进入发现渠道。对 T. brucei 蛋白激酶 AEK1 进行了遗传和化学验证，该激酶在这些药物与人类蛋白激酶不同，敲低后的裂解表型表明：重要的通路由 AEK1 控制，但对其调节或下游效应子一无所知。我们建议填补这一知识空白，我们将测试我们假设调节的特定顺式和反式相互作用。 AEK1 活性，包括探测预测的变构位点并评估候选激活激酶。数据显示，AEK1的抑制导致胞质分裂所需的一种或多种事件的抑制。通过其在细胞环境中的作用，我们将查明 AEK1 被抑制时发生的细胞学缺陷。由于蛋白激酶通过调节下游效应分子发挥其功能，因此 AEK1 必须反映其一种或多种底物的重要性为了识别底物，我们将使用基因。修饰的 AEK1 特异性接受大硫代 ATP 类似物，使其能够特异性硫代磷酸化然后通过转移的硫代磷酸盐的鉴定来捕获底物。通过质谱分析硫代磷酸化肽将允许开发一系列候选底物和共有磷酸化位点序列，可用于识别其他候选者。将审查已识别的分子与已识别的表型过程的关系，以帮助通过揭示调控机制和确定候选底物，确定未来研究的优先顺序。鉴于 AEK1 的高度相似性，该项目将告诉我们 AEK1 的功能途径。锥虫科，我们的许多发现很可能适用于其他锥虫科病原体。