Functional analysis of an essential Trypanosoma brucei protein kinase

布氏锥虫必需蛋白激酶的功能分析

• 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
• 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

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.

锥虫寄生虫导致了世界上一些最具挑战性的被忽视的疾病。人类- 布氏锥虫、克氏锥虫、利什曼原虫致病三联种影响数百人 导致人类非洲锥虫病、恰加斯病和一系列内脏疾病 分别为皮肤损伤。由于没有获得批准的疫苗和治疗方法受到耐药性的挑战， 新药的发现对于治疗、控制和消除这些问题至关重要。 疾病。因此，需要新的经过验证的目标才能进入发现管道。我们最近做了 布鲁氏锥虫蛋白激酶AEK1在基因和化学上都是高度保守的 这些制剂还不同于人类的蛋白激酶。击倒后的裂解表型认为 重要的通路由AEK1控制，但对其调控或下游效应器知之甚少。 我们建议填补这一知识空白。我们将测试我们假设调控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和评估激活的候选激酶。我们的 数据显示，抑制AEK1导致胞质分裂所需的一个或多个事件的抑制。放置 它在细胞内的作用，我们将精确定位当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必须反映其一个或多个底物的重要性。为了鉴定底物，我们将使用一种基因 修饰的AEK1，专门接受大的硫代-ATP类似物，使其特定的硫代磷酸化 底物。然后，底物将被转移的硫代磷酸盐捕获。身份识别 通过质谱学分析硫代磷酸化肽，可以开发出一系列候选底物和 一个共识的磷酸化位点序列，将能够识别其他候选序列。这个 将审查识别的分子与识别的表型过程的关系，以帮助 为今后的研究确定优先次序。通过揭示调节机制和识别候选底物， 这个项目将告诉我们AEK1发挥作用的途径。鉴于AEK1的高度相似性 对于锥虫，我们的许多发现很可能也适用于其他锥虫病原体。