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.

锥虫寄生虫导致一些最具挑战性的被忽视的疾病在世界范围内。人类- 布氏锥虫属的致病性三头虫，克氏锥虫（Trypanosoma cruzi）和利什曼原虫（Leishmania spp.）影响数百 成千上万的人，造成人类非洲锥虫病，恰加斯病，和一系列内脏 皮肤病变，分别。由于没有批准的疫苗和治疗方法受到耐药性的挑战， 递送和功效问题，新药的发现对于治疗、控制和消除这些问题至关重要。 疾病因此，需要新的、经过验证的目标来进入发现管道。我们最近 基因和化学验证了T.布氏蛋白激酶AEK 1，它是高度保守的， 这些试剂还不同于人类蛋白激酶。敲低后的裂解表型表明， 重要的通路由AEK 1控制，但对其调节或下游效应物一无所知。 我们希望填补这一知识空白。我们将测试特定的顺式和反式相互作用，我们假设调节 AEK 1活性，包括探测预测的变构位点和评估候选活化激酶。我们 数据显示AEK 1的抑制导致胞质分裂所需的一个或多个事件的抑制。放置 在细胞环境中的作用，我们将查明AEK 1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， AEK 1必须反映一种或多种底物的重要性。为了识别底物，我们将使用遗传学方法， 修饰的AEK 1特异性接受大的硫代ATP类似物，以使其特异性硫代磷酸化 印刷受体.然后，底物将通过转移的硫代磷酸盐被捕获。识别 硫代磷酸化的肽将允许开发候选底物的列表， 能够鉴定其他候选物的共有磷酸化位点序列。的 鉴定的分子将审查其与鉴定的表型过程的关系，以帮助 优先考虑未来的研究。通过揭示调控机制和识别候选底物， 这个项目将告诉我们AEK 1的功能途径。考虑到AEK 1在不同基因组中的高度相似性， 因此，我们的许多发现可能适用于其他锥虫病原体。