Nutrient Sensing and Hexokinases in T. brucei

描述(申请人提供)：葡萄糖代谢是非洲布氏锥虫感染性生命周期阶段ATP的唯一来源。该途径中的第一种酶己糖激酶的错误调节对寄生虫是有毒的。然而，关于这两个编码这一重要酶活性的基因表达的调节机制，人们知之甚少。这项应用的目标是确定寄生虫在基因表达和酶活性水平上调节己糖激酶酶的机制，以响应不同的环境条件。初步数据表明，锥虫体己糖激动酶是通过调节转录的稳态丰度和表达以及酶低聚体组成的变化来响应环境葡萄糖水平而动态调节的。非洲锥虫转录丰度的调节主要通过转录后机制进行，这是编码在基因3‘UTRs中的信息的结果。影响己糖激酶稳态转录丰度的因素将通过监测携带一系列突变的己糖激酶3‘UTRs的报告基因结构的转录水平来确定。这些结构对基因表达的影响也将通过对报告基因的酶活性进行评分来考虑。在蛋白质水平上，己糖激酶六聚体的组成，寄生虫可以根据生长条件改变，影响酶的活性，包括对调节分子的敏感性。为了了解对调节分子敏感性的差异，将使用定点突变来确定抑制剂结合所需的结构域和残基。将通过在布氏毛滴虫中的表达来评估体外不再受调控的变异体的影响。通过表征己糖激酶表达所需的调控机制，将确定以葡萄糖代谢为靶点的新方法，这是一种必需的寄生虫途径。 公共卫生相关性：拟议的研究对公共健康很重要，因为这里确定的机制将为非洲锥虫迫切需要的治疗开发提供新的目标，同时扩大我们对葡萄糖感应的基本细胞过程的理解，这些主题得到了美国国立卫生研究院的支持。

DESCRIPTION (provided by applicant): Glucose metabolism is the sole source of ATP for the infectious lifecycle stage of the African trypanosome, Trypanosoma brucei. Mis-regulation of the first enzyme in the pathway, hexokinase, is toxic to the parasite. However, little is known concerning the regulatory mechanisms that modulate expression of the two genes that encode this essential enzyme activity. The goal of this application is to identify the mechanisms the parasite employs to regulate hexokinases at the gene expression and enzyme activity levels in response to distinct environmental conditions. Preliminary data indicates the trypanosome hexokinases are dynamically regulated in response to environmental glucose levels through mechanisms that include both modulation of transcript steady-state abundance and expression, as well as changes in enzyme oligomer composition. Regulation of transcript abundance in the African trypanosome occurs primarily via post-transcriptional mechanisms as a result of information encoded in gene 3'UTRs. Elements that influence hexokinase steady-state transcript abundance will be identified by monitoring transcript levels of a reporter gene construct harboring a series of mutated hexokinase 3'UTRs. The impact of these constructs on gene expression will also be considered by scoring enzyme activity of the reporter gene. At the protein level, hexokinase hexamer composition, which the parasite can alter based on growth conditions, influences enzyme activity, including sensitivity to regulatory molecules. To understand the differences in sensitivity to regulatory molecules, site-directed mutagenesis will be used to identify domains and residues required for inhibitor binding. The impact of variants that are engineered to no longer be susceptible to regulation in vitro will be assessed in vivo by expression in T. brucei. Through the characterization of regulatory mechanisms required for hexokinase expression, new means of targeting glucose metabolism, a required parasite pathway, will be identified. PUBLIC HEALTH RELEVANCE: The proposed research is important to public health as mechanisms identified here will yield new targets for desperately needed therapeutic development for the African trypanosome while expanding our understanding of the fundamental cellular process of glucose sensing, topics that are supported by the mission of the NIH.