pH and glucose sensing in Trypanosoma brucei glycosomes

布氏锥虫糖体中的 pH 和葡萄糖传感

• 批准号: 8624128
• 负责人: KENNETH A CHRISTENSEN
• 金额: $ 19.8万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-12 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8624128
• 关键词: Affect; Africa South of the Sahara; African; African Trypanosomiasis; Area; Biology; Blood Circulation; Calcium Signaling; Carbon; Cells; Cessation of life; Chagas Disease; Consumption; Cues; Data; Development; Drug Design; Drug resistance; Endoplasmic Reticulum; Environment; Enzymes; Failure; Fluorescent Probes; Foundations; Glucose; Glycolysis; Glycosome; Goals; Golgi Apparatus; HIV; Housing; Human; Individual; Leishmania; Leishmaniasis; Life; Measurement; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methodology; Mission; Mitochondria; Monitor; Morbidity - disease rate; Nutrient; Organelles; Organism; Parasites; Parasitic Diseases; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Production; Proteins; Public Health; Quantitative Evaluations; Recombinant Proteins; Recombinants; Regulation; Research; Sepsis; Series; Sodium Chloride; Solutions; Source; Staging; Techniques; Temperature; Testing; Toxic effect; Treatment Failure; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomiasis; Work; base; deprivation; design; economic cost; environmental change; enzyme activity; enzyme mechanism; extracellular; glucose metabolism; glucose sensor; hexokinase; in vivo; killings; mortality; novel strategies; peroxisome; public health relevance; ratiometric; response; sensor; small molecule; social; solute; sugar; therapeutic target

PROJECT SUMMARY/ABSTRACT African trypanosomiasis, also called African sleeping sickness, infects tens of thousands of individuals yearly in endemic areas, and is accompanied by continuing high social and economic cost. Existing treatments for trypanosomiasis are woefully inadequate, due both to the emergence of drug resistance that results in high treatment failures rates (30% in some areas), and high toxicity resulting in significant morbidity (10%) and drug-related mortality (5%). There thus remains an outstanding need for rational design of new trypanocidal therapies, particularly those that minimize host toxicity by targeting unique trypanosome biology. Glucose metabolism is the sole source of ATP for the infectious lifecycle stage of the African trypanosome, Trypanosoma brucei, and enzymes central to sugar metabolism are housed in the glycosome, an organelle not found in the parasite's mammalian host(s). Hence, both glycosome function and the control mechanisms governing enzyme activity inside the glycosome are important targets for drug design. We have demonstrated that ATP production in this organism is sensitive to environmentally-influenced changes in glycosomal solution conditions, including pH. Characterizing the intraglycosomal environment is therefore a necessary step in understanding essential glycolytic pathways, and would lay the groundwork for development of anti- trypanosome therapies that target control of glucose metabolism. However, this information is currently lacking at the most basic level. Neither pH nor glucose has been quantified inside the glycosome and their environmentally influenced dynamic range(s) are unknown, a paucity that reflects the historical lack of methodologies to allow quantitative intraglycosomal measurement. Here we propose development of peptide- targeted small molecule and recombinant protein-based sensors to quantitatively determine intraglycosomal pH and glucose levels. Our preliminary data indicates that such sensors can be delivered to the glycosomes of live parasites. Developed sensors can be subsequently modified for measurement of other glycosomal solutes, including ATP, and used to investigate control of glycolysis as a response to other potentially important environmental and developmental conditions, including glucose and divalent salt concentrations, nutrient depletion, and calcium signaling. Resulting findings will illuminate the mechanisms of dynamic regulation of the glycosomal environment, reveal conditions that influence the activity of this essential metabolic pathway, and introduce methodologies likely facilitate a series of new approaches to understanding and testing parasite metabolism. Notably, techniques pioneered in this study can be extended to analysis of other pathogenic kinetoplastid parasites, such as Trypanasoma cruzi and Leishmania spp. that also localize ATP production in glycosomes. In addition, the methodologies can be modified to evaluate the intraorganellar environment in other important subcellular compartments such as the mitochondria, endoplasmic reticulum, and Golgi apparatus. The work is therefore likely to have impact(s) beyond African trypanosomiasis.

项目摘要/摘要 非洲锥虫病，也被称为非洲昏睡病，每年感染数万人。 疾病流行地区，并伴随着持续高额的社会和经济代价。现有的治疗方法 令人遗憾的是，锥虫病是不够的，原因是出现了耐药性，导致了高 治疗失败率(某些地区为30%)，高毒性导致严重发病率(10%)和 与药物有关的死亡率(5%)。因此，仍然需要合理设计新的杀锥虫药 治疗，特别是那些通过靶向独特的锥虫生物学将宿主毒性降至最低的治疗方法。葡萄糖 新陈代谢是非洲锥虫感染生命周期阶段ATP的唯一来源， 布鲁氏锥虫和糖代谢中心的酶被安置在糖体中，而不是细胞器 在寄生虫的哺乳动物宿主(S)中发现。因此，糖体的功能和调控机制 控制糖体内的酶活性是药物设计的重要目标。我们已经证明了 这种生物体中的ATP生产对糖体溶液中受环境影响的变化很敏感 条件，包括pH值。因此，确定糖体内环境的特征是 了解糖酵解的基本途径，将为开发抗肿瘤药物奠定基础 以控制葡萄糖代谢为目标的锥虫疗法。然而，目前缺乏这方面的信息。 在最基本的层面上。糖体内的pH和葡萄糖都没有被量化，它们的 受环境影响的动态范围(S)都是未知的，这一匮乏反映了历史上缺乏 能够进行定量糖体内测量的方法学。在这里，我们提出了多肽的开发- 基于靶向小分子和重组蛋白的传感器定量测定糖体内 PH值和血糖水平。我们的初步数据表明，这种传感器可以被运送到 活的寄生虫。所开发的传感器随后可以被改进以用于其他糖体的测量 包括三磷酸腺苷在内的溶质，用于研究糖酵解的控制，作为对其他潜在的 重要的环境和发育条件，包括葡萄糖和二价盐浓度， 营养耗竭和钙信号。由此产生的发现将阐明动力学的机制 调节糖体环境，揭示影响这一必不可少的活动的条件 代谢途径，并引入方法学可能有助于一系列新的途径来理解 以及测试寄生虫的新陈代谢。值得注意的是，这项研究中首创的技术可以扩展到分析 其他致病动粒体寄生虫，如克氏锥虫和利什曼原虫。这也本地化了 糖体中的三磷酸腺苷的产生。此外，还可以修改方法以评估细胞器内的 其他重要亚细胞隔间的环境，如线粒体、内质网、 和高尔基仪器。因此，这项工作可能会产生超越非洲锥虫病的影响(S)。