Handling Uncertainty in Dynamic Models: The Pentose Phosphate Pathway in Trypanosoma brucei

Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate “leak” must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally.代谢动态模型在潜在药物靶点的识别中具有重要应用价值，针对单细胞生物的相关研究尤具意义。人类非洲锥虫病（human African trypanosomiasis）的病原体布氏锥虫（Trypanosoma brucei）的糖酵解模型，已验证了该研究思路的实用性。本研究将布氏锥虫的戊糖磷酸途径（pentose phosphate pathway, PPP）整合至该糖酵解模型中。该途径同时定位于细胞质（cytosol）与糖体（glycosome）内；若直接将其加入糖酵解模型而未做额外调整，会导致糖体内关键结合磷酸基团的耗竭。为使该模型能够合理反映寄生虫的生理状态，必须解决这一磷酸“泄漏”问题。针对该问题，可归纳出两类主要理论解决方案：（i）在糖体中引入额外的酶促反应；（ii）添加介导细胞质与糖体间结合磷酸基团转运的机制。第一类解决方案的一个典型示例，是在糖体中存在核糖激酶（ribokinase），以利用5-磷酸核糖与ADP再生ATP。针对布氏锥虫核糖激酶的实验表征显示，极低的酶表达量即可满足寄生虫存活需求，这表明还存在其他调控磷酸泄漏的机制。第二类解决方案的示例包括糖体膜上存在ATP:ADP转运体（ATP:ADP exchanger），或是新近报道的通透性孔道（permeability pores）；但目前尚未发现编码此类分子的基因，这阻碍了通过基因操作开展的实验验证。面对这一研究不确定性，本研究提出了一种建模策略，可在系统表征不完整的情境下获取稳健的预测结果。我们通过解析戊糖磷酸途径中某一关键酶的核心功能机制，对该策略进行了演示，并通过实验验证模型预测结果完成了策略的有效性确认。