Quantification of labile heme in live malaria parasites using a genetically encoded biosensor.
Quantification of labile heme in live malaria parasites using a genetically encoded biosensor.
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DOI：
10.1073/pnas.1615195114
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发表时间：
2017-03-14
影响因子：
11.1
通讯作者：
Niles JC
中科院分区：
文献类型：
--
作者：
Abshire JR;Rowlands CJ;Ganesan SM;So PT;Niles JC
疟原虫降解大量的血红蛋白，在一个专门的消化液泡中释放血红素。大部分血红素被隔离在一种惰性晶体中。然而，生物可利用的浓度，不稳定的血红素在寄生虫的细胞质是未知的。我们开发了一种生物传感器，首次提供了对疟疾寄生虫中不稳定血红素浓度的定量分析。我们发现维持约1.6µM不稳定的胞质血红素，包括在与血红蛋白强烈降解相一致的时期。与血红素结合的抗疟药物氯喹会干扰血红素结晶，从而特异性地诱导不稳定血红素的增加。量化疟原虫中不稳定血红素的能力为更好地了解血红素稳态、信号和代谢及其与抗疟疾效力的关系提供了机会。血红素是普遍存在的，但相对而言，人们对这种辅助因子的稳定池的维持知之甚少，这可能确保了其及时的生物利用度，以实现适当的细胞功能。对不稳定血红素的定量分析对于理解大自然如何保护血红素所能获得的多种化学物质，同时最大限度地减少其氧化还原活性造成的细胞损伤至关重要。在这里，我们已经开发并表征了一种基于蛋白质的传感器，该传感器在血红素结合时经历荧光猝灭。通过在人类疟原虫恶性疟原虫中对这种传感器进行遗传编码，我们已经量化了完整的血期疟原虫的胞质不稳定血红素水平。我们的研究结果表明，在整个寄生虫发育过程中，红细胞内稳定地维持着一个不稳定的血红素库（~ 1.6µM），即使在寄生虫大量降解血红蛋白的时期也是如此。我们还发现，与血红素结合的抗疟药物氯喹特异性地增加了不稳定的细胞质血红素，表明这一稳态库的失调，这可能是这类化合物抗疟活性的一个相关组成部分。我们建议在恶性疟原虫的各种环境扰动下使用这种技术可以对基础血红素生物学产生定量的见解。
Malaria parasites degrade substantial quantities of hemoglobin to release heme within a specialized digestive vacuole. Most of this heme is sequestered in an inert crystal. However, the concentration of bioavailable, labile heme in the parasite’s cytosol was unknown. We developed a biosensor to provide the first quantitative insights into labile heme concentrations in malaria parasites. We find that ∼1.6 µM labile cytosolic heme is maintained, including during a period coincident with intense hemoglobin degradation. The heme-binding antimalarial drug, chloroquine, which interferes with heme crystallization, specifically induces an increase in labile heme. The ability to quantify labile heme in malaria parasites opens opportunities for better understanding heme homeostasis, signaling, and metabolism, and its association with antimalarial potency. Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology.