Structural and functional studies on molecular evolution of calcium signaling toolkit

原生动物寄生虫是一种单细胞生物，可导致许多使人衰弱的疾病，包括疟疾、非洲昏睡病和南美锥虫病。当寄生虫感染个体时，它们会引发一系列细胞内反应。随着时间的推移，细胞内钙离子（Ca2+）水平的变化构成了所有含核细胞中关键的Ca2+信号反应，但由于寄生虫与被充分研究的多细胞动物的进化距离很远，因此对介导这些信号的分子蛋白成分（称为Ca2+信号工具箱）及其在寄生虫中的功能知之甚少。迄今为止，比较基因分析表明，寄生虫使用祖先形式的许多蛋白质，这些蛋白质在更进化的动物的Ca2+信号工具包中发现；然而，肌醇1,4,5-三磷酸（IP3）受体（IP3R）和ryanodine受体（RyR）是多细胞动物的关键工具箱成分，尽管小分子引发了类似于这类蛋白质的活性，但尚未在寄生虫中发现。因此，这两个通道是理解单细胞生物中Ca2+信号的缺失环节。我们成功地从引起恰加斯病的克氏锥虫（T. cruzi）单细胞中克隆了一个IP3R祖先蛋白。在克氏锥虫中，IP3Rs控制的Ca2+信号被认为参与了感染过程；因此，克氏锥虫是研究寄生Ca2+信号的良好候选生物。我们建议表征T.克氏IP3R （TcIP3R）的三维结构，就其在寄生虫生命周期中的功能而言，并评估这种Ca2+工具包组件在进化过程中如何被修改。这些数据将使我们能够比较和对比生物世界中的Ca2+信号。我们已经证明，消除TcIP3R对克氏锥虫是致命的，降低TcIP3R水平会减弱这种寄生虫的传染性；因此，我们的研究不仅将为Ca2+信号工具箱的进化提供见解，而且还将为控制这些和其他危及生命的寄生虫提供新的线索。

Protozoan parasites are single cell organisms that cause numerous debilitating diseases including malaria, African sleeping sickness and Chagas disease. When parasites infect an individual they trigger a series of intracellular responses. Changes in intracellular calcium ion (Ca2+) levels over time constitute key Ca2+ signaling responses in all nucleus containing cells, but very little is known about the molecular protein components that mediate these signals known as the Ca2+ signaling toolkit and their functions in parasites due to the large evolutionary distance from well-studied multi-cellular animals. To date, comparative gene analyses have revealed that parasites use ancestral forms of many proteins found in the Ca2+ signaling toolkit of more evolved animals; however, the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyR), critical toolkit components in multi-cellular animals, have not been identified in parasites, despite small molecules eliciting an activity reminiscent of this class of proteins. Thus, these two channels have been a missing link in understanding Ca2+ signaling in unicellular organisms. We successfully cloned an IP3R ancestral protein from the single celled, Trypanosoma cruzi (T. cruzi), a parasite causing Chagas disease. In T. cruzi, Ca2+ signaling controlled by IP3Rs is believed to be involved in the infection process; consequently, T. cruzi is a good candidate organism to study parasitic Ca2+ signaling. We propose to characterize the three-dimensional structure of T. cruzi IP3R (TcIP3R) with respect to its function in parasite life cycles and assess how this Ca2+ toolkit component has been modified during evolution. These data will allow us to compare and contrast Ca2+ signaling within the living world. We have demonstrated that eliminating TcIP3R is lethal to T. cruzi and decreasing TcIP3R levels weakens the infectivity of this parasite; hence, our research will not only provide insights into the evolution of the Ca2+ signaling toolkit, but will also provide new clues to controlling these and other life-threatening parasites.