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体，会导致许多令人衰弱的疾病，包括疟疾、非洲昏睡病和恰加斯病。当寄生虫感染个体时，它们会触发一系列细胞内反应。细胞内钙离子(Ca~(2+))水平随时间的变化构成了所有含核细胞中关键的Ca~(2+)信号反应，但由于与研究良好的多细胞动物的进化距离很远，人们对介导这些信号的分子蛋白成分(称为Ca~(2+)信号工具包)及其在寄生虫中的功能知之甚少。到目前为止，比较基因分析已经揭示了寄生虫使用在更进化的动物的钙信号工具包中发现的许多蛋白质的祖先形式；然而，在多细胞动物中的关键工具包组件1，4，5-三磷酸肌醇(IP3)受体(IP3R)和兰诺定受体(RyR)尚未在寄生虫中被鉴定，尽管小分子引起了这类蛋白质的活性。因此，这两个通道一直是理解单细胞生物体中钙信号的缺失环节。我们成功地从引起恰加斯病的单细胞克氏锥虫(T.ruzi)中克隆了IP3R祖先蛋白。在克氏锥虫中，由IP3Rs控制的钙信号被认为参与了感染过程，因此，克氏锥虫是研究寄生钙信号的一个很好的候选生物。我们建议就其在寄生虫生活史中的功能来描述TcIP3R(TcIP3R)的三维结构，并评估这个钙工具包组件在进化过程中是如何被修改的。这些数据将使我们能够比较和对比生活世界中的钙信号。我们已经证明，消除TcIP3R对克氏锥虫是致命的，降低TcIP3R水平会削弱这种寄生虫的传染性；因此，我们的研究不仅将为钙信号工具包的进化提供见解，还将为控制这些和其他威胁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.