Structural and functional studies on molecular evolution of calcium signaling toolkit

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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.