Protein Interaction Testing of TgCycY Putative Cyclin Protein of Toxoplasma gondii for a Novel Drug Target Identification

The apicomplexan protozoan parasites cause critical health problems and limitation of anti-parasitic drugs is a major problem. Among the apicomplexan parasites, Toxoplasma gondii (T. gondii) is a highly prevalent obligate intracellular protozoan parasite, which seems to rely mostly on proteins that are defined as cyclin and cell cycle kinases that needs to regulate the cell cycle of the tachyzoites. These proteins regulate DNA replication during the cell cycle and lead to moderate cell division which results toxoplasmosis disease. Before targeting the proteins in T. gondii, it is essential to identify these proteins and define their function in the cell cycle via protein-protein interactions. In vitro, protein-protein interaction testing of Toxoplasma gondii ME49 putative TgCycY cyclin via yeast two-hybrid screen was conducted to identify a protein interaction partner. Even though previously tested in vivo analyses showed no direct interaction between TgCycY and TgCrk2, TgCrk2 hypothesized as a potential interacting partner for the TgCycY with the association of a bridging protein. Hypotheses were made based on the ortholog TgCycY-TgCrk2 complex interactions in Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae. Among the identified 8 protein interaction partners from a cDNA library of asynchronous tachyzoite transcriptome that used in the Y2H screen, only TgDJ-1 protein was introduced as the potential interacting partner for the TgCycY. Other protein partners were excluded due to limitations of the yeast-two hybrid screen and lack of information. Based on the previous studies, TgDJ-1 protein has shown function involved in the micronemes secretion and it has introduced as the regulator of the Toxoplasma gondii secretion, motility, and invasion by interacting with TgCDPK1. TgDJ-1 inhibition did not affect the cell cycle of the tachyzoite. A CDK interacting partner for the TgCycY was not detected in this screen. Previously known fact of Cyclin Y function as a substrate for the CDK and the function of the mediating the ortholog CycY-CDK protein-protein interaction via a third protein was applied to the TgCycYTgDJ-1-TgCDPK1 predicted complex. Due to limitations of the current screen and obtained results, indicated hypotheses 1 & 2 cannot be proven and the results direct the conclusion towards the TgCycY having TgDJ-1 as a new protein interacting partner that has existing literature and role of outside the cell cycle regulation. Overall, our project aims to map at the molecular level interactions of putative TgCycY cyclin protein in vitro and identify its function in the T. gondii tachyzoites to introduce it as a novel drug target. Further study regarding the function of TgCycY-TgDJ-1-TgCDPK1 complex is needed to understand the TgCycY role in micronemes secretion and involvement in the tachyzoites