Malaria is one of the deadliest infectious diseases, infecting 300-500 million and killing up to one million people globally every year. It is the world's leading cause of death in children and prevalent in sub-Saharan Africa, South Asia, and parts of South America, adversely impacting economic development in many impoverished countries. Though treatments for malaria do exist, the rising drug-resistances gained by the elusive malaria parasites counteract the efficacy of current treatments and beckon the need for new anti-malarials and new drug targets.
One limiting factor for the development of new antimalarials is our poor understanding of the basic biology of the P. falciparum, the most lethal of the parasite species that cause human malaria. To better understand the parasite biology, we investigate the importance of post-translational modification, specifically ubiquitylation, in regulating Plasmodium biology. On a genome-wide scale, we identify the Plasmodium "ubiquitome", which is the population of proteins that are likely to be modified by ubiquitin. We have found that the Plasmodium ubiquitome takes up a large portion of the entire Plasmodium protein population, which suggests that ubiquitylation is an important aspect of the Plasmodium biology. Furthermore, our findings show that the roles of ubiquitin in the Plasmodium has both conserved and also parasite-specific functions, such as invasion, hemoglobin metabolism, and liver stage-specific purposes.
To fuller expand our understanding of the ubiquitylation pathway in the malaria parasite, we investigate the Plasmodium endoplasmic reticulum-associated degradation (ERAD) pathway, as well as a newly identified duplicated ERAD-like system. In eukaryotic cells, the ERAD pathway serves to recognize misfolded proteins within the ER lumen and label them with ubiquitin for proteasome degradation. Here, we characterize the Plasmodium ERAD system with a combination of localization studies, in vitro biochemical assays, and knockout experiments. Altogether, our findings indicate that the putative ubiquitylating components of the Plasmodium ERAD system localizes to the their expected regions (the ER and cytosol), has ubiquitylating properties and is likely essential to the parasite, leaving the possibility for exploiting the Plasmodium ERAD system for antimalarial targeting.
Lastly, we characterize the duplicated ERAD system and validated that it targets the Plasmodium-specific organelle called the apicoplast. The apicoplast is a four membrane-bound organelle that is essential for the parasite's survival. The functions of the apicoplast (i.e. isoprenoid biosynthesis and fatty acid synthesis) are mostly fulfilled by ~500 nucleus-encoded proteins that are transported to the outermost apicoplast compartment via the secretory pathway. However, how these proteins import into the apicoplast through its multiple membranes remain unclear. Here, by using bioinformatics analysis, cloning techniques, recombinant proteins, biochemical assays and fluorescent microscopy, we have biologically characterized the ERAD-like core ubiquitylating components, showing that they are capable of ubiquitylation, and localize them to the apicoplast. Our data indicates that the ERAD-like system likely has similar translocative properties as the traditional ERAD system of the ER and functions to transport apicoplast-targeted proteins across apicoplast membranes. Because of both the essentiality and specificity of the apicoplast to the malaria parasite, we propose that this apicoplast ERAD-like system would make a solid candidate for anti-malaria drug targeting.
Overall, our investigation validates the importance of ubiquitylation within the Plasmodium biology and also highlights a few potential antimalarial drug targets that are specific to the deadly parasite.