Current interest in the ubiquitination pathway in Plasmodium falciparum was sparked by the discovery that resistance to the frontline antimalarial, artemisinin, is mediated by mutations in the kelch13 or K13 gene. This gene encodes a protein that shares some sequence and conserved domain similarity with a human protein, Keap1, which regulates the ubiquitination of a transcription factor responsible for the control of expression of key genes involved in oxidative stress response. Keap1 performs this function by binding to a multisubunit complex involving a Cullin-family scaffold and a RING (Really Interesting New Gene).
Apart from implications in drug resistance, ubiquitination plausibly plays roles in the parasite progression through the life cycle, intracellular trafficking and control of gene expression. In silico proteomic analyses in Plasmodium have identified most of the components of the ubiquitin system. However, only a few have been functionally characterized.
In this project, four proteins with putative annotations (two Cullins, NEDD8, RBX1) will be investigated. A genetic system, called selection-linked integration (SLI), was used to introduce GFP-tags into the chromosomal copies of these proteins for localization and mislocaliser modules for functional analysis through knock-sideways approaches. Transfections will be done in a gametocyte-producing line to allow study of these proteins in both the asexual and sexual stages. Effect of conditional inactivation of these proteins will be investigated in the absence and presence of dihydroartemisinin (DHA). Moreover, a commercially-generated antibody against one of the putative cullins will be validated. Upon validation, this antibody will be used for immunoprecipitation studies for further studies.
Given that the ubiquitination process and most of the proteins currently annotated to be involved in ubiquitination in P. falciparum are currently unstudied, the results of this project could contribute not only to the study of the mechanism of artemisinin resistance but also to an improved understanding of the basic biology of the parasite.