Title : Artemisinin resistance in P. falciparum: Probing the interacting partners of Kelch13 protein in parasite
Abstract:
Malaria remains a global health issue affecting half of the world's population. The current treatment regimen which includes artemisinin and other combination therapies is being threatened with the rapid emergence of resistance. P. falciparum under drug pr essure has revealed insights into mechanisms of resistance most commonly used antimalarials, such as Chloroq uine, Amodiaquine, Piperaquine, DHFR inhibitors etc. There are currently no 0alternative drugs available to replace Artemisinin. ART resistance has b een shown to be mediated by the Plasmodium Kelch13 ( Pf K13) protein. Pf kelch 13 gene is situated in chromosome 13 and associated with ART resistance , owing to the association of majority of mutations at the kelch BTB/POZ & propeller domain . The present study recombinantly expressed the PfK13-p (BTB/POZ & propeller domain) and generated anti-PfK13-p antibodies for cellular localization and co-immunoprecipitation (co-IP) assays and mass spectroscopy was performed to identify the Pf K13 interacting partn ers. Unique coimmunoprecipitated proteins were identified barring few proteins overlapping with previous studies Protein disulfide isomerase, heat shock proteins, merozoite surface protein 1 (MSP1), L-lactate dehydrogenase, elongation factor 1-alpha. The unique hits of the study were- falcilysin, enolase, phosphoethanolamine N-methyltransferase, glideosome-associated protein 50, fructose-bisphosphate aldolase, adenylate kinase, peptidyl-prolyl cis-trans isomerase, thioredoxin-related protein, putative, 20 kDa chaperonin, ornithine aminotransferase, rhoptry-associated protein 1. The identified proteins were categorized into protein The identified proteins were categorized into protein folding, protein binding/invasion, cellular metabolism and mobility functions. Further,folding, protein binding/invasion, cellular metabolism and mobility functions. Further, bioinformatics bioinformatics proteins identified by thproteins identified by the STRING database represent the e STRING database represent the PfK13PfK13 protein and protein and the respective potential interactors or performing sharedthe respective potential interactors or performing shared functions are shown in network. The functions are shown in network. The minimum interaction score was set to medium confidence level (0.400) and no more than 10 minimum interaction score was set to medium confidence level (0.400) and no more than 10 interactors were selected. PGK (Phosphoglycerate kinase) and Q7KQL9 (Fructoseinteractors were selected. PGK (Phosphoglycerate kinase) and Q7KQL9 (Fructose--biphosphate aldolase) are the two predicted proteins,biphosphate aldolase) are the two predicted proteins, which have been identified via cowhich have been identified via co--IP IP assays. In other experiment, assays. In other experiment, strong binding affinities ofstrong binding affinities of PfPfK13K13--p and two p and two coimmunoprecipitated proteinscoimmunoprecipitated proteins-- Heat Shock Protein 70 and Heat Shock Protein 70 and PfPfFBAP (6.6 and 7.6 µM, FBAP (6.6 and 7.6 μM, respectively) were observed using surface plasmon resonance (SPrespectively) were observed using surface plasmon resonance (SPR).R). Additionally, Additionally, PfPfMSP1 MSP1 formed a complex with formed a complex with PfPfK13K13--p, as evidenced by pullp, as evidenced by pull--down assays.down assays. Interestingly, Interestingly, PfPfKelch13 Kelch13 forms a stable hexamer in Nforms a stable hexamer in N--termini BTBtermini BTB--POZ domain. Further, POZ domain. Further, Using antiUsing anti--PfPfK13K13--p p antibodies, the endogenous antibodies, the endogenous PfPfK13 protein was observed to colocalization with a cytosolic K13 protein was observed to colocalization with a cytosolic markermarker-- PfPfAspRS (aspartyl transferAspRS (aspartyl transfer--RNA synthetase).RNA synthetase). Together, this work identified unique Together, this work identified unique interacting partners of endogenous interacting partners of endogenous PfPfK13 protein, which might have crucial implications in K13 protein, which might have crucial implications in the the PfPfK13 proteK13 protein network and its role in mediating ART resistance.in network and its role in mediating ART resistance.
