Plasmodium palmitoylation machinery engineered in for high-throughput screening of palmitoyl acyl-transferase inhibitors.

Lipid-based palmitoylation is a post-translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, . palmitoylation is catalyzed by 12 putative palmitoyl acyl-transferase enzymes containing the conserved DHHC-CRD (DHHC motif within a cysteine-rich domain) which can serve as a druggable target. However, the paucity of high-throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of , a PTM-null system, to enforce ectopic expression of palmitoyl acyl-transferase in order to study -specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic strains expressing -specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl-transferase activity via acyl-biotin exchange (ABE) and clickable chemistry methods. proteome was found to be palmitoylated in PfDHHC-expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of proteins. Upon treatment with generic inhibitor 2-bromopalmitate (2-BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic strains expressing PfDHHCs can enforce global palmitoylation in the proteome. Interestingly, this finding was corroborated by our palmitoylome profiling, which revealed that out of the total proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high-throughput screening platform.