Plasmodium palmitoylation machinery engineered in E. coli for high‐throughput screening of palmitoyl acyl‐transferase inhibitors

Abstract

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, Plasmodium falciparum. P. falciparum 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 Escherichia coli, a PTM‐null system, to enforce ectopic expression of palmitoyl acyl‐transferase in order to study Plasmodium‐specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium‐specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl‐transferase activity via acyl‐biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC‐expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli 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 E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli 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.

Natural Product Inspired Novel Indole based Chiral Scaffold Kills Human Malaria Parasites via Ionic Imbalance Mediated Cell Death

“…Chemical structures of scaffold 6 and 7 were drawn using ChemDraw Ultra 12.0.2, followed by generation of their energy-minimized 3D-models by using Chem3D Pro 12.0, as described by Yadav, P. et al . 70 . Molecular docking studies were performed by using Autodock Vina Tools 1.5.6 to rationalize the inhibitory activities of scaffolds 6 and 7 against PfATP4 70,71 .…”

Palmitoylated Proteins in Plasmodium falciparum‐Infected Erythrocytes: Investigation with Click Chemistry and Metabolic Labeling

“…and Yadav et al., respectively. [ 23,24 ] Our EM approach further showed the distribution of gold particles, representing palmitoylated proteins, between the parasite and the cytoplasm of the infected erythrocyte (Figure 1e,f). Interestingly, the cytoplasm of erythrocytes infected with ring stage parasites appeared to contain more gold particles than the cytoplasm of erythrocytes infected with trophozoite and schizont stages (Figure 1e).…”

“…[ 18,19 ] This lipid modification has been suggested to play a crucial role in cytoadherence, drug resistance, cell signaling, organelle positioning, cytoskeletal function, development, maturation, and even host cell invasion during the complex life cycle of P. falciparum . [ 6,17,20–25 ] In silico analyses identified an estimated 400 palmitoylated proteins produced in the schizont stages and 12 putative protein acyl‐transferase enzymes (or DHHC enzymes, PfDHHC1‐12) that contain the conserved DHHC motif ([Asp‐His‐His‐Cys] within a cysteine‐rich domain). PfDHHC enzymes catalyze S‐ palmitoylation within the secretory pathway (endoplasmic reticulum and Golgi apparatus) or at unique apicomplexan organelles, such as the inner membrane complex and the rhoptries.…”

“…PfDHHC enzymes catalyze S‐ palmitoylation within the secretory pathway (endoplasmic reticulum and Golgi apparatus) or at unique apicomplexan organelles, such as the inner membrane complex and the rhoptries. [ 17,20–22,24,26 ] Palmitoylation represents the second most prominent PTM after phosphorylation in Plasmodium parasites, which suggests an important role for palmitoylated proteins in parasite development. [ 6 ]…”

Role of RBC membrane protein palmitoylation in regulation of molecular topology and susceptibility toPlasmodium falciparuminvasion

“…The palmitoylation status of RBCs was examined by labelling the cells with Click Tag TM Palmitic Acid Alkyne, a 16-carbon saturated fatty acid group as described in our previously published article 15,31 . Briefly, palmitic acid (Alk-C16, Cayman Chemicals) was dissolved in DMSO to achieve final stock concentration of 20 mM.…”