PtdIns(4,5)P₂ is generated by a novel phosphatidylinositol 4‐phosphate 5‐kinase in the protist parasite Entamoeba histolytica

Abstract

Entamoeba histolytica is an intestinal protist parasite that causes amoebiasis, a major source of morbidity and mortality in developing countries. Phosphoinositides are involved in signalling systems that have a role in invasion and pathogenesis of this parasite. Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) catalyses the generation of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2), a key species of phosphoinositide that regulates various cellular processes. However, phosphatidylinositol phosphate kinase (PIPK) family of enzymes have not been characterized in E. histolytica. Here, we report the identification and characterization of type I PIPK (EhPIPKI) of E. histolytica. Computational analysis revealed homologs of type I and III PIPK family in E. histolytica and the absence of type II PIPK. In spite of low overall sequence identity, the kinase domain was found to be highly conserved. Interestingly, a unique insertion of a tandem repeat motif was observed in EhPIPKI distinguishing it from existing PIPKs of other organisms. Substrate profiling showed that EhPIPKI could phosphorylate at third and fifth hydroxyl positions of phosphatidylinositols, though the predominant substrate was phosphatidylinositol 4‐phosphate (PtdIns(4)P). Furthermore, EhPIPKI underwent intracellular cleavage close to the amino‐terminal, generating two distinct fragments Nter‐EhPIPKI (27p) and Cter‐EhPIPKI (47p). Immunofluorescence and cellular fractionation revealed that the full‐length EhPIPKI and the Cter‐EhPIPKI containing carboxyl‐terminal activation loop were present in the plasma membrane while the Nter‐EhPIPKI was observed in the cytosolic region. In conclusion, E. histolytica has a single EhPIPKI gene that displays novel properties of post‐translational processing, the presence of a repeat domain and substrate specificity not observed in any PIPK enzyme so far.

Lipids in Entamoeba histolytica: Host-Dependence and Virulence Factors

“…PIPK type I produces PI(3,4,5)P 3 from PI(4,5)P 2 . PI(4)P is produced from IP by the action of PI(4)K type III and also, PI can be phosphorylated to generate PI(3)P by class III PI3K (Sharma et al, 2019). Important advances in the lipid knowledge of E. histolytica constitute the findings focused on the inositol group.…”

Understanding anomalous mobility of proteins on SDS‐PAGE with special reference to the highly acidic extracellular domains of human E‐ and N‐cadherins

“…One reason for poor binding of SDS can be the recalcitrance of the protein to unfold under standard conditions of sample preparation for SDS-PAGE. A structurally ultra-stable protein derived from a hyperthermophile can fail to unfold within 2-3 min, or even hours, of boiling in standard SLB, or even under harsher conditions as reported for proteins from Pyrococcus furiosus [12][13][14][15]. (ii) High innate negative charge content.…”

“…Highly acidic proteins can be expected to repel SDS. In a recent series of papers examining the binding of SDS to proteins, it has been observed that the overall charged status of a protein is the primary determinant of the ease with which it can be approached by SDS molecules [1,13,16]. If the pH of a protein's environment is well below its pI, it protein possesses an overall positive charge, and is easily approached by SDS.…”

Novel regulatory roles of PtdIns(4,5)P2generating enzyme EhPIPKI in actin dynamics and phagocytosis ofEntamoeba histolytica

“…The role of PtdIns(4,5)P 2 metabolism has not been studied in relation to processes involving actin cytoskeleton dynamics in E. histolytica . Out of the two members of PIPK family, type I and II that synthesise PtdIns(4,5)P 2 , only type I PIPK (EhPIPKI) is found in E. histolytica and has been recently characterised biochemically (Sharma, Bhattacharya, & Bhattacharya, ). Though EhPIPKI phosphorylates PtdIns(4)P generating PtdIns(4,5)P 2 , it is quite distinct in biochemical properties from PIPKs of other organisms.…”

“…Though EhPIPKI phosphorylates PtdIns(4)P generating PtdIns(4,5)P 2 , it is quite distinct in biochemical properties from PIPKs of other organisms. EhPIPKI has high‐substrate specificity towards PtdIns(4)P, possesses a stretch of tandem repeat motifs in the central region of the kinase domain, and observed to undergo post‐translational cleavage in E. histolytica (Sharma et al, ) . Expression of EhPIPKI was found to be relatively high in phagocytic cell population as compared with the nonphagocytic population of cells (Sateriale et al, 2012).…”