Complementary crosstalk between palmitoylation and phosphorylation events in MTIP regulates its role during Plasmodium falciparum invasion

Abstract

Post-translational modifications (PTMs) including phosphorylation and palmitoylation have emerged as crucial biomolecular events that govern many cellular processes including functioning of motility- and invasion-associated proteins during Plasmodium falciparum invasion. However, no study has ever focused on understanding the possibility of a crosstalk between these two molecular events and its direct impact on preinvasion- and invasion-associated protein-protein interaction (PPI) network-based molecular machinery. Here, we used an integrated in silico analysis to enrich two different catalogues of proteins: (i) the first group defines the cumulative pool of phosphorylated and palmitoylated proteins, and (ii) the second group represents a common set of proteins predicted to have both phosphorylation and palmitoylation. Subsequent PPI analysis identified an important protein cluster comprising myosin A tail interacting protein (MTIP) as one of the hub proteins of the glideosome motor complex in P. falciparum, predicted to have dual modification with the possibility of a crosstalk between the same. Our findings suggested that blocking palmitoylation led to reduced phosphorylation and blocking phosphorylation led to abrogated palmitoylation of MTIP. As a result of the crosstalk between these biomolecular events, MTIP's interaction with myosin A was found to be abrogated. Next, the crosstalk between phosphorylation and palmitoylation was confirmed at a global proteome level by click chemistry and the phenotypic effect of this crosstalk was observed via synergistic inhibition in P. falciparum invasion using checkerboard assay and isobologram method. Overall, our findings revealed, for the first time, an interdependence between two PTM types, their possible crosstalk, and its direct impact on MTIP-mediated invasion via glideosome assembly protein myosin A in P. falciparum. These insights can be exploited for futuristic drug discovery platforms targeting parasite molecular machinery for developing novel antimalarial therapeutics.

Keywords: crosstalk; malaria; myosin A tail interacting protein (MTIP); plasmodium falciparum; post-translational modifications.

Figure 1

A graphical summary.

Figure 2

(A) (I) A Venn diagram representing all the Modifiable proteins (inner subset; dark orange) in the proteome of Plasmodium falciparum 3D7 (outer subset; light blue). (II) A Venn diagram with a more detailed breakdown of the proteins processed or predicted to be processed by palmitoylation and phosphorylation (the red box encapsulates all the dually modifiable proteins) (B) Biological processes enriched by proteins grouped into each of the five k-means clusters of the dually modifiable proteins. (Cluster 1 represented by Red, cluster 2 represented by Yellow, cluster 3 represented by Green, cluster 4 represented by Cyan, and cluster 5 represented by Blue nodes).

Figure 3

(A) STRING-generated Protein–Protein Interaction graph of dually modifiable proteins in k-means cluster 4. The PPI map draws a relationship between the molecular factors of parasitic invasion (blue nodes) and glideosome-mediated motility (larger nodes) in P. falciparum 3D7, which are processed by a crosstalk of Palmitoylation and Phosphorylation. (B) A functional subnetwork of major glideosome and invasion motor complex-related proteins, which has been termed as the infection-associated Motility and Invasion Complex (MIC) (dotted box).

Figure 4

MTIP is an essential merozoite factor guiding parasite motility and erythrocytic invasion in hosts. (A) A Venn diagram showcasing the proteins which are dually modified by palmitoylation and phosphorylation and are essential for parasite survival. (Red fonts within the union of yellow and green ellipses; includes plasmepsin II, adenosine deaminase, calcium-dependent protein kinase 1 and myosin A-tail interacting protein). (B) (I) A STRING-generated PPI network of all the physical interactors constituting the glideosome motor complex. (Blue background) (II) An illustration of the actomyosin motor complex with MTIP as one of the central physical interactors. Revisualized from the published concepts of the glideosome motor unit (Bosch et al., 2006, Saunders et al., 2020). (C) A Protter generated illustration of MTIP showing the colocalization of phosphorylation and palmitoylation sites in the hotspots and MyoA interaction motif (Omasits et al., 2014, Anam et al., 2020).

Figure 5

(A) (I) A schematic representation of acyl biotin exchange analysis to detect palmitoylation of parasite protein coupled with immunoblot analysis. (II) P. falciparum 3D7 lysate blotted using mouse anti-MTIP antibody (1:20,000 dilution in phosphate-buffered saline 0.1% Tween 20). A single band at 24 kDa representing MTIP was observed. (III) The representative immunoblot displayed inputs (upper panel) representing MTIP expression levels in untreated lysates and lysates treated with ST092793 and 2-BMP in presence and absence of HA. Immunoblots represent the MTIP expression in the ABE pulled fraction with and without HA treatment (lower panel). Low intensity of MTIP expression was detected in ST092793 and 2-BMP treated ABE pulled-down fraction in comparison to control. (IV) Bar graph represents the fold change in MTIP band intensity before and after ABE pull down. Two independent experiments have been performed, n = 2. (B) (I) Schematic representation to analyze the dynamic interplay between phosphorylation and palmitoylation status of MTIP. Schizont stage parasite were treated with ST092793, BMP alone. After treatment parasite lysates were subjected to pull-down analysis using phospho-serine antibody, eluate fractions were then probed with anti-MTIP antibodies. (II) Immunoblot represents the input showing equal MTIP expression in untreated and ST092793- and 2-BMP treated samples. (III) In the presence of 50 µM 2-BMP, the phosphorylation of MTIP (measured by pull-down using phospho-Ser antibody followed by probing with anti-MTIP) was predominantly reduced (red box) as compared to MTIP in untreated lane. (IV) Bar graph represents the change in fold intensity before and after pull-down using phospho-Ser antibody. (C) (I) Schematic representation of workflow to analyze the MTIP interaction with MyoA tail. (II, III) The immunoblot showed the synergistic impact of dual PTM on MTIP crosstalk with myosin A tail, in the presence of 50 µM 2-BMP. There was no MTIP band following pull-down using biotinylated myosin A tail as the bait. The graph denotes fold change in intensity of MTIP in comparison to inputs taken before pull-down with myosin A tail in individual lanes. Two independent experiments have been performed.

Figure 6

(A) (I) Schematic of the click chemistry approach for imaging in situ protein palmitoylation in malaria parasite during asexual development. Intraerythrocytic parasites were metabolically labeled with 17-ODYA (palmitic-acid analogue) followed by labeling with Oregon Green 488 in the presence of TCEP and copper sulfate. (II) Clickable metabolic labeling of the P. falciparum parasites following ST092793, 2-BMP, and ST092793+2-BMP treatment. The weak palmitoylation profiles in comparison to untreated parasites, especially in the case of ST092793, were observed. Scale bar indicates 5 µm. (III) Bar graphs represent the mean fluorescence intensity (MFI) and denote the palmitoylation profile in treated and untreated erythrocytes, where 20 cells were used for calculation for two biological replicates. (B) (I) The heat plot of invasion inhibition in 3D7 in the presence of ST092793 and 2-BMP when present alone and in combination (ST092793 + 2-BMP). ST092793 was added horizontally in 96-well plates (0, 0.6, 1.2, 2.5, 5.0, 10, 20, 40 µM) while 2-BMP was added vertically (0, 0.6, 1.2, 2.5, 5.0, 10, 20, 40, 80 µM) in 8*8 format. Dose–response matrices from 0% to 100% indicate different percentages of invasion inhibition. (II) The isobologram analysis of 2-BMP and ST092793 which shows a synergistic effect when used in combination against the 3D7 strain of P. falciparum with FIC index of <1. (III) The dose–response curve for 2-BMP and ST092793 when used alone and in combination. (IV) Giemsa-stained smears from 2-BMP-, ST092793-, and 2BMP+ST092793-treated parasites. Arrowhead indicates the ring formation after successful invasion, while invasion defect was observed in the case of 2-BMP-, ST092793-, and 2BMP + ST092793-treated parasites.