Calcium-dependent phosphorylation of Plasmodium falciparum serine repeat antigen 5 triggers merozoite egress

Abstract

The human malaria parasite proliferates in red blood cells following repeated cycles of invasion, multiplication, and egress. serine repeat antigen 5 (PfSERA5), a putative serine protease, plays an important role in merozoite egress. However, regulation of its activity leading to merozoite egress is poorly understood. In this study, we show that PfSERA5 undergoes phosphorylation prior to merozoite egress. Immunoprecipitation of parasite lysates using anti-PfSERA5 serum followed by MS analysis identified calcium-dependent protein kinase 1 (PfCDPK1) as an interacting kinase. Association of PfSERA5 with PfCDPK1 was corroborated by co-sedimentation, co-immunoprecipitation, and co-immunolocalization analyses. Interestingly, PfCDPK1 phosphorylated PfSERA5 in the presence of Ca and enhanced its proteolytic activity. A PfCDPK1 inhibitor, purfalcamine, blocked the phosphorylation and activation of PfSERA5 both as well as in schizonts, which, in turn, blocked merozoite egress. Together, these results suggest that phosphorylation of PfSERA5 by PfCDPK1 following a rise in cytosolic Ca levels activates its proteolytic activity to trigger merozoite egress.

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

“…and Iyer, G. R. et al . 73,74 . After 1-hour incubation, slides were washed three times with PBS and incubated for 1 hour with Alexa 594-conjugated goat anti-rabbit IgG antibodies diluted 1:500 or Alexa-Fluor 488-conjugated goat anti-rat IgG antibodies diluted 1:200 and mounted with DAPI Antifade reagent (Molecular Devices, USA) for nuclear staining.…”

EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica

“…Immunoprecipitation of trophozoite lysates were performed with both immune and pre-immune control antibodies cross-linked with protein G/S sepharose beads [58, 59] as prescribed by the Pierce Crosslink IP-kit (Thermo Scientific). Briefly, the cells were lysed in lysis buffer (0.025M Tris, 0.15M NaCl, 0.001 M EDTA, 1% NP-40, 5% Glycerol, Protease Inhibitor cocktail (PIC, Roche), pH 7.4) and the lysates were precleared using the control Protein G Sepharose beads.…”

Structure of the Plasmodium falciparum PfSERA5 pseudo‐zymogen

“…This unresolved region corresponds to a site of in vivo phosphorylation, at Thr549. 28 Packing of the molecules within the ASU, and the absence of the 20 interconnecting residues between the prodomain and central domain in this crystal, allow the possibility of a domain-swapped dimer, with the prodomain of one monomer binding the central domain of a second, and vice versa. Specifically, the distance between the last visible C-terminal residue of the prodomain and the first visible N-terminal residue of the central domain could be spanned between domains of one zymogen or in trans across two zymogens ( Figure S1).…”

“…Although the most studied of the family, the exact function of PfSERA5 is still poorly understood with its proteolytic activity being a contentious issue. Despite the high sequence and structural similarity to catalytically active papain‐like proteases, the presence of a serine in place of the cysteine (Ser596) at the putative “catalytic triad” appears to render PfSERA5 catalytically inactive; evidence of weak activity had been observed in early studies, 27,28 although more recent studies report a complete lack of activity 29 . Serine‐containing papain‐like proteases are unique to that of the SERA family.…”