Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

Abstract

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

The Skp1-Cullin1-FBXO1 complex is a pleiotropic regulator required for the formation of gametes and motile forms in Plasmodium berghei

“…By combining the FBXO1-AID/HA and the GD lines, we were able to identify at least three distinct windows of FBXO1 requirement: (i) towards the end of the last round of schizont nuclear division to form merozoites, (ii) before microgametocyte activation for microgamete development and egress and (iii) after macrogametocyte activation to form banana-shaped ookinetes. While we were unable to identify an orthologue of CAND1 that may have evolved beyond our limit of detection, Nedd8 was found enriched in RBX1-HA and SKP1-HA immunoprecipitates suggesting that neddylation is important to regulate SCF FBXO1 function in Plasmodium, as previously suggested 61 . Interestingly, we noticed that FBXO1 levels are dynamically regulated, providing another level of regulation.…”

Plasmodium falciparumcontains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity

“…It is likely that PfWD40A and PfWD40B serve as substrate receptors. Consistent with our previous report of the presence of PfCullin-2 in PfNEDD8 immunoprecipitates, the presence of PfNEDD8 in PfCullin-2/cDDHA immunoprecipitates indicates neddylation of PfCullin-2 [44].…”

“…PfRbx1 was also subcloned into pSMO at StuI/HindIII sites to obtain pSMO-PfRbx1 plasmid and transformed into Rosetta-gami 2(DE3) pLysS cells, which would express it with an N-terminal His-SUMO-tag (HisSUMO/Rbx). The pSMO vector has been reported earlier [44]. The recombinant HisSUMO/Rbx protein was purified from the soluble fraction of IPTGinduced cells by Ni-NTA affinity chromatography under native conditions as has been described earlier [44].…”

“…The pSMO vector has been reported earlier [44]. The recombinant HisSUMO/Rbx protein was purified from the soluble fraction of IPTGinduced cells by Ni-NTA affinity chromatography under native conditions as has been described earlier [44]. Elution fractions containing the pure protein were pooled and concentrated (10 kDa cut off Amicon Ultra centricon) with simultaneous buffer exchange to 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 at 4°C.…”

“…We have previously shown that the P. falciparum cullin homologs are conjugated to NEDD8, which marks the active state of a CRL, and thus suggested active CRLs in P. falciparum [44]. However, it remains to be investigated if Plasmodium cullins form functional complexes, and what are the core components and roles of Plasmodium CRLs during parasite development.…”