Characterization ofPlasmodium falciparumNEDD8 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. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria 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 (Gly75Gly76 mutated to Ala75Ala76) 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 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

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“…80 µl of the eluate was run on a 10% SDS-PAGE gel until the protein ladder completely entered into the resolving gel. The protein band was excised, cut into small pieces, treated with trypsin, peptides were extracted, vacuum dried, and resuspended in 11 µl of 2% formic acid as has been described in detail previously (Bhattacharjee et al, 2020; Sudhakar et al, 2021). 10 µl of the peptide sample was run on the Q-Exactive HF (Thermo Fischer Scientific) for HCD mode fragmentation and LC-MS/MS analysis.…”

“…The resistant colonies along with the BY4741strain as a control were grown overnight in 5 ml of YPD medium at 25°C and 250 rpm. The cultures were harvested at 3000g for 5 minutes at 25°C, and the cell pellets were used for western blot analysis and gDNA isolation as has been described earlier (Bhattacharjee et al, 2020). The integration of transfection cassette was checked by PCR using primers specific for the complete cassette (Sc/PfDDi-5con, Sc/PfDDi-3con), 5’-integration (Sc/PfDDi-5con and PfDDIUbl-R) and 3’-integration (RVP/UBA-F and Sc/pfDDi 3con) loci.…”

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“…Neddylation is carried out by the NEDD8 protein which also functions as an E3 ubiquitin ligase, although neddylation is conserved among eukaryotes, little is known about this protein in Plasmodium and other apicomplexan parasites. Without a C-terminal tail, PfNEDD8 is a 76 amino acid residue protein, making it easily conjugatable ( Rabut and Peter, 2008 ; Bhattacharjee et al., 2020 ). Toxoplasma gondii and P. falciparum also have conserved regions of UCHL3, and PfUCHL3, which have ubiquitin and Nedd8-related activities.…”