https://orcid.org/0000-0003-1164-1965
Figures
Abstract
The ubiquitin-proteasome system (UPS) is essential for Plasmodium falciparum survival and represents a potential target for antimalarial therapies. We utilised a ubiquitin- activity based probe (Ub-Dha) to capture active components of the ubiquitin conjugating machinery during asexual blood-stage development. Several E2 ubiquitin-conjugating enzymes, the E1 activating enzyme, and the HECT E3 ligase PfHEUL were identified and validated through in vitro ubiquitination assays. We also demonstrate selective functional interactions between PfHEUL and a subset of both human and P. falciparum E2s. Additionally, the Ub-Dha probe captured an uncharacterized protein, PF3D7_0811400 (C0H4U0) with no known homology to ubiquitin-pathway enzymes in other organisms. Through structural and biochemical analysis, we validate it as a novel E2 enzyme, capable of binding ubiquitin in a cysteine-specific manner. These findings contribute to our understanding of the P. falciparum UPS, identifying promising novel drug targets and highlighting the evolutionary uniqueness of the Ub-proteasome system in this parasite.
Author summary
Malaria parasites, like all living organisms, need to regulate their proteins carefully to survive and grow. One important way they do this is through a process called ubiquitination, where small tags (ubiquitin) are attached to proteins to control their function or mark them for disposal. This process requires several different enzymes working together like a production line. While we know this system is essential for malaria parasites, we don’t fully understand all the enzymes involved or how they work together. In this study, we used a special molecular tool to identify and study these enzymes in malaria parasites. We discovered how different enzymes in this system interact with each other and validated their functions. Importantly, we identified a completely new type of enzyme that had never been seen before in any organism. Since this ubiquitination system is essential for parasite survival and different from human enzymes, our findings provide new potential targets for antimalarial drug development. Understanding these differences between human and parasite enzymes could help us design drugs that kill the parasites without harming human cells.
Citation: Smith C, Hajisadeghian M, van Noort GJvdH, Deery MJ, Pinto-Fernández A, Kessler BM, et al. (2025) Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium. PLoS Pathog 21(4): e1013032. https://doi.org/10.1371/journal.ppat.1013032
Editor: Vasant Muralidharan, University of Georgia, UNITED STATES
Received: November 21, 2024; Accepted: March 11, 2025; Published: April 18, 2025
Copyright: © 2025 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD056473.
Funding: This work was supported by a Wellcome Trust Doctoral Training Program studentship held by CS and a MRC grant (MR/W025566/1) held by KAT. APF and BMK were supported by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China [grant number: 2018-I2M-2-002]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ubiquitin is a small (8.6 kDa) post-translational protein modifier that, when conjugated to proteins, modulates their activity, fate, and localisation within the cell. The machinery that mediates ubiquitination and deubiquitination of substrates is complex, highly regulated, and functionally conserved across eukaryotes. During the blood-stage of malaria infection, the Plasmodium parasite relies on ubiquitin-driven protein turnover to progress through its life cycle. This process ensures the timely removal of stage-specific proteins, which is essential for parasite development and survival. Additionally, ubiquitination and subsequent degradation of misfolded proteins have been shown to maintain homeostasis during heat shock induced by host fever spikes [1]. More recently, this modification has garnered increased attention due to its involvement in frontline drug resistance. Artemisinin causes oxidative damage to parasite proteins, whose clearance is dependent on the ubiquitin-proteasome system. As such, interference with the ubiquitin pathway by way of proteasome inhibition, augments artemisinin toxicity. Exploiting this synergy could be particularly useful in the context of resistance whereby co-administering a ubiquitin pathway inhibitor could re-establish the potency of artemisinin [2].
The groups of enzymes which constitute the ubiquitin proteasome system (UPS) possess specific conserved domain motifs and architectures that facilitate their function and allow for in silico identification from genomic sequences. Despite the substantial divergence between Apicomplexa and model eukaryotic organisms, there are recognisable homologues to all groups of ubiquitin-proteasome system (UPS) enzymes in Plasmodium, i.e., E1 activating, E2 conjugating, E3 ligating, and deubiquitinating (DUB) enzymes. Two activating enzymes have been identified in P. falciparum parasites, PF3D7_1225800 is found in the cytoplasm and PF3D7_1333200 in the apicoplast [3,4]. In addition, there are 12 putative E2 ubiquitin conjugating enzymes, 4 HECT E3 ligases, 2 types of cullin-RING ligases, 42 putative RING family E3 ligases, and 28 putative deubiquitinating enzymes [5]. Yet, there are approximately 1,500 proteins with unknown function and domain composition in the Plasmodium proteome and several hundred million years of evolutionary divergence between Plasmodium and model organisms. Therefore, there are likely to be highly divergent and novel enzymes with roles in the Plasmodium UPS that have not yet been identified.
Within the UPS, the points of entry (E1 ubiquitin activating enzyme) and exit (the proteasome and DUB enzymes) have been experimentally demonstrated to be essential for parasite survival [3,6,7]. However, the enzymes and processes between these events that confer specificity and regulation to this pathway through differential substrate recognition and ubiquitin chain formation have, to date, been largely overlooked as potential therapeutic targets. Given the greater sequence (and likely functional) divergence of E2 and E3 enzymes between Plasmodium and the human host, the identification and characterisation of these enzymes would provide novel targets and enable species-specific therapeutic intervention.
Activity-based probes (ABPs) are biochemical tools that have been developed to delineate component interactions and interdependencies within the UPS, allowing insight into the spatiotemporal organisation and dynamics of the ubiquitin network. ABPs consist of three parts, each performing a different function: a reactive group whose chemistry allows for covalent bond formation with the desired targets’ active sites in a physiological environment; a recognition element that drives the protein-protein interaction specificity with the desired target; and a reporter tag that allows for identification of the ABP-substrate conjugate [8].
Here, we interrogate the active ubiquitin machinery of asexual, erythrocytic stage Plasmodium falciparum using a biotin-Ub-Dha activity-based probe (ABP) (Fig 1A) for protein capture, enrichment, and identification by mass spectrometry. We identify the P. falciparum E1, several E2 enzymes, a HECT E3 ligase, and a highly enriched unknown protein. We validate these enzymes as bona fide UPS components and determine which E2s serve as functional partners of the identified HECT, PfHEUL, an enzyme that has been implicated in parasite virulence in mouse model systems [9]. We also characterise the unknown protein as a novel E2 enzyme.
A) Ub-Dha reaction scheme. Diagrammatic representation of the bipartite reaction mechanism of Ub-Dha within the ubiquitination cascade. Adenylation activates the latent alkene moiety of dehydroalanine as an electrophile through the activity of the E1 ubiquitin activating enzyme. Pathway 1 represents the nucleophilic attack of the alkene moiety of dehydroalanine that results in stable capture of nucleophile-containing, ubiquitin-binding enzymes via thioether bond formation. Pathway 2 represents the native cascade of E1 to E2 to E3 enzyme by subsequent transthioesterification reaction. Pathway 1 can occur at any stage of the ubiquitination cascade. B) P. falciparum proteins captured by Ub-Dha. Christmas tree plot of biotin-Ub-Dha-trapped proteins from P.falciparum lysate identified by LC/MS/MS. 3 technical repeats were analysed along with the negative control which utilised apyrase to catalyse the conversion of ATP to AMP and inorganic phosphate to effectuate the absence of ATP. The x-axis is fold enrichment and set to start at zero to present enriched hits, and the y-axis is the average iBAQ (intensity-based absolute quantification) values for three technical repeats. Hits highlighted in red are those with putative ubiquitin activity as well as the unknown, Pf3D7_0811400. C) Putative functions of Ub-related proteins captured. Ubiquitin-related proteins identified by LC-MS/MS using the biotin-Ub-Dha probe.
Results
A cascading Ub-derivatised probe captures Ub conjugation machinery in P. falciparum
The Ub-Dha probe demonstrates specificity to ubiquitination enzymes through its requirement for ATP-mediated activation by the E1 activating enzyme [10]. It achieves ubiquitination enzyme capture by either reacting natively through its C-terminal carboxyl group with the active site cysteine of a ubiquitination enzyme, or through its dehydroalanine group which forms an irreversible thioether-linked adduct. The stochasticity of these reactions results in a cascade whereby either the Ub moiety can transverse the ubiquitination machinery by successive transthiolation reactions or at any given step can covalently trap an enzyme by 1,4-addition (Fig 1A).
Mixed-stage asexual Plasmodium falciparum were saponin-extracted from their host erythrocytes, subjected to freeze-thaw lysis and clarified by centrifugation. The resultant lysate was divided between experimental and control samples. The negative control lysate was depleted of residual ATP by the addition of apyrase which catalyses the sequential hydrolysis of ATP to AMP and inorganic phosphate. This lysate was also not supplemented with ATP during the probe incubation period, unlike the experimental biotin-Ub-Dha reaction. The probe was incubated with control and experiment lysates supplemented with ATP to facilitate probe activation. Proteins of interest, now covalently conjugated to the probe were extracted from cell lysate using neutravidin resin and a series of harsh wash conditions. The eluate from this procedure was submitted for protein identification by mass spectrometry. Several ubiquitin-related enzymes were identified by the biotin-Ub-Dha probe: one E1 ubiquitin activating and six E2 conjugating enzymes were enriched compared to background (Fig 1B and 1C). This is in the context of the two known ubiquitin activating enzymes and the 12 predicted ubiquitin conjugating enzymes encoded by P. falciparum, although upon inspection, only 10 of these possess putative catalytic cysteines (S1 Fig). E3 ligases capable of reacting with the electrophilic probes, specifically the four annotated HECT E3 ligases, were not identified above background. However, the HECT E3 ligase PfHEUL was identified by one peptide in one of the technical repeats. A similar number of peptides derived from the Ub-Dha were identified in the ATP supplemented and ATP depleted control experiments, while almost no peptides derived from ubiquitinating enzymes in the control sample. This demonstrated the efficacy of the apyrase treatment in depleting residual ATP and facilitating the reduction of background noise.
P. falciparum E2 conjugating enzymes are active and can undergo autoubiquitination
Several functionally uncharacterised E2 enzymes were identified by activity-based probe; with their putative activity being inferred through their domain homology. PF3D7_0527100, PF3D7_092100, PF3D7_103390, PF3D7_1203900, and PF3D7_1356300 were recombinantly expressed in E.coli with an N-terminal His-tag, then purified by immobilised metal affinity chromatography (IMAC) using nickel-affinity resin followed by size exclusion chromatography (SEC). The Plasmodium E1 activating enzyme was also recombinantly expressed in E.coli using the codon-optimised plasmid kindly donated by the Holder lab and purified as described in [3]. A sixth putative E2 enzyme, PF3D7_0812600, was not included in this panel owing to difficulties in PCR amplification.
Assessment of the E1 and E2 enzyme purity was performed using Coomassie-stained SDS-PAGE (Fig 2A) and by mass spectrometry (S1 Table) to validate absence of E. coli contaminants with ubiquitin activity. For all enzymes expressed and purified, the most prominent band was present at the predicted mass of the representative protein. In the case of PfE1 (PF3D7_1225800), several lower molecular weight bands were observed despite the protein having been exposed to affinity and size exclusion. These bands may represent degradation products.
A) Protein expression and purification. Coomassie stained SDS-PAGE gel of purified proteins: E1 activating enzyme (PF3D7_1225800), E2 conjugating enzymes (PF3D7_0527100, PF3D7_0921000, PF3D7_1033900, PF3D7_1203900, and PF3D7_1356300). B) In vitro autoubiquitination by P.falciparum E2 enzymes. Western blot of ubiquitination reaction catalysed by select PfE2 enzymes, visualised by anti-HA antibody. PfE2 enzymes were incubated with ATP, PfE1, and HA-Ub. The products of the reaction were resolved and visualised to reveal signature ubiquitination patterns formed by the unique activity of the different E2 enzymes.
Functional validation of the E2 enzymes was achieved through in vitro ubiquitination assay in the presence of ATP, HA-tagged ubiquitin, and the Plasmodium ubiquitin activating enzyme [3]. The banding pattern resulting from protein conjugation was resolved by SDS-PAGE and visualised by western blotting using an anti-HA antibody (Fig 2B). The presence of a band at the molecular weight of each E2 enzyme in addition to one or more ubiquitin molecules validated their activity.
Three of the PfE2 enzymes tested (PF3D7_092100, PF3D7_1033900, and PF3D7_1356300) exhibited a clear banding pattern consistent with the conjugation of several ubiquitin proteins, in the absence of an E3 ligase. This phenomenon has been observed with characterised human E2 enzymes [11–13]. This banding pattern can arise through the multi- monoubiquitination of the E2 enzymes, the extension of a monoubiquitinated site to a polyubiquitin chain, or a combination of both. PF3D7_092100, which demonstrated the most distinct ubiquitin conjugate pattern, possesses only 8 lysine residues, and therefore the >10 bands visible at approximate 10 kDa distance must include polyubiquitin chain linkages. Although, the same is not necessarily true for PF3D7_1033900 and PF3D7_1356300 which contain 14 and 20 lysine residues, respectively. A band consistent with free di-ubiquitin (approx. 17 kDa) was also observed except in the case of PF3D7_0527100. This is suggestive of the formation of a polyubiquitin chain linkage at the catalytic cysteine of the PfE2 enzymes which was subsequently released. Conjugation to PF3D7_0527100 was limited to two bands of sizes amounting to approximately the enzyme plus 1 and 2 ubiquitin modifiers, which may indicate the inability of this enzyme to undergo polyubiquitin chain formation. E2 activity was also confirmed to be dependent on the presence of the E1 (S2 Fig).
PfHEUL HECT domain functions with a subset of human and P. falciparum E2 enzymes
The essential ligase PfHEUL was also captured as part of the Ub-Dha screen, albeit by the identification of a single peptide, but demonstrating its presence and functionality during the asexual life cycle stage of P. falciparum. As this enzyme has been implicated in parasite virulence in the mouse model, P. yoelii [9],we were interested in characterising it further in P. falciparum. The sequence of this protein was submitted to bioinformatic analysis by HMMER and BLAST to investigate its function through domain architecture and identification of homologues in model organisms. Owing to the large size of PfHEUL (8591 amino acids, approximately 1 MDa), HMM searches were performed on the N-terminal and C-terminal halves separately. The C-terminal search identified the HECT domain, while the N-terminal search identified a domain of unknown function 908 (DUF908) between residues 176–478. Known DUF908 and HECT domain containing proteins include the homologous HUWE1 E3 ligase (a.k.a. MULE, ARF- BP1, Lasu1, HECTH9) in humans, UPL1/2 in plants, and TOM1 in yeast. Indeed, a BLAST search of PfHEUL returned human HUWE1, A.thaliana UPL1, and S.cerevisiae TOM1 as the top hits. Sequence alignment of these homologues revealed specific conservation of the HECT domain, sharing approximately 50% identity, while upstream there was no discernible sequence similarity (Fig 3A).
A) Comparison of PfHEUL to orthologs in model organisms. Cartoon alignment of PfHEUL with human (Q7Z6Z7), S.cerevisiae (Q03280) and A.thaliana (Q8GY23) orthologs showing conservation of HECT and DUF908 domains. B) In vitro autoubiquitination of PfHEUL HECT domain using human E2 enzymes. Anti-HA western blot representing the in vitro autoubiquitination assay used to validate the activity of the PfHEUL HECT domain using human HA-tagged ubiquitin, E1 activating (UBA1), and E2 conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3). All E2 enzymes except UBE2E1 were capable of functioning in concert with PfHEUL HECT. C) In vitro autoubiquitination of PfHEUL HECT domain using P.falciparum E2 enzymes. Western blots of the in vitro autoubiquitination assay using P. falciparum ubiquitination components: E1 activating enzyme (PF3D7_1225800), indicated E2 conjugating enzymes, and the PfHEUL HECT domain. (Left) Western blot using anti-ubiquitin antibodies to identify ubiquitin conjugated proteins following the ubiquitination reaction. (Right) Western blot using mouse derived PfHEUL HECT antibodies to specifically identify PfHEUL HECT in its native and ubiquitinated states.
To validate the activity and thus function of PfHEUL, the HECT domain comprising residues 8211–8591 was tagged with a carboxy-terminal His-tag and recombinantly expressed in E. coli (S3 Fig). The activity of HECT domains involves the binding of a ubiquitin-loaded E2 conjugating enzyme at the N-lobe of the domain and the subsequent transthiolation reaction at the active site on the C-lobe, where the ubiquitin in transferred from the E2 enzyme onto the ligase cysteine side chain. In vitro and without protein substrate, HECT domains exhibit auto-ubiquitination on their lysine side chains when incubated with upstream components of the ubiquitination cascade [14]. To assess the catalytic function of the HECT domain of PfHEUL, an in vitro ubiquitination assay was performed using commercially available human E1 activating and E2 conjugating components. A panel of five human E2 enzymes was selected based on homology to Plasmodium E2 enzymes, characterised promiscuity, and propensity to act as cognate E2 enzymes for other HECT domain proteins. Human E1 and E2 enzymes were mixed with PfHEUL HECT as indicated and incubated at 25 C for 1 hour, in the presence of ATP. Following co-incubation, SDS-PAGE and western blot showed human UBE2D1, UBE2D2, UBE2D3, and UBE2L3 transferred ubiquitin to the PfHEUL (PF3D7_0826100) HECT domain. This was evidenced by banding patterns of approximate 10 kDa spacing which represented the conjugation of multiple ubiquitin proteins onto the HECT domain itself (Fig 3B). UBE2E1 did not exhibit this pattern of HECT domain autoubiquitination demonstrating that this E2 enzyme does not function in concert with the PfHEUL HECT domain.
The panel of P. falciparum E2 enzymes (Fig 2) were also tested for PfHEUL compatibility. Both anti-Ub and anti-PfHEUL HECT antibodies were used to distinguish between the ubiquitin-conjugated E2 and E3 enzymes, as autoubiquitination of the E2 enzymes was previously observed. In combination, the blots reveal selective compatibility of the Plasmodium enzymes (Fig 3C). Only PF3D7_1356300 was capable of functioning with PfHEUL HECT to bring about polyubiquitination of the HECT domain. The other E2 enzymes only appear to facilitate transfer of a single ubiquitin protein to PfHEUL HECT with progressively diminishing efficiency by PF3D7_1356300, PF3D7_1033900, PF3D7_0527100, PF3D7_0921000, and least of all PF3D7_1203900.
Identification of C8558 as the active site cysteine of PfHEUL
To further characterise the enzymatic activity of the PfHEUL HECT domain, a C>A mutant was generated (corresponding to C558A in the full length PfHEUL), predicted to correspond to the catalytic cysteine residue of HUWE1 by sequence homology (Fig 4A and 4B). The expression and purification procedures for this mutant protein were identical to the wild type protein. Circular dichroism (CD) was used to assess the folded state of the mutant protein relative to the wild type. The acquired spectra were averaged across five measurements, and the resultant curve was smoothed and normalised by protein concentration. The CD spectra of both the wild-type PfHEUL HECT domain and the C8558A mutant were almost identical, indicating the mutation did not affect the HECT domain fold (Fig 4C). The spectral minima and maxima also give insight into the secondary structure of the protein. The generated spectra were submitted to the BeStSel deconvolution algorithm [15] which returned a predicted secondary structure composition of 66% α-helical and 33% β-sheet which is consistent with the mixed secondary structure of helices and sheets in the AlphaFold predicted model (Fig 4A). This result indicates both protein forms are folded, adapt a similar secondary structure, and that the C8558A mutation does not affect the overall secondary structure of the HECT domain.
A) Analysis of PfHEUL HECT domain and catalytic cysteine. AlphaFold predicted structure of PfHEUL HECT domain (residues 8211-8591) (green) overlaid with crystal structure human HUWE1 (PDB:7JQ9) (blue) and with catalytic cysteines marked by the red arrow. B) Alignment of PfHEUL and human HUWE1 HECT domains. Rendered in ESPript with catalytic cysteine demarcated by the red arrow. C) Circular Dichroism spectra of wild type (WT) and C8558A mutant PHEUL HECT recombinant protein. D) In vitro ubiquitination assay. Anti-HA western blot of an in vitro ubiquitination assay using wild type and C8558A mutant PfHEUL HECT domains to demonstrate the contribution of C8558 to the transthiolation of ubiquitin, and subsequent ubiquitination of lysine side chains.
We next sought to assess the contribution of the putative active site cysteine to transthiolation and ubiquitin transfer using the in vitro assay with human E1 and E2 components. UBE2D1 was used as the cognate E2 conjugating enzyme in this assay as it previously exhibited the clearest ubiquitination banding pattern by western blot in the panel of E2 conjugating enzymes tested. When introduced into the in vitro ubiquitination assay using human E1 and E2 components, the autoubiquitination activity of the C8558A mutant was almost completely abolished compared to the wild type domain (Fig 4D). However, there were 2 bands present at the approximate mass of the HECT domain that are possibly the result of a single transthiolation reaction at a neighbouring non-active site cysteine [16].
Identification Pf3D7_0811400, a novel E2 enzyme
An uncharacterised protein (PF3D7_0811400) was highly enriched relative to background, which suggests this protein is capable of binding ubiquitin or its enzymatic-adducts (e.g., E2 ̃Ub) and possesses a sufficiently reactive cysteine side chain permissive to nucleophilic attack by the Dha moiety. No functional domains were identified from the primary sequence of this protein by HMMER search, rather it contains a single DUF2009 (Domain of Unknown Function) domain between residues 118–608.
Analysing the predicted 3D structure of Pf3D7_0811400 revealed two distinct domains. The N-terminal domain (amino acids 1–88) includes five short α-helices (h1-h5, Fig 5) and is connected via a 17-amino-acid linker to the C-terminal DUF2009 domain. We performed a 3D structure search of Pf3D7_0811400 using Foldseek [17] against various databases, including experimental protein crystal structures (PDB) and prediction-based structures (AlphaFold). The structure matched several uncharacterized proteins from other organisms, specifically putative adenylosuccinate lyase (ADSL) in Toxoplasma gondii and an unidentified B-box containing protein in a few organisms such as Synchytrium microbalum and Rhizophagus irregularis (S2 Table). Since many E3 ligases contain B-box domains, we examined this further.
A) Per-residue model confidence scores (pLDDT) ranging from 0 to 100, as predicted by AlphaFold, are color-coded. The overall prediction demonstrates relatively high confidence, with the DUF2009 domain exhibiting the highest confidence scores, indicated by dark blue. In contrast, the N-terminal first alpha-helix and linker residues (low-complexity regions) show the lowest confidence scores, represented by orange. B) The N-terminal α-helices are coloured dark green, while the C-terminal DUF2009 domain is shown in sea green.
A typical B-box domain contains histidine and cysteine residues that coordinate with zinc. Two types of B-boxes exist: B-box1, which spans 50–60 amino acids with a zinc-binding consensus sequence of C-X₂-C-X₇–₁₂C-X₂-C-X₄-C-X₂-[C/H]-X₃–₄-H-X₄–₉-H [C₅(C/H)H₂], and B-box2, which spans 35–45 amino acids with a consensus sequence of C-X₂-H-X₇–₉-C-X₂-[C/D/E]-X₄-C-X₂-C-X₃–₆-H-X₂–₄-[C/H] [CHC(C/D/E)C₂H(C/H)] [18]. Although the DUF2009 domain of PF3D7_0811400 is structurally conserved, it does not possess the typical B-box structure or signature motifs (S4 Fig).
Pf3D7_0811400 contains 13 cysteine residues within its 608-amino-acid sequence. None of these cysteines are located in the N-terminal region, which led us to hypothesise that the N-terminus may be involved in protein-protein interactions, potentially with E3 ligases, while the C-terminus interacts with ubiquitin. Conservation analysis was conducted using a ConSurf model, which estimates the evolutionary conservation of amino acid positions based on phylogenetic relationships among homologous sequences [19]. This analysis revealed that the highest conservation levels are found in the C-terminus of Pf3D7_0811400, particularly around two cysteine residues, C572 and C584. C572 is 100% conserved across all homologues, while C584 shows 95% conservation (S5 Fig). Structural analysis indicated that the thiol side chain of C572 is located in a loop at the base of a conserved and accessible pocket, whereas the side chain of C584 is buried between two α-helices. Protein-protein interaction modelling using AlphaFold suggested that ubiquitin interacts with C572 of PF3D7_0811400 (Fig 6).
All 13 cysteine residues in Pf3D7_0811400 are labelled and highlighted in green. The C-terminal glycine (Gly76) of ubiquitin interacts with C572, which acts as the catalytic cysteine in the core of the catalytic cleft.
The N-terminus of Pf3D7_0811400 is critical for interacting with PfRbx1
Despite its dissimilarity to other E2 and E3 ubiquitin enzymes, PF3D7_0811400 was a particularly robust hit in the mass spectrometry screen. As such, we suspected it to be an E2 Ub conjugating enzyme and set out to test this hypothesis and to understand which domain is essential for its activity. To start with, the five N-terminal α-helices (amino acids 1–88), as predicted by InterPro, were truncated to investigate their role in protein-protein interactions (Fig 7A). HEK293 cells were transfected with either an empty vector, FLAG-tagged PF3D7_0811400, or FLAG-tagged PF3D7_0811400ΔN, an N-terminal truncation missing the first 88 amino acids. Whole cell lysates were subjected to probe labelling by adding biotin-Ub-Dha in the presence of ATP and the human E1 enzyme. The proteins were then co-immunopurified using anti-FLAG magnetic resin and eluted with Laemmli buffer. Western blot analysis with HRP-conjugated streptavidin showed that the biotinylated probe co-immunopurified with both the wild-type and truncated proteins (Fig 7B, top panel). Anti-FLAG immunoblotting confirmed an approximate 10 kDa shift in protein size in the presence of the probe (Fig 7B, bottom panel), indicating that the N-terminal domain is not required for ubiquitin loading Since PF3D7_0811400 was also captured in a PfCullin-2 pulldown we had previously performed [20] and since many E2 enzymes interact with Rbx1 as part of Cullin RING ligase (CRL) complexes, we set out to test this interaction. Myc-tagged PfRbx1 was co-expressed with either FLAG-tagged full-length PF3D7_0811400 or the N-terminal truncation mutant. PfFBXO9 in the presence of PfSkp1 or empty vector were included as positive and negative controls, respectively. Anti-FLAG beads were used to immunopurify all samples and proteins were visualised by anti-FLAG or anti-myc immunoblot. As shown in Fig 7C, PF3D7_0811400 does co-precipitate with PfRbx1, and its N-terminus appears to be critical to mediating this interaction, further supporting its characterisation as an E2.
A) Schematic of Pf3D7_0811400. Domains and truncations used to generate protein for labelling and interaction studies are shown. Five predicted N-terminal α-helices (amino acids 1–88) were truncated to investigate their role in protein-protein interactions. Depicted domains include: FLAG (gray dotted), coils (wide stripes), 17 amino acid linker (white), DUF2009 (narrow stripes). B) Validation of Pf3D7_0811400 interaction with Ub-Dha probe. HEK293T cells were transfected with either an empty vector, FLAG-tagged Pf3D7_0811400, or FLAG-tagged Pf3D7_0811400ΔN. Whole cell lysates were labelled with biotin-Ub-Dha in the presence of ATP and additional E1 enzyme. The proteins were then co-immunopurified on anti-FLAG resin. Proteins were visualised by immunoblot with HRP-conjugated streptavidin (top panel) or Anti-FLAG (bottom panel) to confirm a ~10 kDa shift in protein size in the presence of the probe. C) Pf3D7_0811400 interaction with PfRbx1 via its N-terminal domain. Myc-tagged PfRbx1 was co-expressed with either FLAG-tagged Pf3D7_0811400 WT or the ΔN truncated mutant in HEK293T cells. FLAG-tagged PfFBXO9 in the presence of PfSkp1 was used as a positive control, while an empty vector (EV) in the presence of both PfRbx1 and PfSkp1 served as a negative control. Immunopurification using anti-FLAG beads was performed on all samples and proteins were visualised by anti-myc or anti-FLAG. WCE=whole cell extract. Images of full membranes are included in S6 Fig.
C-terminal end of Pf3D7_0811400 mediates binding to ubiquitin
Since the N-terminus of PF3D7_0811400 was found not to be essential for its interaction with the Ub-Dha probe, we next focused on its C-terminus, which contains the DUF2009 domain and the putative catalytic cysteine C572 as discussed above. It’s been demonstrated that replacing the catalytic cysteine with alanine in some ubiquitin-interacting enzymes, like certain DUBs, can significantly increase their affinity for ubiquitin. However, substituting the cysteine with arginine not only inactivates the enzyme but also prevents ubiquitin binding due to the larger side chain of arginine [21]. We used a C-to-R mutant to repeat the probe-interaction experiment in the presence or absence of ATP. While the wild-type, full-length protein bound the biotin-Ub-Dha probe, the C572R mutation prevents this interaction as demonstrated by both streptavidin and anti-FLAG immunoblots (Fig 8A). To confirm that this interaction is indeed mediated by a direct binding to ubiquitin via the C572R cysteine residue, we repeated the assay but this time used HA-Ub in place of the biotin-Ub-Dha probe. Again, in the presence of ATP, wild-type PF3D7_0811400 was able to bind ubiquitin whereas the C572R enzyme was not (Fig 8B, left panel). Notably, the reaction with the wild-type protein did not result in a ladder as seen in the positive control reactions we ran in parallel using the P. falciparum PF3D7_0921000 E2 and the human E2, UB2R1 (Fig 8B, right panel). Notably, while all identified E2 enzymes share a conserved HPN motif in addition to the catalytic cysteine, this motif is absent in PF3D7_0811400, suggesting a different mechanism of action.
A) Biotin-Ub-Dha-reactivity of wild type and C572R proteins. Wild-type and C572R mutant proteins were expressed in HEK293T cells, purified using anti-FLAG beads and eluted by incubation with a 3XFLAG peptide solution. Purified proteins were subjected to probe labelling in the presence or absence of ATP. (Images of full membranes are included in S7 Fig). B) Ubiquitin-HA-reactivity of wild type and C572R proteins. Purified FLAG-tagged Pf3D7_0811400 or the C572R mutant was used in an in vitro auto-ubiquitination assay. HA-tagged ubiquitin was added to allow detection by anti-HA immunoblot (left panel). The arrow highlights the weak and transient, but highly specific, formation of a thioester-linked E2 ubiquitin (E2~Ub) conjugate in the wild-type E2. PF3D7_0921000, a Plasmodium E2 characterised earlier in this paper, and UB2R1, a human E2, were used as positive controls (right label). C) Comparison of Pf3D7_0811400 with other P. falciparum E2 enzymes. Sequence alignment showing the conserved catalytic HPN motif in all of the described Pf E2 enzymes but not in Pf3D7_0811400.
Exploration of orthology between canonical Plasmodium and human E2s
To classify and name the E2 enzymes characterised in this study, we employed three approaches based on ortholog identification and evolutionary relationships. First, we performed amino acid sequence alignment with model organisms using both the UniProt BLAST tool and NCBI BlastP. Additionally, we utilised the OrthoDB tool [22] to examine evolutionary and functional annotations of orthologs from available genomic data and sequence alignment metrics. While sequence-based homology inference is often successful, many proteins remain unannotated due to the difficulty of detecting distant evolutionary relationships through sequence analysis alone [23]. To address this we conducted a 3D structural search of Pf3D7_0811400 using the Foldseek tool, which offers greater sensitivity for identifying homologous proteins.
As previously mentioned, all the canonical E2s characterised in this study exhibit typical features, including a core catalytic domain (UBC domain) (Fig 9A). This domain adopts an α/β-fold, generally composed of four α-helices and a four-stranded β-meander. Canonical E2s are categorised based on the presence of additional extensions beyond the catalytic core: some E2s consist only of the catalytic domain (Class I), while others have additional N- or C-terminal extensions (Classes II and III, respectively), or both (Class IV) [24]. Based on 3D structural analysis and amino acid sequence alignment, the Plasmodium falciparum E2s identified as part of our study are classified as Class I and Class III, and we have assigned names based on their human homologs (Fig 9B).
A) 3D alignment of the identified PfE2 ligases, illustrating the conserved structure of the catalytic domain typical of E2 enzymes. Each E2 is colour-coded. For PF3D7_0527100 and PF3D7_1033900 solved crystal structures are shown (PDB ID: 2R0J and 2ONU respectively) and for the other E2s structures were modelled. The HPN motif is highlighted in yellow and the catalytic cysteine is shown in phosphor green. B) Left: Schematic representation of E2 enzyme classes based on the presence or absence of extensions. The UBC fold is depicted as a convex black line, and extensions are shown as straight lines. Right: PfE2s are colour-coded according to the class they belong to from the left schematic.
Evolution and specific signatures of Pf3D7_0811400
We performed additional analyses to explore similarities with other DUF2009 containing proteins in other organisms. A search for gene sequence and domain similarities using ORthoDB and NCBI, identified homology to lyases. These enzymes form a large family with essential functions and conserved catalytic structures across eukaryotes. The adenylosuccinate lyase (ADSL) superfamily is a well-characterised member of this group that catalyses the conversion of adenylosuccinate to AMP and fumarate as part of the purine nucleotide cycle.
Despite a low overall sequence identity (~15–30%) among ADSL superfamily members, three conserved regions (C1–C3) define these enzymes, with C3 typically containing the signature sequence GSSxxPxKxN [25]. When comparing PF3D7_0811400 to typical ADSL enzymes, this specific signature was absent. A low level of similarity was observed in amino acids 483–512 (GSSMIHMGDTNIPNTFMFIDKYTQVPRILN), possibly explaining this protein’s annotation as an ADSL superfamily member (S8 Fig). However, the absence of the catalytic regions would suggest that this protein does not retain lyase function. Moreover, given the presence of a canonical ADSL enzyme (PF3D7_0206700) in the Plasmodium genome, it is unlikely that PF3D7_0811400 possesses the same adenylosuccinate lyase activity.
To explore structural similarity in more depth, we aligned the top 128 proteins from our previous Foldseek search (S1 Table) revealing the conservation of several residues around the catalytic cysteine (FDGSGX₇GSCIDGRLTS)(S8 Fig). This unique signature may define a new family of noncanonical E2 enzyme, something we are currently exploring through structural and mutagenesis studies to determine the role these residues may play in the enzyme’s activity.
Discussion
The ubiquitin landscape of Plasmodium has been predicted in silico, but biochemical exploration is limited [5,20,26–29]. A profile of the ubiquitin pathway acting at the pathology-causing stage of P. falciparum is a useful and important platform from which to initiate programmes of study and screening, using the ubiquitin enzymes identified as viable therapeutic targets. Previous work has demonstrated the functional activity of the E1 activating enzyme in vitro and in vivo [3] and the E2 enzyme PF3D7_1203900 in vitro [26]. The E2 enzyme PF3D7_0527100 was also shown to be active in vitro and negatively regulated through S106 phosphorylation by PfPK9 [30], with subsequent work demonstrating its involvement in K63 polyubiquitin linkage formation in vivo [31]. Several structures of Plasmodium E2 ubiquitination enzymes have been solved as part of larger structural elucidation programmes [32], including: PF3D7_0527100 (2R0J) in complex with PF3D7_0305700 (2Q0V; 3E95), PF3D7_1033900 (2ONU), PF3D7_1345500 (2H2Y), and the P.vivax homologue (2FO3). Together, the structural data and the functional validation presented in this study provide a basis for structure-guided drug design.
One putative E3 ligase (PF3D7_0826100 PfHEUL) was identified using ABPs in this study, albeit with low confidence - although its presence during asexual development is supported by transcriptomic data [33]. There may be other factors limiting the Ub-Dha probe reactivity to these enzymes and their subsequent detection. Protein levels may be transient and low abundance making peptide detection by mass spectrometry challenging. The cascading mechanism of Ub-Dha leads to attrition of the ubiquitin machinery following capture of enzymes at each stage. Thus, the capture of E3 ligase may happen at levels below the detection limit of this approach.
There are a total of 4 HECT domain-containing E3 ligases annotated in the Plasmodium falciparum genome. Of the four, only PfHEUL was found to be essential by piggyBac mutagenesis in P. falciparum [34]. Based on the structure of distant homologues in humans and fungi, PfHEUL may adopt a large solenoid structure where the HECT domain catalyses ubiquitin ligation to specific substrates recruited by the extensive protein binding modules upstream of the catalytic domain [35,36]. Mutations in this enzyme have been linked to pyrimethamine resistance, however the mechanism has not been investigated. The position of these mutations were found upstream of the HECT domain and likely alter protein recruitment as opposed to ubiquitin transfer [37,38]. This class of E3 ubiquitin ligases has also proven to be tractable and specific targets for small molecule inhibition, with several sites of inhibition identified across the HECT domain [39,40]. Together, these factors suggest PfHEUL would be a suitable target for antimalarial therapeutic intervention.
PfHEUL is one of the largest proteins in the P.falciparum proteome. At 1 MDa, direct study of the protein in its entirety is problematic owing to issues arising in cloning and recombinant protein production. To circumvent these obstacles the approximately 45 kDa catalytic HECT domain was examined in isolation. The sequence of the HECT domain of this protein is highly conserved across Plasmodium species, indicating the functional importance this catalytic domain plays within this E3 ligase and across Plasmodium biology. The protein sequence upstream of the HECT domain is less conserved, which suggests species-specific variation with regards to substrate recruitment.
Within the activity assay, all tested E2 enzymes were capable of conjugating ubiquitin, although unique polyubiquitination patterns were observed most clearly with PF3D7_092100, PF3D7_103390, and PF3D7_1356300. E2 enzymes have been demonstrated to catalyse auto(poly)ubiquitination in the absence of E3 enzymes, however, the physiological relevance of this is unclear [13]. It may represent the ability of these E2 enzymes to form polyubiquitin chains at their active site which they could transfer en masse in vivo, as opposed to a model where monoubiquitination of a substrate lysine primes the protein for successive ubiquitin chain extension by single ubiquitin proteins.
PF3D7_0527100 (PfUBC13) and its homologues are associated with K63 polyubiquitination, although no extensive polyubiquitination pattern was produced by this enzyme in the in vitro assay [31]. There are ubiquitin conjugating enzyme variants (UEVs) that lack the active site cysteine and are thus non-catalytic; homologues of PfUBC13 have been found to associate with UEVs to modulate their E2 activity and linkage specificity [41]. There are two examples of these UEVs in Plasmodium: PF3D7_0305700 (PfMMS13) and PF3D7_1243700. As mentioned, the co-structure of PfUBC13:PfMMS2 has been solved, demonstrating a stable interaction. The formation of this complex may be required for ubiquitin polymerisation (via K63).
Studies of the Plasmodium falciparum protein interactome have shown physical association of PfMMS2 with several E2 enzymes: ubiquitin-conjugating enzymes PF3D7_0527100, PF3D7_092100, PF3D7_1203900, PF3D7_1356300, as well as SUMO- conjugating enzyme PF3D7_0915100, and NEDD8-conjugating enzyme PF3D7_1245300 [42]. Future studies of the utility of these accessory E2 enzymes in concert with catalytic E2 enzymes may reveal functional modulation relative to the E2 enzyme alone, expanding the Plasmodium ubiquitin and ubiquitin- like small modifier landscape.
The E2 superfamily, classified into 17 families in eukaryotes, typically shares a conserved 150-amino-acid core known as the Ubiquitin Conjugation (UBC) domain, which includes a catalytic cysteine essential for thioester bond formation with ubiquitin. In catalytically active E2s, the active site generally contains an acidic residue, usually aspartate, or a phosphorylatable serine, known as the conserved E2 serine/aspartate (CES/D) site. Together with the catalytic cysteine and the His-Pro-Asn (HPN) motif, this CES/D motif forms a defining fingerprint of the E2 superfamily, crucial to their role in the ubiquitination cascade [43]. For instance, in the human E2, Ube2I, key residues such as Asn85 of the HPN motif, along with Tyr87 and Asp127, are essential for lowering the pKa of substrate lysine, facilitating its nucleophilic attack on the E2Ub thioester bond [44]. While active E2s are regulated by phosphorylation at their catalytic site, inactive members, like UEVs, lack this conserved catalytic cysteine. However, families 5 and 13, classified as Noncanonical Ubiquitin Conjugating Enzymes (NCUBE), show unique structural reorganisations around the catalytic cleft and do not consistently maintain the HPN motif [45].
In our study, we identified a novel active E2 enzyme that lacks the HPN motif, the CES/D site, and other typical E2 features, making it unlike any E2 characterised to date. With a molecular weight of ~71 kDa and a DUF2009 domain, this enzyme may represent a new NCUBE family member. Although the presence of a catalytic cysteine has been reported in a few DUF containing proteins such as DUF62 [46], its presence within the DUF2009 domain is unprecedented and could provide new insights into E2 enzyme function, suggesting that similar E2s may exist in other organisms. This discovery also marks the first identification of an entire active site within a DUF2009 domain, opening avenues for further study of this protein family. From an evolutionary perspective, the high similarity of Pf3D7_0811400’s DUF2009 domain in many B-box proteins, particularly in certain fungal parasites, suggests that the active site within this domain has been evolutionarily conserved. In contrast, the protein-protein interaction site at the N-terminus appears to have undergone significant evolutionary shifts. It has likely transitioned from a metal-binding B-box domain to coiled helices as an interaction interface, adapting the protein’s function to meet the specific requirements of the parasite under natural selection pressures. This highlights the evolutionary flexibility of E2 enzymes, enabling them to respond to environmental and functional demands. Of note, this motif is present in a variety of species, including fungi, oomycetes, apicomplexa, green, red, and brown algae, and even a predicted B-box protein in flowering plants (F2E0E1_HORVV) suggesting an evolutionary branching event.We have also previously captured this protein in a co-immunoprecipitation experiment with P. falciparum Cullin-4 and Rbx-1 [20], suggesting it may function in association with Cul4 CRLs.
Characterizing the ubiquitin pathway of P. falciparum is essential for developing innovative therapeutic strategies, such as Targeted Protein Degradation (TPD). TPD leverages the cell’s proteolytic systems to selectively degrade disease-causing proteins, offering advantages over traditional inhibitors [47]. Among these strategies, PROteolysis TArgeting Chimeras (PROTACs) have emerged as promising tools to induce ubiquitination and degradation of target proteins [48]. While PROTACs could address challenges in antiparasitic drug development, including drug resistance and targeting “undruggable” proteins, as far as we know, no malaria-specific PROTACs have been developed to date, likely due to the limited understanding of the UPS in protozoan parasites [49].
To design an effective malaria-specific PROTAC, identifying and characterizing unique Plasmodium-specific UPS components is crucial. While E3 ligases have been widely explored for targeted degradation, recent studies highlight the potential of E2 ubiquitin-conjugating enzymes as complementary tools for UPS hijacking [50]. In addition to the HECT E3 ligase (PfHEUL) characterized in this study, the novel, Plasmodium-specific E2 enzyme represents a promising target for pathogen-specific protein degradation with minimal host toxicity. Further characterization of its interactions with E3 ligases and substrates is necessary to fully exploit its therapeutic potential. Expanding proteome-editing tools in this way broadens the target landscape and enhances strategies for functional protein depletion, benefiting both fundamental research and clinical applications [48].
The results detailed in this study demonstrate the applicability of ubiquitin probes for the identification and study of (de)ubiquitination machinery in Plasmodium. The validation of several ubiquitin E2 enzymes and their capacity for autoubiquitination has given insight into the machinery of the Plasmodium ubiquitin pathway and provided the foundation for the characterisation of novel antimalarial drug targets.
Materials and methods
Asexual parasite stage culture
Plasmodium falciparum (strain NF54) was cultured essentially as described in [51]. Briefly, parasites were cultured within O+ human erythrocytes provided by anonymous health donors, via the National Health Service (NHS) Blood and Transplant, at 5% haematocrit in RPMI 1640 complete medium containing L- glutamine and 25 mM HEPES (Sigma), supplemented with 0.2% sodium bicarbonate, 0.5% Albumax II (Gibco), 0.36 mM hypoxanthine (Sigma) and 50 mg/L gentamicin sulphate (Melford) under a 90% N2/5% O2/5% CO2 gaseous atmosphere at 37°C, within a hypoxic incubator chamber (Billups-Rothenberg). Parasites were maintained at a parasitaemia of 0.2-10%, calculated by counting the percentage of parasitized erythrocytes in Hemacolor (Sigma) stained thin blood smears using light microscopy.
Infected erythrocytes at a parasitaemia of 8–10% were collected by centrifugation, and washed twice in 10 v:v PBS. A volume of 0.2% (w/v) saponin equivalent to the starting culture was used to resuspend the cell pellet, and incubated for 10 minutes at 4°C. Following centrifugation at 3,200 g, the supernatant was removed, and the parasite pellet was washed three times in ice cold PBS. Following the final wash, the supernatant was aspirated, and the pellet flash-frozen in liquid nitrogen and stored at -80°C until use.
Methods for ubiquitin-Dha activity-based protein profiling (ABPP)
1 L of P.falciparum culture at 8% parasitaemia was grown and harvested. Frozen parasite pellets were resuspended in lysis buffer (50 mM HEPES pH 8, 100 mM NaCl, 1 mM DTT, 1% w/v N-octyl glucoside, Complete Mini protease inhibitor). The cell suspension was lysed by 5 freeze-thaw cycles with agitation. The soluble fraction was separated by centrifugation (13,000 xg for 30 minutes at 4C), and protein concentration measured by the Pierce BCA protein assay kit (ThermoFisher) following the manufacturer’s instructions.
The procedure for the biotin-Ub-Dha probe used was based on the method described in [10]. For use with the biotin-Ub-Dha probe, the supernatant was split in two (experimental and control) and 10 mg total protein was used in each condition. To the control sample, 2 units of apyrase (Sigma-Aldrich) were incubated with the lysate for 30 minutes at 37°C to deplete residual ATP. 10 μg of biotin-Ub-Dha was added to both lysates, and 10 mM Mg-ATP was added only to the experimental lysate. Both lysates were subsequently incubated for 90 minutes at 37°C with agitation; within this time the experimental lysate was supplemented with 1 mM Mg-ATP every 15 minutes. The final reaction volume totalled 200 μL.
Following incubation, 20 μL pre-washed Pierce Neutravidin resin (ThermoFisher) was added and incubated for 3 hours at 4°C with rotation. Beads were collected by centrifugation and washed 4 times with a change of vessel before the final wash. (Wash 1: 2% SDS. Wash 2: 50 mM HEPES pH 7.5, 1 mM EDTA, 500 mM NaCl, 1% Triton-X 100, 0.1% sodium deoxycholate. Wash 3: 10 mM Tris pH 8, 1 mM EDTA, 0.5% NP 40, 250 mM LiCl. Wash 4: 50 mM Tris pH 8, 50 mM NaCl). The beads were collected by centrifugation and resuspended in a 1x LDS buffer (ThermoFisher) before incubation at 95°C for 10 minutes. The eluent was collected and processed for LC-MS/MS.
Proteomics sample preparation
The eluates were prepared for mass spectrometry (MS) analysis following a protocol as described previously [52]. Briefly, immunoprecipitated sample eluates were diluted to 175 μL with ultra-pure water and reduced with 5 μL of DTT (200 mM in 0.1 M Tris, pH 7.8) for 30 minutes at 37°C. The samples were then alkylated with 20 μL of iodoacetamide (100 mM in 0.1 M Tris, pH 7.8) for 15 minutes at room temperature, protected from light.
Protein precipitation was performed using a double methanol/chloroform extraction method. Protein samples were treated with 600 μL of methanol, 150 μL of chloroform, and 450 μL of water, followed by vigorous vortexing. Samples were centrifuged at 17,000 × g for 3 minutes, and the upper aqueous phase was removed. Proteins were pelleted by adding 450 μL of methanol and centrifuging at 17,000 × g for 6 minutes. The supernatant was discarded, and the extraction process was repeated. The precipitated proteins were resuspended in 50 μL of 6 M urea and diluted to less than 1 M urea with 250 μL of 20 mM HEPES (pH 8.0) buffer. Protein digestion was conducted by adding trypsin (Worthington; from a 1 mg/mL stock in 1 mM HCl) at a ratio of 1:100, rocking at 12 rpm at room temperature overnight. Following digestion, the samples were acidified to 1% trifluoroacetic acid, desalted on C18 solid-phase extraction cartridges (SEP-PAK plus, Waters), dried, and re-suspended in buffer A.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using a Dionex Ultimate 3000 nano-ultra high-pressure reverse-phase chromatography system coupled online to a Q Exactive (Thermo Scientific) mass spectrometer, as described previously [53]. In brief, the samples were separated on an EASY-Spray PepMap RSLC C18 column (500 mm × 75 μm, 2 μm particle size, Thermo Scientific) over a 60-minute gradient of 2–35% acetonitrile in 5% dimethyl sulfoxide (DMSO), 0.1% formic acid at a flow rate of 250 nL/min. MS1 scans were acquired at a resolution of 60,000 at 200 m/z, and the top 12 most abundant precursor ions were selected for high collision dissociation (HCD) fragmentation. Data was collected for one biological repeat and three technical repeats with intensity-based absolute quantification (iBAQ) values averaged across the technical repeats.
To analyse recombinant protein expressed from E.coli, peptide separation was performed at a 300 nL/min flow rate with a top 20 data-dependent acquisition (DDA) method, 35,000 resolution for MS1, and a scan range of m/z 380–1500.
Data analysis
Raw MS data was used as input for analysis using MaxQuant software v.2.0.3.1. Parameters were set as follows: carbamidomethyl (C) as a fixed modification, and oxidation (M) and deamidation (NQ) as variable modifications; label min ratio count was set to 1 and deamidation was included as a modification used in protein quantification, while discard unmodified counterpart peptides was unchecked; the FTMS MS/MS was set to 0.05 Da and the ITMS MS/MS match tolerance was set to 0.6 Da; and iBAQ was checked. The PlasmoDB v61 proteome fasta file was used as the reference proteome file.
The resultant ProteinGroup output file was input into Perseus v1.6.15.0 for further analysis using the iBAQ values. Column values were filtered based on categorical column: only identified by site, reverse, and potential contaminant. Samples were grouped as control or experimental by categorical row annotation. iBAQ values were transformed by log2(x), and invalid values filtered with a minimum requirement of 2 valid values per group. Invalid values were imputed from a normal distribution: width (0.3), down shift (1.8). A scatter plot was produced using fold change as the x-axis and log10(average intensity) as the y-axis, both calculated from the iBAQ values. A significance B outlier test was performed on this plot to identify enriched hit proteins at a threshold p<0.05.
Following data acquisition from E.coli produced recombinant protein, raw files were converted to mgf format and searched using Mascot (version 3.0.0) against the cRAP contaminant database, Uniprot Escherichia coli, and Plasmodium falciparum protein databases, assuming trypsin digestion. Mascot search parameters included 20 ppm parent ion tolerance, 0.1 Da fragment ion tolerance, carbamidomethylation of cysteine as a fixed modification, and deamidation of asparagine/glutamine and oxidation of methionine as variable modifications.Protein identification was validated using Scaffold (version 5.1.1), with Peptide Prophet (95% confidence) and Protein Prophet (99% confidence) algorithms, with a minimum of 2 identified peptides.
Protein expression and purification
pET28a+ plasmids encoding His-tagged Plasmodium falciparum proteins were heat shocked into C41 E.coli and selected by kanamycin. 2xYT media was inoculated with a transformed colony, and protein expression induced with IPTG at OD600 = 0.8. Following overnight growth, culture was harvested by centrifugation, and mechanically lysed in 50 mM Tris pH 8, 500 mM NaCl, 0.5 mM TCEP supplemented with Complete protease inhibitor tablets and benzonase, using a cell disruptor (Constant Systems). Soluble protein was taken following clarification by centrifugation (40,000 xg for 1 hour at 4C) and 0.22 µm membrane. Proteins were subject to Ni-affinity by HisTrap Excel column and size exclusion chromatography by either S75 16/60 or S200 10/300 column, in conjunction with an AKTA system (Cytiva). A desalting step was incorporated into the workflow at the point of elution from the IMAC resin, such that upon imidazole-mediated elution the protein was minimally exposed to the high salt concentration of the elution buffer. Eluted fractions were subject to analysis by SDS-PAGE and Coomassie staining, before aliquoting and storage at -80°C following flash-freezing.
Expression and purification of Pf3D7_0811400
The Pf3D7_0811400 cDNA sequence was codon-optimized for human expression using the Twist Biosciences Beta Codon Optimization Tool, and an N-terminal FLAG-tagged construct was synthesised and cloned into the pTwist vector under the CMV promoter (pTWIST CMV; Twist Biosciences). For protein expression, 10 µg of DNA was transfected into HEK293T cells at 85% confluency in a 10 cm cell culture plate, using FuGENE HD transfection reagent (E2311, Promega). After 36 hours, cells were harvested and lysed using Triton lysis buffer with protease inhibitor cocktail (11836170001, Roche) (LBI). The lysate was subjected to immunoprecipitation by incubating with Anti-FLAG M2 Magnetic Beads (M8823, Sigma-Aldrich) for 2 hours at 4°C on a rotating platform. The magnetic beads were then washed three times with LBI and twice with LB. Purified proteins were eluted by incubating the beads in a 3xFLAG Peptide (F4799, Sigma-Aldrich) solution in TBS for 1 hour at 4°C while rotating. The magnetic beads were separated using a SureBeads Magnetic Rack (1614916, Bio-Rad), and the purified protein was snap-frozen and stored at -80°C.
SDS-PAGE and Western blot
SDS-PAGE was performed using NuPAGE 4–12% Bis-Tris precast gels using MOPS or MES buffer (ThermoFisher). 4x LDS buffer containing 5% β-mercaptoethanol was added to samples in a 1:3 ratio, this mixture was then heated to 95°C for 10 minutes prior to gel loading. The gel was run at 180V for 40–60 minutes. Coomassie staining of SDS-PAGE gels was performed using InstantBlue gel stain following the manufacturer’s instructions (Expedion).
SDS-PAGE gels were transferred to methanol-activated PVDF membrane by Trans- Blot SD semi-dry transfer at a constant voltage of 10V for 1 hour (BioRad). Membranes were washed with TBST before blocking in 5% milk (TBST) for 1 hour with agitation. Primary (and conjugated) antibodies: rat anti-HA-peroxidase clone 3F10 high affinity (Roche), mouse anti-His clone 1B7G5 (Proteintech), mouse anti-Ub VU-1 (LifeSensors), or mouse anti-PfHEUL HECT (kindly provided by Andrew Blagborough) were incubated with the membrane at a 1:1000 dilution (5% milk-TBST) for 1 hour at room temperature. Following incubation, membranes were washed 3 times with TBST. For the unconjugated antibody, the membrane was incubated again in 5% milk (TBST) containing a 1:5000 dilution of secondary antibody goat anti-mouse-HRP (ThermoFisher) for 1 hour at room temperature. Following antibody incubations the membranes were washed three times in TBST. Probed membranes were visualised using ECL western blotting substrate (Promega) and Azure c500 gel imaging systems.
Ubiquitination assays
To test the auto-ubiquitination activity of the PfE2 enzymes, 5 µM of P. falciparum protein (PF3D7_0527100, PF3D7_0921000, PF3D7_1033900, PF3D7_1203900, PF3D7_1356300) was incubated with 25 µM HA-Ub and 100 nM HsUBA1 (E1), 10 mM ATP, and the reaction buffer components of the Ubiquitin conjugation initiation kit (K-995, R&D systems) following the manufacturer’s instructions. For ubiquitination studies of Pf3D7_0811400, the Abcam Ubiquitination Assay Kit (ab139467) was used according to the manufacturer’s instructions. Biotin-Ub-Dha and HA-Ub were substituted for the ubiquitin solution provided in the kit for probe labelling and the E2 auto-ubiquitination assay, respectively.
To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 µM of the recombinant HECT domain were incubated with 2.5 µM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).
To assess the cooperativity of the P.falciparum E1, E2, and HECT E3 domain activities, 100 nM of PfE1 (PF3D7_1225800), 5 µM PfE2 enzymes (PF3D7_0527100, PF3D7_0921000, PF3D7_1033900, PF3D7_1203900, PF3D7_1356300), and 5 µM of PfHEUL (PF3D7_0826100) HECT domain were incubated with 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems). All reactions were incubated at 37C for 1 hour. The reactions were quenched with 1X LDS sample buffer (ThermoFisher) supplemented with 50 mM DTT. Samples were analysed by SDS-PAGE and western blot as described above.
In-silico analysis
Sequence alignment and residue conservation were conducted using Clustal Omega [54] and visualised with ESPript 3 [55]. Protein 3D structure predictions and protein-protein interaction modelling were carried out using AlphaFold3, a deep-learning-based method [56]. Structural figures were generated using Pymol and UCSF ChimeraX [57], and structural alignments were performed with the RCSB PDB Pairwise Structure Alignment tool [58]. To identify orthologs, the Consurf web server [59] was employed, which also colour-coded the input PDB file based on the computed conservation score for each amino acid residue.
Supporting information
S1 Fig. Alignment of P. falciparum putative E2 enzymes.
MUSCLE sequence alignment of PfE2 enzymes rendered in ESPript. All annotated PfE2 enzymes contain a conserved cysteine residue (blue arrow), here at numbered residue 94, except two putative UEV enzymes PF3D7_1243700 and PF3D7_0305700.
https://doi.org/10.1371/journal.ppat.1013032.s001
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S2 Fig. Validation of E2 activity control blots.
A) Anti-HA blot to demonstrate that E2 activity is ablated in the absence of E1 enzyme for all HIS-E2 enzymes shown in Fig 2. PfE2 enzymes are HIS-tagged and ubiquitin is HA-tagged. B) Additional control showing ablation of E1 activity in absence of Mg-ATP by anti-HA blot to detect ubiquitin. C-D) A repeat of panels A and B but probed with anti-HIS to demonstrate presence of E2 enzymes.
https://doi.org/10.1371/journal.ppat.1013032.s002
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S3 Fig. Expression and purification of PfHEUL HECT domain.
A) Western blot of His-tag PfHEUL HECT (̃45 kDa) expression trial under varying incubation temperature and IPTG concentration. Bacteria were chemically lysed and the soluble and insoluble fractions separated by centrifugation prior to resuspension and SDS-PAGE. B) Nickel affinity and desalting workflow assessing input lysate, wash steps, and elution. PfHEUL was eluted maximally by the third elution fraction, and these fractions were pooled and subjected to SEC. This sample demonstrated a single band at the expected molecular weight of PfHEUL.
https://doi.org/10.1371/journal.ppat.1013032.s003
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S4 Fig. A. AlphaFold-predicted 3D-structure alignment of Pf3D7_0811400 (green) and Sphaeroforma arctica B box-type domain-containing protein (silver).
While the C-terminal DUF2009 domain shows a high level of similarity between the two, the B-box domain (highlighted in dark grey) is absent in the N-terminus of Pf3D7_0811400. B. Amino acid sequence alignment of the B-box domain from the B box-type domain-containing protein with the corresponding N-terminal region of Pf3D7_0811400. Cysteines and histidines involved in zinc interaction, indicated by red arrows, are present in the B-box protein but absent in Pf3D7_0811400.
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S5 Fig. Consurf output for Pf3D7_0811400.
The Consurf web server was used to identify orthologs based on the protein sequence of Pf3D7_0811400. The output was used to colour-code the input PDB structural file (accessed through the AlphaFold web server) based on the computed conservation score for each amino acid residue. Residues are colour-rendered according to the conservation index, with annotations corresponding to the key.
https://doi.org/10.1371/journal.ppat.1013032.s005
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S6 Fig. Full membranes for Fig 7 immunoblots.
A) Full membranes for Fig 7B streptavidin-HRP (left) and anti-FLAG (right) immunoblots. FLAG-tagged PfFBXO9 served as an additional negative control. B) Full membranes for Fig 7C. The whole cell extract (WCE) membrane was sequentially probed with both anti-FLAG and anti-myc antibodies. The immunopurified proteins were also probed with both antibodies, but on separate membranes (middle image is anti-FLAG and right image is anti-myc). FLAG-tagged Pf3D7_0811400 WT (indicated by**); ΔN N-terminal truncated mutant (indicated by***); FLAG-tagged PfFBXO9 (indicated by*).
https://doi.org/10.1371/journal.ppat.1013032.s006
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S7 Fig. Full membranes for Fig 8A.
Panels A and B show the full membrane for western blots shown in Fig 8A. Here, a positive control is also included, PF3D7_0921000, a P. falciparum E2 enzyme characterised earlier in this paper. Arrows indicate the signals corresponding to probe interactions with each E2. The FLAG and HIS blots represent the loading of PF3D7_0811400 and PF3D7_0921000, respectively.
https://doi.org/10.1371/journal.ppat.1013032.s007
(TIF)
S8 Fig. Orthology analysis of PF3D7_0811400.
A) 3D structural alignment of the human and Plasmodium ADSL enzymes with the second DUF2009 domain of PF3D7_0811400. While there is high structural similarity between the ADSL enzymes of Plasmodium (gray) and human (blue), PF3D7_0811400 (green) shows only a limited match. Catalytic histidine (Hs H159/ Pf H173) and Catalytic serine (Hs S289/ Pf S298) are conserved in ADSL enzymes but not in DUF2009 domain. Also C3 loop also known as Fumarate lyase, conserved site which is critical for the enzyme activity is absent in PF3D7_0811400. B) Conservation of the catalytic region in 128 eukaryotic proteins with similar structure. Protein sequences were downloaded from UniProt and aligned using MUSCLE, with the alignment visualized in Jalview. Conservation is shown as a histogram, with ‘*’ indicating fully conserved columns (score of 11 using default amino acid property grouping) and ‘+’ marking columns with mutations that preserve all properties (score of 10). C) Classification of the 128 selected proteins based on their corresponding organisms carrying this conserved structure. Data were obtained from the NCBI Taxonomy Browser, and the Common Tree was generated using the NCBI Taxonomy Common Tree tool.
https://doi.org/10.1371/journal.ppat.1013032.s008
(TIF)
S1 Table. Purity Validation of Recombinant Proteins.
Assessment of protein purity by mass spectrometry: the purified proteins were subjected to MS/MS analysis to identify potential E. coli ubiquitin enzyme contamination that could interfere with subsequent ubiquitination assays.
https://doi.org/10.1371/journal.ppat.1013032.s009
(XLSX)
S2 Table. Foldseek analysis.
Protein structure alignments of Pf3D7_0811400 against large protein structure collections by Foldseek Server.
https://doi.org/10.1371/journal.ppat.1013032.s010
(XLSX)
Acknowledgments
We thank Dr. Andrew Blagborough for helping generate the anti-PfHEUL antibody. We are very grateful for expert help with mass spectrometry analysis by the Discovery Proteomics Facility led by Iolanda Vendrell and Roman Fischer (University of Oxford).
References
- 1. Horrocks P, Newbold CI. Intraerythrocytic polyubiquitin expression in Plasmodium falciparum is subjected to developmental and heat-shock control. Mol Biochem Parasitol. 2000;105(1):115–25. pmid:10613704
- 2. Sutherland CJ, Henrici RC, Artavanis-Tsakonas K. Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing. FEMS Microbiol Rev. 2021;45(3):fuaa056. pmid:33095255
- 3. Green JL, Wu Y, Encheva V, Lasonder E, Prommaban A, Kunzelmann S, et al. Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development. PLoS Pathog. 2020;16(6):e1008640. pmid:32569299
- 4. Spork S, Hiss JA, Mandel K, Sommer M, Kooij TWA, Chu T, et al. An unusual ERAD-like complex is targeted to the apicoplast of Plasmodium falciparum. Eukaryot Cell. 2009;8(8):1134–45. pmid:19502583
- 5. Ponts N, Yang J, Chung D-WD, Prudhomme J, Girke T, Horrocks P, et al. Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence. PLoS One. 2008;3(6):e2386. pmid:18545708
- 6. Gantt SM, Myung JM, Briones MR, Li WD, Corey EJ, Omura S, et al. Proteasome inhibitors block development of Plasmodium spp. Antimicrob Agents Chemother. 1998;42(10):2731–8. pmid:9756786
- 7. Arora P, Narwal M, Thakur V, Mukhtar O, Malhotra P, Mohmmed A. A Plasmodium falciparum ubiquitin-specific protease (PfUSP) is essential for parasite survival and its disruption enhances artemisinin efficacy. Biochem J. 2023;480(1):25–39. pmid:36511651
- 8. Hewings DS, Flygare JA, Bogyo M, Wertz IE. Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights. FEBS J. 2017;284(10):1555–76. pmid:28196299
- 9. Nair SC, Xu R, Pattaradilokrat S, Wu J, Qi Y, Zilversmit M, et al. A Plasmodium yoelii HECT-like E3 ubiquitin ligase regulates parasite growth and virulence. Nat Commun. 2017;8(1):223. pmid:28790316
- 10. Mulder MPC, Witting K, Berlin I, Pruneda JN, Wu K-P, Chang J-G, et al. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. Nat Chem Biol. 2016;12(7):523–30. pmid:27182664
- 11. Cook BW, Lacoursiere RE, Shaw GS. Recruitment of Ubiquitin within an E2 Chain Elongation Complex. Biophys J. 2020;118(7):1679–89. pmid:32101714
- 12. Middleton AJ, Day CL. From seeds to trees: how E2 enzymes grow ubiquitin chains. Biochem Soc Trans. 2023;51(1):353–62. pmid:36645006
- 13. David Y, Ziv T, Admon A, Navon A. The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines. J Biol Chem. 2010;285(12):8595–604. pmid:20061386
- 14. Kamadurai HB, Qiu Y, Deng A, Harrison JS, Macdonald C, Actis M, et al. Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. Elife. 2013;2:e00828. pmid:23936628
- 15. Micsonai A, Moussong É, Wien F, Boros E, Vadászi H, Murvai N, et al. BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy. Nucleic Acids Res. 2022;50(W1):W90–8. pmid:35544232
- 16. Pandya R, Partridge J, Love K, Schwartz T, Ploegh H. A structural element within the HUWE1 HECT domain modulates self-ubiquitination and substrate ubiquitination activities. J Biol Chem. 2010;285:5664–73.
- 17. van Kempen M, Kim SS, Tumescheit C, Mirdita M, Lee J, Gilchrist CLM, et al. Fast and accurate protein structure search with Foldseek. Nat Biotechnol. 2024;42(2):243–6. pmid:37156916
- 18.
Anthony M, Massiah M. Zinc-binding B-box domains with RING folds serve critical roles in the protein ubiquitination pathways in plants and animals. Ubiquitin Proteasome System - Current Insights into Mechanism Cellular Regulation and Disease. IntechOpen; 2019.
- 19. Yariv B, Yariv E, Kessel A, Masrati G, Chorin AB, Martz E, et al. Using evolutionary data to make sense of macromolecules with a “face-lifted” ConSurf. Protein Sci. 2023;32(3):e4582. pmid:36718848
- 20. Rashpa R, Smith C, Artavanis-Tsakonas K, Brochet M. A multistage Plasmodium CRL4WIG1 ubiquitin ligase is critical for the formation of functional microtubule organization centers in microgametocytes. mBio. 2024;15(10):e0167224. pmid:39207167
- 21. Morrow ME, Morgan MT, Clerici M, Growkova K, Yan M, Komander D, et al. Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins. EMBO Rep. 2018;19(10):e45680. pmid:30150323
- 22. Kriventseva EV, Tegenfeldt F, Petty TJ, Waterhouse RM, Simão FA, Pozdnyakov IA, et al. OrthoDB v8: update of the hierarchical catalog of orthologs and the underlying free software. Nucleic Acids Res. 2015;43(Database issue):D250-6. pmid:25428351
- 23. Mahlich Y, Steinegger M, Rost B, Bromberg Y. HFSP: high speed homology-driven function annotation of proteins. Bioinformatics. 2018;34(13):i304–12. pmid:29950013
- 24. van Wijk SJL, Timmers HTM. The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J. 2010;24(4):981–93. pmid:19940261
- 25. Tsai M, Koo J, Yip P, Colman RF, Segall ML, Howell PL. Substrate and product complexes of Escherichia coli adenylosuccinate lyase provide new insights into the enzymatic mechanism. J Mol Biol. 2007;370(3):541–54. pmid:17531264
- 26. Chung D-WD, Ponts N, Prudhomme J, Rodrigues EM, Le Roch KG. Characterization of the ubiquitylating components of the human malaria parasite’s protein degradation pathway. PLoS One. 2012;7(8):e43477. pmid:22912882
- 27. Karpiyevich M, Adjalley S, Mol M, Ascher DB, Mason B, van Noort GJ van der Heden, et al. Nedd8 hydrolysis by UCH proteases in Plasmodium parasites. PLoS Pathog. 2019;15(10):e1008086. pmid:31658303
- 28. Artavanis-Tsakonas K, Weihofen WA, Antos JM, Coleman BI, Comeaux CA, Duraisingh MT, et al. Characterization and structural studies of the Plasmodium falciparum ubiquitin and Nedd8 hydrolase UCHL3. J Biol Chem. 2010;285(9):6857–66. pmid:20042598
- 29. Artavanis-Tsakonas K, Misaghi S, Comeaux CA, Catic A, Spooner E, Duraisingh MT, et al. Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum. Mol Microbiol. 2006;61(5):1187–95. pmid:16925553
- 30. Philip N, Haystead TA. Characterization of a UBC13 kinase in Plasmodium falciparum. Proc Natl Acad Sci U S A. 2007;104(19):7845–50. pmid:17452636
- 31. Maneekesorn S, Knuepfer E, Green JL, Prommana P, Uthaipibull C, Srichairatanakool S, et al. Deletion of Plasmodium falciparum ubc13 increases parasite sensitivity to the mutagen, methyl methanesulfonate and dihydroartemisinin. Sci Rep. 2021;11(1):21791. pmid:34750454
- 32. Vedadi M, Lew J, Artz J, Amani M, Zhao Y, Dong A, et al. Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms. Mol Biochem Parasitol. 2007;151(1):100–10. pmid:17125854
- 33. Painter HJ, Chung NC, Sebastian A, Albert I, Storey JD, Llinás M. Genome-wide real-time in vivo transcriptional dynamics during Plasmodium falciparum blood-stage development. Nat Commun. 2018;9(1):2656. pmid:29985403
- 34. Zhang M, Wang C, Otto TD, Oberstaller J, Liao X, Adapa SR, et al. Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science. 2018;360(6388):eaap7847. pmid:29724925
- 35. Grabarczyk D, Petrova O, Deszcz L, Kurzbauer R, Murphy P, Ahel J, et al. HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain. Nature Chemical Biology. 2021;17:1084–92.
- 36. Hunkeler M, Jin CY, Ma MW, Monda JK, Overwijn D, Bennett EJ, et al. Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition. Mol Cell. 2021;81(17):3468–3480.e7. pmid:34314700
- 37. Cowell AN, Istvan ES, Lukens AK, Gomez-Lorenzo MG, Vanaerschot M, Sakata-Kato T, et al. Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics. Science. 2018;359(6372):191–9. pmid:29326268
- 38. Park DJ, Lukens AK, Neafsey DE, Schaffner SF, Chang H-H, Valim C, et al. Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite. Proc Natl Acad Sci U S A. 2012;109(32):13052–7. pmid:22826220
- 39. Chen D, Gehringer M, Lorenz S. Developing Small-Molecule Inhibitors of HECT-Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities. Chembiochem. 2018;19(20):2123–35. pmid:30088849
- 40. Weber J, Polo S, Maspero E. HECT E3 Ligases: A Tale With Multiple Facets. Front Physiol. 2019;10:370. pmid:31001145
- 41. Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, et al. Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J Cell Biol. 2005;170(5):745–55. pmid:16129784
- 42. Hillier C, Pardo M, Yu L, Bushell E, Sanderson T, Metcalf T, et al. Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality. Cell Rep. 2019;28(6):1635–1647.e5. pmid:31390575
- 43. Cook B, Shaw G. Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes. Biochem J. 2012;445:167–74.
- 44. Knipscheer P, Sixma T. Divide and conquer: the E2 active site. Nature Structural & Molecular Biology. 2006;13:474–6.
- 45. Valimberti I, Tiberti M, Lambrughi M, Sarcevic B, Papaleo E. E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft. Sci Rep. 2015;5:14849. pmid:26463729
- 46. Agarwal V, Miles ZD, Winter JM, Eustáquio AS, El Gamal AA, Moore BS. Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse. Chem Rev. 2017;117(8):5619–74. pmid:28106994
- 47. Zhong G, Chang X, Xie W, Zhou X. Targeted protein degradation: advances in drug discovery and clinical practice. Signal Transduct Target Ther. 2024;9(1):308. pmid:39500878
- 48. He M, Cao C, Ni Z, Liu Y, Song P, Hao S, et al. PROTACs: great opportunities for academia and industry (an update from 2020 to 2021). Signal Transduct Target Ther. 2022;7(1):181. pmid:35680848
- 49. Espinoza-Chávez RM, Salerno A, Liuzzi A, Ilari A, Milelli A, Uliassi E, et al. Targeted Protein Degradation for Infectious Diseases: from Basic Biology to Drug Discovery. ACS Bio Med Chem Au. 2022;3(1):32–45. pmid:37101607
- 50. Taylor J, Barrett N, Martinez Cuesta S, Cassidy K, Pachl F, Dodgson J. Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes. Commun Biol. 2024;7:1179.
- 51. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193(4254):673–5. pmid:781840
- 52. Pinto-Fernández A, Davis S, Schofield A, Scott H, Zhang P, Salah E, et al. Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics. Frontiers in Chemistry. 2019;7:592.
- 53. Fischer R, Kessler BM. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics. 2015;15(7):1224–9. pmid:25515006
- 54. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539. pmid:21988835
- 55. Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42(Web Server issue):W320–4. pmid:24753421
- 56. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630(8016):493–500. pmid:38718835
- 57. Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, et al. UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. 2023;32(11):e4792. pmid:37774136
- 58. Bittrich S, Segura J, Duarte JM, Burley SK, Rose Y. RCSB protein Data Bank: exploring protein 3D similarities via comprehensive structural alignments. Bioinformatics. 2024;40(6):btae370. pmid:38870521
- 59. Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 2016;44(W1):W344-50. pmid:27166375