{"paper_id":"059f4564-3ecd-415a-ab2d-028ada511ee8","body_text":"A role for the poly-asparagine repeat in the Plasmodium histone acetyltransferase, PfGCN5 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A role for the poly-asparagine repeat in the Plasmodium histone acetyltransferase, PfGCN5 Kelly Rubiano, Aaron Morris, Francisca De Luna Vitorino, Benjamin A. Garcia, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7490809/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Plasmodium falciparum possesses one of the most AT-rich genomes in nature (80.6%). A consequence is an asparagine-rich proteome. A quarter of P. falciparum proteins possess poly-asparagine repeats that can extend more than 100 residues. The role of these repeats has remained a mystery in the biology of this parasite. Here, we find that the poly-asparagine repeat-containing N terminus of the histone acetyltransferase PfGCN5 associates with the C-terminal catalytic domain after cleavage in the nucleus. Deletion of the repeat destabilizes the N-terminal polypeptide, leading to impaired parasite development and growth, particularly under stress conditions. Using high-resolution mass spectrometry and western blotting analysis, we uncovered a profound effect of the poly-asparagine repeat on acetylation of histones H3, H3.3, and H4. These findings suggest that the poly-asparagine repeat contributes to PfGCN5 acetyltransferase activity, a role previously attributed solely to its C-terminal domain. This report of a function for a poly-asparagine repeat in P. falciparum expands our understanding of a pervasive characteristic of its proteome. Biological sciences/Microbiology/Parasitology/Parasite biology Biological sciences/Microbiology/Parasitology/Parasite evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The human malaria parasite, Plasmodium falciparum , exhibits one of the most AT-rich genomes (80.6%) sequenced to date, and among the Plasmodium species infecting humans, it outruns its peers 1 – 3 . As expected, amino acid residues with high AT content, such as asparagine (AAT/AAC) and lysine (AAA/AAG), are enriched in the P. falciparum proteome 4 . However, codon bias analyses established a preference for asparagine over lysine residues despite equivalent AT-richness (83.3%) 4,5 . Remarkably, asparagine (N) residues are found in tandem predominantly in intragenic regions, forming low-complexity, intrinsically disordered regions in about 24% of the P. falciparum proteome, when considering at least 30 N in an 80-residue window 4 . P. falciparum’s poly-N repeats are either perfect or imperfect. When imperfect, they are typically interrupted by polar residues, such as serine, aspartic acid and glutamic acid, as well as the aromatic residue tyrosine. These repeats average 37 residues but can exceed 100 residues in length 6 . Since poly-asparagine repeats in P. falciparum proteins do not cluster in specific protein families, metabolic pathways, or parasite stages, the functional contribution of these repeats has been elusive 7 . Structural mapping of poly-N repeats suggests that they are unlikely to adopt globular folds due to their flexible and hydrophilic nature, which favors protrusion from protein cores. This raises the possibility that poly-N repeats enhance protein function, adopt new functions, or confer protein stability without interfering with basal protein activity 4 , 5 . Furthermore, poly-N repeats exhibit an intrinsic propensity to aggregate that increases with length and temperature 8 . Thus, the evolutionary forces shaping the P. falciparum proteome, an organism that withstands febrile cycles, are of interest. To date, only two P. falciparum proteins containing poly-N repeats have been experimentally characterized. In the first case, the parasite chaperone PfHsp110, but not its human or yeast counterparts, efficiently prevents heat shock (HS)-induced aggregation of the parasite’s putative CDK2-regulatory subunit, which contains 83 consecutive N residues, highlighting the robust specialization that parasite chaperones have evolved 9 . In the second case, removal of the 28-residue poly-N tract from the essential proteasome clamp subunit, Rpn6, had no effect on protein expression, function, or parasite viability at 37°C or 41°C, suggesting that some poly-N repeats may lack functional properties 10 . Given the large group of poly-N repeat-containing proteins that remain uncharacterized in P. falciparum , we sought to investigate the role of an imperfect repeat of 98 residues containing 81 N, located in the N terminus of the parasite’s histone acetyltransferase GCN5 (PfGCN5). GCN5 is highly conserved among eukaryotes with two well-characterized C-terminal domains: a histone acetyltransferase (HAT) domain, which catalyzes the transfer of acetyl groups to lysine residues, and a bromodomain (BrD), which binds acetylated substrates to support enzymatic processivity 11 . GCN5 participates in the megadalton SAGA complex, where, together with ADA2, ADA3, and SGF29, it forms the HAT catalytic module of the complex 12 , 13 . In P. falciparum , GCN5 is essential and regulates the expression of a subset of genes involved in development, invasion, and stress response 14 – 17 . Seminal work on PfGCN5 has underscored its pivotal role in catalyzing histone acetylation marks associated with euchromatin 16 , 18 , 19 . Deletion of the BrD from PfGCN5 leads to decreased histone acetylation and altered chromatin architecture, resulting in defective intraerythrocytic development and dysregulated sexual commitment 16 , 17 . Bromodomain deletion lines also exhibit impaired maturation of gametocytes and mosquito stages 17 . Therefore, PfGCN5 holds a critical role in modifying the chromatin landscape across the parasite's life cycle. Our findings show that the N-terminal fragment containing the poly-N repeat in PfGCN5 contributes to the HAT activity of the protein following cleavage, by associating with C-terminal catalytic domain. We characterized a poly-N repeat deletion line and found reduced histone acetylation and impaired parasite growth, particularly under stress conditions. We explored the nucleocytoplasmic live-cell dynamics of the N-terminal polypeptide and determined that it is destabilized when the poly-N repeat is removed. Furthermore, we have expanded the known repertoire of acetylation marks catalyzed by PfGCN5’s activity to H3.3 and H4. Results The N terminus of PfGCN5 containing the poly-asparagine repeat is important for parasite growth GCN5 is a highly conserved protein across eukaryotes, with most of the homology restricted to its C terminus, where the HAT domain and the BrD are located (Supplemental figure 1) . Yeast and Tetrahymena GCN5 have short N termini with less than 200 amino acids upstream of the HAT domain. In contrast, metazoans express two GCN5 isoforms from alternative splicing: a short version that is highly homologous to the yeast GCN5 and a long variant whose extended N-terminus displays a P300/CBP-associated factor (PCAF)-homology domain 20–23 . The PCAF homology domain has been implicated in the recruitment of transcriptional activators, including two other HAT proteins, P300 and CBP, recognition of nucleosomal substrates, and it has been shown to possess E3 ubiquitin ligase activity 20,24 . The PfGCN5 (PF3D7_0823300) is produced as a single transcript and exhibits the longest N-terminal extension described to date. This extension contains no recognizable domains except for a 98-residue imperfect repeat containing 81 N (spanning from amino acid position 143 to 240) (Figure 1, Supplemental figure 2) . The related Apicomplexan parasite, Toxoplasma gondii , encodes two GCN5 homologs (TgGCN5A/B) that are expressed as single transcripts and exhibit extended N-terminal extensions as well. However, these N-terminal extensions share little sequence similarity with PfGCN5 25,26 . Sequence alignment of GCN5 homologs across Plasmodium species revealed that those naturally exposed to febrile temperatures, such as P. falciparum, P. vivax, P. knowlesi, P. gallinaceum, and P. relictum, possess N-terminal low complexity intrinsically disordered regions (LC-IDRs) consisting of poly-N or glycine-serine (GS)-rich domains (Figure 1, Supplemental figure 2) . In contrast, GCN5 homologs from Plasmodium species that do not experience fever, such as P. yoelii and P. berghei , display shorter and less conspicuous repetitive sequences rich in N, G, E, and A . Notably, avian malaria parasites ( P. gallinaceum and P. relictum) survive their host's body temperature, ranging between 38.5- 43.8 °C, and Plasmodium (P. berghei and yoelii)- infected mice experience hypothermia instead of hyperthermia 27–29 . These observations led us to hypothesize that the extended LC-IDRs in the N terminus of Plasmodium spp. naturally exposed to fever may confer a survival advantage in their hosts. ' To test this hypothesis, we generated a rapamycin-inducible PfGCN5 conditional knockout line (PfGCN5 LoxP ) using the DiCre recombinase system 30 . Specifically, we integrated a Saccharomyces cerevisiae -recoded version of the pfgcn5 coding sequence, C-terminally GFP-3HA tagged and flanked by LoxP sites at the endogenous locus, using CRISPR/Cas9 (Supplemental figure 3A) 31 . By employing an NF54 attB line expressing a dimerizable Cre recombinase, we could excise the entire gene upon rapamycin (RAP) addition (Supplemental figure 3B ) 32,33 . Similar to previous reports 15,34 , before excision, we observed nuclear localization of PfGCN5 throughout the intraerythrocytic cycle and extensive processing of the full-length protein (190 kDa), resulting in multiple fragments (Supplemental figure 3C,D) . Of note, the protein is synthesized starting at the early ring stage and is almost completely processed by the end of the intraerythrocytic cycle. Following RAP addition (0-3 hours post-invasion, early ring stage), knockout (KO) parasites stalled at the trophozoite stage and were unable to undergo schizogony, resulting in cell death (Figure 2A,B) . Time-course western blotting analysis showed that protein expression was reduced by approximately 50% in RAP-treated cultures, highlighting the stability of these fragments (Figure 2C) . We then attempted to rescue the PfGCN5 KO with a series of C-terminally FLAG-tagged constructs expressed in trans from the attB locus under the control of the native PfGCN5 promoter (Figure 2D) 32 . Growth and development could be fully rescued by the introduction of a second copy of the WT pfgcn5 gene (Figure 2D,E) . Interestingly, while P. vivax ( Pv) GCN5 fully rescued the growth of PfGCN5 KO parasites, P. yoelii (Py) GCN5 supported only partial rescue, displaying ~30% growth over three replication cycles (Figure 2D,E) . To evaluate whether this growth phenotype was due to the N terminus of PyGCN5, we made a chimeric protein containing the first 676 aa from the PfGCN5 N terminus, including the poly-N repeat, and the C terminus from PyGCN5. Given the widespread processing of PfGCN5, we determined the length of the PfGCN5 N terminus based on the conserved motif IKNI/M/LR found at amino acid residue 677 (Supplemental figure 2) . This chimeric protein was able to fully rescue the growth of the PfGCN5 KO (Figure 2D,E). Finally, we observed that all GCN5 homologs and the PyChimera were well expressed, localized to the nucleus, and processed (Figure 2F and Supplemental figure 4) . Taking together these results suggest that the N termini of PfGCN5 and PvGCN5, containing poly-N or GS-rich repeats, respectively, are important for P. falciparum parasite survival. The poly-N repeat in PfGCN5’s N terminus is required for normal parasite growth To dissect whether the growth-related role of PfGCN5 N terminus lies in its poly-N repeat, we engineered three C-terminally FLAG-tagged PfGCN5 rescue constructs, whose poly-N repeat was incrementally shortened from its C-terminal end (Figure 3A) . These deletions retained varying lengths of the 98-residue repeat containing 81N found in the WT protein: deletion #1 (Del1) retains 48 N, deletion #2 (Del2) 23 N, and deletion #3 (Del3) 7 N. Upon RAP treatment, unlike the death phenotype of PfGCN5 KO parasites, the deletion lines could progress through the first cycle without any notable defects, presenting abnormal trophozoite and schizont morphologies only in the second cycle (Figure 3B) . Similar to the PyGCN5 rescue line, all three deletion lines partially rescued the growth of the PfGCN5 KO line, exhibiting about 30% growth relative to the WT rescue line over three replication cycles (Figure 3C) . Western blot analysis confirmed that all deletion constructs were expressed at comparable levels to the WT protein and underwent normal proteolytic processing (Figure 3D) . Based on the consistent phenotype, proper protein level expression, and processing across the deletion lines, we selected one line (Del3) for the subsequent analysis. As in the WT, the Del3 rescue line showed nuclear localization of PfGCN5 (Figure 3E) . Altogether, these data indicate that the poly-N repeat in PfGCN5 is required for normal parasite growth. The poly-N repeat in PfGCN5 facilitates cell recovery after stress. The observation that poly-N or GS-rich repeats are found in GCN5 homologs from Plasmodium spp. that are naturally subjected to fever but absent in those that do not encounter such conditions prompted us to investigate the potential link between these LC-IDRs and stress. Numerous reports in P. falciparum and other systems have emphasized GCN5’s ability to act as a master regulator of stress by deploying transcriptional cascades that enable stress management 14,15,35–37 . Specifically, HS, low glucose, and dihydroartemisinin (DHA) have been shown to induce higher PfGCN5 expression 14,15,38 . Additionally, restricting PfGCN5 overexpression heightens the parasite’s susceptibility to stress 14,15 . With this in mind, we subjected our WT and Del3 rescue lines to HS (41 °C for 6h), room temperature (RT) (~25 °C for 12h), and DHA (100 nM for 90 min) starting 24h after a 3h RAP pulse at the 0-3h ring stage 39–41 . We then returned the cultures to standard conditions (37°C, drug washed out) for 6 days. Exposure to HS and RT exacerbated the growth defect in RAP-treated Del3 parasites in comparison to untreated Del3 and WT rescue parasites (Figure 4A,B) . Exposure to DHA rendered rapamycin-induced Del3 parasites unable to recover (Figure 4C) . Intriguingly, Del3 parasites also exhibited poor recovery even in the absence of rapamycin. This dominant-negative effect could result from the increased expression of PfGCN5 during stress, leading to the displacement of the WT protein by the truncated version in SAGA-like complexes. These results support the hypothesis that LC-IDRs in the N terminus of PfGCN5 contribute to the parasite’s adaptive transcriptional regulation. PfGCN5 histone acetyltransferase activity is supported by its poly-N repeat PfGCN5 retained HAT activity when its C-terminal region, comprising the HAT and BrD or the HAT domain alone, was used to acetylate free H3 at lysine residues 9 and 14 (H3K9ac and H3K14ac) from calf thymus 18 . Subsequent studies confirmed this activity in parasites 16 . Although P. falciparum encodes four histone variants (H2A.Z, H2B.Z, H3.3, and CenH3) besides the canonical core histones, PfGCN5’s role in these variants is unknown, except for H2B.Z, whose acetylation levels were lower in a PfGCN5 BrD deletion line 17,42 . To investigate if the poly-N repeat in PfGCN5 could play a role in histone acetylation, we examined H3K9ac levels in synchronized parasites treated with or without rapamycin at 0-3h post-invasion. Samples were harvested at 12-15h, 24-27h, 32-35h, and 45-48h post-invasion during the first intraerythrocytic cycle post-RAP addition. While we observed slight decreases in the level of H3K9ac at 45-48h in the PfGCN5 LoxP and Del3 lines treated with RAP, there were no significant differences across lines at any of the evaluated time points (Figure 5A,B left side) . Encouraged by this trend, and the observation that RAP-treated deletion lines exhibited a fitness cost only during the second cycle, we extended these studies to the second intraerythrocytic cycle post-rapamycin addition. In agreement with previous studies, we found significant changes in H3K9ac at all time points during the second cycle in RAP-induced PfGCN5 KO and Del3 parasites (Figure 5A,B right side) . Note that PfGCN5 KO parasites die in the first cycle, so acetylation levels in the second cycle cannot be assessed. In light of the limitations of western blot analysis for quantitation and the limited availability of anti-histone antibodies for examining other histone modifications, we opted for a quantitative, high-resolution mass spectrometry approach. To achieve this, we purified core histones using an acid extraction method from synchronized cultures at 32-35h and 45-48h of the first intraerythrocytic cycle, and at 32-35h of the second intraerythrocytic cycle post-RAP induction, which occurred at 0-3h post-invasion of the first intraerythrocytic cycle. Interestingly, we found significant differences for H3K9ac, H3K9acK14ac, H3K18ac, H3K23ac, H3K18acK23ac, H3K27ac, H3.3K9acK14ac, and H4K8acK16ac at 45-48h of the first intraerythrocytic cycle and/or at 35-38h of the second intraerythrocytic cycle (Figure 6) . We did not observe any differences in H3K9ac levels at 32-25h of the first intraerythrocytic cycle, consistent with our western blot results. The non-significant trend towards decreased H3K9ac in the western blot studies became significant differences at 45-48h of the first intraerythrocytic cycle for the MS analysis, underscoring the technique's sensitivity. Notably, H2B.Z was not detected, and di- and tri-methylation of H3K4 did not show significant differences across lines, in contrast to the western blot findings of a previous paper 16 . Overall, these findings confirm and broaden our understanding of the extensive role of PfGCN5 in chromatin remodeling and gene expression. More critically, they suggest that the poly-N repeat contributes to the HAT activity of PfGCN5. PfGCN5 is processed in the nucleus Recent studies have proposed that PfGCN5 is trafficked via the ER-Golgi to the digestive vacuole, where it is processed by the cysteine protease, falcipain 3, before translocating to the nucleus 34,43 . We, however, did not find evidence of this. We treated asynchronous PfGCN5 LoxP parasites with the cysteine protease inhibitor, E64D (10uM), the secretory pathway inhibitor Brefeldin A (5 µg/mL), or DMSO for three hours 44,45 . While parasites treated with E64D displayed an enrichment of the full-length protein as previously reported, PfGCN5 nuclear localization remained unchanged (Figure 7A,B) 34 . This outcome suggests that the full-length protein is processed in the nucleus. Furthermore, we observed that PfGCN5 processing and trafficking are insensitive to Brefeldin A treatment, indicating an ER-Golgi-independent trafficking pathway. The importin α/β machinery mediates a major nuclear transport pathway. Given that TgGCN5A enters the nucleus via direct interaction with the adaptor molecule importin-α 26 , we sought to test whether PfGCN5 uses this pathway 26 . We treated asynchronous PfGCN5 LoxP parasites with 10 μM or 25 μM ivermectin for three hours. Treatment of P. falciparum parasites with ivermectin has been shown to block this pathway 46 . We found that ivermectin-treated parasites not only exhibit stabilization of the full-length protein but also redistribution of the PfGCN5 signal to the cytoplasm, in stark contrast to DMSO-treated parasites (Figure 7C, D) . The poly-N repeat confers stability to the N-terminal polypeptide post-cleavage and interacts with the C-terminal catalytic domain We wanted to tag the N-terminus of PfGCN5 to track its kinetics. After several unsuccessful attempts, we adopted the NanoLuc® Binary Technology, NanoBiT (Promega), for higher-resolution monitoring of the poly-N repeat. For this, we first engineered a stable parasite line expressing the larger NanoLuc subunit, LgBiT, using the piggyBac transposon-mediated genomic integration 47 . Growth assays showed no significant differences between a WT and the LgBiT-expressing line (Supplemental figure 5A) . Expression and localization of LgBiT were verified by western blot and IFA, respectively, revealing an 18 kDa cytosolic protein that is consistent with findings in other systems (Supplemental figure 5B, C) 48 . We then used this LgBiT line to integrate a C-terminally FLAG-tagged second copy of PfGCN5 containing the 11-amino acid smaller NanoLuc subunit, HiBiT, in the poly N-repeat (position marked by arrowhead in Figure 1A) at the non-essential attB locus, resulting in the LgBiT-HiBiT line. To regulate expression, this second copy of PfGCN5 was placed under the control of the endogenous promoter. Insertion of HiBiT did not impair parasite growth in comparison to the WT line (Supplemental figure 5A) . Similarly, we attempted to introduce the truncated version of PfGCN5, Del3, tagged with HiBiT in the LgBiT line, but were unsuccessful. Therefore, we introduced this second copy into the PfGCN5 LoxP line. This line also had normal growth (Supplemental figure 5A) . Luminescence-based detection of the WT HiBiT-tagged protein by western blot revealed the presence of the 190 kDa full-length protein, and processed forms of 120, 110, and 50 kDa that were sequentially produced through the parasite intraerythrocytic cycle (Figure 8A) . Notably, all these fragments were also detected by another group using polyclonal antibodies that recognize the N-terminal region of PfGCN5, encompassing amino acid residues 9-25 14 . In contrast, in the Del3-HiBiT line, HiBiT western blotting failed to recognize bands, indicating that the N-terminal polypeptide is rapidly degraded in the Del3 line (Figure 8B) . IFAs showed that the poly-N repeat containing fragments in the LgBiT-HiBiT line exhibit a higher concentration in the nucleus with weak cytoplasmic signal in trophozoite and schizont stages (Figure 8C) . To investigate the dynamics of the PfGCN5 N terminus in the cell, we performed cellular fractionation studies using the LgBiT-HiBiT line. Interestingly, the full-length, the 120 kDa, and the 110 kDa forms remain in the nucleus, while the 50 kDa fragment containing the poly-N repeat is exported to the cytosol (Figure 8D) . To further explore the live-cell kinetics of the poly-N-repeat containing fragments, we took advantage of the high affinity between the LgBiT and HiBiT subunits and employed a cell-permeable substrate (furimazine) to measure the real-time binding activity. We first confirmed that the LgBiT protein is nuclear impermeable even in the presence of the PfGCN5-HiBiT protein by performing cellular fractionation (Supplemental figure 5D) . Since LgBiT was found to be exclusively cytosolic, it acts as a cytosolic sensor for HiBiT-tagged poly-N repeat-containing fragments (Figure 8E) . Consistent with our earlier observations in figures 8C and D, cytosolic levels of HiBiT-tagged fragments rise dramatically midway through the cycle, after the 50kDa fragment is generated (Figure 8F) . Given that the deletion of the poly-N repeat resulted in the destabilization of the N-terminal polypeptide post-cleavage and a reduction in histone acetylation levels, a function catalyzed by the C-terminal HAT domain, we wondered if the N-terminal polypeptide containing the poly-N repeat could associate with the C-terminal fragments. To address this question, we pulled down the C-terminal fragments by the FLAG tag and blotted for HiBiT luminescence (Figure 8G) . C-terminal FLAG-tagged forms could co-precipitate all HiBiT-tagged fragments, suggesting interaction. Notably, the 50 kDa HiBiT-tagged piece, previously shown to be exported to the cytosol, was more abundant in the flow-through than in the IP fraction, leaving the 120 kDa and 110 kDa fragments as the potential interactors of the catalytic domain. Additionally, we asked whether N-terminal peptides could be identified in C-terminal pull-downs by mass spectrometry and if shortening the length of the poly-N repeat in PfGCN5 disturbs complex formation or other interactions. To do this, we performed co-immunoprecipitation analysis using anti-FLAG magnetic beads across the WT and deletion rescue lines in the absence of rapamycin. LC-MS/MS analysis identified N-terminal peptides when pulling down from the WT rescue line but not from the deletion rescue lines, confirming the association between N- and C-terminal fragments in the WT line ( Supplemental figure 6 ). While five out of nine PfGCN5 complex members were found in both the WT and Del3 rescue lines, the stoichiometry of one of its members was dysregulated in the deletion lines (Supplemental table 1) 16 . Specifically, the Nucleosome Assembly Protein (NAPS; PF3D7_0919000) had a threefold increase. Furthermore, the adaptor protein 14-3-3I (PF3D7_0818200) and another PHD-domain-containing protein (PF3D7_0310200), previously found significantly enriched in PfGCN5 pull-downs, were enriched several folds in the deletion lines 16 . These findings indicate that the N-terminal domain containing the poly-N repeat interacts with the C-terminal catalytic domain post-cleavage. Such an association could promote the participation of NAPS in the SAGA-like complex. Discussion In this study, we report a functional role for a poly-N repeat in a P. falciparum protein. With approximately 1,300 proteins containing poly-N repeats, this is the most prevalent characteristic of the P. falciparum proteome that has remained elusive in the biology of this deadly parasite. While the function of such repeats may vary across individual proteins, our findings suggest an important regulatory function in this instance, which opens a new area of research in parasite biology. We highlighted the existence of extended LC-IDRs in the N terminus of GCN5 homologs from Plasmodium species that are naturally exposed to fever, in stark contrast to those that do not encounter this stress. Remarkably, PvGCN5, which carries a GS-rich repeat, rescued the growth of a PfGCN5 KO line, and deletion of the poly-N repeat from PfGCN5 phenocopied the growth profile of the PyGCN5 (short higher-complexity repeat) rescue line. This suggests that the functional contribution of the N-terminal LC-IDR does not depend strictly on N residues but rather on the biophysical properties shared by polar and uncharged amino acid repeats. Thus, Plasmodium species may utilize synonymous “amino acid grammar” to achieve similar biological outcomes within their shared host. Mounting data in other systems has demonstrated the capacity for polar tracts rich in N, Q, G, and S to phase separate through dynamic multivalent interactions 49 – 51 . This phenomenon aids cellular organization, transcriptional regulation, genome maintenance, and complex formation, among others 52 , 53 . Whether LC-IDRs composed of poly-N repeats in P. falciparum phase separate remains to be solved. Intriguingly, 7 out 9 proteins forming the PfGCN5 saga-like complex also contain prominent poly-N repeats 16 . Some of these may enhance interaction among complex members, provide better anchoring to histones, or allow transient interactions during stress conditions. Indeed, we found that the Del3 protein failed to maintain the stoichiometry levels of one complex member (NAPS) in comparison to the WT protein. Furthermore, we observed that 14-3-3I and a PHD-containing protein were several-fold enriched in the Del3 line. 14-3-3I is a scaffold protein that binds to phosphorylated serine/threonine residues, modulating the function, localization, and stability of its binding partners, as well as stabilizing protein complexes by facilitating protein-protein interactions 54 , 55 . Given that PfGCN5 is extensively phosphorylated, we speculate that 14-3-3I may maintain the complex in tight engagement by binding to PfGCN5 and members of the SAGA-like complex simultaneously 56 Finally, the function of the PHD-containing protein could replace that of the PHD2 since both proteins bind to methylated histones. It has been proposed that poly-N repeats in P. falciparum can behave as “tRNA sponges” by slowing down ribosomal translation, giving the expected limited availability of asparaginylated tRNAs 57 . However, we demonstrated that the PfGCN5 Del3 truncated protein is expressed at the same level as the WT second copy, suggesting that translation efficiency did not depend on the amount of N residues. Extensive truncation studies in the N-terminal extension of TgGCN5A/B, identified divergent nuclear localization signals embedded in intrinsically disordered regions 58 . In PfGCN5, we demonstrated that, like the WT, the Del3 mutant PfGCN5 localizes to the nucleus, indicating that the poly-N repeat in PfGCN5 does not mediate nuclear trafficking. Together, we established that reducing the length of the poly-N repeat in PfGCN5 does not affect expression levels, processing, or localization. Substantial data support the essential role of GCN5 during stress by remodeling chromatin structure through histone acetylation and by binding to a subset of genes directly implicated in stress tolerance, in both P. falciparum and other organisms 14 , 15 , 59 , 60 . We showed that the Del3 line is highly susceptible to heat, cold, and drug stress, suggesting that the poly-N repeat aids PfGCN5 activity during stress, but further investigation is needed to uncover the molecular mechanism behind this. Cellular fractionation studies examining the N terminus, in conjunction with observations from E64D and Ivermectin treatments, demonstrated that the full-length PfGCN5 is exported to the nucleus, likely via the importin α/β transport machinery, where a cysteine protease subsequently processes it. Luminescence-based western blotting coupled with live-cell kinetics using a split nano-luciferase system allowed us to conclude that once in the nucleus, the N-terminus containing the poly-N repeat is cleaved into 120 kDa and 110 kDa processed forms. Later, midway through the intraerythrocytic cycle, a 50 kDa fragment is produced and exported back to the cytosol. Given that PfGCN5 has cytosolic non-histone substrates, it is possible that such bidirectional trafficking of the poly-N repeat contributes to this 38 , 61 . Inspection of N- and C-terminal blots indicates that PfGCN5 is alternatively cleaved, producing alternative 120 kDa fragments from the N- or C-terminus. This work determined that the poly-N repeat stabilizes the N-terminal polypeptide after cleavage from the catalytic domain. Extensive data demonstrate that LC-IDRs are often targets of post-translational modifications, which subsequently modulate stability by promoting conformational changes, protein interactions, or mediating subcellular localization 62 , 63 . Alternatively, the flexible nature of the poly-N repeat could facilitate transient interactions that favor stability 64 . Only H3K9ac, H3K14ac, H3K4me3, and H2B.Zac had been described as modifications catalyzed by PfGCN5 16,17 . Here, we extended the post-translational landscape mediated by PfGCN5 to H3K9acK14ac, H3K18ac, H3K23ac, H3K18acK23ac, H3K27ac, H3.3K9ac, H3.3K9acK14ac, and H4K8acK16ac. Although we did not find significant changes for H3.3K9ac, a conserved trend was observed between PfGCN5 LoxP parasites treated with or without rapamycin and the WT and Del3 complemented lines. We did not find evidence that PfGCN5 promotes methylation of H3. While histone acetylation of H3 and H3.3 has been linked to parasite development, stress response, and antigenic variation, H4 acetylation has been associated with both parasite development and DNA repair 65 , 66 . Moreover, we identified several concomitant histone acetylation marks associated with PfGCN5 activity, indicating that it plays a crucial role in writing the “chromatin code”. Histone acetylation changes in the Del3 line underpin the importance of the poly-N repeat during its stay in the nucleus. Interestingly, we detected interaction between the C-terminal fragments carrying the catalytic domain and the N-terminal polypeptide containing the poly-N repeat post-cleavage. Such interaction occurs with the N-terminal 120 kDa and/or 110 kDa fragments, which only reside in the nucleus, and are apparent during the first half of the cycle. The GCN5 N/C-terminal fragments could interact directly or could associate through other components of the SAGA-like complex. Nevertheless, it is clear that the poly-N repeat stabilizes the N-terminal fragment of PfGCN5 and is important for C-terminal histone acetylation activity in vivo . A structure of this complex would be revealing. Materials and methods Reagents All primers were obtained from Integrated DNA Technologies. The list of primers used in this study can be found in Appendix A, Table 1A. Gene blocks corresponding to pvgcn5, and recoded pfgcn5 and pygcn5 coding sequences were synthetized by Genewiz. Restriction enzymes and Gibson Assembly® Master Mix were purchased from New England Biolabs. For site-directed mutagenesis, we used the Quick-Change Lightning kit from Agilent. The rabbit anti-HA antibody and the mouse anti-flag magnetic beads were obtained from Millipore Sigma. The rat anti-FLAG antibody was obtained from Novus Biologicals. The mouse anti-PMV antibody was previously described 67 . The mouse anti-H3 and H3K9ac were obtained from Epigentek and Active Motif, respectively. Mouse anti-LgBiT and anti-HiBiT antibodies were obtained from Promega. Rapamycin, Brefeldin A, E64D, Dihydroartemisinin, Saponin, Sorbitol, WR99210, and the stain for thin smears (Hemacolor®) were purchased from Millipore Sigma. Generation of plasmids A S. cerevisiae recoded version of pfgcn5 was inserted at the AsiSI restriction site of the pSN054 plasmid, previously described by Polino et al. 2020 68 .The GFP sequence amplified with primers KR24 and KR25, was inserted at the AsiSI restriction site in the pSN054. The AsiSI restriction site was restored upon cloning, allowing for a S. cerevisiae recoded version of pfgcn5 amplified with primers KR137 and KR138 to be in frame with GFP and 3xHA, upon Gibson assembly. The 490 bp immediately upstream of the pfgcn5’s start codon amplified with primer pair KR135 and KR136, and 792 bp downstream of the stop codon amplified with KR47 and KR21, were used as the left (LHR) and right homologous (RHR) region, respectively. The column-purified LHR and RHR were fused to restriction sites FseI and I-SceI, respectively, using Gibson assembly. This resulted in the pSN054_PfGCN5-GFP-3HA-LoxP plasmid that was used to modify the pfgcn5 locus. For generating rescue constructs, we used the pEOE-2X-attP-3xFlag described elsewhere 69 . An upstream region, extending 987 bp from the pfgcn5 start codon, was amplified with primer pair KR104 and KR105 and utilized as a promoter. GCN5 variants amplified with the indicated primers in Supplemental table 2 were introduced at the AvrII restriction site. All amplicons were introduced using In-Fusion cloning. To make the deletion constructs and HiBiT insertion, site-directed mutagenesis was employed using primers KR333, KR334, KR335, and KR300. For generating the LgBiT line, we used the pTEOE vector previously described and inserted the LgBiT amplicon at the XhoI site using in-fusion cloning 70 . The LgBiT ORF was amplified from the LgBiT expression vector from Promega using primers KR256 and KR258. For CRISPR/Cas9 editing, we used the Cas9-encoding pAIO3 plasmid, previously described 71 . Primers and the corresponding reverse complement needed to make gRNAs 45 were annealed in a thermal cycler and inserted into the AvrII restriction site of pAIO3 by In-Fusion cloning. All plasmids and their parasite integration products were analyzed by PCR and sequencing. Parasite culture, transfection, selection, and synchronization NF54 attB parasites expressing Cre recombinase 32 , 33 were maintained in human red blood cells (3% hematocrit) prepared in RPMI 1640 supplemented with AlbuMAX (2.5 g/L), sodium pyruvate (110 mg/L), hypoxanthine (15 mg/L), HEPES (1.19 g/L), sodium bicarbonate (2.52 g/L), glucose (2 g/L), and gentamycin (10 ug/L). For CRISPR/Cas9 editing of the endogenous pfgcn5 locus, we used cultures with more than 5% ring stages and transfected parasites via electroporation using a Bio-Rad Gene Pulser. For complementation of the PfGCN5 LoxP line, plasmids containing WT, GCN5 homologs, PyChimera or deletion constructs were independently co-transfected with a Bxb1 integrase plasmid for integration at the non-essential locus cg6 72 . For rapamycin-mediated excision, we used a concentration of 20 nM for 24 hours. While the PfGCN5 LoxP line was maintained in 2.5 µg/mL blasticidin (BSD), all rescue lines were kept in a medium containing 2.5 µg/mL BSD and 5 nM WR99210 (WR). The LgBiT or LgBiT-HiBiT lines were supplemented with 12.5 µg/mL DSM1 or 12.5 µg/mL DSM1 and 5 nM WR, respectively, for selection. Parasites were cloned by limiting dilution seeding ~ 0.5 parasites per well in a 96-well plate. For synchronization, asynchronous cultures with more than 5% schizont stages were allowed to run through a MACS LD magnet column (Miltenyi Biotec). After a wash with warmed media, the column was removed from the magnet and eluted into a 15 mL conical tube. Purified schizonts were allowed to egress for 3 hours in uninfected red blood cells. Newly infected red blood cells were purified by incubation with 5% sorbitol for 10 min. Growth curves and flow cytometry Parasites were seeded at a starting parasitemia of 1% with or without rapamycin and plated in triplicate on a 96-well plate. Every other day, the media was changed, and the parasitemia was measured by flow cytometry using 50,000 events recorded per sample in an Attune NxT Flow cytometer. Cells were stained with acridine orange diluted 1:25 in PBS. At day 4, after parasitemia was recorded, cultures were diluted 1:5 to prevent overgrowth. Cumulative parasitemia was back calculated based on the dilution factor. Measured parasitemia at day 0 was subtracted from the final parasitemia obtained on day 6 to control for differences at the start of the experiment. The average parasitemia on day 6 for the PfGCN5 KO line in the absence of rapamycin was considered 100% growth. The percentage growth for the rescue lines was calculated as the ratio between the average parasitemia from each line and the parasitemia from the PfGCN5 KO line on day 6. Western blotting To visualize the PfGCN5 endogenous protein, we processed at least 5 mL of culture at 5% parasitemia and lysed it in PBS containing 0.035% saponin at 4°C for 5 min. Saponin pellets were then resuspended in 1x sample buffer containing beta-mercaptoethanol and boiled at 99°C. After centrifugation at 14,000 rpm in a microfuge at 4°C for 10 min, a fraction of the supernatant was subjected to SDS-PAGE and immunoblotting. To visualize the GCN5 variants, we processed at least 20 mL of culture at 5% parasitemia and lysed as described above. Saponin pellets were resuspended in an NP40-containing lysis buffer (0.1% NP40, 50mM Tris, and 150 mM NaCl, 1x HALT protease inhibitor) and subjected to three cycles of freezing and thawing, followed by sonication. After spinning at 14,000 rpm at 4°C for 10 min, protein lysates were incubated with mouse anti-FLAG magnetic beads overnight at 4°C. Elution was performed in boiling 2x sample buffer containing beta-mercaptoethanol. Flow-throughs were used to visualize various loading controls upon western blotting. Primary antibodies included rabbit anti-HA (1:1,000), rat anti-FLAG (1:1,000), mouse anti-histone H3 (1:1,000), mouse anti-H3K9ac (1:1,000), mouse anti-LgBiT (1:1,000), mouse anti-HiBiT (1:1,000), rabbit anti-HAD1 (1:1,000), and mouse anti-PMV (1:500). In each case, corresponding IRDye conjugated secondary antibodies were used at 1:10,000 dilution. An Odyssey imaging system (Licor) was utilized to visualize blots. Immunofluorescence assays Cells were fixed and permeabilized in Hemacolor® fixing solution (product number 1.11955) for 10 seconds and then rinsed three times in PBS. Blocking was performed using 3% BSA in PBS for one hour at room temperature or overnight at 4°C. Dilutions used for primary antibodies are as follows: rabbit anti-HA (1:500), rat anti-FLAG (1:500), rabbit anti-aldolase (1:500), mouse anti-LgBiT (1:250), and mouse anti-HiBiT (1:500). Secondary antibodies, Alexa Fluor 488 or 555 (Life Technologies), were used at a 1:2,000 dilution. ProLong antifade and 4’,6’-diamidino-2-phenylindole (DAPI) (Invitrogen) were used to mount cells. Images were taken in a Zeiss Imager M2 Plus wide-field fluorescence microscope. Cytoplasmic and nuclear fractionation Parasites were lysed in half of the culture volume with cold 0.035% saponin for 4 min at 4°C and washed with cold 1x PBS. Parasite pellets were then resuspended in cytoplasmic lysis buffer (25mM Tris-HCl pH 7.5, 10mM NaCl, 1.5mM MgCl2, 1% Igepal, halt protease inhibitor cocktail, 1mM PMSF, 50mM sodium fluoride and 1mM sodium orthovanadate) and incubated on ice for 30 minutes. For complete and gentle homogenization, samples were macerated in a cold glass douncer and centrifuged at 13,300 rpm for 10 min at 4C. Supernatants were saved as cytoplasmic fractions, and the remaining pellets were lysed in 0.1% Igepal buffer containing 150mM NaCl and 50mM Tris HCl pH 7.6 for immunoprecipitations or 1x sample buffer containing beta-mercaptoethanol for western blots. Immunoprecipitation Saponin pellets were resuspended in 0.1% Igepal buffer (150mM NaCl and 50mM Tris HCl pH 7.6) containing Halt™ protease inhibitor cocktail EDTA-free (ThermoFisher, cat. number 78425), 1mM PMSF, 50mM sodium fluoride and 1mM sodium orthovanadate. After three cycles of freezing and thawing, samples were sonicated and spun at 13,500 rpm for 10min at 4C. The recovered supernatant was incubated overnight at 4C with mouse-anti FLAG magnetic beads (Sigma, cat. number M8823) or mouse-anti HiBiT magnetic beads (cat. number CS3278A08). Antigen-conjugated beads were magnetized and washed two times with 1X TBS. The antigen was eluted in boiling 2X sample buffer containing beta-mercaptoethanol. In some cases, elution was performed using 0.1M Glycine pH 2.0 in continuous rotation at room temperature for 15 minutes. Once the eluted antigen was recovered the pH was neutralized using 1M Tris pH 8.0. Histone extraction Approximately 10 mL of synchronous cultures at 5% parasitemia and 3% hematocrit were treated with cold 0.035% saponin in PBS and then washed with cold PBS. Cell pellets were washed twice with a nuclear extraction buffer (15 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 5 mM MgCl 2 , 1 mM CaCl 2 , 250 mM sucrose, 500 uM AEBSF, 1 mM DTT, 5 nM microcystin, 10 mM sodium butyrate, and 1x HALT protease cocktail inhibitor). Briefly, washed cell pellets were treated with the above-described nuclear extraction buffer containing 0.3% NP-40 and incubated on ice for 30 minutes, followed by homogenization in a chilled douncer. After centrifugation at 2,000g at 4°C for 10 minutes, the supernatant (cytosolic fraction) was removed, and the pellet (nuclear fraction) was washed twice in the nuclear extraction buffer. Following centrifugation as above, the pellet was resuspended in chilled 0.2 M H 2 SO 4 and incubated with constant rotation for 2 hours at 4°C. Posterior to centrifugation at 3,400g at 4°C for 10 minutes, solubilized histones were then treated with 100% trichloroacetic acid (~ 25% of the total volume) and incubated overnight at 4°C. Precipitated histones were then washed with cold acetone containing 0.1% HCl, followed by a final wash with ice-cold acetone before resuspending extracted histones in 50mM ammonium bicarbonate. Mass spectrometry identification of histone acetylation modifications The histones were extracted and prepared for chemical derivatization and digestion as described previously 73 , 74 . In brief, the lysine residues from histones were derivatized with the propionylation reagent (1:2 reagent:sample ratio) containing acetonitrile and propionic anhydride (3:1), and the solution pH was adjusted to 8.0 using ammonium hydroxide. The propionylation was performed twice and the samples were dried on speed vac. The derivatized histones were then digested with trypsin at a 1:50 ratio (wt/wt) in 50 mM ammonium bicarbonate buffer at 37°C overnight. The N-termini of histone peptides were derivatized with the propionylation reagent twice and dried on speed vac. The peptides were desalted with the self-packed C18 stage tip. The purified peptides were then dried and reconstituted in 0.1% formic acid. An LC-MS/MS system consisted of a Vanquish Neo UHPLC coupled to an Orbitrap Exploris 240 (Thermo Scientific) was used for peptide analysis. Histones peptide samples were maintained at 7°C on sample tray in LC. Separation of peptides was carried out on an Easy-Spray™ PepMap™ Neo nano-column (2 µm, C18, 75 µm X 150 mm) at room temperature with a mobile phase. The chromatography conditions consisted of a linear gradient from 2 to 32% solvent B (0.1% formic acid in 100% acetonitrile) in solvent A (0.1% formic acid in water) over 48 min and then 42 to 98% solvent B over 12 min at a flow rate of 300 nL/min. The mass spectrometer was programmed for data-independent acquisition (DIA). One acquisition cycle consisted of a full MS scan, 35 DIA MS/MS scans of 24 m/z isolation width starting from 295 m/z to reach 1100 m/z. Typically, full MS scans were acquired in the Orbitrap mass analyzer across 290–1100 m/z at a resolution of 60,000 in positive profile mode with an auto maximum injection time and an AGC target of 300%. MS/MS data from HCD fragmentation was collected in the the Orbitrap. These scans typically used an NCE of 30, an AGC target of 1000%, and a maximum injection time of 60 ms. Histone MS data were analyzed with EpiProfile 75 . HiBiT blotting Proteins were transferred to PDVF membranes followed by 5 washes with 0.1% TBS-T. Wash buffer was discarded, and a solution containing the LgBiT protein diluted 200-fold in the 1X Nano-Glo® buffer was added and incubated overnight at 4°C with gentle rocking. The next day, after allowing membranes to equilibrate to room temperature, furimazine (substrate) was added at a 1:500 dilution and incubated for 5 minutes before imaging. Luminescence assays Live-cell kinetics of cytosolic HiBiT-tagged fragments were determined using the Nano-Glo® Live Cell Assay System (Promega, cat. number N2011) as per manufacturer instructions. Briefly, 100uL of cell cultures at 3% hematocrit and 5% parasitemia were mixed with 25uL of the Nano-Glo® buffer containing the substrate at 1:20 dilution in opaque white 96-well plates. After gently mixing by hand, luminescence was immediately measured in a Perkin Elmer EnVision 2103 microplate reader. Total levels of HiBiT-tagged fragments were determined using the Nano-Glo® HiBiT Lytic Detection System (Promega, cat. number N3030) as per manufacturer instructions. An equal volume of cell cultures at 3% hematocrit and 5% parasitemia were mixed with the lytic buffer containing the LgBiT protein and substrate at a 1:100:50 dilution. Plates were incubated in a dark environment at room temperature for 10 min before measuring luminescence in a Perkin Elmer EnVision 2103 microplate reader. Declarations Contributions K.R. and D.E.G. conceived and designed the study. K.R. performed experiments, acquired and analyzed data. A. M. and S. M. acquired data. F.D.L.V. and B.G. performed high-resolution mass spectrometry identification of histone acetylation. K.R. and D.E.G. wrote the manuscript. Acknowledgments This work was supported by the American Heart Association predoctoral fellowship (23PRE1026393) provided to K. R. We thank Dr. Eva Istvan and Dr. Muhammad Hasan for helpful suggestions, Barbara Vaupel for her assistance with cloning, Dr. David Fidock for the NF54 attB line, Dr. Josh Beck for the NF54-attB-DiCre expressing line, Audrey Odom John for anti-HAD1 antiserum, and the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center for LC/MS data acquisition and analysis. References Gardner MJ et al (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419:498–511 Hamilton WL et al (2017) Extreme mutation bias and high AT content in Plasmodium falciparum. Nucleic Acids Res 45:1889–1901 Su X, Lane KD, Xia L, Sá JM, Wellems TE (2019) Plasmodium genomics and genetics: new insights into malaria pathogenesis, drug resistance, epidemiology, and evolution. Clin Microbiol Rev 32:e00019–e00019 Singh GP et al (2004) Hyper-expansion of asparagines correlates with an abundance of proteins with prion-like domains in Plasmodium falciparum. Mol Biochem Parasitol 137:307–319 Pizzi E, Frontali C (2001) Low-complexity regions in Plasmodium falciparum proteins. Genome Res 11:218–229 Zilversmit MM et al (2010) Low-complexity regions in Plasmodium falciparum: missing links in the evolution of an extreme genome. Mol Biol Evol 27:2198–2209 Muralidharan V, Goldberg DE (2013) Asparagine repeats in Plasmodium falciparum proteins: good for nothing? PLoS Pathog 9:e1003488 Lu X, Murphy RM (2015) Asparagine repeat peptides: aggregation kinetics and comparison with glutamine repeats. Biochemistry 54:4784–4794 Muralidharan V, Oksman A, Pal P, Lindquist S, Goldberg DE (2012) Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nat Commun 3:1–10 Muralidharan V, Oksman A, Iwamoto M, Wandless TJ, Goldberg DE (2011) Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag. Proceedings of the National Academy of Sciences 108, 4411–4416 Josling GA, Selvarajah SA, Petter M, Duffy MF (2012) The role of bromodomain proteins in regulating gene expression. Genes vol. 3 320–343 Preprint at https://doi.org/10.3390/genes3020320 Grant PA et al (1998) A Subset of TAFIIs Are Integral Components of the SAGA Complex Required for Nucleosome Acetylation and Transcriptional Stimulation. Cell 94:45–53 Sun J et al (2018) Structural basis for activation of SAGA histone acetyltransferase Gcn5 by partner subunit Ada2. Proc Natl Acad Sci U S A 115:10010–10015 Rawat M et al (2021) Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum. Scientific Reports 2021 11:1 11, 1–13 Lucky AB et al (2023) Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses. Antimicrob Agents Chemother 67 Miao J et al (2021) A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum. PLoS Pathog 17:e1009351 Tang J et al (2025) PfGCN5 is essential for Plasmodium falciparum survival and transmission and regulates Pf H2B.Z acetylation and chromatin structure. Nucleic Acids Res 53 Fan Q, An L, Cui L (2004) Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling. Eukaryot Cell 3:264–276 Cui L et al (2007) PfGCN5-mediated histone H3 acetylation plays a key role in gene expression in Plasmodium falciparum. Eukaryot Cell 6:1219–1227 Xu W, Edmondson DG, Roth SY (1998) Mammalian GCN5 and P/CAF acetyltransferases have homologous amino-terminal domains important for recognition of nucleosomal substrates. Mol Cell Biol 18:5659–5669 Smith ER et al (1998) Cloning of Drosophila GCN5: Conserved Features among Metazoan GCN5 Family Members. Nucleic Acids Res 26 http://ulrec3.unil.ch:80/ Martel A, Brar H, Mayer BF, Charron JB (2017) Diversification of the histone acetyltransferase GCN5 through alternative splicing in brachypodium distachyon. Front Plant Sci 8 Haque ME et al (2021) The GCN5: Its biological functions and therapeutic potentials. Clinical Science vol. 135 Preprint at https://doi.org/10.1042/CS20200986 Toma-Fukai S et al (2020) Crystal structure of GCN5 PCAF N-terminal domain reveals atypical ubiquitin ligase structure. J Biol Chem 295:14630–14639 Bhatti MM, Livingston M, Mullapudi N, Sullivan WJ (2006) Pair of unusual GCN5 histone acetyltransferases and ADA2 homologues in the protozoan parasite Toxoplasma gondii. Eukaryot Cell 5:62–76 Bhatti MM, Sullivan WJ (2005) Histone acetylase GCN5 enters the nucleus via importin-α in protozoan parasite Toxoplasma gondii. J Biol Chem 280:5902–5908 Prinzinger R, Preßmar A, Schleucher E (1991) Body temperature in birds. Comp Biochem Physiol Physiol 99:499–506 Amani V et al (1998) Cloned lines of Plasmodium berghei ANKA differ in their abilities to induce experimental cerebral malaria. Infect Immun 66:4093–4099 Cumnock K et al (2018) Host Energy Source Is Important for Disease Tolerance to Malaria. Curr Biol 28:1635–1642e3 Collins CR et al (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol 88:687–701 Collins CR et al (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol 88:687 Adjalley SH, Lee MCS, Fidock DA (2010) A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. Methods Mol Biol 634:87–100 Fierro MA, Hussain T, Campin LJ, Beck JR (2023) Knock-sideways by inducible ER retrieval enables a unique approach for studying Plasmodium-secreted proteins. Proc Natl Acad Sci U S A 120:e2308676120 Bhowmick K et al (2020) Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway that is essential for its function. J Cell Sci 133 Johnsson A, Xue-Franzén Y, Lundin M, Wright APH (2006) Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes. Eukaryot Cell 5:1337–1346 Gan L, Wei Z, Yang Z, Li F, Wang Z (2021) Updated mechanisms of GCN5—the monkey king of the plant kingdom in plant development and resistance to abiotic stresses. Cells vol. 10 Preprint at https://doi.org/10.3390/cells10050979 Xue-Franzén Y et al (2010) Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles. BMC Genomics 11 Rawat M, Malhotra R, Shintre S, Pani S, Karmodiya K (2020) Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum. J Biosci 45 Bridgford JL et al (2018) Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome. Nature Communications 2018 9:1 9, 1–9 Lu KY et al (2020) Phosphatidylinositol 3-phosphate and hsp70 protect plasmodium falciparum from heat-induced cell death. Elife 9:1–27 Lavrentieva A et al (2025) Viability of Plasmodium falciparum parasites in human plasma under different storage conditions. Vox Sang 120:149–154 Miao J et al (2006) The malaria parasite Plasmodium falciparum histones: Organization, expression, and acetylation. Gene 369:53–65 Nagar P et al (2024) Plasmodium falciparum cysteine protease Falcipain 3: A potential enzyme for proteolytic processing of histone acetyltransferase PfGCN5. Biotechnol Appl Biochem 71:1304–1315 Banerjee R et al (2002) Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Proc Natl Acad Sci U S A 99:990–995 Mukherjee S, Nguyen S, Sharma E, Goldberg DE (2022) Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X. Nature Communications 2022 13:1 13, 1–14 Panchal M et al (2014) Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP. Cell Death Dis 5 Balu B et al (2009) piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome. BMC Microbiol 9:83 Oh-hashi K, Furuta E, Fujimura K, Hirata Y (2017) Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells. Biochem Biophys Rep 12:40–45 March ZM, King OD, Shorter J (2016) Prion-like domains as epigenetic regulators, scaffolds for subcellular organization, and drivers of neurodegenerative disease. Brain Research vol. 1647 9–18 Preprint at https://doi.org/10.1016/j.brainres.2016.02.037 Jung JH et al (2020) A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis. Nature 585:256–260 Franzmann TM, Alberti S (2019) Prion-like low-complexity sequences: Key regulators of protein solubility and phase behavior. J Biol Chem 294:7128–7136 Holehouse AS, Kragelund BB (2024) The molecular basis for cellular function of intrinsically disordered protein regions. Nature Reviews Molecular Cell Biology vol. 25 187–211 Preprint at https://doi.org/10.1038/s41580-023-00673-0 Boeynaems S et al (2018) Protein phase separation: a new phase in cell biology. Trends Cell Biol 28:420–435 Obsil T, Obsilova V (2011) Structural basis of 14-3-3 protein functions. Seminars in Cell and Developmental Biology vol. 22 663–672 Preprint at https://doi.org/10.1016/j.semcdb.2011.09.001 Tzivion G, Shen YH, Zhu J (2001) 14-3-3 Proteins; bringing new definitions to scaffolding. Oncogene vol. 20 6331–6338 Preprint at https://doi.org/10.1038/sj.onc.1204777 Alam MM et al (2015) Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion. Nat Commun 6 Filisetti D et al (2013) Aminoacylation of plasmodium falciparum trnaasn and insights in the synthesis of asparagine repeats. J Biol Chem 288:36361–36371 Dixon SE, Bhatti MM, Uversky VN, Dunker AK, Sullivan WJ (2011) Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B. Mol Biochem Parasitol 175:192–195 Chao X et al (2024) Histone Acetyltransferase GCN5 Regulates Rice Growth and Development and Enhances Salt Tolerance. Rice Sci. 10.1016/j.rsci.2024.06.002 Gaupel AC, Begley TJ, Tenniswood M (2015) Gcn5 Modulates the Cellular Response to Oxidative Stress and Histone Deacetylase Inhibition. J Cell Biochem 116:1982–1992 Ankita Tehlan PNRPK (2025) B. A. K. I. K. N. S. S. K. D. Plasmodium falciparum acetyltransferase GCN5 acts as a dual regulator of essential glycolytic enzyme phosphoglycerate mutase. FEBS J Bah A, Forman-Kay JD (2016) Modulation of intrinsically disordered protein function by post-translational modifications. J Biol Chem 291:6696–6705 Van Der Lee R et al (2014) Classification of intrinsically disordered regions and proteins. Chemical Reviews vol. 114 6589–6631 Preprint at https://doi.org/10.1021/cr400525m Chakrabarti P, Chakravarty D (2022) Intrinsically disordered proteins/regions and insight into their biomolecular interactions. Biophys Chem 283 Gupta DK, Patra AT, Zhu L, Gupta AP, Bozdech Z (2016) DNA damage regulation and its role in drug-related phenotypes in the malaria parasites. Sci Rep 6:23603 Fraschka SA-K, Henderson RWM, Bártfai R (2016) H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum. Sci Rep 6:31965 Banerjee R et al (2002) Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Proceedings of the National Academy of Sciences 99, 990–995 Polino AJ, Nasamu AS, Niles JC, Goldberg DE (2020) Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin v. ACS Infect Dis 6:738–746 Mukherjee S, Nguyen S, Sharma E, Goldberg DE (2022) Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X. Nat Commun 13 Sigala PA, Crowley JR, Henderson JP, Goldberg DE (2015) Deconvoluting heme biosynthesis to target blood-stage malaria parasites. Elife 4 Nessel T et al (2020) EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole. Cell Microbiol 22 Adjalley SH, Lee MCS, Fidock DA (2010) Springer,. A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. in In Vitro Mutagenesis Protocols 87–100 Sidoli S, Bhanu NV, Karch KR, Wang X, Garcia BA (2016) Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: From histone extraction to data analysis. Journal of Visualized Experiments (2016) Bhanu NV, Sidoli S, Garcia BA (2020) A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry. Bio Protoc 10:e3756 Yuan ZF et al (2015) Epiprofile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol Cell Proteomics 14:1696–1707 Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7490809\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":509204425,\"identity\":\"9abfef76-3ddf-4888-84cb-3325b448e42f\",\"order_by\":0,\"name\":\"Kelly Rubiano\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kelly\",\"middleName\":\"\",\"lastName\":\"Rubiano\",\"suffix\":\"\"},{\"id\":509204426,\"identity\":\"2c11f83a-4327-4b77-abf2-c246b7964591\",\"order_by\":1,\"name\":\"Aaron Morris\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Biology, Washington University in St. Louis, MO, USA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Aaron\",\"middleName\":\"\",\"lastName\":\"Morris\",\"suffix\":\"\"},{\"id\":509204427,\"identity\":\"0097b44b-1112-4ec5-acf3-529433ee1468\",\"order_by\":2,\"name\":\"Francisca De Luna Vitorino\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-8543-2299\",\"institution\":\"Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Francisca\",\"middleName\":\"De Luna\",\"lastName\":\"Vitorino\",\"suffix\":\"\"},{\"id\":509204428,\"identity\":\"97a80d40-e0de-4689-83f7-65f8ba05f6c4\",\"order_by\":3,\"name\":\"Benjamin A. Garcia\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0003-3596-4750\",\"institution\":\"Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Benjamin\",\"middleName\":\"A.\",\"lastName\":\"Garcia\",\"suffix\":\"\"},{\"id\":509204429,\"identity\":\"8878b01a-8b30-4beb-a4a0-73fa84ffcdee\",\"order_by\":4,\"name\":\"Sumit Mukherjee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Biology, Texas State University, San Marcos, TX, USA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sumit\",\"middleName\":\"\",\"lastName\":\"Mukherjee\",\"suffix\":\"\"},{\"id\":509204424,\"identity\":\"5b75a55d-1c03-435c-9888-5433a61dbdc9\",\"order_by\":5,\"name\":\"Daniel E. Goldberg\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYDCCAwyMB3gbgAx2EMFgAcQJBLUwQLTwHACpliBFi0QCkVr4zi8+cODtDpt8+cg3ZpI/f0gw8LPnGODVInnjWcLBuWfSLDfezjGT5gHaItnzBr8WgxtnDA7zth02MJydu00a5DCDGwRsgWr5b2A48+w2yR9ALfYEtZzvAWk5YCAvwbtNAuQwAwmCfmED+SXZwIAn/7M1T5oEj8SZZwV4tfCdP3zwwdsddgby7ccSb/6wsZHjb0/egFcLJDpALjwAoXnwKwcBfqhS+QbCakfBKBgFo2CEAgDjpk1YSf0FvwAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0003-3529-8399\",\"institution\":\"Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Daniel\",\"middleName\":\"E.\",\"lastName\":\"Goldberg\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-08-29 18:05:36\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7490809/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7490809/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":90571169,\"identity\":\"5a059e64-afba-4511-9f8b-8aa3229630d7\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:07\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":294149,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLow-complexity and higher-complexity repeats in the N termini of plasmodial GCN5 homologs. A) \\u003c/strong\\u003eLow-complexity regions containing poly-N or GS-repeats in parasites naturally encountering fever during their intraerythrocytic life cycle, highlighted in pink. Short repetitive repeats of higher complexity found in parasites that do not encounter fever during their intraerythrocytic life cycle, highlighted in light blue. Thermometer cartoons in red and blue indicate whether hosts experience fever or not, respectively. Black line represents the sequence deleted in Del1; blue line represents additional sequence deleted in Del2; orange line depicts additional sequence deleted in Del3 lines. Therefore, the deleted region in Del3 encompasses the combined length of the three lines. The purple triangle at amino acid 238 represents the HiBiT insertion site. \\u003cstrong\\u003eB) \\u003c/strong\\u003eSequence logo of short repeats in \\u003cem\\u003eP. yoelii \\u003c/em\\u003eand \\u003cem\\u003eP. berghei\\u003c/em\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/24053253a2327c29f768c23e.png\"},{\"id\":90571174,\"identity\":\"0f99cf87-0cf4-4017-ac57-da6f96be7444\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:07\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":246264,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe N terminus of PfGCN5 containing the poly-asparagine domain is required for normal parasite growth. A) \\u003c/strong\\u003eGiemsa-stained thin smears from PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites treated with or without rapamycin (RAP). \\u003cstrong\\u003eB) \\u003c/strong\\u003eGrowth of PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites treated with or without RAP, measured by flow cytometry every two days over 6 days. Mean values from three independent experiments are shown, and error bars represent standard deviations. Data were analyzed statistically by a two-tailed Student’s test, and the p-value\\u003cem\\u003e \\u003c/em\\u003eis shown on the graph. \\u003cstrong\\u003eC) \\u003c/strong\\u003eRepresentative western blot from three biological replicates illustrating the differences in protein expression of PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites treated with or without RAP during the first intraerythrocytic cycle. hpi: hours post-infection. An anti-HA antibody was used to recognize C-terminally processed forms of endogenous PfGCN5, and an anti-histone H3 antibody was used as a loading control. Right panel: PfGCN5 expression was quantified by densitometry after normalizing the HA signal to that of H3. Shown is the fold change between -RAP/+RAP. Mean ± SD of three biological replicates. \\u003cstrong\\u003eD) \\u003c/strong\\u003eSchematic representation of plasmodial GCN5 homologs and chimeric construct used to rescue the PfGCN5 KO, depicting the conserved histone acetyltransferase (HAT) domain and bromodomain (BrD) in grey and conserved motif expanding from amino acid 677 to 747 in black used to delimit the PfGCN5 N terminus for the chimera. Homologs from \\u003cem\\u003ePlasmodium \\u003c/em\\u003espp. such as \\u003cem\\u003eP. falciparum \\u003c/em\\u003eand \\u003cem\\u003eP. vivax \\u003c/em\\u003edisplay N-terminal low-complexity repeats highlighted in pink. In contrast, \\u003cem\\u003eP. yoelii \\u003c/em\\u003edisplays\\u003cem\\u003e \\u003c/em\\u003ea\\u003cem\\u003e \\u003c/em\\u003eshort, repetitive sequence of higher complexity, indicated in dark blue. \\u003cstrong\\u003eE) \\u003c/strong\\u003eGrowth of GCN5 variant rescue lines in the presence or absence of RAP relative to the control (PfGCN5\\u003csup\\u003eLoxP \\u003c/sup\\u003eparasites in the absence of RAP) after three intraerythrocytic cycles. The distribution of three or four independent experiments is shown. Dashed lines inside violins represent, from top to bottom, the third quartile, the median, and the first quartile. Data were analyzed statistically by one-way ANOVA with Tukey’s multiple comparison post hoc test. *\\u003cem\\u003ep\\u003c/em\\u003e=\\u0026lt;0.05, \\u003cem\\u003e**\\u003c/em\\u003e**\\u003cem\\u003ep\\u003c/em\\u003e = \\u0026lt;0.0001. \\u003cstrong\\u003eF) \\u003c/strong\\u003eImmunofluorescence analysis of all homolog rescue lines in the absence of RAP, showing colocalization between the endogenous PfGCN5 identified with anti-HA antibodies and the second copy proteins identified with anti-FLAG antibodies. Shown is one of three biological replicates. Pv: \\u003cem\\u003eP. vivax\\u003c/em\\u003e, Py: \\u003cem\\u003eP. yoelii\\u003c/em\\u003e, PyCh: PyChimera.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/2f0f83a050b63998b5cd0f4f.png\"},{\"id\":90571172,\"identity\":\"e047b754-6fa0-4a2a-95cd-230e71b051f8\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:07\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":215965,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe poly-N domain in PfGCN5 is required for normal parasite growth. A) \\u003c/strong\\u003eSchematic representation of deletion constructs. The pink boxes illustrate the quantity of asparagine residues that remain: 48 N in Del1, 23 N in Del2, and 7 N in Del3 out of 81 N in the WT copy. Numbers above are the boundary residues of the repeat. PfGCN5 constructs were expressed under the control of the native promoter as second copies in the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003econditional knockout line. \\u003cstrong\\u003eB) \\u003c/strong\\u003eGiemsa-stained thin smears of deletion rescue lines treated with RAP across two life cycles. Red arrowheads point to aberrant cells. \\u003cstrong\\u003eC) \\u003c/strong\\u003eGrowth of WT and deletion rescue lines relative to the control (?)after three intraerythrocytic cycles. The distribution of three independent experiments is shown. Dashed lines inside violins represent, from top to bottom, the third quartile, the median, and the first quartile. Data were analyzed statistically by one-way ANOVA with Tukey’s multiple comparisons post hoc test \\u003cem\\u003e**\\u003c/em\\u003e**\\u003cem\\u003ep\\u003c/em\\u003e = \\u0026lt;0.0001. \\u003cstrong\\u003eD) \\u003c/strong\\u003eRepresentative western blot from three biological replicates showing FLAG-tagged GCN5 protein levels in WT and deletion rescue lines. HAD1: loading control. \\u003cstrong\\u003e\\u0026nbsp;E) \\u003c/strong\\u003eImmunofluorescence analysis showing nuclear localization for both WT and truncated PfGCN5 (Del3), identified with anti-FLAG antibodies. DAPI and aldolase were used as markers for the nucleus and cytoplasm, respectively. Shown is one biological replicate from three.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/fedc879e390b968fb9949588.png\"},{\"id\":90571171,\"identity\":\"20e706f7-b8ed-424e-9ff2-a75d85b6a7eb\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:07\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":69466,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe PfGCN5 deletion line is susceptible to stress. \\u003c/strong\\u003eSynchronized 0-3h post-invasion PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites expressing a PfGCN5 WT or deletion (Del3) second copy construct were treated with or without RAP and then 24h later were exposed to heat shock (HS, 41°C, 6h), room temperature (RT, 25C, 12h), or dihydroartemisinin (DHA, 100 nM, 90 min) before return to standard conditions.\\u0026nbsp; Final parasitemia after three erythrocytic cycles, normalized to that of the WT rescue line grown in the absence of RAP. The distribution of three independent experiments is shown. Dashed lines inside violins represent, from top to bottom, the third quartile, the median, and the first quartile. Data were analyzed statistically by one-way ANOVA with Tukey’s multiple comparison post hoc test. *p=0.0161, **p=0.0061, ***p=0.0010, ****p = \\u0026lt;0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/b733bbb3b6ff91711381dd21.png\"},{\"id\":90571180,\"identity\":\"cede976a-916b-49f9-bbd1-3999589c3f39\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:07\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":204082,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePoly-N repeat deletion affects PfGCN5-mediated acetylation of H3K9ac.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eA) \\u003c/strong\\u003eUnmodified H3 and\\u003cstrong\\u003e \\u003c/strong\\u003eH3K9ac levels identified with anti-histone H3 and anti-H3K9ac specific antibodies in protein lysates from parasites treated with or without RAP.\\u003cstrong\\u003e \\u003c/strong\\u003eWT, wild-type PfGCN5 rescue; D3, deletion3 rescue. Representative western blot from three biological replicates.\\u003cstrong\\u003e B) \\u003c/strong\\u003eQuantification of the fold change between normalized H3K9ac levels to unmodified H3 from RAP-treated and non-treated parasites from three biological replicates. Data from the 1\\u003csup\\u003est\\u003c/sup\\u003e cycle were analyzed statistically by one-way ANOVA with Tukey’s multiple comparison post hoc test. Data from the 2\\u003csup\\u003end\\u003c/sup\\u003e cycle was analyzed statistically by a two-tailed Student’s t-test (KO parasites were dead by the second cycle and thus excluded from these analysis). *\\u003cem\\u003ep\\u003c/em\\u003e=0.039, **\\u003cem\\u003ep=\\u003c/em\\u003e0.0032.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/39e6e3987231e7631d5fa68d.png\"},{\"id\":90571186,\"identity\":\"bd783158-95d5-4896-b81b-3fe20c9989fa\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:08\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":294281,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHistone post-translational modifications identified by mass spectrometry.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAcid-extracted core histones were subjected to propionylation and trypsin digestion before LC-MS/MS analysis. The total abundance of each modified peptide was calculated based on the sum of all unmodified and modified peptide forms. The ratio of each peptide was determined as its abundance relative to the total abundance. Data from three biological replicates were analyzed statistically by one-way ANOVA with Fisher’s multiple comparisons post hoc test. *p=0.0152, **p=0.0022, ***p=0.0005, ****p=\\u0026lt;0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/5d3866de4ed2025c412e960d.png\"},{\"id\":90571188,\"identity\":\"20fbebef-3e3a-4ca2-8079-6c45763c63a8\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:08\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":145699,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePfGCN5 does not require processing prior to nuclear localization.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eA) \\u003c/strong\\u003eRepresentative anti-HA immunoblot from three biological replicates showing processing of the endogenous PfGCN5 protein in the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e line in the absence of rapamycin. Arrows represent the molecular mass of the C-terminal fragments after subtracting the mass of the GFP tag. \\u003cstrong\\u003eB) \\u003c/strong\\u003eImmunofluorescence analysis showing cells from the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e line in the absence of rapamycin, treated with E64D (10uM), brefeldin A (BFA, 0.5ug/mL), or DMSO. Three biological replicates were performed. \\u003cstrong\\u003eC) \\u003c/strong\\u003eRepresentative western blot from two biological replicates showing enrichment of the full-length protein upon treatment of parasites with 10 μM or 25 μM ivermectin in comparison to the DMSO control. Arrows represent the molecular mass of the C-terminal fragments identified with an anti-HA antibody after subtracting the mass of GFP. \\u003cstrong\\u003eD) \\u003c/strong\\u003eLocalization of PfGCN5 in cells treated with ivermectin or DMSO. DAPI and anti-HA antibodies were used to localize the nucleus and endogenous PfGCN5 protein, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/c82b4f0b02abd5912e07dff7.png\"},{\"id\":90571185,\"identity\":\"b0c17a79-7d0c-41bb-b4cd-168d4dd63cf5\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:08\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":230640,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eIntracellular dynamics of the N-terminal polypeptide of PfGCN5. A)\\u003c/strong\\u003e \\u0026nbsp;Time-course luminescence blotting of protein lysates from the LgBiT-HiBiT and \\u003cstrong\\u003eB)\\u003c/strong\\u003e Del3-HiBiT lines. Representative blot from three biological replicates. PM V: anti-plasmepsin V loading control. \\u003cstrong\\u003eC)\\u003c/strong\\u003e Localization of PfGCN5 HiBiT-tagged fragments by IFA using an anti-HiBiT antibody. DAPI and aldolase were utilized to stain the nucleus and cytoplasm, respectively. Shown is one biological replicate from three. \\u003cstrong\\u003eD)\\u003c/strong\\u003eLuminescence blotting of fractionated LgBiT-HiBiT parasites at 20-23h hpi. Anti-HAD1 and anti-histone H3-specific antibodies were used as markers of the cytosolic “C” and nuclear “N” fractions, respectively. “I”= input. Shown is one biological replicate from three. \\u003cstrong\\u003eE) \\u003c/strong\\u003eIllustration of cytosolic recognition of HiBiT-tagged fragments by the LgBiT subunit. The nucleus-impermeable LgBiT protein cannot probe nuclear fragments tagged with HiBiT. \\u003cstrong\\u003eF)\\u003c/strong\\u003e Live-cell detection of cytosolic HiBiT-tagged fragments throughout the intraerythrocytic life cycle. Mean values from four independent experiments are shown, and error bars represent standard deviations. \\u003cstrong\\u003eG)\\u003c/strong\\u003eImmunoprecipitation of N- and C-terminal fragments was performed with monoclonal mouse anti-HiBiT or anti-FLAG antibodies conjugated to magnetic beads, respectively. After detection of FLAG signal, the same membrane was used in luminescence-based detection of HiBiT . Representative blot from three independent replicates. IP = Immunoprecipitate, FT= Flow-through. Labels to the left indicate the tags used for western blotting.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/32fd41ab4a0beba353ee4738.png\"},{\"id\":92726409,\"identity\":\"6bc1271c-0a28-4685-8297-72da4a1783e5\",\"added_by\":\"auto\",\"created_at\":\"2025-10-03 14:44:56\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3185138,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/45f5780e-05c1-4f5e-ac4f-c4128e58badd.pdf\"},{\"id\":90571173,\"identity\":\"9f236445-4368-4eee-8e8f-3b51c186f537\",\"added_by\":\"auto\",\"created_at\":\"2025-09-04 08:24:07\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3571421,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary Information\",\"description\":\"\",\"filename\":\"SupplementalfiguresKR082925.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7490809/v1/bb3a4d713be1218e6238a086.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003eA role for the poly-asparagine repeat in the \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePlasmodium\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e histone acetyltransferase, PfGCN5\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe human malaria parasite, \\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e, exhibits one of the most AT-rich genomes (80.6%) sequenced to date, and among the \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species infecting humans, it outruns its peers\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR2\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. As expected, amino acid residues with high AT content, such as asparagine (AAT/AAC) and lysine (AAA/AAG), are enriched in the \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteome\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. However, codon bias analyses established a preference for asparagine over lysine residues despite equivalent AT-richness (83.3%)\\u003csup\\u003e4,5\\u003c/sup\\u003e. Remarkably, asparagine (N) residues are found in tandem predominantly in intragenic regions, forming low-complexity, intrinsically disordered regions in about 24% of the \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteome, when considering at least 30 N in an 80-residue window\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eP. falciparum\\u0026rsquo;s\\u003c/em\\u003e poly-N repeats are either perfect or imperfect. When imperfect, they are typically interrupted by polar residues, such as serine, aspartic acid and glutamic acid, as well as the aromatic residue tyrosine. These repeats average 37 residues but can exceed 100 residues in length\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. Since poly-asparagine repeats in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteins do not cluster in specific protein families, metabolic pathways, or parasite stages, the functional contribution of these repeats has been elusive\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. Structural mapping of poly-N repeats suggests that they are unlikely to adopt globular folds due to their flexible and hydrophilic nature, which favors protrusion from protein cores. This raises the possibility that poly-N repeats enhance protein function, adopt new functions, or confer protein stability without interfering with basal protein activity\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Furthermore, poly-N repeats exhibit an intrinsic propensity to aggregate that increases with length and temperature\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Thus, the evolutionary forces shaping the \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteome, an organism that withstands febrile cycles, are of interest.\\u003c/p\\u003e\\u003cp\\u003eTo date, only two \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteins containing poly-N repeats have been experimentally characterized. In the first case, the parasite chaperone PfHsp110, but not its human or yeast counterparts, efficiently prevents heat shock (HS)-induced aggregation of the parasite\\u0026rsquo;s putative CDK2-regulatory subunit, which contains 83 consecutive N residues, highlighting the robust specialization that parasite chaperones have evolved\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. In the second case, removal of the 28-residue poly-N tract from the essential proteasome clamp subunit, Rpn6, had no effect on protein expression, function, or parasite viability at 37\\u0026deg;C or 41\\u0026deg;C, suggesting that some poly-N repeats may lack functional properties\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. Given the large group of poly-N repeat-containing proteins that remain uncharacterized in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e, we sought to investigate the role of an imperfect repeat of 98 residues containing 81 N, located in the N terminus of the parasite\\u0026rsquo;s histone acetyltransferase GCN5 (PfGCN5). GCN5 is highly conserved among eukaryotes with two well-characterized C-terminal domains: a histone acetyltransferase (HAT) domain, which catalyzes the transfer of acetyl groups to lysine residues, and a bromodomain (BrD), which binds acetylated substrates to support enzymatic processivity\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. GCN5 participates in the megadalton SAGA complex, where, together with ADA2, ADA3, and SGF29, it forms the HAT catalytic module of the complex\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. In \\u003cem\\u003eP. falciparum\\u003c/em\\u003e, GCN5 is essential and regulates the expression of a subset of genes involved in development, invasion, and stress response\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR15 CR16\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Seminal work on PfGCN5 has underscored its pivotal role in catalyzing histone acetylation marks associated with euchromatin\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. Deletion of the BrD from PfGCN5 leads to decreased histone acetylation and altered chromatin architecture, resulting in defective intraerythrocytic development and dysregulated sexual commitment\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Bromodomain deletion lines also exhibit impaired maturation of gametocytes and mosquito stages\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, PfGCN5 holds a critical role in modifying the chromatin landscape across the parasite's life cycle.\\u003c/p\\u003e\\u003cp\\u003eOur findings show that the N-terminal fragment containing the poly-N repeat in PfGCN5 contributes to the HAT activity of the protein following cleavage, by associating with C-terminal catalytic domain. We characterized a poly-N repeat deletion line and found reduced histone acetylation and impaired parasite growth, particularly under stress conditions. We explored the nucleocytoplasmic live-cell dynamics of the N-terminal polypeptide and determined that it is destabilized when the poly-N repeat is removed. Furthermore, we have expanded the known repertoire of acetylation marks catalyzed by PfGCN5\\u0026rsquo;s activity to H3.3 and H4.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe N terminus of PfGCN5 containing the poly-asparagine repeat is important for parasite growth\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGCN5 is a highly conserved protein across eukaryotes, with most of the homology restricted to its C terminus, where the HAT domain and the BrD are located \\u003cstrong\\u003e(Supplemental figure 1)\\u003c/strong\\u003e. Yeast and Tetrahymena GCN5 have short N termini with less than 200 amino acids upstream of the HAT domain. In contrast, metazoans express two GCN5 isoforms from alternative splicing: a short version that is highly homologous to the yeast GCN5 and a long variant whose extended N-terminus displays a P300/CBP-associated factor (PCAF)-homology domain \\u003csup\\u003e20\\u0026ndash;23\\u003c/sup\\u003e. The PCAF homology domain has been implicated in the recruitment of transcriptional activators, including two other HAT proteins, P300 and CBP, recognition of nucleosomal substrates, and it has been shown to possess E3 ubiquitin ligase activity\\u003csup\\u003e20,24\\u003c/sup\\u003e. The PfGCN5 (PF3D7_0823300) is produced as a single transcript and exhibits the longest N-terminal extension described to date. This extension contains no recognizable domains except for a 98-residue imperfect repeat containing 81 N (spanning from amino acid position 143 to 240) \\u003cstrong\\u003e(Figure 1, Supplemental figure 2)\\u003c/strong\\u003e. The related Apicomplexan parasite, \\u003cem\\u003eToxoplasma gondii\\u003c/em\\u003e, encodes two GCN5 homologs (TgGCN5A/B) that are expressed as single transcripts and exhibit extended N-terminal extensions as well. However, these N-terminal extensions share little sequence similarity with PfGCN5\\u003csup\\u003e25,26\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eSequence alignment of GCN5 homologs across \\u003cem\\u003ePlasmodium\\u0026nbsp;\\u003c/em\\u003especies revealed that those naturally exposed to febrile temperatures, such as \\u003cem\\u003eP. falciparum, P. vivax, P. knowlesi, P. gallinaceum,\\u0026nbsp;\\u003c/em\\u003eand\\u003cem\\u003e\\u0026nbsp;P. relictum,\\u003c/em\\u003e possess N-terminal low complexity intrinsically disordered regions (LC-IDRs) consisting of poly-N or glycine-serine (GS)-rich domains \\u003cstrong\\u003e(Figure 1, Supplemental figure 2)\\u003c/strong\\u003e. In contrast, GCN5 homologs from \\u003cem\\u003ePlasmodium\\u0026nbsp;\\u003c/em\\u003especies that do not experience fever, such as\\u003cem\\u003e\\u0026nbsp;P. yoelii\\u0026nbsp;\\u003c/em\\u003eand \\u003cem\\u003eP. berghei\\u003c/em\\u003e, display shorter and less conspicuous repetitive sequences rich in N, G, E, and A .\\u0026nbsp;Notably, avian malaria parasites (\\u003cem\\u003eP. gallinaceum\\u0026nbsp;\\u003c/em\\u003eand \\u003cem\\u003eP. relictum)\\u0026nbsp;\\u003c/em\\u003esurvive their host\\u0026apos;s body temperature, ranging between 38.5- 43.8 \\u0026deg;C, and \\u003cem\\u003ePlasmodium (P. berghei and yoelii)-\\u003c/em\\u003einfected mice experience hypothermia instead of hyperthermia\\u003csup\\u003e27\\u0026ndash;29\\u003c/sup\\u003e. These observations led us to hypothesize that the extended LC-IDRs in the N terminus of \\u003cem\\u003ePlasmodium\\u0026nbsp;\\u003c/em\\u003espp. naturally exposed to fever may confer a survival advantage in their hosts. \\u0026apos;\\u003c/p\\u003e\\n\\u003cp\\u003eTo test this hypothesis, we generated a rapamycin-inducible PfGCN5 conditional knockout line (PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e) using the DiCre recombinase system\\u003csup\\u003e30\\u003c/sup\\u003e. Specifically, we integrated a \\u003cem\\u003eSaccharomyces cerevisiae\\u003c/em\\u003e-recoded version of the \\u003cem\\u003epfgcn5\\u003c/em\\u003e coding sequence, C-terminally GFP-3HA tagged and flanked by LoxP sites at the endogenous locus, using CRISPR/Cas9 \\u003cstrong\\u003e(Supplemental figure 3A)\\u003c/strong\\u003e\\u003csup\\u003e31\\u003c/sup\\u003e.\\u0026nbsp;By employing an NF54 attB\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003eline expressing a dimerizable Cre recombinase, we could excise the entire gene upon rapamycin (RAP) addition \\u003cstrong\\u003e(Supplemental figure 3B\\u003c/strong\\u003e)\\u003csup\\u003e32,33\\u003c/sup\\u003e. Similar to previous reports\\u003csup\\u003e15,34\\u003c/sup\\u003e, before excision, we observed nuclear localization of PfGCN5 throughout the intraerythrocytic cycle and extensive processing of the full-length protein (190 kDa), resulting in multiple fragments \\u003cstrong\\u003e(Supplemental figure 3C,D)\\u003c/strong\\u003e. Of note, the protein is synthesized starting at the early ring stage and is almost completely processed by the end of the intraerythrocytic cycle. Following RAP addition (0-3 hours post-invasion, early ring stage), knockout (KO) parasites stalled at the trophozoite stage and were unable to undergo schizogony, resulting in cell death \\u003cstrong\\u003e(Figure 2A,B)\\u003c/strong\\u003e. Time-course western blotting analysis showed that protein expression was reduced by approximately 50% in RAP-treated cultures, highlighting the stability of these fragments \\u003cstrong\\u003e(Figure 2C)\\u003c/strong\\u003e. We then attempted to rescue the PfGCN5 KO with a series of C-terminally FLAG-tagged constructs expressed \\u003cem\\u003ein trans\\u003c/em\\u003e from the \\u003cem\\u003eattB\\u003c/em\\u003e locus under the control of the native PfGCN5 promoter \\u003cstrong\\u003e(Figure 2D)\\u003c/strong\\u003e\\u003csup\\u003e32\\u003c/sup\\u003e. Growth and development could be fully rescued by the introduction of a second copy of the WT \\u003cem\\u003epfgcn5\\u003c/em\\u003e gene \\u003cstrong\\u003e(Figure 2D,E)\\u003c/strong\\u003e. Interestingly, while \\u003cem\\u003eP. vivax (\\u003c/em\\u003ePv) GCN5 fully rescued the growth of PfGCN5 KO parasites, \\u003cem\\u003eP. yoelii\\u003c/em\\u003e (Py) GCN5 supported only partial rescue, displaying ~30% growth over three replication cycles \\u003cstrong\\u003e(Figure 2D,E)\\u003c/strong\\u003e. To evaluate whether this growth phenotype was due to the N terminus of PyGCN5, we made a chimeric protein containing the first 676 aa from the PfGCN5 N terminus, including the poly-N repeat, and the C terminus from PyGCN5. Given the widespread processing of PfGCN5, we determined the length of the PfGCN5 N terminus based on the conserved motif IKNI/M/LR found at amino acid residue 677 \\u003cstrong\\u003e(Supplemental figure 2)\\u003c/strong\\u003e. This chimeric protein was able to fully rescue the growth of the PfGCN5 KO \\u003cstrong\\u003e(Figure 2D,E).\\u003c/strong\\u003e Finally, we observed that all GCN5 homologs and the PyChimera were well expressed, localized to the nucleus, and processed \\u003cstrong\\u003e(Figure 2F and Supplemental figure 4)\\u003c/strong\\u003e. Taking together these results suggest that the N termini of PfGCN5 and PvGCN5, containing poly-N or GS-rich repeats, respectively, are important for \\u003cem\\u003eP. falciparum\\u003c/em\\u003e parasite survival.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe poly-N repeat in PfGCN5\\u0026rsquo;s N terminus is required for normal parasite growth\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo dissect whether the growth-related role of PfGCN5 N terminus lies in its poly-N repeat, we engineered three C-terminally FLAG-tagged PfGCN5 rescue constructs, whose poly-N repeat was incrementally shortened from its C-terminal end \\u003cstrong\\u003e(Figure 3A)\\u003c/strong\\u003e. These deletions retained varying lengths of the 98-residue repeat containing 81N found in the WT protein: deletion #1 (Del1) retains 48 N, deletion #2 (Del2) 23 N, and deletion #3 (Del3) 7 N. Upon RAP treatment, unlike the death phenotype of PfGCN5 KO parasites, the deletion lines could progress through the first cycle without any notable defects, presenting abnormal trophozoite and schizont morphologies only in the second cycle \\u003cstrong\\u003e(Figure 3B)\\u003c/strong\\u003e. Similar to the PyGCN5 rescue line, all three deletion lines partially rescued the growth of the PfGCN5 KO line, exhibiting about 30% growth relative to the WT rescue line over three replication cycles \\u003cstrong\\u003e(Figure 3C)\\u003c/strong\\u003e. Western blot analysis confirmed that all deletion constructs were expressed at comparable levels to the WT protein and underwent normal proteolytic processing \\u003cstrong\\u003e(Figure 3D)\\u003c/strong\\u003e. Based on the consistent phenotype, proper protein level expression, and processing across the deletion lines, we selected one line (Del3) for the subsequent analysis. As in the WT, the Del3 rescue line showed nuclear localization of PfGCN5 \\u003cstrong\\u003e(Figure 3E)\\u003c/strong\\u003e. Altogether, these data indicate that the poly-N repeat in PfGCN5 is required for normal parasite growth.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe poly-N repeat in PfGCN5 facilitates cell recovery after stress.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe observation that poly-N or GS-rich repeats are found in GCN5 homologs from \\u003cem\\u003ePlasmodium spp.\\u003c/em\\u003e that are naturally subjected to fever but absent in those\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003ethat do not encounter such conditions prompted us to investigate the potential link between these LC-IDRs and stress. Numerous reports in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e and other systems have emphasized GCN5\\u0026rsquo;s ability to act as a master regulator of stress by deploying transcriptional cascades that enable stress management\\u003csup\\u003e14,15,35\\u0026ndash;37\\u003c/sup\\u003e. Specifically, HS, low glucose, and dihydroartemisinin (DHA) have been shown to induce higher PfGCN5 expression\\u003csup\\u003e14,15,38\\u003c/sup\\u003e. Additionally, restricting PfGCN5 overexpression heightens the parasite\\u0026rsquo;s susceptibility to stress \\u003csup\\u003e14,15\\u003c/sup\\u003e. With this in mind, we subjected our WT and Del3 rescue lines to HS (41 \\u0026deg;C for 6h), room temperature (RT) (~25 \\u0026deg;C for 12h), and DHA (100 nM for 90 min) starting 24h after a 3h RAP pulse at the 0-3h ring stage\\u003csup\\u003e39\\u0026ndash;41\\u003c/sup\\u003e. We then returned the cultures to standard conditions (37\\u0026deg;C, drug washed out) for 6 days. Exposure to HS and RT exacerbated the growth defect in RAP-treated Del3 parasites in comparison to untreated Del3 and WT rescue parasites \\u003cstrong\\u003e(Figure 4A,B)\\u003c/strong\\u003e. Exposure to DHA rendered rapamycin-induced Del3 parasites unable to recover \\u003cstrong\\u003e(Figure 4C)\\u003c/strong\\u003e. Intriguingly, Del3 parasites also exhibited poor recovery even in the absence of rapamycin. This dominant-negative effect could result from the increased expression of PfGCN5 during stress, leading to the displacement of the WT protein by the truncated version in SAGA-like complexes. These results support the hypothesis that LC-IDRs in the N terminus of PfGCN5 contribute to the parasite\\u0026rsquo;s adaptive transcriptional regulation. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePfGCN5 histone acetyltransferase activity is supported by its poly-N repeat\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePfGCN5 retained HAT activity when its C-terminal region, comprising the HAT and BrD or the HAT domain alone, was used to acetylate free H3 at lysine residues 9 and 14 (H3K9ac and H3K14ac) from calf thymus\\u003csup\\u003e18\\u003c/sup\\u003e. Subsequent studies confirmed this activity in parasites\\u003csup\\u003e16\\u003c/sup\\u003e. Although \\u003cem\\u003eP. falciparum\\u003c/em\\u003e encodes four histone variants (H2A.Z, H2B.Z, H3.3, and CenH3) besides the canonical core histones, PfGCN5\\u0026rsquo;s role in these variants is unknown, except for H2B.Z, whose acetylation levels were lower in a PfGCN5 BrD deletion line\\u003csup\\u003e17,42\\u003c/sup\\u003e. To investigate if the poly-N repeat in PfGCN5 could play a role in histone acetylation, we examined H3K9ac levels in synchronized parasites treated with or without rapamycin at 0-3h post-invasion. Samples were harvested at 12-15h, 24-27h, 32-35h, and 45-48h post-invasion during the first intraerythrocytic cycle post-RAP\\u0026nbsp;addition. While we observed slight decreases in the level of H3K9ac at 45-48h in the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e and Del3 lines treated with RAP, there were no significant differences across lines at any of the evaluated time points \\u003cstrong\\u003e(Figure 5A,B left side)\\u003c/strong\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEncouraged by this trend, and the observation that RAP-treated deletion lines exhibited a fitness cost only during the second cycle, we extended these studies to the second intraerythrocytic cycle post-rapamycin addition. In agreement with previous studies, we found significant changes in H3K9ac at all time points during the second cycle in RAP-induced PfGCN5 KO and Del3 parasites \\u003cstrong\\u003e(Figure 5A,B right side)\\u003c/strong\\u003e. Note that PfGCN5 KO parasites die in the first cycle, so acetylation levels in the second cycle cannot be assessed. In light of the limitations of western blot analysis for quantitation and the limited availability of anti-histone antibodies for examining other histone modifications, we opted for a quantitative, high-resolution mass spectrometry approach. To achieve this, we purified core histones using an acid extraction method from synchronized cultures at 32-35h and 45-48h of the first intraerythrocytic cycle, and at 32-35h of the second intraerythrocytic cycle post-RAP induction, which occurred at 0-3h post-invasion of the first intraerythrocytic cycle. Interestingly, we found significant differences for H3K9ac, H3K9acK14ac, H3K18ac, H3K23ac, H3K18acK23ac, H3K27ac, H3.3K9acK14ac, and H4K8acK16ac at 45-48h of the first intraerythrocytic cycle and/or at 35-38h of the second intraerythrocytic cycle \\u003cstrong\\u003e(Figure 6)\\u003c/strong\\u003e. We did not observe any differences in H3K9ac levels at 32-25h of the first intraerythrocytic cycle, consistent with our western blot results. The non-significant trend towards decreased H3K9ac in the western blot studies became significant differences at 45-48h of the first intraerythrocytic cycle for the MS analysis, underscoring the technique\\u0026apos;s sensitivity. Notably, H2B.Z was not detected, and di- and tri-methylation of H3K4 did not show significant differences across lines, in contrast to the western blot findings of a previous paper\\u003csup\\u003e16\\u003c/sup\\u003e. Overall, these findings confirm and broaden our understanding of the extensive role of PfGCN5 in chromatin remodeling and gene expression. More critically, they suggest that the poly-N repeat contributes to the HAT activity of PfGCN5.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePfGCN5 is processed in the nucleus\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRecent studies have proposed that PfGCN5 is trafficked via the ER-Golgi to the digestive vacuole, where it is processed by the cysteine protease, falcipain 3, before translocating to the nucleus\\u003csup\\u003e34,43\\u003c/sup\\u003e. We, however, did not find evidence of this. We treated asynchronous PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites with the cysteine protease inhibitor, E64D (10uM), the secretory pathway inhibitor Brefeldin A (5 \\u0026micro;g/mL), or DMSO for three hours\\u003csup\\u003e44,45\\u003c/sup\\u003e. While parasites treated with E64D displayed an enrichment of the full-length protein as previously reported, PfGCN5 nuclear localization remained unchanged \\u003cstrong\\u003e(Figure 7A,B)\\u003c/strong\\u003e\\u003csup\\u003e34\\u003c/sup\\u003e. This outcome suggests that the full-length protein is processed in the nucleus. Furthermore, we observed that PfGCN5 processing and trafficking are insensitive to Brefeldin A treatment, indicating an ER-Golgi-independent trafficking pathway.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe importin \\u0026alpha;/\\u0026beta; machinery mediates a major nuclear transport pathway. Given that TgGCN5A enters the nucleus via direct interaction with the adaptor molecule importin-\\u0026alpha; \\u003csup\\u003e26\\u003c/sup\\u003e, we sought to test whether PfGCN5 uses this pathway\\u003csup\\u003e26\\u003c/sup\\u003e. We treated asynchronous PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites with 10 \\u0026mu;M or 25 \\u0026mu;M ivermectin for three hours. Treatment of \\u003cem\\u003eP. falciparum\\u003c/em\\u003e parasites with ivermectin has been shown to block this pathway \\u003csup\\u003e46\\u003c/sup\\u003e. We found that ivermectin-treated parasites not only exhibit stabilization of the full-length protein but also redistribution of the PfGCN5 signal to the cytoplasm, in stark contrast to DMSO-treated parasites \\u003cstrong\\u003e(Figure 7C, D)\\u003c/strong\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe poly-N repeat confers stability to the N-terminal polypeptide post-cleavage and interacts with the C-terminal catalytic domain\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe wanted to tag the N-terminus of PfGCN5 to track its kinetics. After several unsuccessful attempts, we adopted the NanoLuc\\u0026reg; Binary Technology, NanoBiT (Promega), for higher-resolution monitoring of the poly-N repeat. For this, we first engineered a stable parasite line expressing the larger NanoLuc subunit, LgBiT, using the \\u003cem\\u003epiggyBac\\u0026nbsp;\\u003c/em\\u003etransposon-mediated genomic integration\\u003csup\\u003e47\\u003c/sup\\u003e. Growth assays showed no significant differences between a WT and the LgBiT-expressing line \\u003cstrong\\u003e(Supplemental figure 5A)\\u003c/strong\\u003e. Expression and localization of LgBiT were verified by western blot and IFA, respectively, revealing an 18 kDa cytosolic protein that is consistent with findings in other systems \\u003cstrong\\u003e(Supplemental figure 5B, C)\\u003c/strong\\u003e\\u003csup\\u003e48\\u003c/sup\\u003e. We then used this LgBiT line to integrate \\u0026nbsp;a C-terminally FLAG-tagged second copy of PfGCN5 containing the 11-amino acid smaller NanoLuc subunit, HiBiT, in the poly N-repeat (position marked by arrowhead in Figure 1A) at the non-essential \\u003cem\\u003eattB\\u0026nbsp;\\u003c/em\\u003elocus, resulting in the LgBiT-HiBiT line. To regulate expression, this second copy of PfGCN5 was placed under the control of the endogenous promoter. Insertion of HiBiT did not impair parasite growth in comparison to the WT line \\u003cstrong\\u003e(Supplemental figure 5A)\\u003c/strong\\u003e. Similarly, we attempted to introduce the truncated version of PfGCN5, Del3, tagged with HiBiT in the LgBiT line, but were unsuccessful. Therefore, we introduced this second copy into the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e line. This line also had normal growth \\u003cstrong\\u003e(Supplemental figure 5A)\\u003c/strong\\u003e. Luminescence-based detection of the WT HiBiT-tagged protein by western blot revealed the presence of the 190 kDa full-length protein, and processed forms of 120, 110, and 50 kDa that were sequentially produced through the parasite intraerythrocytic cycle \\u003cstrong\\u003e(Figure 8A)\\u003c/strong\\u003e. Notably, all these fragments were also detected by another group using polyclonal antibodies that recognize the N-terminal region of PfGCN5, encompassing amino acid residues 9-25 \\u003csup\\u003e14\\u003c/sup\\u003e. In contrast, in the Del3-HiBiT line, HiBiT western blotting failed to recognize bands, indicating that the N-terminal polypeptide is rapidly degraded in the Del3 line \\u003cstrong\\u003e(Figure 8B)\\u003c/strong\\u003e. IFAs showed that the poly-N repeat containing fragments in the LgBiT-HiBiT line exhibit a higher concentration in the nucleus with weak cytoplasmic signal in trophozoite and schizont stages \\u003cstrong\\u003e(Figure 8C)\\u003c/strong\\u003e. To investigate the dynamics of the PfGCN5 N terminus in the cell, we performed cellular fractionation studies using the LgBiT-HiBiT line. Interestingly, the full-length, the 120 kDa, and \\u0026nbsp;the 110 kDa forms remain in the nucleus, while the 50 kDa fragment containing the poly-N repeat is exported to the cytosol \\u003cstrong\\u003e(Figure 8D)\\u003c/strong\\u003e. To further explore the live-cell kinetics of the poly-N-repeat containing fragments, we took advantage of the high affinity between the LgBiT and HiBiT subunits and employed a cell-permeable substrate (furimazine) to measure the real-time binding activity. We first confirmed that the LgBiT protein is nuclear impermeable even in the presence of the PfGCN5-HiBiT protein by performing cellular fractionation \\u003cstrong\\u003e(Supplemental figure 5D)\\u003c/strong\\u003e. Since LgBiT was found to be exclusively cytosolic, it acts as a cytosolic sensor for HiBiT-tagged poly-N repeat-containing fragments \\u003cstrong\\u003e(Figure 8E)\\u003c/strong\\u003e. Consistent with our earlier observations in figures 8C and D, cytosolic levels of HiBiT-tagged fragments rise dramatically midway through the cycle, after the 50kDa fragment is generated \\u003cstrong\\u003e(Figure 8F)\\u003c/strong\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eGiven that the deletion of the poly-N repeat resulted in the destabilization of the N-terminal polypeptide post-cleavage and a reduction in histone acetylation levels, a function catalyzed by the C-terminal HAT domain, we wondered if the N-terminal polypeptide containing the poly-N repeat could associate with the C-terminal fragments. To address this question, we \\u0026nbsp;pulled down the C-terminal fragments by the FLAG tag and blotted for HiBiT luminescence \\u003cstrong\\u003e(Figure 8G)\\u003c/strong\\u003e. \\u0026nbsp;C-terminal FLAG-tagged forms could co-precipitate all HiBiT-tagged fragments, suggesting interaction. Notably, the 50 kDa HiBiT-tagged piece, previously shown to be exported to the cytosol, was more abundant in the flow-through than in the IP fraction, leaving the 120 kDa and 110 kDa fragments as the potential interactors of the catalytic domain. Additionally, we asked whether N-terminal peptides could be identified in C-terminal pull-downs by mass spectrometry and if shortening the length of the poly-N repeat in PfGCN5 disturbs complex formation or other interactions. To do this, we performed co-immunoprecipitation analysis using anti-FLAG magnetic beads across the WT and deletion rescue lines in the absence of rapamycin. LC-MS/MS analysis identified N-terminal peptides when pulling down from the WT rescue line but not from the deletion rescue lines, confirming the association between N- and C-terminal fragments in the WT line (\\u003cstrong\\u003eSupplemental figure 6\\u003c/strong\\u003e). While five out of nine PfGCN5 complex members were found in both the WT and Del3 rescue lines, the stoichiometry of one of its members was dysregulated in the deletion lines \\u003cstrong\\u003e(Supplemental table 1)\\u003c/strong\\u003e\\u003csup\\u003e16\\u003c/sup\\u003e. Specifically, the Nucleosome Assembly Protein (NAPS; PF3D7_0919000) had a threefold increase. Furthermore, the adaptor protein 14-3-3I (PF3D7_0818200) and another PHD-domain-containing protein (PF3D7_0310200), previously found significantly enriched in PfGCN5 pull-downs, were enriched several folds in the deletion lines \\u003csup\\u003e16\\u003c/sup\\u003e. These findings indicate that the N-terminal domain containing the poly-N repeat interacts with the C-terminal catalytic domain post-cleavage. Such an association could promote the participation of NAPS in the SAGA-like complex.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this study, we report a functional role for a poly-N repeat in a \\u003cem\\u003eP. falciparum\\u003c/em\\u003e protein. With approximately 1,300 proteins containing poly-N repeats, this is the most prevalent characteristic of the \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteome that has remained elusive in the biology of this deadly parasite. While the function of such repeats may vary across individual proteins, our findings suggest an important regulatory function in this instance, which opens a new area of research in parasite biology.\\u003c/p\\u003e\\u003cp\\u003eWe highlighted the existence of extended LC-IDRs in the N terminus of GCN5 homologs from \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species that are naturally exposed to fever, in stark contrast to those that do not encounter this stress. Remarkably, PvGCN5, which carries a GS-rich repeat, rescued the growth of a PfGCN5 KO line, and deletion of the poly-N repeat from PfGCN5 phenocopied the growth profile of the PyGCN5 (short higher-complexity repeat) rescue line. This suggests that the functional contribution of the N-terminal LC-IDR does not depend strictly on N residues but rather on the biophysical properties shared by polar and uncharged amino acid repeats. Thus, \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species may utilize synonymous \\u0026ldquo;amino acid grammar\\u0026rdquo; to achieve similar biological outcomes within their shared host. Mounting data in other systems has demonstrated the capacity for polar tracts rich in N, Q, G, and S to phase separate through dynamic multivalent interactions\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR50\\\" citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e. This phenomenon aids cellular organization, transcriptional regulation, genome maintenance, and complex formation, among others\\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e. Whether LC-IDRs composed of poly-N repeats in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e phase separate remains to be solved. Intriguingly, 7 out 9 proteins forming the PfGCN5 saga-like complex also contain prominent poly-N repeats \\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. Some of these may enhance interaction among complex members, provide better anchoring to histones, or allow transient interactions during stress conditions. Indeed, we found that the Del3 protein failed to maintain the stoichiometry levels of one complex member (NAPS) in comparison to the WT protein. Furthermore, we observed that 14-3-3I and a PHD-containing protein were several-fold enriched in the Del3 line. 14-3-3I is a scaffold protein that binds to phosphorylated serine/threonine residues, modulating the function, localization, and stability of its binding partners, as well as stabilizing protein complexes by facilitating protein-protein interactions \\u003csup\\u003e\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e. Given that PfGCN5 is extensively phosphorylated, we speculate that 14-3-3I may maintain the complex in tight engagement by binding to PfGCN5 and members of the SAGA-like complex simultaneously\\u003csup\\u003e\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e\\u003c/sup\\u003e Finally, the function of the PHD-containing protein could replace that of the PHD2 since both proteins bind to methylated histones.\\u003c/p\\u003e\\u003cp\\u003eIt has been proposed that poly-N repeats in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e can behave as \\u0026ldquo;tRNA sponges\\u0026rdquo; by slowing down ribosomal translation, giving the expected limited availability of asparaginylated tRNAs \\u003csup\\u003e\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u003c/sup\\u003e. However, we demonstrated that the PfGCN5 Del3 truncated protein is expressed at the same level as the WT second copy, suggesting that translation efficiency did not depend on the amount of N residues. Extensive truncation studies in the N-terminal extension of TgGCN5A/B, identified divergent nuclear localization signals embedded in intrinsically disordered regions\\u003csup\\u003e\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e\\u003c/sup\\u003e. In PfGCN5, we demonstrated that, like the WT, the Del3 mutant PfGCN5 localizes to the nucleus, indicating that the poly-N repeat in PfGCN5 does not mediate nuclear trafficking. Together, we established that reducing the length of the poly-N repeat in PfGCN5 does not affect expression levels, processing, or localization.\\u003c/p\\u003e\\u003cp\\u003eSubstantial data support the essential role of GCN5 during stress by remodeling chromatin structure through histone acetylation and by binding to a subset of genes directly implicated in stress tolerance, in both \\u003cem\\u003eP. falciparum\\u003c/em\\u003e and other organisms \\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e\\u003c/sup\\u003e. We showed that the Del3 line is highly susceptible to heat, cold, and drug stress, suggesting that the poly-N repeat aids PfGCN5 activity during stress, but further investigation is needed to uncover the molecular mechanism behind this.\\u003c/p\\u003e\\u003cp\\u003eCellular fractionation studies examining the N terminus, in conjunction with observations from E64D and Ivermectin treatments, demonstrated that the full-length PfGCN5 is exported to the nucleus, likely via the importin α/β transport machinery, where a cysteine protease subsequently processes it. Luminescence-based western blotting coupled with live-cell kinetics using a split nano-luciferase system allowed us to conclude that once in the nucleus, the N-terminus containing the poly-N repeat is cleaved into 120 kDa and 110 kDa processed forms. Later, midway through the intraerythrocytic cycle, a 50 kDa fragment is produced and exported back to the cytosol. Given that PfGCN5 has cytosolic non-histone substrates, it is possible that such bidirectional trafficking of the poly-N repeat contributes to this\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e\\u003c/sup\\u003e. Inspection of N- and C-terminal blots indicates that PfGCN5 is alternatively cleaved, producing alternative 120 kDa fragments from the N- or C-terminus.\\u003c/p\\u003e\\u003cp\\u003eThis work determined that the poly-N repeat stabilizes the N-terminal polypeptide after cleavage from the catalytic domain. Extensive data demonstrate that LC-IDRs are often targets of post-translational modifications, which subsequently modulate stability by promoting conformational changes, protein interactions, or mediating subcellular localization\\u003csup\\u003e\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e\\u003c/sup\\u003e. Alternatively, the flexible nature of the poly-N repeat could facilitate transient interactions that favor stability\\u003csup\\u003e\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eOnly H3K9ac, H3K14ac, H3K4me3, and H2B.Zac had been described as modifications catalyzed by PfGCN5\\u003csup\\u003e16,17\\u003c/sup\\u003e. Here, we extended the post-translational landscape mediated by PfGCN5 to H3K9acK14ac, H3K18ac, H3K23ac, H3K18acK23ac, H3K27ac, H3.3K9ac, H3.3K9acK14ac, and H4K8acK16ac. Although we did not find significant changes for H3.3K9ac, a conserved trend was observed between PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e parasites treated with or without rapamycin and the WT and Del3 complemented lines. We did not find evidence that PfGCN5 promotes methylation of H3. While histone acetylation of H3 and H3.3 has been linked to parasite development, stress response, and antigenic variation, H4 acetylation has been associated with both parasite development and DNA repair\\u003csup\\u003e\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, we identified several concomitant histone acetylation marks associated with PfGCN5 activity, indicating that it plays a crucial role in writing the \\u0026ldquo;chromatin code\\u0026rdquo;. Histone acetylation changes in the Del3 line underpin the importance of the poly-N repeat during its stay in the nucleus. Interestingly, we detected interaction between the C-terminal fragments carrying the catalytic domain and the N-terminal polypeptide containing the poly-N repeat post-cleavage. Such interaction occurs with the N-terminal 120 kDa and/or 110 kDa fragments, which only reside in the nucleus, and are apparent during the first half of the cycle. The GCN5 N/C-terminal fragments could interact directly or could associate through other components of the SAGA-like complex. Nevertheless, it is clear that the poly-N repeat stabilizes the N-terminal fragment of PfGCN5 and is important for C-terminal histone acetylation activity \\u003cem\\u003ein vivo\\u003c/em\\u003e. A structure of this complex would be revealing.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eReagents\\u003c/h2\\u003e\\u003cp\\u003eAll primers were obtained from Integrated DNA Technologies. The list of primers used in this study can be found in Appendix A, Table\\u0026nbsp;1A. Gene blocks corresponding to pvgcn5, and recoded pfgcn5 and pygcn5 coding sequences were synthetized by Genewiz. Restriction enzymes and Gibson Assembly\\u0026reg; Master Mix were purchased from New England Biolabs. For site-directed mutagenesis, we used the Quick-Change Lightning kit from Agilent. The rabbit anti-HA antibody and the mouse anti-flag magnetic beads were obtained from Millipore Sigma. The rat anti-FLAG antibody was obtained from Novus Biologicals. The mouse anti-PMV antibody was previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e\\u003c/sup\\u003e. The mouse anti-H3 and H3K9ac were obtained from Epigentek and Active Motif, respectively. Mouse anti-LgBiT and anti-HiBiT antibodies were obtained from Promega. Rapamycin, Brefeldin A, E64D, Dihydroartemisinin, Saponin, Sorbitol, WR99210, and the stain for thin smears (Hemacolor\\u0026reg;) were purchased from Millipore Sigma.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eGeneration of plasmids\\u003c/h3\\u003e\\n\\u003cp\\u003eA \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e recoded version of \\u003cem\\u003epfgcn5\\u003c/em\\u003e was inserted at the AsiSI restriction site of the pSN054 plasmid, previously described by Polino et al. 2020\\u003csup\\u003e68\\u003c/sup\\u003e .The GFP sequence amplified with primers KR24 and KR25, was inserted at the AsiSI restriction site in the pSN054. The AsiSI restriction site was restored upon cloning, allowing for a S. cerevisiae recoded version of pfgcn5 amplified with primers KR137 and KR138 to be in frame with GFP and 3xHA, upon Gibson assembly. The 490 bp immediately upstream of the pfgcn5\\u0026rsquo;s start codon amplified with primer pair KR135 and KR136, and 792 bp downstream of the stop codon amplified with KR47 and KR21, were used as the left (LHR) and right homologous (RHR) region, respectively. The column-purified LHR and RHR were fused to restriction sites FseI and I-SceI, respectively, using Gibson assembly. This resulted in the pSN054_PfGCN5-GFP-3HA-LoxP plasmid that was used to modify the pfgcn5 locus. For generating rescue constructs, we used the pEOE-2X-attP-3xFlag described elsewhere\\u003csup\\u003e\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e\\u003c/sup\\u003e. An upstream region, extending 987 bp from the pfgcn5 start codon, was amplified with primer pair KR104 and KR105 and utilized as a promoter. GCN5 variants amplified with the indicated primers in \\u003cb\\u003eSupplemental table 2\\u003c/b\\u003e were introduced at the AvrII restriction site. All amplicons were introduced using In-Fusion cloning. To make the deletion constructs and HiBiT insertion, site-directed mutagenesis was employed using primers KR333, KR334, KR335, and KR300. For generating the LgBiT line, we used the pTEOE vector previously described and inserted the LgBiT amplicon at the XhoI site using in-fusion cloning \\u003csup\\u003e\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e\\u003c/sup\\u003e. The LgBiT ORF was amplified from the LgBiT expression vector from Promega using primers KR256 and KR258. For CRISPR/Cas9 editing, we used the Cas9-encoding pAIO3 plasmid, previously described \\u003csup\\u003e\\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e\\u003c/sup\\u003e. Primers and the corresponding reverse complement needed to make gRNAs 45 were annealed in a thermal cycler and inserted into the AvrII restriction site of pAIO3 by In-Fusion cloning. All plasmids and their parasite integration products were analyzed by PCR and sequencing.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eParasite culture, transfection, selection, and synchronization\\u003c/h2\\u003e\\u003cp\\u003eNF54 attB parasites expressing Cre recombinase\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e were maintained in human red blood cells (3% hematocrit) prepared in RPMI 1640 supplemented with AlbuMAX (2.5 g/L), sodium pyruvate (110 mg/L), hypoxanthine (15 mg/L), HEPES (1.19 g/L), sodium bicarbonate (2.52 g/L), glucose (2 g/L), and gentamycin (10 ug/L). For CRISPR/Cas9 editing of the endogenous \\u003cem\\u003epfgcn5\\u003c/em\\u003e locus, we used cultures with more than 5% ring stages and transfected parasites via electroporation using a Bio-Rad Gene Pulser. For complementation of the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e line, plasmids containing WT, GCN5 homologs, PyChimera or deletion constructs were independently co-transfected with a Bxb1 integrase plasmid for integration at the non-essential locus \\u003cem\\u003ecg6\\u003c/em\\u003e \\u003csup\\u003e72\\u003c/sup\\u003e. For rapamycin-mediated excision, we used a concentration of 20 nM for 24 hours. While the PfGCN5\\u003csup\\u003eLoxP\\u003c/sup\\u003e line was maintained in 2.5 \\u0026micro;g/mL blasticidin (BSD), all rescue lines were kept in a medium containing 2.5 \\u0026micro;g/mL BSD and 5 nM WR99210 (WR). The LgBiT or LgBiT-HiBiT lines were supplemented with 12.5 \\u0026micro;g/mL DSM1 or 12.5 \\u0026micro;g/mL DSM1 and 5 nM WR, respectively, for selection. Parasites were cloned by limiting dilution seeding\\u0026thinsp;~\\u0026thinsp;0.5 parasites per well in a 96-well plate. For synchronization, asynchronous cultures with more than 5% schizont stages were allowed to run through a MACS LD magnet column (Miltenyi Biotec). After a wash with warmed media, the column was removed from the magnet and eluted into a 15 mL conical tube. Purified schizonts were allowed to egress for 3 hours in uninfected red blood cells. Newly infected red blood cells were purified by incubation with 5% sorbitol for 10 min.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eGrowth curves and flow cytometry\\u003c/h2\\u003e\\u003cp\\u003eParasites were seeded at a starting parasitemia of 1% with or without rapamycin and plated in triplicate on a 96-well plate. Every other day, the media was changed, and the parasitemia was measured by flow cytometry using 50,000 events recorded per sample in an Attune NxT Flow cytometer. Cells were stained with acridine orange diluted 1:25 in PBS. At day 4, after parasitemia was recorded, cultures were diluted 1:5 to prevent overgrowth. Cumulative parasitemia was back calculated based on the dilution factor. Measured parasitemia at day 0 was subtracted from the final parasitemia obtained on day 6 to control for differences at the start of the experiment. The average parasitemia on day 6 for the PfGCN5 KO line in the absence of rapamycin was considered 100% growth. The percentage growth for the rescue lines was calculated as the ratio between the average parasitemia from each line and the parasitemia from the PfGCN5 KO line on day 6.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eWestern blotting\\u003c/h2\\u003e\\u003cp\\u003eTo visualize the PfGCN5 endogenous protein, we processed at least 5 mL of culture at 5% parasitemia and lysed it in PBS containing 0.035% saponin at 4\\u0026deg;C for 5 min. Saponin pellets were then resuspended in 1x sample buffer containing beta-mercaptoethanol and boiled at 99\\u0026deg;C. After centrifugation at 14,000 rpm in a microfuge at 4\\u0026deg;C for 10 min, a fraction of the supernatant was subjected to SDS-PAGE and immunoblotting. To visualize the GCN5 variants, we processed at least 20 mL of culture at 5% parasitemia and lysed as described above. Saponin pellets were resuspended in an NP40-containing lysis buffer (0.1% NP40, 50mM Tris, and 150 mM NaCl, 1x HALT protease inhibitor) and subjected to three cycles of freezing and thawing, followed by sonication. After spinning at 14,000 rpm at 4\\u0026deg;C for 10 min, protein lysates were incubated with mouse anti-FLAG magnetic beads overnight at 4\\u0026deg;C. Elution was performed in boiling 2x sample buffer containing beta-mercaptoethanol. Flow-throughs were used to visualize various loading controls upon western blotting. Primary antibodies included rabbit anti-HA (1:1,000), rat anti-FLAG (1:1,000), mouse anti-histone H3 (1:1,000), mouse anti-H3K9ac (1:1,000), mouse anti-LgBiT (1:1,000), mouse anti-HiBiT (1:1,000), rabbit anti-HAD1 (1:1,000), and mouse anti-PMV (1:500). In each case, corresponding IRDye conjugated secondary antibodies were used at 1:10,000 dilution. An Odyssey imaging system (Licor) was utilized to visualize blots.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eImmunofluorescence assays\\u003c/h2\\u003e\\u003cp\\u003eCells were fixed and permeabilized in Hemacolor\\u0026reg; fixing solution (product number 1.11955) for 10 seconds and then rinsed three times in PBS. Blocking was performed using 3% BSA in PBS for one hour at room temperature or overnight at 4\\u0026deg;C. Dilutions used for primary antibodies are as follows: rabbit anti-HA (1:500), rat anti-FLAG (1:500), rabbit anti-aldolase (1:500), mouse anti-LgBiT (1:250), and mouse anti-HiBiT (1:500). Secondary antibodies, Alexa Fluor 488 or 555 (Life Technologies), were used at a 1:2,000 dilution. ProLong antifade and 4\\u0026rsquo;,6\\u0026rsquo;-diamidino-2-phenylindole (DAPI) (Invitrogen) were used to mount cells. Images were taken in a Zeiss Imager M2 Plus wide-field fluorescence microscope.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCytoplasmic and nuclear fractionation\\u003c/h2\\u003e\\u003cp\\u003eParasites were lysed in half of the culture volume with cold 0.035% saponin for 4 min at 4\\u0026deg;C and washed with cold 1x PBS. Parasite pellets were then resuspended in cytoplasmic lysis buffer (25mM Tris-HCl pH 7.5, 10mM NaCl, 1.5mM MgCl2, 1% Igepal, halt protease inhibitor cocktail, 1mM PMSF, 50mM sodium fluoride and 1mM sodium orthovanadate) and incubated on ice for 30 minutes. For complete and gentle homogenization, samples were macerated in a cold glass douncer and centrifuged at 13,300 rpm for 10 min at 4C. Supernatants were saved as cytoplasmic fractions, and the remaining pellets were lysed in 0.1% Igepal buffer containing 150mM NaCl and 50mM Tris HCl pH 7.6 for immunoprecipitations or 1x sample buffer containing beta-mercaptoethanol for western blots.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eImmunoprecipitation\\u003c/h2\\u003e\\u003cp\\u003eSaponin pellets were resuspended in 0.1% Igepal buffer (150mM NaCl and 50mM Tris HCl pH 7.6) containing Halt\\u0026trade; protease inhibitor cocktail EDTA-free (ThermoFisher, cat. number 78425), 1mM PMSF, 50mM sodium fluoride and 1mM sodium orthovanadate. After three cycles of freezing and thawing, samples were sonicated and spun at 13,500 rpm for 10min at 4C. The recovered supernatant was incubated overnight at 4C with mouse-anti FLAG magnetic beads (Sigma, cat. number M8823) or mouse-anti HiBiT magnetic beads (cat. number CS3278A08). Antigen-conjugated beads were magnetized and washed two times with 1X TBS. The antigen was eluted in boiling 2X sample buffer containing beta-mercaptoethanol. In some cases, elution was performed using 0.1M Glycine pH 2.0 in continuous rotation at room temperature for 15 minutes. Once the eluted antigen was recovered the pH was neutralized using 1M Tris pH 8.0.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eHistone extraction\\u003c/h2\\u003e\\u003cp\\u003eApproximately 10 mL of synchronous cultures at 5% parasitemia and 3% hematocrit were treated with cold 0.035% saponin in PBS and then washed with cold PBS. Cell pellets were washed twice with a nuclear extraction buffer (15 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 5 mM MgCl\\u003csub\\u003e2\\u003c/sub\\u003e, 1 mM CaCl\\u003csub\\u003e2\\u003c/sub\\u003e, 250 mM sucrose, 500 uM AEBSF, 1 mM DTT, 5 nM microcystin, 10 mM sodium butyrate, and 1x HALT protease cocktail inhibitor). Briefly, washed cell pellets were treated with the above-described nuclear extraction buffer containing 0.3% NP-40 and incubated on ice for 30 minutes, followed by homogenization in a chilled douncer. After centrifugation at 2,000g at 4\\u0026deg;C for 10 minutes, the supernatant (cytosolic fraction) was removed, and the pellet (nuclear fraction) was washed twice in the nuclear extraction buffer. Following centrifugation as above, the pellet was resuspended in chilled 0.2 M H\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e and incubated with constant rotation for 2 hours at 4\\u0026deg;C. Posterior to centrifugation at 3,400g at 4\\u0026deg;C for 10 minutes, solubilized histones were then treated with 100% trichloroacetic acid (~\\u0026thinsp;25% of the total volume) and incubated overnight at 4\\u0026deg;C. Precipitated histones were then washed with cold acetone containing 0.1% HCl, followed by a final wash with ice-cold acetone before resuspending extracted histones in 50mM ammonium bicarbonate.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eMass spectrometry identification of histone acetylation modifications\\u003c/h2\\u003e\\u003cp\\u003eThe histones were extracted and prepared for chemical derivatization and digestion as described previously \\u003csup\\u003e\\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e74\\u003c/span\\u003e\\u003c/sup\\u003e. In brief, the lysine residues from histones were derivatized with the propionylation reagent (1:2 reagent:sample ratio) containing acetonitrile and propionic anhydride (3:1), and the solution pH was adjusted to 8.0 using ammonium hydroxide. The propionylation was performed twice and the samples were dried on speed vac. The derivatized histones were then digested with trypsin at a 1:50 ratio (wt/wt) in 50 mM ammonium bicarbonate buffer at 37\\u0026deg;C overnight. The N-termini of histone peptides were derivatized with the propionylation reagent twice and dried on speed vac. The peptides were desalted with the self-packed C18 stage tip. The purified peptides were then dried and reconstituted in 0.1% formic acid. An LC-MS/MS system consisted of a Vanquish Neo UHPLC coupled to an Orbitrap Exploris 240 (Thermo Scientific) was used for peptide analysis. Histones peptide samples were maintained at 7\\u0026deg;C on sample tray in LC. Separation of peptides was carried out on an Easy-Spray\\u0026trade; PepMap\\u0026trade; Neo nano-column (2 \\u0026micro;m, C18, 75 \\u0026micro;m X 150 mm) at room temperature with a mobile phase. The chromatography conditions consisted of a linear gradient from 2 to 32% solvent B (0.1% formic acid in 100% acetonitrile) in solvent A (0.1% formic acid in water) over 48 min and then 42 to 98% solvent B over 12 min at a flow rate of 300 nL/min. The mass spectrometer was programmed for data-independent acquisition (DIA). One acquisition cycle consisted of a full MS scan, 35 DIA MS/MS scans of 24 m/z isolation width starting from 295 m/z to reach 1100 m/z. Typically, full MS scans were acquired in the Orbitrap mass analyzer across 290\\u0026ndash;1100 m/z at a resolution of 60,000 in positive profile mode with an auto maximum injection time and an AGC target of 300%. MS/MS data from HCD fragmentation was collected in the the Orbitrap. These scans typically used an NCE of 30, an AGC target of 1000%, and a maximum injection time of 60 ms. Histone MS data were analyzed with EpiProfile \\u003csup\\u003e\\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e75\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eHiBiT blotting\\u003c/h2\\u003e\\u003cp\\u003eProteins were transferred to PDVF membranes followed by 5 washes with 0.1% TBS-T. Wash buffer was discarded, and a solution containing the LgBiT protein diluted 200-fold in the 1X Nano-Glo\\u0026reg; buffer was added and incubated overnight at 4\\u0026deg;C with gentle rocking. The next day, after allowing membranes to equilibrate to room temperature, furimazine (substrate) was added at a 1:500 dilution and incubated for 5 minutes before imaging.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eLuminescence assays\\u003c/h2\\u003e\\u003cp\\u003eLive-cell kinetics of cytosolic HiBiT-tagged fragments were determined using the Nano-Glo\\u0026reg; Live Cell Assay System (Promega, cat. number N2011) as per manufacturer instructions. Briefly, 100uL of cell cultures at 3% hematocrit and 5% parasitemia were mixed with 25uL of the Nano-Glo\\u0026reg; buffer containing the substrate at 1:20 dilution in opaque white 96-well plates. After gently mixing by hand, luminescence was immediately measured in a Perkin Elmer EnVision 2103 microplate reader. Total levels of HiBiT-tagged fragments were determined using the Nano-Glo\\u0026reg; HiBiT Lytic Detection System (Promega, cat. number N3030) as per manufacturer instructions. An equal volume of cell cultures at 3% hematocrit and 5% parasitemia were mixed with the lytic buffer containing the LgBiT protein and substrate at a 1:100:50 dilution. Plates were incubated in a dark environment at room temperature for 10 min before measuring luminescence in a Perkin Elmer EnVision 2103 microplate reader.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eContributions\\u003c/h2\\u003e\\u003cp\\u003eK.R. and D.E.G. conceived and designed the study. K.R. performed experiments, acquired and analyzed data. A. M. and S. M. acquired data. F.D.L.V. and B.G. performed high-resolution mass spectrometry identification of histone acetylation. K.R. and D.E.G. wrote the manuscript.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgments\\u003c/h2\\u003e\\u003cp\\u003eThis work was supported by the American Heart Association predoctoral fellowship (23PRE1026393) provided to K. R. We thank Dr. Eva Istvan and Dr. Muhammad Hasan for helpful suggestions, Barbara Vaupel for her assistance with cloning, Dr. David Fidock for the NF54 \\u003cem\\u003eattB\\u003c/em\\u003e line, Dr. Josh Beck for the NF54-attB-DiCre expressing line, Audrey Odom John for anti-HAD1 antiserum, and the Proteomics \\u0026amp; Mass Spectrometry Facility at the Danforth Plant Science Center for LC/MS data acquisition and analysis.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eGardner MJ et al (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419:498\\u0026ndash;511\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHamilton WL et al (2017) Extreme mutation bias and high AT content in Plasmodium falciparum. Nucleic Acids Res 45:1889\\u0026ndash;1901\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSu X, Lane KD, Xia L, S\\u0026aacute; JM, Wellems TE (2019) Plasmodium genomics and genetics: new insights into malaria pathogenesis, drug resistance, epidemiology, and evolution. Clin Microbiol Rev 32:e00019\\u0026ndash;e00019\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSingh GP et al (2004) Hyper-expansion of asparagines correlates with an abundance of proteins with prion-like domains in Plasmodium falciparum. Mol Biochem Parasitol 137:307\\u0026ndash;319\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePizzi E, Frontali C (2001) Low-complexity regions in Plasmodium falciparum proteins. Genome Res 11:218\\u0026ndash;229\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZilversmit MM et al (2010) Low-complexity regions in Plasmodium falciparum: missing links in the evolution of an extreme genome. Mol Biol Evol 27:2198\\u0026ndash;2209\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMuralidharan V, Goldberg DE (2013) Asparagine repeats in Plasmodium falciparum proteins: good for nothing? PLoS Pathog 9:e1003488\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLu X, Murphy RM (2015) Asparagine repeat peptides: aggregation kinetics and comparison with glutamine repeats. Biochemistry 54:4784\\u0026ndash;4794\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMuralidharan V, Oksman A, Pal P, Lindquist S, Goldberg DE (2012) Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nat Commun 3:1\\u0026ndash;10\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMuralidharan V, Oksman A, Iwamoto M, Wandless TJ, Goldberg DE (2011) Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag. \\u003cem\\u003eProceedings of the National Academy of Sciences\\u003c/em\\u003e 108, 4411\\u0026ndash;4416\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eJosling GA, Selvarajah SA, Petter M, Duffy MF (2012) The role of bromodomain proteins in regulating gene expression. \\u003cem\\u003eGenes\\u003c/em\\u003e vol. 3 320\\u0026ndash;343 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3390/genes3020320\\u003c/span\\u003e\\u003cspan address=\\\"10.3390/genes3020320\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGrant PA et al (1998) A Subset of TAFIIs Are Integral Components of the SAGA Complex Required for Nucleosome Acetylation and Transcriptional Stimulation. Cell 94:45\\u0026ndash;53\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSun J et al (2018) Structural basis for activation of SAGA histone acetyltransferase Gcn5 by partner subunit Ada2. Proc Natl Acad Sci U S A 115:10010\\u0026ndash;10015\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eRawat M et al (2021) Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum. \\u003cem\\u003eScientific Reports 2021 11:1\\u003c/em\\u003e 11, 1\\u0026ndash;13\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLucky AB et al (2023) Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses. Antimicrob Agents Chemother 67\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMiao J et al (2021) A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum. PLoS Pathog 17:e1009351\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eTang J et al (2025) PfGCN5 is essential for Plasmodium falciparum survival and transmission and regulates Pf H2B.Z acetylation and chromatin structure. Nucleic Acids Res 53\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFan Q, An L, Cui L (2004) Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling. Eukaryot Cell 3:264\\u0026ndash;276\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCui L et al (2007) PfGCN5-mediated histone H3 acetylation plays a key role in gene expression in Plasmodium falciparum. Eukaryot Cell 6:1219\\u0026ndash;1227\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXu W, Edmondson DG, Roth SY (1998) Mammalian GCN5 and P/CAF acetyltransferases have homologous amino-terminal domains important for recognition of nucleosomal substrates. Mol Cell Biol 18:5659\\u0026ndash;5669\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSmith ER et al (1998) Cloning of Drosophila GCN5: Conserved Features among Metazoan GCN5 Family Members. Nucleic Acids Res 26 \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://ulrec3.unil.ch:80/\\u003c/span\\u003e\\u003cspan address=\\\"http://ulrec3.unil.ch:80/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMartel A, Brar H, Mayer BF, Charron JB (2017) Diversification of the histone acetyltransferase GCN5 through alternative splicing in brachypodium distachyon. Front Plant Sci 8\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHaque ME et al (2021) The GCN5: Its biological functions and therapeutic potentials. \\u003cem\\u003eClinical Science\\u003c/em\\u003e vol. 135 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1042/CS20200986\\u003c/span\\u003e\\u003cspan address=\\\"10.1042/CS20200986\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eToma-Fukai S et al (2020) Crystal structure of GCN5 PCAF N-terminal domain reveals atypical ubiquitin ligase structure. J Biol Chem 295:14630\\u0026ndash;14639\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBhatti MM, Livingston M, Mullapudi N, Sullivan WJ (2006) Pair of unusual GCN5 histone acetyltransferases and ADA2 homologues in the protozoan parasite Toxoplasma gondii. Eukaryot Cell 5:62\\u0026ndash;76\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBhatti MM, Sullivan WJ (2005) Histone acetylase GCN5 enters the nucleus via importin-α in protozoan parasite Toxoplasma gondii. J Biol Chem 280:5902\\u0026ndash;5908\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePrinzinger R, Pre\\u0026szlig;mar A, Schleucher E (1991) Body temperature in birds. Comp Biochem Physiol Physiol 99:499\\u0026ndash;506\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAmani V et al (1998) Cloned lines of Plasmodium berghei ANKA differ in their abilities to induce experimental cerebral malaria. Infect Immun 66:4093\\u0026ndash;4099\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCumnock K et al (2018) Host Energy Source Is Important for Disease Tolerance to Malaria. Curr Biol 28:1635\\u0026ndash;1642e3\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCollins CR et al (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol 88:687\\u0026ndash;701\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCollins CR et al (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol 88:687\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAdjalley SH, Lee MCS, Fidock DA (2010) A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. Methods Mol Biol 634:87\\u0026ndash;100\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFierro MA, Hussain T, Campin LJ, Beck JR (2023) Knock-sideways by inducible ER retrieval enables a unique approach for studying Plasmodium-secreted proteins. Proc Natl Acad Sci U S A 120:e2308676120\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBhowmick K et al (2020) Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway that is essential for its function. J Cell Sci 133\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eJohnsson A, Xue-Franz\\u0026eacute;n Y, Lundin M, Wright APH (2006) Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes. Eukaryot Cell 5:1337\\u0026ndash;1346\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGan L, Wei Z, Yang Z, Li F, Wang Z (2021) Updated mechanisms of GCN5\\u0026mdash;the monkey king of the plant kingdom in plant development and resistance to abiotic stresses. \\u003cem\\u003eCells\\u003c/em\\u003e vol. 10 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3390/cells10050979\\u003c/span\\u003e\\u003cspan address=\\\"10.3390/cells10050979\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXue-Franz\\u0026eacute;n Y et al (2010) Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles. BMC Genomics 11\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eRawat M, Malhotra R, Shintre S, Pani S, Karmodiya K (2020) Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum. J Biosci 45\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBridgford JL et al (2018) Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome. \\u003cem\\u003eNature Communications 2018 9:1\\u003c/em\\u003e 9, 1\\u0026ndash;9\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLu KY et al (2020) Phosphatidylinositol 3-phosphate and hsp70 protect plasmodium falciparum from heat-induced cell death. Elife 9:1\\u0026ndash;27\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLavrentieva A et al (2025) Viability of Plasmodium falciparum parasites in human plasma under different storage conditions. Vox Sang 120:149\\u0026ndash;154\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMiao J et al (2006) The malaria parasite Plasmodium falciparum histones: Organization, expression, and acetylation. Gene 369:53\\u0026ndash;65\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eNagar P et al (2024) Plasmodium falciparum cysteine protease Falcipain 3: A potential enzyme for proteolytic processing of histone acetyltransferase PfGCN5. Biotechnol Appl Biochem 71:1304\\u0026ndash;1315\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBanerjee R et al (2002) Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Proc Natl Acad Sci U S A 99:990\\u0026ndash;995\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMukherjee S, Nguyen S, Sharma E, Goldberg DE (2022) Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X. \\u003cem\\u003eNature Communications 2022 13:1\\u003c/em\\u003e 13, 1\\u0026ndash;14\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePanchal M et al (2014) Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP. Cell Death Dis 5\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBalu B et al (2009) piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome. BMC Microbiol 9:83\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eOh-hashi K, Furuta E, Fujimura K, Hirata Y (2017) Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells. Biochem Biophys Rep 12:40\\u0026ndash;45\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMarch ZM, King OD, Shorter J (2016) Prion-like domains as epigenetic regulators, scaffolds for subcellular organization, and drivers of neurodegenerative disease. \\u003cem\\u003eBrain Research\\u003c/em\\u003e vol. 1647 9\\u0026ndash;18 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.brainres.2016.02.037\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.brainres.2016.02.037\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eJung JH et al (2020) A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis. Nature 585:256\\u0026ndash;260\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFranzmann TM, Alberti S (2019) Prion-like low-complexity sequences: Key regulators of protein solubility and phase behavior. J Biol Chem 294:7128\\u0026ndash;7136\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHolehouse AS, Kragelund BB (2024) The molecular basis for cellular function of intrinsically disordered protein regions. \\u003cem\\u003eNature Reviews Molecular Cell Biology\\u003c/em\\u003e vol. 25 187\\u0026ndash;211 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/s41580-023-00673-0\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/s41580-023-00673-0\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBoeynaems S et al (2018) Protein phase separation: a new phase in cell biology. Trends Cell Biol 28:420\\u0026ndash;435\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eObsil T, Obsilova V (2011) Structural basis of 14-3-3 protein functions. \\u003cem\\u003eSeminars in Cell and Developmental Biology\\u003c/em\\u003e vol. 22 663\\u0026ndash;672 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.semcdb.2011.09.001\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.semcdb.2011.09.001\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eTzivion G, Shen YH, Zhu J (2001) 14-3-3 Proteins; bringing new definitions to scaffolding. \\u003cem\\u003eOncogene\\u003c/em\\u003e vol. 20 6331\\u0026ndash;6338 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/sj.onc.1204777\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/sj.onc.1204777\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlam MM et al (2015) Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion. Nat Commun 6\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFilisetti D et al (2013) Aminoacylation of plasmodium falciparum trnaasn and insights in the synthesis of asparagine repeats. J Biol Chem 288:36361\\u0026ndash;36371\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eDixon SE, Bhatti MM, Uversky VN, Dunker AK, Sullivan WJ (2011) Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B. Mol Biochem Parasitol 175:192\\u0026ndash;195\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eChao X et al (2024) Histone Acetyltransferase GCN5 Regulates Rice Growth and Development and Enhances Salt Tolerance. Rice Sci. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1016/j.rsci.2024.06.002\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.rsci.2024.06.002\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGaupel AC, Begley TJ, Tenniswood M (2015) Gcn5 Modulates the Cellular Response to Oxidative Stress and Histone Deacetylase Inhibition. J Cell Biochem 116:1982\\u0026ndash;1992\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAnkita Tehlan PNRPK (2025) B. A. K. I. K. N. S. S. K. D. Plasmodium falciparum acetyltransferase GCN5 acts as a dual regulator of essential glycolytic enzyme phosphoglycerate mutase. FEBS J\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBah A, Forman-Kay JD (2016) Modulation of intrinsically disordered protein function by post-translational modifications. J Biol Chem 291:6696\\u0026ndash;6705\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eVan Der Lee R et al (2014) Classification of intrinsically disordered regions and proteins. \\u003cem\\u003eChemical Reviews\\u003c/em\\u003e vol. 114 6589\\u0026ndash;6631 Preprint at \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1021/cr400525m\\u003c/span\\u003e\\u003cspan address=\\\"10.1021/cr400525m\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eChakrabarti P, Chakravarty D (2022) Intrinsically disordered proteins/regions and insight into their biomolecular interactions. Biophys Chem 283\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGupta DK, Patra AT, Zhu L, Gupta AP, Bozdech Z (2016) DNA damage regulation and its role in drug-related phenotypes in the malaria parasites. Sci Rep 6:23603\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFraschka SA-K, Henderson RWM, B\\u0026aacute;rtfai R (2016) H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum. Sci Rep 6:31965\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBanerjee R et al (2002) Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. \\u003cem\\u003eProceedings of the National Academy of Sciences\\u003c/em\\u003e 99, 990\\u0026ndash;995\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePolino AJ, Nasamu AS, Niles JC, Goldberg DE (2020) Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin v. ACS Infect Dis 6:738\\u0026ndash;746\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMukherjee S, Nguyen S, Sharma E, Goldberg DE (2022) Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X. Nat Commun 13\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSigala PA, Crowley JR, Henderson JP, Goldberg DE (2015) Deconvoluting heme biosynthesis to target blood-stage malaria parasites. Elife 4\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eNessel T et al (2020) EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole. Cell Microbiol 22\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAdjalley SH, Lee MCS, Fidock DA (2010) Springer,. A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. in \\u003cem\\u003eIn Vitro Mutagenesis Protocols\\u003c/em\\u003e 87\\u0026ndash;100\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSidoli S, Bhanu NV, Karch KR, Wang X, Garcia BA (2016) Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: From histone extraction to data analysis. \\u003cem\\u003eJournal of Visualized Experiments\\u003c/em\\u003e (2016)\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBhanu NV, Sidoli S, Garcia BA (2020) A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry. Bio Protoc 10:e3756\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eYuan ZF et al (2015) Epiprofile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol Cell Proteomics 14:1696\\u0026ndash;1707\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7490809/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7490809/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e possesses one of the most AT-rich genomes in nature (80.6%). A consequence is an asparagine-rich proteome. A quarter of \\u003cem\\u003eP. falciparum\\u003c/em\\u003e proteins possess poly-asparagine repeats that can extend more than 100 residues. The role of these repeats has remained a mystery in the biology of this parasite. Here, we find that the poly-asparagine repeat-containing N terminus of the histone acetyltransferase PfGCN5 associates with the C-terminal catalytic domain after cleavage in the nucleus. Deletion of the repeat destabilizes the N-terminal polypeptide, leading to impaired parasite development and growth, particularly under stress conditions. Using high-resolution mass spectrometry and western blotting analysis, we uncovered a profound effect of the poly-asparagine repeat on acetylation of histones H3, H3.3, and H4. These findings suggest that the poly-asparagine repeat contributes to PfGCN5 acetyltransferase activity, a role previously attributed solely to its C-terminal domain. This report of a function for a poly-asparagine repeat in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e expands our understanding of a pervasive characteristic of its proteome.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A role for the poly-asparagine repeat in the Plasmodium histone acetyltransferase, PfGCN5\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-04 08:24:02\",\"doi\":\"10.21203/rs.3.rs-7490809/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"834263e6-6f22-4b19-ad73-71759a6edf82\",\"owner\":[],\"postedDate\":\"September 4th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":54179050,\"name\":\"Biological sciences/Microbiology/Parasitology/Parasite biology\"},{\"id\":54179051,\"name\":\"Biological sciences/Microbiology/Parasitology/Parasite evolution\"}],\"tags\":[],\"updatedAt\":\"2026-04-15T23:35:19+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-09-04 08:24:02\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7490809\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7490809\",\"identity\":\"rs-7490809\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}