The Plasmodium falciparum RING Finger Protein PfRNF1 Forms an Interaction Network with Regulators of Sexual Development

Abstract

RNA-binding E3 ubiquitin ligases (RBULs) provide a link between RNA metabolic processes and the ubiquitin proteasome system (UPS). In humans, RBULs are involved in various biological processes, such as cell proliferation and differentiation, as well as sexual development. To date, little is known about their role in the protozoan parasite Plasmodium falciparum, the causative agent of malaria tropica. We previously identified a novel P. falciparum RBUL, the RING finger E3 ligase PfRNF1, which is highly expressed during gametocyte development. Here, we conducted BioID-based proximity interaction studies to unveil the PfRNF1 interactome. We show that in immature gametocytes, PfRNF1 forms an interaction network that is mainly composed of RNA-binding proteins, including the translational repressors DOZI and CITH and members of the CCR4-NOT complex, as well as UPS-related proteins. In particular, PfRNF1 interacts with recently identified regulators of sexual development like the zinc finger protein PfMD3, with which it shares the majority of interactors. The common interactome of PfRNF1 and PfMD3 comprises several uncharacterized proteins predominantly expressed in male or female gametocytes. Our results demonstrate that PfRNF1 engages with RNA-binding proteins crucial for sex determination in gametocytes, thereby linking posttranscriptional regulation with the UPS.

1. Introduction

Malaria, caused by Plasmodium parasites, leads to 597,000 deaths annually, with Plasmodium falciparum being responsible for the most lethal infections [1]. Malaria pathogenesis is linked to the proliferating blood stages of the parasite, while the sexual stages, particularly the gametocytes, play a critical role in disease transmission by Anopheles mosquitoes. The sexual phase of P. falciparum starts with sexual commitment, which occurs in a small fraction of asexual blood-stage parasites and is triggered by environmental factors such as nutrient depletion. This process is particularly regulated by the Apetala 2 (Ap2) transcription factor AP2-G and leads to gametocytogenesis. During asexual blood-stage replication, the AP2-G-encoding gene is silenced by heterochromatin protein 1 (HP1) but becomes activated following the removal of HP1 by gametocyte development protein 1 (GDV1). AP2-G then enables sexual commitment and the development of gametocytes through the cascading activation of gametocyte-specific genes (e.g., reviewed in [2,3,4]).

AP2-G reprograms asexual blood-stage parasites for sexual development, but how it drives male and female gametocyte differentiation is unclear. Plasmodium lacks sex chromosomes, suggesting that epigenetic and epitranscriptomic factors control sex determination. Several AP2 proteins were shown to regulate sex identity in gametocytes, including AP2-FG and AP2-O3, which promote female gene profiles while repressing male genes, and AP2-G5, which modulates male development [5,6,7,8]. Furthermore, RNA-binding proteins (RBPs) play a critical role during gametocyte development by regulating posttranscriptional processes. Some plasmodial RBPs, such as DOZI (development of zygote inhibited), CITH (worm CAR-I and fly Trailer Hitch), and PUF2 (Pumilio and Fem-3 binding factor 2), repress transcripts in female gametocytes and store them for later use following parasite transmission to mosquitoes, when the zygote needs to develop in the mosquito midgut (e.g., [9,10,11,12]; reviewed in [13,14]).

A still under-investigated group of RBPs with roles in gametocytogenesis are zinc finger proteins (ZFPs) (reviewed in [15]). In general, ZFPs, specifically those bearing C3H1 motifs, play critical roles in RNA metabolic processes like mRNA splicing, polyadenylation, export, and translation, as well as ubiquitination and transcriptional repression (e.g., reviewed in [16,17]). Recently, we characterized two C3H1-ZFPs with important functions during the development of male P. falciparum gametocytes, PfMD3 (male development protein 3) and PfZNF4 (zinc finger protein 4). Both ZFPs were originally identified by us during a transcriptomic screen for genes deregulated upon treatment of gametocytes with the histone deacetylase inhibitor Trichostatin A (TSA) [18]. While parasites deficient in PfMD3 are impaired in male gametocyte maturation, PfZNF4 deficiency blocks male gametogenesis through the downregulation of male-enriched genes, particularly those associated with axoneme formation [19,20]. The plasmodial MD3 is part of a group of sexual regulators that were identified during a global screen of barcoded P. berghei mutants and predicted to be important for the development of male and female gametocytes [21].

Another ZFP identified during the screening for TSA-deregulated genes of P. falciparum gametocytes is the RING finger domain-containing RNA-binding E3 ubiquitin ligase (RBUL) PfRNF1 [18]. Generally, RBULs are key players in linking the RNA metabolism with the ubiquitin–proteasome system (UPS), and in humans, they are involved in various biological processes, such as cell proliferation and differentiation, as well as sexual development (reviewed in [22,23]). Here, we identified the PfRNF1 interactome during the gametocytogenesis of P. falciparum using BioID-based interaction studies and demonstrated a comprehensive interaction network of PfRNF1 with gametocyte-specific RBPs and sexual development regulators.

2. Results

PfRNF1 is a 136-kDa protein with a C-terminal RING zinc finger domain (Figure 1a). AlphaFold protein structure analysis predicted a globular protein with several central helices and a conserved C-terminal ring finger domain (Figure 1b). Analysis of single-cell transcriptomics using publicly available data from the Malaria Cell Atlas revealed low numbers of pfrnf1-expressing cells in the sexual commitment and stalk phase of gametocyte development and increasing numbers of pfrnf1-positive cells during branching and in gametocytes of male and female identity (Figure 1c). Semi-quantitative RT-PCR using RNA from rings, trophozoites, and schizonts, as well as from immature, mature, and activated gametocytes, showed high pfrnf1 transcript levels in gametocytes compared to the those in asexual blood stages, with particularly high levels in immature gametocytes (Figure 1d and Figure S1a).

The stage specificity and subcellular localization of PfRNF1 were investigated using existing anti-PfRNF1.1 and anti-PfRNF1.2 antibodies (Figure S1b). Western blotting of lysates generated from rings, trophozoites, and schizonts as well as from immature and mature gametocytes highlighted prominent PfRNF1 levels in immature gametocytes, while only weak bands were detected in the other parasite stages (Figure 1e). Indirect immunofluorescence assays (IFAs) localized PfRNF1 to the cytoplasm and nucleus of the developing and activated gametocytes and confirmed peak PfRNF1 levels in immature stage II gametocytes (Figure 1f and Figure S2). The expression data were in accord with our previous reports on PfRNF1 [18].

To determine the PfRNF1 interaction network, we generated a transgenic line episomally expressing a PfRNF1-GFP-BirA fusion protein. Blood-stage parasites were transfected with the vector pARL-PfRNF1-pffnpa-GFP-BirA [20,24], whereby the expression of PfRNF1-GFP-BirA was controlled by the gametocyte-specific pffnpa promotor (Figure S3a). Diagnostic PCR confirmed the presence of the respective vector in the transgenic line (Figure S3b). IFA using anti-GFP antibody demonstrated the presence of GFP-tagged PfRNF1 in the maturing gametocytes (Figure S4a). Western blot analysis confirmed the presence of the PfRNF1-GFP-BirA fusion protein with an expected molecular weight of ~200 kDa in gametocyte lysates of line PfRNF1-pffnpa-GFP-BirA (Figure S4b). Protein biotinylation in the immature gametocytes was verified by Western blotting, following incubation of the transgenic parasites with 50 µM biotin for 24 h. Immunoblotting with alkaline phosphatase-conjugated streptavidin resulted in multiple bands of potential biotinylated proteins, including a band running at ~200 kDa, likely representing biotinylated PfRNF1-GFP-BirA (Figure S4c). No prominent bands were detected in lysates of biotin-treated WT NF54 parasites.

Immature gametocytes of line PfRNF1-pffnpa-GFP-BirA were treated with 50 µM biotin for 24 h and subjected to mass spectrometry-based proximity-dependent biotin identification (BioID-MS) to identify the PfRNF1 interactome. BioID-MS resulted in the identification of 233 significantly enriched hits in immature PfRNF1-pffnpa-GFP-BirA gametocytes (Tables S1 and S2). For further analyses, we excluded proteins with predicted signal peptides, which are likely to be secreted, resulting in a total of 226 putative interactors. These included components of the ribosomal subunits and the UPS, proteins involved in translation initiation (eIFs) and repression (e.g., CITH, DOZI), as well as in mRNA decay (e.g., CCR4-NOT components).

The putative PfRNF1 interactors were subjected to STRING-based analyses to investigate protein–protein interaction networks using the Markov Clustering algorithm. A total of 15 clusters were identified, 8 of which included ≥ 4 proteins (Figure 2a; Table S3). The most prominent cluster comprised ribosomal proteins and involved a satellite cluster of proteasomal components (red cluster). Two proteins stood out from this cluster, showing multiple interactions with other cluster members, i.e., the NOC3 (nucleolar complex-associated protein 3) domain-containing protein PF3D7_1466800 (henceforth termed PfNOC3DP) and the nuclear export mediator factor PfNEMF (PF3D7_1202600). Further clusters included proteins with roles in chromatin organization (olive cluster), nuclear transport (green cluster), and mRNA decay (light green cluster); two smaller clusters contained proteins related to splicing, i.e., three SR proteins (medium sea green cluster) and three spliceosomal U4/U6.U5 tri-snRNP components (orchid cluster). These clusters together indicated a strong link between PfRNF1 and RNA metabolic processes. A CCR4-NOT complex cluster (yellow cluster) linked to the RBP PfPUF1 and a cluster of three translation initiation components (cyan cluster) together with clusters of heat shock proteins (dark goldenrod cluster), and prefoldin subunits (medium purple cluster) additionally connected PfRNF1 with processes of proteostasis. Other clusters of the interactome network included components of glycolytic processes (brown cluster) and V-type ATPases (hot pink cluster).

During a recent study on the C3H1-ZFP PfMD3, we identified PfRNF1 as its interactor by BioID analysis and validated the protein–protein interaction via co-immunoprecipitation assays [20]; here, PfMD3 was conversely identified as an interactor of PfRNF1. We therefore compared the interactomes of PfRNF1 (226 proteins) and PfMD3 (98 proteins) and identified 84 interactors shared by both ZFPs (Figure 2b; Table S2). To investigate the potential sex specificity of the interactors shared by PfRNF1 and PfMD3, we visually analyzed their transcriptomic profiles, which are publicly available at the Malaria Cell Atlas database. Of the 84 interactors, transcripts of 11 proteins were particularly abundant in gametocytes of both sexes; 5 were highly abundant in female and 4 in male gametocytes (Figure 2c). Three interactors had high transcript levels in gametocytes but higher transcript levels in the asexual blood stages; 61 interactors had comparable transcript levels in asexual blood stages and gametocytes or were solely expressed in the asexual blood stages. Notably, the shared interactors found in gametocytes included the majority of the recently identified group of regulators of male and female gametocyte development, i.e., GD1, FD1, FD2, FD4, and MD2 [21]. Gene ontology (GO) enrichment analyses assigned the shared interactors in particular to the biological processes of translation and regulation of RNA stability (Figure 2d).

Transcriptomic profiling was performed in more detail for PfRNF1, PfMD3, and 15 selected interactors using the Malaria Cell Atlas database (Figure 3a). PfRNF1 and PfMD3 transcript expression was depicted in gametocytes during development independently of sex (Figure 3b). Interactors of the two bait proteins that were particularly expressed in female gametocytes according to visual interpretation from the respective Malaria Cell Atlas UMAP plot included, in addition to PfFD2, PfFD4, and the CCR4-NOT component PfNOT2, two unknown proteins, namely, PF3D7_0825900 (henceforth termed female gametocyte protein PfFGP1) and the above-mentioned PfNOC3DP (Figure 3c; Table S2). The four proteins that were transcriptionally highly expressed in males included the RBP PfPUF1, the structural inner membrane complex (IMC)-associated protein PfPIP1, the Kelch domain-containing protein PF3D7_1131600 (henceforth termed PfKelchDP), and a yet unknown protein, PF3D7_0602000 (henceforth termed protein of developing gametocytes PfPDG2). Notably, PfPDG2 was previously described as a C3H1-ZFP [15,25]; however, a distinct zinc finger domain could not be annotated. The 11 proteins, which were highly expressed in both male and female gametocytes, included PfRNF1, PfZNF4, PfFD1, PfMD3, PfMDV1 (male development gene 1), and the structural IMC proteins PfPIP2 and PfPIP3. Further proteins in male and female gametocytes were the MKT1 domain-containing protein PF3D7_1003700 (henceforth termed PfMKT1DP), the C3H1-ZFP PF3D7_0522900 (henceforth termed PfZFP-G1), the SUZ domain-containing protein PF3D7_0218200 (henceforth termed PfSUZDP), and a yet unknown Plasmodium protein, PF3D7_1416600 (henceforth termed protein of developing gametocytes PfPDG1). Three proteins exhibited high transcript expression in gametocytes as well as in asexual blood-stage parasites, namely, PfGD1, Pfa35-2, and ornithine aminotransferase PfOAT (Figure 3c, Table S2). Notably, PfMD2 appeared to be expressed in two transcript variants, both of which showed low expression levels and were thus not included in the evaluation.

3. Discussion

Our combined data show that PfRNF1 is an RBUL of gametocytes that forms a comprehensive interaction network with other ZFPs and recently identified regulators of gametocyte development. The expression of PfRNF1 starts in the stalk phase of gametocyte development and continues during branching and the early sex identity phase with peak levels at stage II gametocyte stages. As previously shown by us, the pfrnf1-encoding gene associates with acetylated H3K9, and both PfRNF1-specific transcript and protein levels increase following treatment of gametocytes with TSA [18], suggesting that its expression during sexual development is epigenetically regulated.

The most prominent interactors of PfRNF1 can be divided in two groups. The first group comprises components of the UPS, like proteasome subunits, the ubiquitin-like protein PF3D7_0922100, and the ubiquitin-specific protease PF3D7_0904600. The second group comprises various types of RBPs. These include the ALBA family members ALBA1, ALBA3, and ALBA4, which have functions in mRNA homeostasis and translational regulation [26,27,28], as well as components of the CCR4-NOT core complex like CAF1, CAF40, NOT1-G, NOT1, and NOT2. The CCR4-NOT complex is a conserved large, multifunctional assembly of proteins that function in mRNA decay [29]. The plasmodial components of the CCR4-NOT complex have mainly been studied in P. yoelii. It was demonstrated by loss-of-function studies that PyCCR4-1, PyNOT1-G, and PfCAF1 play crucial roles during gametocyte development and gametogenesis by regulating mRNAs important for these processes [30,31]. Other RBPs that interact with PfRNF1 are associated with translational repression, such as CITH, DOZI, and PABP1. These RBPs store mRNAs that encode proteins required for the development of the mosquito midgut stages in cytosolic granules, and the transcripts are only introduced to protein synthesis at the onset of gametogenesis (reviewed in [14]). A further interacting RBP is PfPUF1. Notably, its deficiency leads to a sharp decline in late-stage gametocytes and a sex-ratio shift towards males [32]. Considering the fact that single-cell transcriptomics assign PfPUF1 particularly to the male branch, a function of PfPUF1 in repressing male transcripts can be considered.

PfRNF1 also interacts with the C3H1-ZFP PfMD3 [20], a regulator of male development. We recently showed that a lack of PfMD3 significantly impairs gametocyte maturation and leads to a sex-ratio shift towards females [20]. We now demonstrate that both ZFPs, PfRNF1 and PfMD3, share the majority of interactors. The majority of the shared interactors have predicted functions in translation and RNA stability and include in particular members of gametocyte development regulators, as originally identified in P. berghei, i.e., GD1, FD1, FD2, FD4, and MD2 [21] as well as PfZNF4, a C3H1-ZFP crucial for male gametogenesis [19]. To be highlighted is the putative interaction of PfRNF1 and PfMD3 with PfGD1, a regulator of female gametocyte development. P. berghei parasites lacking the orthologous PbGD1 show a sex-ratio shift towards males, comparable to the above-mentioned loss-of-function phenotype of PfPUF1. Co-immunoprecipitation assays using PbGD1 as bait revealed several interactors that were also shared between PfRNF1 and PfMD3, such as the RBPs CITH, PUF1, NOT-1G, PABP1, PDG2, and MKT1DB; the sex development regulators FD1, FD2, and FD4; and the ATP-dependent RNA helicase DBP1 and 14-3-3I (PF3D7_0818200) [21]. It is worth mentioning that in yeast, MKT1 is a posttranscriptional regulator that interacts with the poly(A)-binding protein Pab1 to regulate the translation of the mating-type switching endonuclease HO [33], suggesting a comparable role of MKT1DB during the sex determination of P. falciparum gametocytes. PfRNF1 was also identified in a protein interaction network with the male development regulator PfMD1, which is also an interactor of PfMD3. PfMD1 is a component of cytoplasmic granules and involved in male gametocyte development, with the N-terminus of the regulator being crucial for a male fate, while the LOTUS domain at the C-terminus guides male gametocytogenesis [34]. The combined data pinpoint PfRNF1 as part of a network composed of RBPs and translational regulators involved in sex determination, most likely by promoting and repressing or degrading transcripts important for male or female fate. In accord with these findings, a recent single-cell transcriptomics analysis reported that the PfRNF1-encoding gene is targeted by AP2-G5, a transcription factor regulating male development [8].

In conclusion, we provide evidence that PfRNF1 is a multifunctional RBUL that links the UPS with RBPs to control the posttranscriptional machinery of P. falciparum gametocytes. We hypothesize that PfRNF1 is part of a regulatory network that balances the threshold traits of gene products required for sex identity during the branching phase of gametocyte development. In humans, RBULs are currently investigated as novel targets for anticancer therapy (reviewed in [35]), which gives rise to hope that plasmodial RBULs could represent target structures for antimalarials and transmission-blocking agents in future studies.

4. Materials and Methods

4.1. Gene Identifiers

The following PlasmoDB gene IDs were assigned to the genes and proteins examined in this study: PfRNF1 (PF3D7_0314700); Pf39 (PF3D7_1108600); Pfs230 (PF3D7_0209000); PfAMA1 (PF3D7_1133400); PfCCp2 (PF3D7_1455800); PfFBPA (PF3D7_1444800); PfFD1 (PF3D7_1241400); PfFD2 (PF3D7_1146800); PfFD4 (PF3D7_1220000); PfFGP1 (PF3D7_0825900); PfFNPA (PF3D7_1451600); PfGD1 (PF3D7_0927200); PfKelchDP (PF3D7_1131600); PfMD3 (PF3D7_0315600); PfMSP1 (PF3D7_0930300); PfMKT1DP (PF3D7_1003700); PfNOC3DP (PF3D7_1466800); PfNOT2 (PF3D7_1128600); PfPDG1 (PF3D7_1416600); PfPDG2 (PF3D7_0602000); PfPUF1 (PF3D7_0518700); PfSUZDP (PF3D7_0218200); PfZFP-G1 (PF3D7_0522900); PfZNF4 (PF3D7_1134600).

4.2. Antibodies

The following primary antibodies were used in the study: mouse anti-GFP (Roche, Basel, Switzerland); rabbit anti-Pfs230 (BioGenes, Berlin, Germany); rabbit anti-Pf39 (Davids Biotechnology, Regensburg, Germany); rabbit anti-PfMSP-1 (ATCC, Manassas, VA, USA); mouse anti-PfRNF1.1 [20]; mouse anti-PfRNF1.2 [18]. The following dilutions were used: 1) IFA: rabbit anti-Pfs230 (1:500), rabbit anti-Pf39 (1:200), mouse anti-PfRNF1.1 (1:20), mouse anti-PfRNF1.2 (1:20), mouse anti-GFP (1:200), rabbit anti-PfMSP1 (1:100); 2) Western blotting: rabbit anti-Pf39 (1:10,000), mouse anti-GFP (1:500), mouse anti-PfRNF1.2 (1:500).

4.3. Parasite Culture

The gametocyte-producing strain P. falciparum NF54 (termed WT NF54) was used in the experiments. The cultivation of parasites and the purification of gametocytes were performed as described previously (e.g., [20,36]). Human erythrocyte concentrate and serum were purchased from the transfusion medicine department of the University Hospital Aachen, Germany. The work with human blood was approved by the University Hospital Aachen Ethics commission (EK007/13); serum samples were pooled, and the donors remained anonymous.

4.4. Generation of Line PfRNF1-pffnpa-GFP-BirA

The PfRNF1-pffnpa-GFP-BirA parasite line was generated using the vector pARL-pffnpa-GFP-BirA as described previously [20,24,36]. PfRNF1-pffnpa-GFP-BirA-forward-primer (primer 1; Figure S3a) and PfRNF1-pffnpa-GFP-BirA-reverse-primer were used for gene amplification (for primer sequences, see Table S4). The presence of the vector in the transfectant line was confirmed by diagnostic PCR (Figure S3b) using the above forward primer (primer 1), as well as pARL-GFP-BirA-reverse-primer (primer 2; Figure S3a; for primer sequences, see Table S4). The amplification of the fructose bisphosphate aldolase-encoding gene pffbpa was used as loading control, as described previously [19].

4.5. Semi-Quantitative RT-PCR

To determine the transcript expression of PfRNF1, total RNA was isolated from rings, trophozoites, schizonts, and immature and mature gametocytes as well as gametocytes at 30 min post-activation, and semi-quantitative RT-PCR was performed as described previously [19] using pfrnf1-specific primers for transcript amplification (for primer sequences, see Table S4). Stage purity was verified by the amplification of the asexual blood-stage transcript pfama1 (apical membrane antigen 1) and the gametocyte-specific transcript pfccp2 (LCCL domain-containing protein 2); the amplification of the pffbpa transcript (fructose bisphosphate aldolase) served as positive and loading control. Potential gDNA contamination was excluded by pffbpa amplification using RNA samples lacking reverse transcriptase (for primer sequences, see Table S4).

4.6. Western Blotting

Parasite lysates of lines PfRNF1-pffnpa-GFP-BirA and WT NF54 were prepared and subjected to Western blotting as described previously [20,24,36]. PfRNF1 was detected by immunoblotting with mouse anti-PfRNF1.2 antibody, and PfRNF1-GFP-BirA was detected using mouse anti-GFP antibody. Immunoblotting with antibodies against the endoplasmic reticulum-resident protein Pf39 served as a loading control. Lysates of non-infected red blood cells or WT NF54 served as negative controls. For the detection of primary antibodies, goat anti-mouse and anti-rabbit alkaline phosphatase-conjugated secondary antibodies (1:5000; Sigma-Aldrich, Taufkirchen, Germany) were used. Biotinylated proteins were labeled using alkaline phosphatase-conjugated streptavidin (1:1000; Sigma-Aldrich, Taufkirchen, Germany).

4.7. Indirect Immunofluorescence Assay

Methanol-fixed monolayers of blood-stage parasites of lines PfRNF1-pffnpa-GFP-BirA and WT NF54 were subjected to IFA as described previously [20,36]. PfRNF1 was detected by immunolabeling with anti-PfRNF1.1 and anti-PfRNF1-2 antibodies, and PfRNF1-GFP-BirA was detected using mouse anti-GFP antibody. Asexual blood stages and gametocytes were highlighted by anti-PfMSP1 and anti-Pfs230 antisera; sera from non-immunized mice were used for negative control. For the detection of primary antibodies, goat anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 (Invitrogen, Karlsruhe, Germany) were used. The parasite nuclei were stained with Hoechst 33342 (1:5000; Invitrogen, Karlsruhe, Germany).

4.8. BioID-MS Analysis

Percoll-enriched immature gametocytes of lines PfRNF1-pffnpa-GFP-BirA and WT NF54 were treated with 50 µM biotin for 24 h. The cells were subsequently harvested, processed by single-pot solid-phase-enhanced sample preparation, and subjected to liquid chromatography-mass spectrometry analysis, followed by label-free quantification as described previously [20,24,36]. BioID-MS was performed on three independent streptavidin-purified protein samples with three technical replicates for each sample. Only peptides with a minimum length of 7 amino acids were considered. Proteins had to be identified by at least two peptides and present in all three biological replicates with at least a two-fold enrichment compared to the controls. Statistical analysis of the data was conducted using Student’s t-test, which was corrected by the Benjamini–Hochberg (BH) method for multiple hypothesis testing (FDR of 0.01). Proteins with a putative signal peptide were excluded from further investigations.

4.9. Bioinformatics

The 3D structure of PfRNF1 was predicted using the AlphaFold program (https://alphafold.ebi.ac.uk; see entry O97260; accessed on 19 December 2024 [37,38]). Gene expression, protein function, and GO term analysis were performed using the database PlasmoDB (http://plasmoDB.org; accessed on 13 January 2025 [23]). Transcriptomic profiling was carried out using the Malaria Cell Atlas database (https://www.malariacellatlas.org; accessed on 13 January 2025 [39]) with UMAP settings. Network analysis was conducted using the STRING database (version 11.0; https://string-db.org; accessed on 20 December 2024 [40]) utilizing the Markov Clustering algorithm and default settings.

4.10. Data Availability

The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org; accessed on 2 June 2025) via the jPOST partner repository with the dataset identifiers PXD040384 for ProteomeXchange and JPST002050 for jPOST.