Biology subjects

Pradel, G.

Publications and source records attributed to Pradel, G..

7 recordsLinked to original sources

bioRxiv2024

Plasmodium falciparum SET10 is a histone H3 lysine K18 methyltransferase that participates in a chromatin modulation network crucial for intraerythrocytic development

Lifecycle progression of the malaria parasite Plasmodium falciparum requires precise tuning of gene expression including histone methylation. The histone methyltransferase PfSET10 was previously described as a H3K4 methyltransferase involved in var gene regulation, making it a prominent antimalarial target. In this study, we investigate the role of PfSET10 in the blood stages of P. falciparum in more detail, using tagged PfSET10-knockout (KO) and -knockdown (KD) lines. We demonstrate a nuclear localization of PfSET10 with peak protein levels in schizonts. PfSET10 deficiency results in reduced intraerythrocytic growth, but has no effect on gametocyte formation. When the PfSET10-KO line is screened for histone methylation variations, lack of PfSET10 renders the parasites unable to mark H3K18me1, while no significant changes in the H3K4 methylation status are observed. Comparative transcriptomic profiling of PfSET10-KO schizonts demonstrates the upregulation of transcripts particularly encoding proteins linked to erythrocyte invasion and multigene family proteins, suggesting a repressive function of the histone methylation mark. TurboID coupled with mass spectrometry further reveals an extensive nuclear PfSET10 interaction network with roles in transcriptional regulation, DNA replication and repair, chromatin remodeling and mRNA processing. Main interactors of PfSET10 include ApiAP2 transcription factors, chromatin modulators like PfMORC and PfISWI, mediators of RNA polymerase II, and DNA replication licensing factors. The combined data pinpoint PfSET10 as a histone H3 lysine K18 methyltransferase of the P. falciparum blood stages that regulates nucleic acid metabolic processes as part of a comprehensive chromatin modulation network. ImportanceThe fine-tuned regulation of DNA replication and transcription is particularly crucial for the rapidly multiplying blood stages of malaria parasites and proteins involved in these processes represent important drug targets. This study demonstrates that contrary to previous reports the histone methyltransferase PfSET10 of the malaria parasite Plasmodium falciparum methylates histone 3 at lysine K18, a histone mark to date not well understood. Deficiency of PfSET10 due to genetic knockout affects genes involved in intraerythrocytic development. Furthermore, in the nuclei of blood stage parasites, PfSET10 interacts with various protein complexes crucial for DNA replication, remodeling and repair, as well as for transcriptional regulation and mRNA processing. In summary, this study highlights PfSET10 as a H3K18 methyltransferase with critical functions in chromatin maintenance during the development of P. falciparum in red blood cells.

microbiology↗

bioRxiv2023

The Plasmodium falciparum CCCH zinc finger protein MD3 regulates male gametocytogenesis through its interaction with RNA-binding proteins

Malaria transmission to mosquitoes is dependent on the formation of gametocytes. When fully matured, gametocytes are able to transform into gametes in the mosquitos midgut, a process accompanied with their egress from the enveloping erythrocyte. Gametocyte maturation and gametogenesis require a well-coordinated gene expression programme that involves a wide spectrum of regulatory proteins, ranging from histone modifiers to transcription factors to RNA-binding proteins. Here, we investigated the role of the CCCH-zinc finger protein MD3 in P. falciparum gametocytogenesis. MD3 was originally identified by us as an epigenetically regulated protein of immature gametocytes and recently shown to be involved in male development in a barcode-based screen in P. berghei. We here show that MD3 is mainly present in the cytoplasm of immature male P. falciparum gametocytes. Parasites deficient of MD3 are impaired in gametocyte maturation and male gametocyte exflagellation. BioID analysis in combination with co-immunoprecipitation assays unveiled an interaction network of MD3 with RNA-binding proteins like PABP1 and ALBA3, with translational initiators, regulators and repressors like elF4G, PUF1, NOT1 and CITH, and with other regulators of gametocytogenesis, including ZNF4, MD1 and GD1. We conclude that MD3 is part of a regulator complex crucial for post-transcriptional fine-tuning of male gametocytogenesis.

microbiology↗

bioRxiv2023

Proximity interaction analysis of the Plasmodium falciparum putative ubiquitin ligase PfRNF1 reveals a role in RNA regulation

Some proteins have acquired both ubiquitin ligase activity and RNA-binding properties and are therefore known as RNA-binding Ubiquitin ligases (RBULs). These proteins provide a link between the RNA metabolism and the ubiquitin proteasome system (UPS). The UPS is a crucial protein surveillance system of eukaryotes primarily involved in the selective proteolysis of proteins which are covalently marked with ubiquitin through a series of steps involving ubiquitin E1 activating, E2 conjugating and E3 ligating enzymes. The UPS also regulates other key cellular processes such as cell cycle, proliferation, cell differentiation, transcription and signal transduction. While RBULs have been characterized in other organisms, little is known about their role in Plasmodium falciparum, the causative agent of the deadliest human malaria, malaria tropica. In this study, we characterized a previously identified putative P. falciparum RING finger E3 ligase PfRNF1. We show that the protein is highly expressed in sexual stage parasites and mainly present in immature male gametocytes. Using proximity interaction studies with parasite lines expressing PfRNF1 tagged with the Biotin ligase BirA, we identified an interaction network of PfRNF1 in both the asexual blood stages and gametocytes composed mainly of ribosomal proteins, RNA-binding proteins including translational repressors such DOZI, CITH, PUF1 and members of the CCR4-NOT complex, as well as proteins of the UPS such as RPN11, RPT1 and RPT6. Our interaction network analysis reveals PfRNF1 as a potential RNA-binding E3 ligase which links RNA dependent processes with protein ubiquitination to regulate gene expression. ImportanceRBULs provide a link between RNA-mediated processes with the ubiquitin system. Only a few RBULs have been identified and none has been characterized in the malaria parasite P. falciparum. In this study, we unveiled the interactome of the putative P. falciparum E3 ligase PfRNF1. We show that PfRNF1 interacts with both proteins of the ubiquitin system as well as RNA-binding proteins therefore indicating that it is a putative RBUL which links RNA regulation with the ubiquitin system in P. falciparum.

cell biology↗

bioRxiv2023

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole ruptures prior to the erythrocyte membrane. Membrane rupture requires the exocytosis of specialized secretory vesicles of the parasites; i.e. the osmiophilic bodies (OBs) involved in rupturing the parasitophorous vacuole membrane, and vesicles (here termed g-exonemes) that harbour the perforin-like protein PPLP2 required for erythrocyte lysis. While several OB proteins are known, like G377 and MDV1/Peg3, the protein composition of the g-exonemes remains unclear. Here, we used high- resolution imaging and BioID methods to study the two types of egress vesicles in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the g-exonemes and that exocytosis of the g-exonemes, but not of the OBs, is calcium-sensitive. Further, the two types of vesicles exhibit distinct proteomes. In addition to known egress-related proteins, our analyses revealed novel components of OBs and g-exonemes, including proteins involved in vesicle trafficking. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

molecular biology↗

bioRxiv2022

Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes of Plasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated these proteins as mitochondrial residents as they were found to be localized as punctate foci. We further validated PfPHBs as organellar proteins residing in the Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

molecular biology↗

bioRxiv2022

The Plasmodium falciparum CCCH zinc finger protein ZNF4 plays an important role in gametocyte exflagellation through the regulation of male gametocyte enriched transcripts

CCCH zinc finger proteins (ZFPs) function mainly as RNA-binding proteins (RBPs) where they play a central role in the mRNA metabolism. Over 27 CCCH-ZFPs are encoded in the genome of the human malaria parasite Plasmodium falciparum, the causative agent of malaria tropica. However, little is known about their functions. In this study, we characterize one member of the PfCCCH-ZFP named ZNF4. We show that ZNF4 is highly expressed in mature gametocytes where it predominantly localizes to the cytoplasm. Targeted gene disruption of ZNF4 showed no significant effect in asexual blood stage replication and gametocyte development while male gametocyte exflagellation was significantly impaired leading to reduced malaria transmission in the mosquito. Comparative transcriptomics between wildtype (WT) and the ZNF4-deficient line (ZNF4-KO) demonstrated the de-regulation of about 473 genes (274-upregulated and 199 down-regulated) in mature gametocytes. Most of the down-regulated genes show peak expression in mature gametocyte with male enriched genes associated to the axonemal dynein complex formation and cell projection organization highly affected, pointing to the phenotype in male gametocyte exflagellation. Up-regulated genes are associated to ATP synthesis. Our combine data therefore indicate that ZNF4 is a CCCH zinc finger protein which plays an important role in male gametocyte exflagellation through the regulation of male gametocyte enriched genes. Author SummaryCCCH ZFPs have gained significant interest due to their ability to interact with RNA and control different RNA metabolic processes. The role of CCCH ZFPs in the malaria parasite Plasmodium falciparum has not been well studied. In this study we report the functional characterization of a PfCCCH-ZFP named ZNF4. Using mouse anti-ZNF4 antisera and a ZNF4-HA tag parasite line we show that the protein is mainly expressed in the cytosol of mature gametocytes. To determine the function of ZNF4, we generated a ZNF4 knockout parasite line and we found that the asexual blood stages and gametocytes developed normally while the ability of the male gametocytes to exflagellate as well as the further development of the parasite in the mosquito was significantly impaired. Interestingly, when the transcriptome of mature gametocytes of the ZNF4-KO was compared to the WT, down-regulated genes were mainly male gametocyte enriched genes associated to processes involved in gametocyte exflagellation such axonemal dynein complex formation and cell projection organization indicating that ZNF4 plays an important role in male gametocyte exflagellation through the regulation of male gametocyte enriched transcripts.

cell biology↗

bioRxiv2019

A patatin-like phospholipase is crucial for gametocyte induction in the malaria parasite Plasmodium falciparum

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on erythrocytic replication, gametocyte maturation and gametogenesis. However, blood stage parasites lacking PNPLA1 were severely impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2. Because PC synthesis is pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis, we hypothesize that the high PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

cell biology↗