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Nuclear phosphatase PfNIF2 governs erythrocyte invasion and genome integrity through invasion-related proteins and SR1 dephosphorylation

ABSTRACT: PfNIF2 is a nuclear protein phosphatase belonging to the NIF family in Plasmodium falciparum. It is characterized by a conserved ‘DLDET’ motif, which is essential for its catalytic activity. PfNIF2 primarily localizes to the perinuclear or nuclear compartment and shows peak expression during the schizont stage of the parasite's lifecycle. Genetic disruption of the pfnif2 gene significantly impaired the efficiency of merozoite invasion, resulting in a 1.65-fold reduction (P < 0.05). It also increased susceptibility to artemisinin derivatives, with the IC50 of dihydroartemisinin decreasing from 12.5 nM to 7.9 nM (P < 0.001), although the asexual development of the parasites remained viable. Site-directed mutagenesis confirmed that aspartate 65 within the ‘D63LD65ET’ motif is critical for enzymatic function and invasion competence. Transcriptomic profiling revealed extensive dysregulation of invasion-related genes, including RON4 and MSP1, in schizont-stage PfNIF2Del parasites. Integrated co-immunoprecipitation, LC-MS/MS, and phosphoproteomic analyses demonstrated that PfNIF2 interacts with DNA repair machinery, such as SR1, SUN2, and MSH2-1. It plays a role in dephosphorylating RNA-binding proteins and invasion effectors. Notably, the expression of a phosphoablative variant of serine/arginine-rich splicing factor 1 (SR1-S205A/S208A) in PfNIF2Del parasites substantially rescued defects in DNA damage repair, alternative splicing fidelity, artemisinin sensitivity, and erythrocyte invasion. These findings establish PfNIF2 as a multifunctional regulator that bridges nuclear RNA processing, genome integrity maintenance, and host cell invasion in malaria parasites.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE301593 | GEO | 2025/08/13

REPOSITORIES: GEO

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Project description:Plasmodium falciparum, the most pathogenic human malaria parasite, infects millions of human beings and causes a serious public health threat. Currently, the most potent anti-malarial drugs are artemisinin and its derivatives1,2. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge3. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources4. Once activated, artemisinins are converted into highly reactive carbon-centered radicals5,6 that attack macromolecules through alkylation and propagate a series of damages, eventually leading to parasite death7,8. Even though several parasite proteins have been reported as the targets of artemisinin9,10, the exact mechanism of artemisinin action is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted. Here, we have developed an unbiased chemical proteomics approach to directly probe the mechanism of action of artemisinin in P. falciparum in situ. An alkyne-tagged artemisinin analogue coupled with biotin enables selective purification and identification of 124 artemisinin covalent-binding protein targets, many of which are involved in essential biological processes of the parasite. In vitro assays confirm the specific artemisinin binding and inhibition of selected targets. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using the alkyne-tagged artemisinin coupled with a fluorescent dye to monitor its protein binding, we showed that heme, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The extremely high level of heme released from the hemoglobin digestion by the parasite make artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages) compared to parasites at early ring stage, which have low level of heme, mainly from endogenous synthesis. The ‘blood eating’ nature of the parasite with the release of large amounts of heme confers artemisinin with extremely high specificity against the parasites, with minimum side effects towards healthy red blood cells. Taken together, our results established a unifying model to explain the action and specificity of artemisinin in parasite killing. Our findings could facilitate the development of better alternative strategies to treat malaria in times of emerging artemisinin resistance11,12.

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Nuclear phosphatase PfNIF2 governs erythrocyte invasion and genome integrity through invasion-related proteins and SR1 dephosphorylation

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