Biology subjects

Kirkman, L.

Publications and source records attributed to Kirkman, L..

5 recordsLinked to original sources

bioRxiv2025

Stage-specific regulation of the Plasmodium falciparum proteasome activity reveals adaptive rewiring in artemisinin resistance

The ubiquitin-proteasome system (UPS) is essential for Plasmodium falciparum to maintain protein homeostasis, adapt to proteotoxic stress, and regulate parasite growth and stage transitions. The proteolytic 20S proteasome core is the central component of the UPS, where unfolded protein substrates are degraded into oligopeptides. Mechanisms regulating malaria parasite proteasome activity are poorly understood and have not been thoroughly studied. This knowledge gap is especially critical in the context of artemisinin (ART) resistance, where parasite survival depends on an enhanced stress response, including a greater reliance on the UPS. Here, we profiled proteasome activity and abundance across the parasite intraerythrocytic developmental cycle (IDC) in both ART-sensitive (ART-S) Dd2 and ART-resistant (ART-R) Dd2K13R539T parasites. We uncovered striking stage-specific regulation: proteasome activity was abundant in the ring stage, decreased in trophozoites, and then peaked in schizonts. Furthermore, ART-R Dd2K13R539T parasites exhibited higher ring-stage proteasome activity than ART-S Dd2, despite reduced proteasome abundance, suggesting a unique adaptive rewiring of proteasome function. To study proteasome regulation in the parasite, we manipulated proteasome abundance in Dd2 and Dd2K13R539T by creating a conditional knockdown of PfUMP1, a conserved proteasome maturation factor. PfUMP1 depletion disrupted 20S assembly, decreased proteasome activity, and led to parasite death. These experiments uncovered two key features of proteasome regulation in P. falciparum: (1) the absence of canonical transcriptional regulation of proteasome genes in response to downregulation of proteasome activity, and (2) ART-R parasites exhibit a ring-stage specific increased sensitivity to proteasome downregulation. Together, our findings reveal a previously unrecognized layer of proteasome regulation in malaria parasites and how, as part of their survival adaptations to decreased hemoglobin uptake associated with ART resistance, these parasites alter proteasome function to survive. This work reinforces the therapeutic potential of the proteasome as a stage- and resistance-specific antimalarial target.

microbiology↗

bioRxiv2025

Recombinant proteasome provides new avenues for anti-malarial drug development.

The Plasmodium falciparum 20S proteasome (Pf20S) has emerged as a promising antimalarial target. Development of therapeutics to this target has previously relied on native purifications of Pf20S, which is challenging and has limited the scope of previous efforts. Here, we report an effective recombinant Pf20S platform to facilitate drug discovery. Proteasome assembly was carried out in insect cells by co-expressing all fourteen subunits along with the essential chaperone homolog, Ump1. Unexpectedly, the isolated proteins consisted of both a mature and an immature complex. Cryo-EM analysis of the immature complexes revealed structural insights detailing how Ump1 and the propeptides of the {beta}2 and {beta}5 subunits coordinate {beta}-ring assembly, which differ from human and yeast homologs. Biochemical validation confirmed that {beta}1, {beta}2, and {beta}5 subunits of the mature proteasome were catalytically active. Clinical proteasome inhibitors, bortezomib, carfilzomib and marizomib were potent but lacked Pf20S selectivity. However, the tripeptide-epoxyketone J-80 inhibited Pf20S {beta}5 with an IC50 of 22.4 nM and 90-fold selectivity over human {beta}5. Structural studies using cryo-EM elucidated the basis for the selective binding of J-80. Further evaluation of novel Pf20S-selective inhibitors such as the reversible TDI-8304 and irreversible analogs, 8304-vinyl sulfone and 8304-epoxyketone, confirmed their potency and selectivity over the human constitutive proteasome. This recombinant Pf20S platform facilitates detailed biochemical and structural studies, accelerating the development of selective antimalarial therapeutics.

biochemistry↗

bioRxiv2025

Plasmodium falciparum DNA repair dynamics reveal unique roles for TLS polymerases and PfRad51 in genome diversification

The haploid malaria parasite, Plasmodium falciparum, evolved a unique cohort of DNA repair pathways enabling the parasite to survive in a vertebrate host red blood cell and the mosquito vector. P. falciparum chromosomes are partitioned into a highly conserved core genome and remarkably diverse, largely subtelomeric regions that contain genes encoding important parasite virulence factors. The molecular mechanisms that maintain this chromosomal structure have not been identified. Here, we describe specific DNA repair pathways that differentiate between hypervariable subtelomeric and conserved core regions of the genome. Based on our previous work, we hypothesized that there are potentially important interactions between translesion (TLS) and homologous recombination (HR) pathways for the diversification of multicopy gene families in P. falciparum. Thus, we created knockout parasite lines of the DNA repair enzymes: PfRad51 and the TLS polymerases PfPol{zeta} and PfRev1. We identified that irradiation hypersensitivity varied across the cell cycle for TLS{Delta} parasites and was uniform across the erythrocytic cycle for PfRad51{Delta} parasites, highlighting the variable roles of these pathways. However, important interactions between these pathways were found when we studied directed double strand break (DSB) repair, which revealed a difference in the DNA damage response according to chromosomal location. PfRad51 was essential for HR-mediated repair in the core genome. In contrast, we identified a Rad51 independent homology-directed repair in all three of our knockout lines when a DSB was made in the subtelomeric region of the chromosome. We propose that this differential DNA damage response maintains the distinction in diversification across the chromosome.

microbiology↗

bioRxiv2020

Telomere length dynamics in response to DNA damage in malaria parasites

Malaria remains a major cause of morbidity and mortality within the developing world. Recent work has implicated chromosome end stability and the repair of DNA breaks through telomere healing as potent drivers of variant antigen diversification, thus associating basic mechanisms for maintaining genome integrity with aspects of host-parasite interactions. Here we applied long-read sequencing technology to precisely examine the dynamics of telomere addition and chromosome end stabilization in response to double strand breaks within subtelomeric regions. We observed that the process of telomere healing induces the initial synthesis of telomere repeats well in excess of the minimal number required for end stability. However once stabilized, these newly created telomeres appear to function normally, eventually returning to a length nearing that of intact chromosome ends. These results parallel recent observations in humans, suggesting an evolutionarily conserved mechanism for chromosome end repair.

cell biology↗

bioRxiv2019

A histone methyltransferase inhibitor can reverse epigenetically acquired drug resistance in the malaria parasite Plasmodium falciparum

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC50 concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, reversing acquired resistance to the antimalarial compound blasticidin S that is transported through PSAC. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.\n\nImportanceDrug resistance is a major concern for the treatment of infectious diseases throughout the world. For malaria, a novel mechanism of resistance was recently described in which epigenetic modifications led to a resistance phenotype that is rapidly reversible, thus reducing the fitness cost that is often associated with genetic mutations that lead to resistance. The possibility of this type of resistance arising in a natural setting is particularly troubling since parasites could rapidly switch to and from a resistant phenotype, thus making it especially difficult to combat. Here we show that application of a histone methyltransferase inhibitor can rapidly reverse the epigenetic changes that lead to drug resistance, thereby causing parasites to revert to a drug sensitive phenotype. This is a novel application of drugs that target epigenetic modifiers and lends additional support for ongoing efforts to develop drugs against malaria that target the histone modifiers of the parasite.

microbiology↗