Biology subjects

Wallis, C.

Publications and source records attributed to Wallis, C..

7 recordsLinked to original sources

bioRxiv2026

Mutations within and outside the PfFNT transport pathway drive MMV007839 resistance in Plasmodium falciparum

Plasmodium falciparum, the parasite responsible for the majority of malaria cases and deaths worldwide, poses a major global health challenge due to its ability to rapidly develop resistance to antimalarial drugs. To circumvent existing resistance pathways, it is of interest to identify both new inhibitors and new therapeutic targets within the parasite. One such target is the Plasmodium falciparum Formate-Nitrite Transporter (PfFNT), which mediates essential lactate and proton (H+) export from the parasite cytosol during the intra-erythrocytic stage. Several inhibitors of PfFNT have been identified that block lactate/H transport and/or kill parasites in vitro, including MMV007839, which binds within the substrate transport pathway as revealed by cryo- EM structures. However, three resistance-conferring mutations have previously been identified following prolonged in vitro exposure of parasites to PfFNT inhibitors. Here, we performed further in vitro evolution experiments with MMV007839 in P. falciparum and identified two additional mutations in PfFNT, V200L and L198M, the latter of which is located outside the substrate transport pathway. These mutations are associated with 6-fold and 13-fold changes in the IC50 of MMV007839. Using molecular dynamics simulations, and thermodynamic integration, we show that these mutations, together with previously identified resistance mutations, destabilise inhibitor binding within the transport pathway; and we suggest the molecular mechanisms underlying this destabilisation. Finally, using both molecular docking and additional simulations, we suggest new sites where additional mutations that confer resistance to PfFNT inhibitors may arise. Author SummaryMalaria parasites are becoming resistant to existing drugs, creating an urgent need for new treatments. One promising drug target is the Plasmodium falciparum Formate-Nitrite Transporter (PfFNT), which the parasite needs to remove waste products and survive during infection. Although some compounds that block PfFNT have been identified, several mutations in the protein can make these compounds ineffective. To better understand how resistance might develop, we exposed malaria parasites to the PfFNT inhibitor MMV007839 in the laboratory and identified two new resistance-related mutations, one of which lies outside the MMV007839-binding site. We also used computer models to predict other regions where resistance-conferring mutations could occur. These findings help assess PfFNT as a drug target and provide guidance for designing new drugs that are less likely to be undermined by resistance.

biophysics↗

bioRxiv2026

Targeted epigenetic repression of oncogenic transcription factors via CRISPR/dCas9 locus-specific silencing

Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we implement precision molecular medicine and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high genomic selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive malignancy affecting children and adolescents. We describe the development of a non-viral and programmable polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in patient-derived xenografts (PDXs) of EWS. Moreover, silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. To our knowledge, we describe the first non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which can be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

genomics↗

bioRxiv2026

A proton transfer mechanism in the malaria parasite lactate/H+ symporter reveals a channel-like transporter without conformational changes

The malaria parasite Plasmodium falciparum relies on anaerobic glycolysis for energy during the intraerythrocytic stage, producing lactate and H+ that must be extruded via its formate-nitrite transporter (PfFNT) to prevent death by cytosolic acidification and swelling. Unlike bacterial homologues classified as channels, PfFNT mediates saturable lactate transport and was classified as a transporter, raising questions about its transport mechanism and what distinguishes channels from transporters. Here, we combine over 720 s of molecular dynamics simulations, quantum chemical calculations, and transport assays in Xenopus laevis oocytes to characterize the transport cycle of PfFNT. We show a transport mechanism in which protonation of a central histidine (H230) enables lactate binding, and proton transfer from H230 to lactate is required to release the substrate as neutral lactic acid. This mechanism is supported by data indicating that neutral compounds such as lactamide do not enter the cavity, while anions like iodide, which are unlikely to protonate under physiological conditions, bind in the cavity but are not released and thus block lactate transport. As neither lactate nor lactic acid can diffuse passively through the protein, it is the requirement for proton transfer defines PfFNT as a lactate/H+ transporter rather than a channel. Notably, the PfFNT transport process occurs without significant protein conformational changes. Furthermore, our data indicates that under acidic conditions, small neutral molecules such as formic acid can passively diffuse through the protein. This suggests PfFNT challenges standard definitions of channels and transporters and differs from existing examples of channel-like transporters as it i) operates as a transporter without conformational changes and ii) can switch between transporter and channel modes within the same transport pathway.

biophysics↗

bioRxiv2025

AlphaFold can be used to predict the oligomeric states of proteins

Homooligomerisation is a prevalent and important process that many proteins undergo to form the quaternary structures required for biological function. However, determining oligomeric states and structures experimentally remains technically challenging and time-consuming for many proteins. Here, we show that the protein structure prediction tools AlphaFold2-Multimer and AlphaFold3 can be used to quickly and accurately predict oligomeric states and structures for a range of soluble and membrane proteins. Using a benchmark set of 40 proteins, we provide optimal parameters for minimizing computational cost while maintaining accuracy. We further extended this analysis to a large dataset of over 1,000 proteins using AlphaFold2-Multimer and observe comparable overall performance but find that accurate oligomeric state prediction remains challenging for proteins that lack close structural representatives in the AlphaFold training set. Together, our results suggest both the utility and current limitations of AlphaFold-based oligomeric state prediction, highlight cases where multiple physiologically relevant assemblies may be plausible, and provide practical guidance for applying these methods to proteins lacking experimental structural data.

biophysics↗

bioRxiv2025

KIDINS220 and InsP8 safeguard the stepwise regulation of phosphate exporter XPR1

XPR1 is emerging as the only known inorganic phosphate (Pi) exporter in humans, critical for Pi homeostasis, with its activity stimulated by inositol pyrophosphate InsP8 and regulated by neuronal scaffold protein KIDINS220. Our structural studies reveal InsP8 specifically activates XPR1 in a stepwise manner, involving profound SPX domain movements. Each XPR1 subunit functions with four gating states, in which Pi permeates a constriction site via a "knock-kiss-kick" process. In contrast, KIDINS220 delicately stabilizes XPR1 in a closed conformation through multiple mechanisms, one of which involves trapping the XPR1 1 helix--critical for InsP8 binding--within an interaction hub. InsP8 serves as a key to release KIDINS220s restraint, reinforcing a "key-to-locks" mechanism to safeguard the stepwise activation. Additionally, our study provides direct structural insights into XPR1-associated neuronal disorders and highlights the evolutionary conservation and divergence among XPR1 orthologues, offering a comprehensive understanding of Pi homeostasis across species.

molecular biology↗

bioRxiv2022

Pro-resolving lipid mediators within brain esterified lipid pools are reduced in female rats chronically exposed to traffic-related air pollution or genetically susceptible to Alzheimer's Disease phenotype

Traffic-related air pollution (TRAP) is a risk factor for Alzheimers disease (AD) where neuroinflammation underlies disease progression and pathogenesis. Unresolved inflammation in AD is known to be exacerbated by brain deficits in unesterified pro-resolving lipid mediators enzymatically synthesized from polyunsaturated fatty acids. Recently, we reported that in the brain, unesterified pro-resolving lipid mediators which are bioactive, can also be supplied from less bioactive esterified lipid pools such as neutral lipids (NLs) and phospholipids (PLs). It is not known whether esterified pro-resolving lipid mediators are affected by AD pathology and exacerbated by TRAP exposure. In the present study we addressed this data gap using TgF344-AD male and female rats that express human AD risk genes and their wildtype littermates exposed to filtered air (FA) or TRAP from 1 to 15 months of age. Esterified lipid mediators within NLs and PLs were quantified by mass-spectrometry. We observed a significant reduction in pro-resolving lipid mediators in both NLs and PLs of female TgF344-AD rats compared to wildtype controls. TRAP exposure also reduced pro-resolving lipids in the female brain, mainly in PL pools, but did not exacerbate changes observed in TgF344-AD rats. Minimal changes were observed in males. Our findings indicate that AD genotype and chronic TRAP exposure result in sex-specific deficits in brain esterified pro-resolving lipid mediators, the pool that supplies free and bioactive lipid mediators. These data provide new information on lipid-mediated mechanisms regulating impaired inflammation resolution in AD, and show for the first time that chronic TRAP exposure targets the same lipid network implicated in AD.

neuroscience↗

bioRxiv2019

Genomes from Bacteria Associated with the Canine Oral Cavity: a Test Case for Automated Genome-Based Taxonomic Assignment

Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach was less subjective, and required far less labor. The primary differences in the remaining taxonomic assignments related to proposed taxonomy changes by the GTDB team.

bioinformatics↗