Biology subjects

Farrell, B.

Publications and source records attributed to Farrell, B..

8 recordsLinked to original sources

bioRxiv2026

Dynamic hinge-motion of PfRIPR revealed by malaria invasion inhibitory antibodies

The PfPCRCR complex is essential for invasion of human erythrocytes by the deadliest malaria parasite, Plasmodium falciparum. Antibodies against each subunit of PfPCRCR prevent erythrocyte invasion and the PfRH5 component is currently the most advanced blood-stage malaria vaccine. Central within PfPCRCR is PfRIPR. This complex molecule contains a core and a flexible tail and allows PfPCRCR to bridge the parasite and erythrocyte during invasion. In this study, we generated a small panel of human monoclonal antibodies against PfRIPR. We structurally characterised four PfRIPR tail-binding antibodies in complex with PfRIPR fragments. We show that growth-inhibitory antibody RP.012 induces a kink in the PfRIPR tail while non-inhibitory antibodies do not. Furthermore, we show that these four antibodies modulate each other, either through antagonism or by acting synergistically. These studies have implications for the design of PfRIPR-based vaccine immunogens and indicate that the tail of PfRIPR undergoes essential conformational changes during erythrocyte invasion.

immunology↗

bioRxiv2024

Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3

Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RTKs) presents additional challenges. Oncogenic fusions of RTKs with heterologous partners represent a particularly difficult-to-express protein subtype due to their high flexibility, aggregation propensity and the lack of a known method for extraction within the native lipid environment. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 hours. The resultant protein was reconstituted using SMA copolymers with a specific yield of 300 {micro}g/mL of lysate. Functionally, the protein demonstrated significant kinase domain phosphorylation (t<0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins.

biochemistry↗

bioRxiv2024

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5

Despite recent success in licencing of two malaria vaccines, there continues to be an urgent need for improved vaccine immunogens as the world aims for malaria eradication. The invasion of erythrocytes by Plasmodium falciparum is an essential step in the life cycle of the parasite, preceding symptoms of disease and parasite transmission. Antibodies which target the PfRH5 protein are highly effective at preventing erythrocyte invasion and the most effective growth-inhibitory antibodies bind to a single epitope. Here we used Rosetta-based protein design to produce a focused synthetic immunogen on which this epitope is presented on a small scaffold. Structural biology and biophysics were used to demonstrate that the immunogen is correctly folded and binds neutralising monoclonal antibodies with at least nanomolar affinity. In immunised rats, the immunogen induced PfRH5-targeting antibodies that inhibit parasite growth at a thousand-fold lower concentration that those induced through immunisation with PfRH5. Finally, we show that priming with the focused immunogen and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens. One Sentence SummaryRational, structure-guided design has been used to produce a vaccine immunogen in which the epitope for the most growth-inhibitory monoclonal antibody targeting PfRH5 has been correctly presented on a synthetic scaffold for use in a vaccine to protect from blood-stage malaria.

immunology↗

bioRxiv2023

Natural malaria infection elicits rare but potent neutralizing antibodies to the blood-stage antigen RH5

Plasmodium falciparum RH5 is the most advanced blood-stage malaria vaccine candidate and is under evaluation for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection. Here, we found that RH5-reactive B cells were rare in malaria-exposed individuals despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from these individuals were extensively mutated but mostly targeted non-neutralizing epitopes, in contrast to antibodies from RH5-vaccinated, malaria-naive individuals. However, infection-derived MAD8-151 and MAD8-502 were among the most potent neutralizers out of 186 antibodies isolated from both cohorts and target the same epitopes as the most effective vaccine-induced antibodies. Binding to basigin receptor-proximal epitopes was the primary factor governing the potency of RH5-specific antibodies from both natural infection and vaccination, followed by the strength of binding. These results indicate a clear strategy for the development of next-generation RH5 vaccines for use in malaria-endemic regions.

immunology↗

bioRxiv2023

Structure of the PfRCR complex which bridges the malaria parasite and erythrocyte during invasion

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The five-component PfPCRCR complex, containing PfRH5, PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies or nanobodies against each of these five conserved proteins, making them the leading blood stage malaria vaccine candidates. However, little is known about the molecular mechanism by which PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. This reveals that PfRIPR consists of an ordered multi-domain core flexibly linked to an elongated tail. We test the hypothesis that PfRH5 opens to insert into the membrane, but instead show that a rigid, disulphide-locked PfRH5 can mediate efficient erythrocyte invasion. Finally, we show that the elongated tail of PfRIPR, which is the target of growth- neutralising antibodies, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. Therefore, a modular PfRIPR is linked to the merozoite membrane through an elongated tail, while its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides novel insight into the mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

microbiology↗

bioRxiv2022

Evaluating the differential expression of TAM family receptors and efferocytosis activities in differentiated and polarized THP-1 macrophage

Tissue homeostasis is tightly balanced between cell death and renewal. Each day, as many as 1011 cells die in the human body that need to be removed and replaced. The clearance of apoptotic cells, termed efferocytosis, is crucial to tissue homeostasis. Central to this process is macrophage-mediated efferocytosis. Apart from general phagocytosis, efferocytosis is apoptotic cell-specific, which aids in clearing dead cells and preventing the accumulation of self-antigens released by the apoptotic cells. The TAM family receptor kinases (TYRO3, AXL, MERTK) and ligands (GAS6 and PROS) play important roles in engaging the apoptotic cells to initiate efferocytic engulfment and the downstream cellular responses. Dysregulated efferocytic function in macrophages is associated with human diseases such as atherosclerosis, lupus, lung fibrosis, and cancer. Conversely, understanding the regulation and molecular mechanisms of macrophage efferocytosis can potentially lead to beneficial treatments for the above diseases. Despite numerous efferocytosis studies that use primary and cell line-derived macrophages, there has not been a thorough characterization of a cell line system that can be reliably used for efferocytosis assays. Consequently, many macrophage efferocytosis assays reported do not clearly distinguish efferocytosis from phagocytosis. Here we evaluated the THP-1 cell line as a potential human macrophage cell line system for efferocytosis studies. Consequently, many macrophage efferocytosis assays reported do not clearly distinguish efferocytosis from phagocytosis. Through the study we examined the differential expression of the TAM family receptors and their ligands in the various THP-1 macrophage differentiation and polarization states. We also characterized the THP-1 cell line as a reliable system for performing in vitro efferocytosis studies.

cell biology↗

bioRxiv2018

Electromechanics and Volume Dynamics in Non-excitable Tissue Cells

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis and embryogenesis. How the cell size is set, maintained, and changed over a cells lifetime is not well understood. In this work we focus on how the volume of non-excitable tissue cells is coupled to the cell membrane electrical potential and the concentration of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole cell patch clamp results in a 30 percent increase in cell volume, while hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high potassium solution causes volume increase by up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and in the concentration of permeable ions in the cell surrounding. The model quantitatively predicts that the cell volume is determined by the total amount of intracellular ion and protein content.

biophysics↗

bioRxiv2017

Catch-slip behavior observed upon rupturing membrane-cytoskeleton bonds

Cells are capable of cytoskeleton remodeling in response to environmental cues at the plasma membrane. The propensity to remodel in response to a mechanical stimulus is reflected in part by the lifetime of the membrane-cytoskeleton bonds upon application of a tensile loading rate. We measure the lifetime and force to rupture membrane-cytoskeleton linkages of a head and neck squamous cell carcinoma (HNSCC) cell line, HN-31 by applying a tensile loading rate (< 60 pN/s) with a handle bound to a cell, while monitoring the displacement of the handle at 2 kHz after averaging. We observe the lifetime increases with loading rate, rf to a maximum after which it decreases with further increase in rf. This biphasic relationship appears insensitive to drugs that target microtubule assembly, but is no longer detectable, i.e., lifetime is independent of rf in cells with reduced active Rho-GTPases. The loading rate-time relationship resembles catch-slip behavior reported upon applying tensile loads to separate protein complexes. Under small loads the bonds catch to increase lifetimes, under larger loads their lifetime shortens and they dissociate in a slip-like manner. Our data conforms to a model that considers the membrane-cytoskeleton bonds exhibit a load-dependent conformational change and dissociate via two pathways. We also find the membrane-cytoskeleton linkages strengthen with stationary compressive load, FSC (|FSC| < 40 pN), and conclude this metastatic cell line responds to small mechanical stimuli by promoting cytoskeleton remodeling as evident by observing F-actin within the membrane nanotube (10 {micro}m length) formed after bond rupture.

biophysics↗