Biology subjects

Sharpe, S.

Publications and source records attributed to Sharpe, S..

10 recordsLinked to original sources

bioRxiv2026

Heparin nucleates and promotes tropoelastin coacervation through a transient interaction driven by domain 36

Elastin is the extracellular matrix (ECM) protein responsible for the elastic recoil property of certain tissues including the skin, arteries, and lung. Elastic fibre assembly begins with the coacervation of soluble monomeric tropoelastin and is driven by interactions with other ECM components such as glycosaminoglycans (GAGs). Previous research shows that GAG interactions can promote tropoelastin coacervation but lack structural and mechanistic details of this interaction. In this study, we describe the key interactions between tropoelastin and heparin using NMR spectroscopy and coacervation experiments. We propose a mechanism in which substoichiometric GAGs can act as a nucleating scaffold, primarily through transient multivalent interactions with domain 36 of tropoelastin, reducing the energetic barrier for coacervation. Our results provide the first detailed molecular view of tropoelastin-GAG interactions and support a role for negatively charged GAGs in modulating tropoelastin coacervation and thus initiating elastic fibre assembly.

biochemistry↗

bioRxiv2026

A free energy landscape screen reveals the disordered conformational ensemble of tropoelastin

Understanding how proteins explore their conformational energy landscapes is essential for linking sequence to function, yet current ensemble methods are limited by sampling inefficiency and poor scalability to large disordered systems. Here we introduce a free energy landscape screen (FELS), a conceptually different approach that replaces sampling-centric ensemble fitting with broad exploration of energy landscapes, screening thousands of landscape shapes--from highly funneled to flat and rugged. By systematically biasing and evaluating large conformer pools according to contact propensities derived from experiment, FELS efficiently identifies sets of conformers that best reproduce experimental data and highlights candidates for structural refinement, without being restricted by chain length or amount of disorder. To demonstrate the power of this approach we applied it to a previously intractable system, human tropoelastin (hTE), a [~]700-residue precursor of elastin. FELS provides the first experimentally defined atomistic view of the hTE conformational ensemble, revealing that this protein is intrinsically disordered yet exhibits distinct local secondary structure and specific, transient medium- and long-range contacts that organize its ensemble. These findings reconcile long-standing conflicting models and demonstrate that FELS provides a general, experimentally driven framework for mapping conformational energy landscapes of large proteins across the continuum between structural order and disorder.

biochemistry↗

bioRxiv2026

Human CD1c-autoreactive T cells recognise Mycobacterium tuberculosis-infected antigen-presenting cells and display cytotoxic effector programmes

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from infection globally, yet the contribution of non-classical T-cell pathways to human immunity remains poorly defined. CD1c-autoreactive T-cells, which recognise self-lipids presented by the antigen-presenting molecule CD1c, are frequent in human blood, but their role during infection is unclear. Here, we investigate how CD1c-expressing antigen-presenting cells (APCs) and Mtb infection shape CD1c-autoreactive T-cell responses using engineered human APC systems, complemented by single-cell transcriptomic profiling to define the ex vivo phenotypic landscape of these T-cells. CD1c is present within human TB granulomas, whereas Mtb down-modulates CD1c expression on infected APCs, consistent with an immune evasion strategy. CD1c-autoreactive T-cells respond more strongly to Mtb-infected CD1c+ APCs than to uninfected cells, exhibiting enhanced activation, cytotoxicity, and diverse cytokine secretion via CD1c-dependent recognition. Under in vitro conditions, these T-cells reduce relative Mtb burden in infected phagocytes. Single-cell RNA-sequencing reveals cytotoxic effector-memory programmes and expression of antimicrobial molecules, providing a mechanistic basis for these responses. Together, these findings define a human CD1c-restricted T-cell response to Mtb-infected APCs and identify autoreactive CD1c-restricted T-cells as a candidate cellular axis for lipid-directed immunity in TB.

immunology↗

bioRxiv2025

Ligand-responsive groove remodelling in human and macaque CD1d reveals a conserved MHC-like gating mechanism

CD1d presents lipid antigens to invariant natural killer T (iNKT) cells. We determined a high-resolution crystal structure of human CD1d bound to -galactosylceramide (-GalCer) at 1.76 [A], enabling detailed investigation of ligand-sensitive conformational flexibility at Phe84, a conserved aromatic residue that caps the F' groove. Electron density at Phe84 revealed multiple side-chain conformations, suggestive of ligand-induced plasticity. Molecular dynamics simulations indicated that the canonical rotamer is energetically favoured in the absence of a stabilising groove-occupying ligand. To assess conservation of this putative gating mechanism, we solved the first CD1d structure from a non-human primate, rhesus macaque CD1d--GalCer, at 1.83 [A] resolution. In contrast to the human complex, Phe84 in macaque CD1d adopted a fixed conformation. As this aromatic residue is conserved across CD1 isoforms and CD1d-expressing species, and mirrors gating residues in MHC class I that regulate peptide accommodation, our findings support a shared evolutionary strategy for managing antigen diversity. These data provide critical insight into the mechanisms of antigen presentation by CD1 molecules. Significance StatementThis study reveals that Phe84, a conserved aromatic residue in CD1d, may act as a ligand-responsive gate modulating F' groove accessibility. This conditional plasticity could enable binding of structurally diverse lipid antigens and appears conserved across CD1 isoforms. The mechanism parallels class I MHC, where gating residues regulate peptide presentation, suggesting an evolutionarily shared strategy for accommodating antigen diversity.

immunology↗

bioRxiv2024

An amyloidogenic fragment of the SARS CoV-2 envelope protein promotes serum amyloid A misfolding and fibrillization

SARS CoV-2 infection can affect a surprising number of organs in the body and cause symptoms such as abnormal blood coagulation, fibrinolytic disturbances, and neurodegeneration. Our study delves into the intricate pathogenic potential of a SARS-CoV-2 envelope protein peptide, shedding light on its implications for multi-organ effects and amyloid formation. Specifically, we focus on the peptide SK9 or 54SFYVYSRVK62 derived from the C-terminus of human SARS coronavirus 2 envelope protein. We demonstrate that SK9 containing peptides readily form classic amyloid structures consistent with predictions of amyloid aggregation algorithms. In vivo, overexpression of proteases such as neutrophil elastase during inflammation can potentially lead to C-terminal peptides containing SK9. We also demonstrate that SK9 can promote the fibrillization of SAA, a protein marker of acute inflammation. Our investigations reveal that the aromatic residues Phe2 and Tyr3 of SK9 play a pivotal role in its amyloidogenic function. We show that the primary sites of SK9-SAA binding lie in the amyloidogenic hotspots of SAA itself. Our results highlight two possible complications of SARS CoV-2 infection in individuals with hyper-inflammation either due to amyloids arising from SK9 containing peptides or SK9-induced AA amyloidosis.

biochemistry↗

bioRxiv2023

Structural studies of a serum amyloid A octamer that is primed to scaffold lipid nanodiscs

Serum amyloid A (SAA) is a highly conserved acute-phase protein that acts on multiple pro-inflammatory pathways during the inflammatory response and is used as a biomarker of inflammation. It has also been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol. In patients with chronic inflammatory diseases, elevated SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to high-density lipoprotein (HDL), stabilizing SAA and altering its functional properties, likely through altered accessibility of protein-protein interaction sites on SAA. While high-resolution structures for lipid-free forms of SAA have been reported, their relationship with the lipid or HDL-bound forms of the protein, has not been established. We used multiple biophysical techniques, including SAXS, TEM, SEC-MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid-free and lipid-bound forms of SAA. SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring-like structures reported for lipid-free ApoA-I. These SAA octamers represent a previously uncharacterized structure for lipid-free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA-I. SAA-lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid-free SAA octamer, suggesting that relatively few conformational rearrangements are required for lipid binding. This study suggests a new model for SAA-lipid interactions and provides new insight into the ability of SAA to stabilize protein-lipid nanodiscs or even replace ApoA-I on HDL particles during inflammation.

biochemistry↗

bioRxiv2021

Induction of specific antibodies, IgG-secreting plasmablasts and memory B cells following BCG vaccination

Many tuberculosis (TB) vaccine candidates are designed as a boost to BCG; an understanding of the BCG-induced immune response is therefore critical, and the opportunity to relate this to circumstances where BCG does protect may direct the design of more efficacious vaccines. While the T cell response to BCG vaccination has been well-characterised, little is known about the B cell and antibody response. We demonstrate BCG vaccine-mediated induction of specific antibodies in different human populations and macaque species which represent important preclinical models for TB vaccine development. We observe a strong correlation between antibody titres in serum versus plasma with modestly higher titres in serum. We also report for the first time the rapid and transient induction of antibody-secreting plasmablasts following BCG vaccination, together with a robust and durable memory B cell response in humans. Finally, we demonstrate a potential contribution of the antibody response to BCG vaccine-mediated control of mycobacterial growth in vitro. Taken together, our findings indicate that the humoral immune response in the context of BCG vaccination merits further attention to determine whether TB vaccine candidates could benefit from the induction of humoral as well as cellular immunity.

immunology↗

bioRxiv2020

mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines to meet a worldwide need. mRNA-based vaccines are the most promising technology for rapid and safe SARS-CoV-2 vaccine development and production. We have designed CVnCoV, a lipid-nanoparticle (LNP) encapsulated, sequence optimised mRNA-based SARS-CoV-2 vaccine that encodes for full length, pre-fusion stabilised Spike protein. Unlike other mRNA-based approaches, CVnCoV exclusively consists of non-chemically modified nucleotides and can be applied at comparatively low doses. Here we demonstrate that CVnCoV induces robust humoral and cellular responses in non-human primates (NHPs). Animals vaccinated with 8 g of CVnCoV were protected from challenge infection with SARS-CoV-2. Comprehensive analyses of pathological changes in challenged animals via lung histopathology and Computed Tomography (CT) scans gave no indication of enhanced disease upon CVnCoV vaccination. These results demonstrate safety, immunogenicity, and protective efficacy of CVnCoV in NHPs that extend our previously published preclinical data and provide strong support for further clinical testing in ongoing phase 2b/3 efficacy studies.

immunology↗

bioRxiv2020

Immunological and pathological outcomes of SARS-CoV-2 challenge after formalin-inactivated vaccine immunisation of ferrets and rhesus macaques

There is an urgent requirement for safe and effective vaccines to prevent novel coronavirus disease (COVID-19) caused by SARS-CoV-2. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferret and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine in ferrets or unvaccinated macaques. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen at seven days post infection in ferrets. This increased lung pathology was observed early in the infection (day 7) but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

pathology↗

bioRxiv2019

The genomic basis of evolved virus resistance is dependent on environmental resources

Parasites impose strong selection on their hosts, but the level of resistance evolved may be constrained by the availability of resources. However, studies identifying the genomic basis of such resource mediated selection are rare, particularly in non-model organisms. Here, we investigated the role of nutrition in the evolution of resistance to a DNA virus (PiGV), and associated trade-offs, in a lepidopteran pest species (Plodia interpunctella). Through selection experiments and whole genome sequencing we identify putative mechanisms of resistance that depend on the nutritional environment during selection. We find that the evolution of resistance is specific to diet, with adaptation to a low nutrition diet constraining resistance when challenged with the pathogen on a high nutrition diet. Resistance in a low nutrition environment is negatively correlated with growth rate, consistent with an established trade-off between immunity and development. Whole genome resequencing of the host shows that resistance mechanisms are highly polygenic and suggests evidence for trade-offs at the genetic level. Critically when populations evolve in high resource conditions, resistance is linked to metabolic and immune pathways, however it is more closely associated with cytoskeleton organisation when selected under low nutrition. Our results emphasise the importance of resources on the evolution of resistance.

evolutionary biology↗