Biology subjects

Roberts, A. R. E.

Publications and source records attributed to Roberts, A. R. E..

5 recordsLinked to original sources

bioRxiv2024

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-alpha-inhibitor regulate crosslinking of the hyaluronan matrix

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter--inhibitor (II) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC*HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric Half-PTX3 and a construct missing the 24 N-terminal residues ({Delta}1-24-PTX3). SAXS determined a length of [~]520 [A] for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC*HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC*HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC*HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

biochemistry↗

bioRxiv2024

Fungi activate Toll-1 dependent immune evasion to induce cell loss in the host brain

Fungi evolve within the host, ensuring their own nutrition and reproduction, at the expense of host health. They intervene in hosts brain function, to alter host behaviour and induce neurodegeneration. In humans, fungal infections are emerging as drivers of neuroinflammation, neurodegenerative diseases and psychiatric disorders. However, how fungi alter the host brain is unknown. Fungi trigger an innate immune response mediated by the Toll-1/TLR receptor, the adaptor MyD88 and the transcription factor Dif/NF{kappa}B, that induce the expression of antimicrobial peptides (AMPs). However, in the nervous system, Toll-1/TLR could also drive an alternative pathway involving the adaptor Sarm, which causes cell death instead. Sarm is the universal inhibitor of MyD88 and could drive immune evasion. The entomopathogenic fungus Beauveria bassiana is well-known to activate Toll-1 signalling in innate immunity in Drosophila. In fruit-flies, the adaptor Wek links Toll-1 to Sarm. Thus, here we asked whether B. bassiana could damage the Drosophila brain via Toll-1, Wek and Sarm. We show that exposure to B. bassiana reduced fly lifespan and impaired locomotion. B. bassiana entered the brain and induced the up-regulation of AMPs, as well as wek and sarm, within the brain. Exposure to B. bassiana caused neuronal and glial loss in the adult Drosophila brain. Importantly, RNAi knockdown of Toll-1, wek or sarm concomitantly with infection prevented B. bassiana induced cell loss. By contrast, over-expression of wek or sarm was sufficient to cause dopaminergic neuron loss in the absence of infection. These data show that B. bassiana caused cell loss in the host brain via Toll-1/Wek/Sarm signalling driving immune evasion. We conclude that pathogens can benefit from an innate immunity receptor to damage the host brain. A similar activation of Sarm downstream of TLRs in response to fungal infections could underlie psychiatric and neurodegenerative diseases in humans.

neuroscience↗

bioRxiv2023

Competitive specific anchorage of molecules onto surfaces: quantitative control of grafting densities and contamination by free anchors

The formation of surfaces decorated with biomacromolecules such as proteins, glycans or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes, and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest, and present effective guidelines towards optimizing incubation conditions for the quantitative control of surface densities. Focussing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

biophysics↗

bioRxiv2022

A quantitative method for sizing hyaluronan and other glycosaminoglycans on surfaces

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift ({Delta}D) and the negative frequency shift (-{Delta}f) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from [~]2 kDa tetrasaccharides to [~]1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the {Delta}D/-{Delta}f ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

biophysics↗

bioRxiv2021

Nuclear cholesterol is required for transcriptional repression by BASP1

Lipids are present within the cell nucleus where they engage with factors involved in gene regulation. Cholesterol associates with chromatin in vivo and stimulates nucleosome packing in-vitro, but its effects on specific transcriptional responses are not clear. Here we show that the lipidated WT1 transcriptional corepressor, BASP1, interacts with cholesterol in the cell nucleus through a conserved cholesterol interaction motif. We demonstrate that BASP1 directly recruits cholesterol to the promoter region of WT1 target genes. Mutation of BASP1 to ablate its interaction with cholesterol or the treatment of cells with drugs that block cholesterol biosynthesis inhibit the transcriptional repressor function of BASP1. We find that the BASP1-cholesterol interaction is required for BASP1-dependent chromatin remodelling and the direction of transcription programs that control cell differentiation. Our study uncovers a mechanism for gene-specific targeting of cholesterol where it is required to mediate transcriptional repression. SignificanceCholesterol is present within the cell nucleus where it associates with chromatin but to date, a direct role for cholesterol in nuclear processes has not been identified. We demonstrate that the transcriptional repressor BASP1 directly interacts with cholesterol within the cell nucleus through a consensus cholesterol interaction motif. BASP1 recruits cholesterol to the promoter region of target genes where it is required to mediate chromatin remodelling and transcriptional repression. Our work demonstrates that nuclear cholesterol plays a direct role in transcriptional regulation.

biochemistry↗