Biology subjects

Wright, G. S. A.

Publications and source records attributed to Wright, G. S. A..

3 recordsLinked to original sources

bioRxiv2025

Reliable repurposing of antibody interactome inside the cell

In biology proximity is paramount and eighty-five percent of the human proteome has at least one documented interacting monoclonal antibody. These molecules penetrate the cytoplasm poorly and are very often non-functional within the cell. Sequence analysis of 106 antibody variable domains alongside the cytoplasmic human proteome shows charge and isoelectric point are characteristics ill adapted to intracellular monodispersity. Characterisation of forty-five single-chain variable fragment (scFv) intrabodies expressed in human cells confirmed charge to have the greatest impact on solubility. We created new interdomain linkers, optimised scFv domain orientation and found variable heavy domain framework sites to be generally positively charged, and promote insolubility, but be amenable to optimisation. This is applied in combination to reduce the search space and refine the products of AI-led inverse folding to create highly soluble, abundant and thermally stable intrabodies that maintain parent antibody epitope recognition. Over six hundred intrabody sequences are described targeting sixty cytoplasmic proteins with linear, conformational, post-translational modification or oligomeric state specificity. Interactions were validated for p53, -synuclein, SOD1, polyQ, FUS/TLS, UCHL1 and GFP. This approach removes obstacles hindering intracellular repurposing of the vast sequenced antibody interactome with applications relevant to many human disease states.

molecular biology↗

bioRxiv2025

A biological PROTAC for α-synuclein.

The accumulation of misfolded and aggregation-prone proteins is the hallmark of neurodegenerative diseases such as Parkinsons disease and amyotrophic lateral sclerosis (ALS). -Synuclein aggregation drives Parkinsons disease pathology, and is a suitable target for selective protein clearance. Biological proteolysis targeting chimeras (bioPROTACs) aim to eliminate disease-causing intracellular proteins using host cell ubiquitination and degradation functions. Here, we describe a bioPROTAC comprising the E3 ubiquitin ligase domain of CHIP (carboxy terminus of Hsc70-interacting protein) fused to NbSyn87, a nanobody specific for -synuclein. Co-expression with -synuclein resulted in a significant decrease in the abundance or complete degradation of both wild-type and Parkinsons disease-associated mutant -synuclein (A53T, A53V, and G51D) dependent on cell type. The bioPROTAC also significantly reduced abundance of insoluble -synuclein aggregates. In contrast, CHIP-based bioPROTACs targeting superoxide dismutase 1 (SOD1) or LIM domain only 2 (LMO2) failed to degrade their targets and in some instances, increased target abundance due to stabilising interactions with the recognition domain. These findings demonstrate key parameters for consideration during BioPROTAC design including target half-life, bioPROTAC solubility, recognition domain binding affinity, molecular chaperone activity, and interdomain linker optimisation. This work demonstrates the use of CHIP-based bioPROTACs for therapeutic degradation of -synuclein in the synucleinopathies and provides insights for future targeted degrader development.

molecular biology↗

bioRxiv2025

The role of Akkermansia muciniphila sulfatases in colonic mucinutilisation

Akkermansia muciniphila, an obligate mucin degrader, is a major member of the human colonic microbiota and has been associated positive health outcomes. Mucins are complex glycoproteins that contain heavily sulfated O-glycans and form the protective colonic mucus layer. Bacterial carbohydrate sulfatases are required to metabolise these heavily sulfated mucin glycans and excessive bacterial foraging has been associated with several diseases. Sulfatases have been linked with inflammatory bowel disease, making these microbiota enzymes potential drug targets. A. muciniphila expresses carbohydrate sulfatases that can act on colonic mucins yet their roles in its metabolism remain opaque. Our data reveal that A. muciniphila requires glycopeptides/protein forms of colonic mucin for metabolism and its sulfatases have unique adaptations compared to Bacteroides species. Localisation studies reveal that desulfation of N-acetyl-D-glucosamine, but not D-galactose, is exclusively periplasmic. A cell surface sulfatase has a novel carbohydrate binding module that binds to colonic mucin. This paints a contrasting picture of sulfated mucin metabolism by Akkermansia muciniphila versus Bacteroides species. These data will be important for understanding the contexts for Akkermansia muciniphilas positive health correlations.

biochemistry↗