Biology subjects

Bulleid, N. J.

Publications and source records attributed to Bulleid, N. J..

3 recordsLinked to original sources

bioRxiv2026

A cotranslational role for selenocysteine in productive protein folding

Selenocysteine (Sec) is generally regarded as the catalytic determinant that distinguishes selenoproteins from their cysteine (Cys)-containing counterparts. Consequently, the biological significance of Sec has largely been attributed to its unique chemical properties within mature proteins. Here, we uncover an unexpected role for Sec in protein biogenesis. Using the endoplasmic reticulum selenoprotein SELENOM as a model, we show that replacement of Sec with Cys redirects the protein into an alternative assembly pathway, resulting in formation of a stable domain-swapped homodimer. Biochemical and structural analyses demonstrate that this phenotype cannot be explained solely by altered active-site chemistry, as dimerisation persists following extensive active-site mutagenesis. Remarkably, reintroduction of Sec at alternative positions suppresses dimer formation, including when Sec is placed within a cleaved signal peptide and is absent from the mature protein. Thus, identical protein sequences can adopt distinct folding outcomes depending on whether Sec incorporation occurred during biosynthesis. These findings reveal a previously unrecognised role for Sec in productive protein folding and raise the possibility that evolutionary retention of the Sec incorporation pathway reflects advantages not only in catalysis but also in protein folding.

cell biology↗

bioRxiv2024

The chemical chaperone 4-phenylbutyric acid rescues molecular cell defects of COL3A1 mutations that cause vascular Ehlers Danlos Syndrome

PurposeVascular Ehlers Danlos Syndrome (vEDS) is a connective tissue disorder caused by COL3A1 mutations for which there are no treatments due to a limited understanding of underlying mechanisms. We aimed to address this critical knowledge gap, focusing on collagen folding, to establish if targeting protein folding represents a potential therapeutic approach. MethodsWe performed a mechanistic analysis of two novel COL3A1 glycine mutations, G189S and G906R, using primary patient fibroblast cultures, and performed pre-clinical proof-of-concept treatments using FDA-approved chemical chaperones targeting protein folding and/or degradation. ResultsCOL3A1 mutations caused secretion of misfolded collagen III and intracellular collagen retention, leading to matrix defects and endoplasmic reticulum (ER) stress, with increased severity for the more C-terminal mutation. Promoting ER protein folding capacity through the chemical chaperone 4-phenylbutyric acid rescued the ER stress, thermostability of secreted collagen, matrix defects and apoptosis. Optimising treatment duration and dosage helped overcome allele-dependent treatment efficacy. In contrast, protein degradation alone or combined with targeting protein folding did not increase efficacy. ConclusionER stress is a molecular mechanism in vEDS that can be influenced by the position of COL3A1 mutation, and promoting protein folding is a putative mechanism-based therapeutic approach that can rescue intra- and extracellular defects.

genetics↗

bioRxiv2021

A Cytosolic Reductase Pathway is Required for Complete N-Glycosylation of an STT3B-Dependent Acceptor Site.

N-linked glycosylation of proteins entering the secretory pathway is an essential post-translational modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide bond, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is dependent on the STT3A-containing oligosaccharide transferase. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

cell biology↗