Biology subjects

Bryce, R.

Publications and source records attributed to Bryce, R..

3 recordsLinked to original sources

bioRxiv2025

A Proteolytic Switch: USP5 controls SDE2 function via UBL-directed cleavage

Proteolytic processing is a critical regulatory mechanism in eukaryotic cells, yet the molecular identities and mechanisms underlying these events often remain elusive. Silencing Defective 2 (SDE2) is an essential human protein involved in multiple aspects of genome regulation, including DNA repair, ribosome biogenesis, and mRNA splicing. SDE2 is expressed with an N-terminal ubiquitin-like domain (SDE2UBL) which must be proteolytically cleaved to release the functional C-terminal domain (SDE2CT). This cleavage not only activates SDE2CT but also marks it for subsequent degradation, highlighting the importance of this tightly regulated processing. Despite the crucial role of this cleavage event, the human protease responsible has remained unknown. Here, we identify that the deubiquitinating enzyme, ubiquitin-specific protease 5 (USP5), catalyses the cleavage of SDE2. Using an integrated workflow combining biochemical assays, proteomic profiling, and mass spectrometry, we demonstrate that USP5 selectively processes SDE2 in vitro and in cell. To validate the specificity of this interaction, we engineered SDE2UBL into an activity-based probe, and developed a cellular reporter assay, both of which confirmed USP5 as the primary effector. Biophysical analysis further revealed that SDE2UBL binds to USP5 with similar characteristics to ubiquitin, albeit with reduced affinity, supporting a mechanism of substrate mimicry. Together, these findings uncover a novel regulatory axis for SDE2 function, highlighting an underappreciated role for DUBs in regulating protein maturation events. They also establish a versatile approach for identifying and validating substrate-protease interactions with broader implications for the study of post-translational regulation.

biochemistry↗

bioRxiv2020

LRRC8A regulates hypotonicity-induced NLRP3 inflammasome activation

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1{beta} and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered a therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but the identity of which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of Cl- channels in the regulation of NLRP3 activation. Specifically we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to Cl- channel inhibitors, suggesting there are additional and specific Cl- sensing and regulating mechanisms controlling NLRP3.

immunology↗

bioRxiv2019

Lead optimisation of dehydroemetine for repositioned use in malaria

Drug repositioning offers an effective alternative to de novo drug design to tackle the urgent need for novel anti-malarial treatments. The anti-amoebic compound, emetine dihydrochloride, has been identified as a potent in-vitro inhibitor of the multi-drug resistant strain K1 of Plasmodium falciparum (IC50: 47 nM + 2.1 nM). 2,3-dehydroemetine, a synthetic analogue of emetine dihydrochloride has been claimed to have less cardiotoxic effects than emetine. The structures of two diastereoisomers of 2,3-dehydroemetine were modelled on the reported emetine binding site on cryo-EM structure 3J7A and it was found that (-)-R,S-dehydroemetine mimicked the bound pose of emetine more closely than (-)-S,S-dehydroisoemetine. (-)-R,S-dehydroemetine was also found to be highly potent against the multi-drug resistant K1 strain of P. falciparum in comparison with (-)-S,S-dehydroisoemetine, which loses its potency due to the change of configuration at C-1. In addition to its effect on the asexual erythrocytic stages of P. falciparum, the compounds exhibited gametocidal properties with no cross-resistance against any of the multi-drug resistant strains tested. Drug interaction studies showed (-)-R,S-dehydroemetine to have synergistic antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride, and (-)-R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and displayed atovoquone-like activity on the mitochondrial membrane potential.

microbiology↗