Biology subjects

Campbell, L. J.

Publications and source records attributed to Campbell, L. J..

6 recordsLinked to original sources

bioRxiv2026

Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

Deubiquitylases modulate cellular processes by cleaving monoubiquitin or polyubiquitin chains. The ARISC-RAP80 complex partners with BRCA1-BARD1 to form the BRCA1-A super-complex, which recognizes K63-linked ubiquitin chains at DNA damage sites. ARISC-RAP80 contains multiple ubiquitin-binding sites, yet how these influence recognition and cleavage of K63-polyubiquitylated substrates remains unknown. We discover that a composite three-subunit interface allows ARISC-RAP80 to position K63-linked polyubiquitin chains in its catalytic site. Substrate recognition is further supported by RAP80 and non-catalytic ubiquitin-binding sites that impose a compact conformation to K63-polyubiquitylated substrates. This mechanism exploits the inherent flexibility of long ubiquitin chains and differs considerably from other deubiquitylases. Structure-guided mutagenesis validate ubiquitin chain interactions, and cell-based assays demonstrate a functional role of the observed interfaces in chromatin recruitment. Our findings define mechanisms of polyubiquitin chain decoding and cleavage by ARISC-RAP80, linking ubiquitin reading and erasing functions to BRCA1-A mediated DNA damage responses.

biochemistry↗

bioRxiv2024

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation

Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions.

biochemistry↗

bioRxiv2023

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) encodes a divergent class 1b XRN1-resistant RNA element.

Flavivirids are small, enveloped, positive-sense RNA viruses from the Flaviviridae family with genomes between [~]9-13kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. To date, however, no flavivirus-like or flavivirid has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the United Kingdom, utilising high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid [~]11.2kb in length. The virus encodes a single open reading frame of 3456 amino acids and 5 and 3 untranslated regions (UTRs) of 227 and 666nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, 95.64-97.39% pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of putative secondary structures in the 3'-UTR of RaTV indicated a potential exoribonuclease-resistant RNA (xrRNA) structure identified in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro XRN1 digestion assay showing that RaTV likely forms a divergent but functionally homologous XRN1-resistant xrRNA.

microbiology↗

bioRxiv2023

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4AK20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural, and single molecule imaging approaches. We find that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed-AFM to provide real-time visualization of BRCA1-BARD1 complex recognising chromatin, we show a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiqiutin ligase catalytic activity. Multivalent interactions across nucleosomes explains how BRCA1-BARD1 can recognize chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

molecular biology↗

bioRxiv2022

Network analysis reveals the molecular bases of statin pleiotropy that vary with genetic background

Many approved drugs are pleiotropic, for example statins, whose main cholesterol lowering activity is complemented by anticancer and pro-diabetogenic mechanisms involving poorly characterized genetic interaction networks. We investigated these using the Saccharomyces cerevisiae genetic model where most genetic interactions known are limited to the statin-sensitive S288C genetic background. We therefore broadened our approach by investigating gene interactions to include two statin-resistant UWOPS87-2421 and Y55 genetic backgrounds. Networks were functionally focused by selection of HMG1 and BTS1 mevalonate pathway genes for detecting genetic interactions. Networks, multi-layered by genetic background, were analysed for modifying key genes using network centrality (degree, betweenness, closeness), pathway enrichment, functional community modules and gene ontology. Statin treatment induces the unfolded protein response and we found modifying genes related to dysregulated endocytosis and autophagic cell death. To translate results to human cells, human orthologues were searched for other drugs targets, thus identifying candidates for synergistic anticancer bioactivity.

systems biology↗

bioRxiv2017

Disease mediated changes to life history and demography threaten the survival of European amphibian populations

Infectious diseases can influence the life history strategy of their hosts and such influences subsequently impact the demography of infected populations, reducing viability independently of increased mortality or morbidity. Amphibians are the most threatened group of vertebrates and emerging infectious diseases play a large role in their population declines. Viruses of genus Ranavirus are responsible for one of the deadliest of these diseases. To date no work has evaluated the impact of ranaviruses on host life-history post metamorphosis or population demographic structure at the individual level. In this study, we used skeletochronology and morphology to evaluate the impact of ranaviruses on the demography of populations of European common frog (Rana temporaria) in the United Kingdom. We compared ecologically similar populations that differed only in their historical presence or absence of ranaviral disease. Our results suggest that ranaviruses are associated with shifts in the age structure of infected populations, potentially caused by increased adult mortality and associated shifts in the life history of younger age classes. Population projection models indicate that such age truncation could heighten the vulnerability of frog populations to stochastic environmental challenges. Our individual level data provide further compelling evidence that the emergence of infectious diseases can alter host demography, subsequently increasing population vulnerability to additional stressors.

ecology↗