Biology subjects

Elliott, P. R.

Publications and source records attributed to Elliott, P. R..

6 recordsLinked to original sources

bioRxiv2026

DNA-induced conformational changes in SPRTN relieve its auto-inhibitory effect on protease activity

The DNA-dependent metalloprotease SPRTN has emerged as a key enzyme in the proteolysis of DNA-protein crosslinks (DPCs), thereby protecting us against genome instability, accelerated ageing, and cancer. DNA and ubiquitin chains serve as the primary activator and catalyst of SPRTN proteolysis, respectively, but how they promote SPRTN activation and activity remains incompletely understood. To address this question, we developed a highly sensitive multi-turnover fluorescence resonance energy transfer (FRET) assay to monitor SPRTN proteolysis in real time. We found that the auto-cleaved N-terminal SPRTN fragment, comprising the metalloprotease domain (MPD), zinc-binding domain (ZBD), and basic region (BR), is highly stable, enzymatically active, and retains ubiquitin-dependent activation. Interestingly, the MPD alone exhibits basal intrinsic activity that is independent of both DNA activation and ubiquitin avidity effect. We show that ZBD and MPD together exert steric regulation: ZBD maintains MPD in an autoinhibited state, while MPD largely prevents ZBD from binding DNA. BR, together with DNA, is essential to relieve ZBD-mediated inhibition of MPD. Using a site-trapping approach, we demonstrate that the ZBD-BR-DNA trinity induces an open conformation of the SPRTN N-terminus in cis, thereby releasing autoinhibition. MPD and BR together restrict the DNA-binding stoichiometry of ZBD, enabling SPRTN to function efficiently in proximity to DNA despite its low abundance in vivo. Collectively, our work overturns the long-standing dogma that SPRTN autocleavage inactivates the enzyme and reveals how DNA-induced conformational changes in SPRTN fine-tune its protease activity, providing a prerequisite for subsequent ubiquitin activation and rapid proteolysis of DPCs.

biochemistry↗

bioRxiv2025

KDM7-mediated oxygen sensing reprograms chromatin to enhance hypoxia tolerance in the root

Roots frequently encounter low oxygen (hypoxia) from soil compaction or water saturation and must adapt to this stress. We investigated how root tip cells sense hypoxia and adjust the meristem epigenome to activate genes that promote tolerance and growth under oxygen limitation. In Arabidopsis root tips, hypoxia tolerance was linked to increased trimethylation of histone H3 at lysine 4 (H3K4me3). We also found that group 7 demethylases (KDM7s) are directly inhibited by hypoxia, and that genetic inactivation of KDM7s, like hypoxia, induces expression of genes essential for meristem survival under oxygen deprivation. We propose that KDM7s function as root-specific oxygen sensors that prime and support hypoxia tolerance.

plant biology↗

bioRxiv2025

obABPP-HT*: A Precision-Engineered Activity Proteomics Pipeline for the Streamlined Discovery of Deubiquitinase Inhibitors

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.

biochemistry↗

bioRxiv2024

The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks

DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognises its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs still needs to be discovered. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain named the Ubiquitin interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains. SPRTN binding to ubiquitin chains via USD leads to [~] 67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. This study reveals the ubiquitination of DPCs is the key signal for SPRTNs substrate specificity and rapid proteolysis.

biochemistry↗

bioRxiv2022

Sex-specific deubiquitylation drives immune-related neurodegeneration in Drosophila

Risk of neurodegenerative disease such as late onset Alzheimers is linked to aberrant ubiquitinylation and accumulation of non-degraded proteins in brain cells. A glial network of innate immune genes modulates inflammatory responses to such protein deposition. However, vulnerability differs between the sexes. Here, we show that the Drosophila homologue of the deubiquitylase Trabid can align the sex-specific aspects of neurodegenerative phenotypes with changes in ubiquitylation and inflammatory activity. An enzymatically null Trabid in flies, caused sex-specific changes in locomotion, sleep patterns, brain histology and ultimately, lifespan. These changes were underscored by altered ubiquitin and proteome enrichment profiles and the same enzymatic activity as its human counterpart. When the sex-determination gene transformer was silenced in astrocytes or immunocompetent tissues, sex differences were significantly reduced. Our results indicate that Trabid underscores sex-specificity in disease neurology, by controlling the balance between ubiquitylation and inflammation.

immunology↗

bioRxiv2022

Structural basis for antagonism of the ubiquitin ligase BIRC6 by SMAC

Apoptosis, a form of genetically programmed cell death, can be triggered by either internal or external signals ultimately activating caspases, a family of proteases1. Certain members of the inhibitors of apoptosis (IAP) family are sentinel proteins preventing untimely cell death by inhibiting caspases. IAPs are in turn regulated by antagonists including second mitochondria-derived activator of caspase (SMAC). Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP, possesses dual E2/E3 ubiquitin ligase activity and is implicated in apoptosis via caspase inhibition2-7. How this is achieved remains unknown. Here we show BIRC6 directly restricts activated caspase-3, and ubiquitinates activated caspases-3, -7 and -9 working exclusively with the non-canonical E1, UBA6. Importantly, we show SMAC supresses both mechanisms. Cryo-electron microscopy (cryo-EM) structures of BIRC6 alone and in complex with SMAC reveal BIRC6 exists as an anti-parallel dimer with a substrate-binding module juxtaposed to the catalytic domain at each end, and we identify multiple highly conserved unannotated domains important for architecture and function. Through our structural, biochemical and biophysical findings, we discover SMAC engages BIRC6 at multiple sites resulting in a sub-nanomolar affinity enabling SMAC to competitively displace caspases, thus antagonising BIRC6-mediated caspase inhibition.

biochemistry↗