Biology subjects

Jones, H. B. L.

Publications and source records attributed to Jones, H. B. L..

4 recordsLinked to original sources

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↗

bioRxiv2025

USP30 inhibition improves mitochondrial health through both PINK1-dependent and independent mechanisms

Mitochondrial dysfunction is a key feature of many pathologies, including Parkinsons disease. The selective vulnerability of dopaminergic neurons is thought to be influenced by mitochondrial dysfunction and mutations in the mitophagy regulating proteins PINK1 and Parkin that are known to cause early-onset Parkinsonism in an autosomal recessive manner. Augmentation of mitophagy through inhibition of USP30 may be a viable therapeutic strategy for a number of diseases including Parkinsons. USP30 inhibition has been demonstrated to augment PINK1/PRKN mitophagy but also potentiate basal mitophagy to support the removal of dysfunctional mitochondria. Therefore, long-term de-regulation of mitophagy has been proposed to lead to mitochondrial depletion. We have used an integrated approach across cell lines, primary neurons and iPSC-derived dopaminergic neuronal cultures to assess the short and long-term effects of USP30 inhibition on mitochondrial health and neuronal activity. We investigated the dependence of USP30 inhibition phenotypes on the PINK1/Parkin pathway using genetic ablation and in iPSC-derived neurons from Parkinsons patients with PINK1 or PRKN mutations. Loss of USP30 through CRISPR/Cas9-mediated knockout resulted in increased basal and depolarisation-induced mitophagy in SH-SY5Y cells. Loss of USP30 or pharmacological inhibition altered mitochondrial morphology and led to increases in membrane potential and ATP levels with decreased oxygen consumption, suggesting that USP30 loss results in a more efficient mitochondrial network. These changes in morphology were found to be independent of PINK1 or Parkin. Chronic pharmacological inhibition of USP30 or CRISPRi-mediated knockdown of USP30 did not impact dopaminergic neuronal activity, as assessed by electrophysiological profiling, but did potentiate depolarisation-induced mitophagy in primary and iPSC-derived neuronal cultures. We observed minimal changes in mitophagy levels in iPSC-derived dopaminergic neurons from Parkinsons patients with PINK1 or PRKN mutations that were independent of the ability to produce p65Ub. Importantly, within this experimental paradigm, pharmacological USP30 inhibition increased depolarisation-induced mitophagy in both PINK1 and PRKN patients to the same extent as control neurons. These results support a role for USP30 in modulating the trigger threshold for mitophagy and suggest that USP30 inhibitors may be beneficial in patients with impairments in PINK1/Parkin-mediated mitophagy.

neuroscience↗

bioRxiv2024

Integrative Proximal-Ubiquitomics Profiling for Deubiquitinase and E3 Ligase Substrate Discovery Applied to USP30

Increasing interest in deubiquitinases (DUBs) and ubiquitin E3 ligases as drug targets to modulate critical molecular pathways in disease is driven by the discovery of specific cellular roles of these enzymes. Key to this is the identification of DUB or E3 ligase substrates. While global cellular ubiquitination changes upon perturbation of DUB/E3 ligase activity can be studied using mass spectrometry-based proteomic methods, these datasets include indirect and downstream ubiquitination events. To enrich for direct substrates of DUB/E3 ligase enzymes, we have combined proximity-labelling methodology (APEX2) and subsequent ubiquitination enrichment (based on the K-{varepsilon}-GG motif) to form a proximal-ubiquitome workflow. We have applied this technology to identify altered ubiquitination events in the proximity of the DUB ubiquitin specific protease 30 (USP30) upon its inhibition. We show ubiquitination events previously linked to USP30 on TOMM20 and FKBP8 and the previously undescribed candidate substrate LETM1, which is deubiquitinated in a USP30-dependent manner.

molecular biology↗

bioRxiv2022

ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA and DDA mass spectrometry methods

Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling), that in combination with DIA MS methods allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day.

biochemistry↗