Biology subjects

Clague, M. J.

Publications and source records attributed to Clague, M. J..

16 recordsLinked to original sources

bioRxiv2026

HRS dephosphorylation at membrane contact sites promotes sorting within multivesicular endosomes

HRS is a receptor tyrosine kinase substrate and component of the ESCRT machinery. It enables sorting of ubiquitylated cargo into multivesicular endosomes (MVEs) for lysosomal degradation but also functions in receptor recycling. We show that co-depletion of its ESCRT-0 binding partners, STAM1 and STAM2, recapitulates defects in EGF receptor (EGFR) sorting onto intraluminal vesicles (ILVs) but does not mirror the increased MVE size evident after HRS depletion. Using mutagenesis of the endogenous gene or introduction of APEX2-tagged HRS variants, we find that HRS Y329/334 phosphorylation is dispensable for EGFR sorting and MVE size control. AnnexinA1 mediates endosomal contact with the ER and promotes EGF-stimulated ILV formation. We show that reduced ILV formation on AnnexinA1 depletion is accompanied by increased HRS phosphorylation, likely through reduced HRS dephosphorylation by ER-localised PTP1B. Conversely, Y329/334 mutation renders ILV formation insensitive to AnnexinA1 depletion. Our data suggest that rapid HRS dephosphorylation at ER:MVE contacts promotes efficient ILV formation.

cell biology↗

bioRxiv2025

Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation.

Induced protein proximity offers powerful new routes to modulate protein fate. While proteolysis-targeting chimeras (PROTACs) promote degradation through E3 ligase recruitment, the converse principle--targeted protein stabilisation or enhancement via deubiquitylase (DUB) recruitment--is only beginning to emerge. The immune checkpoint receptor CTLA4, whose deficiency causes severe autoimmunity, undergoes rapid ubiquitin-dependent lysosomal degradation, making it one of the most short-lived transmembrane proteins. Using an inducible "RapTag" system, which brings together tagged proteins through rapalog-mediated FKBP-FRB dimerisation, we show that enforced proximity to the broad-specificity DUB USP15 markedly increases total and cell-surface CTLA4 levels. Controlled expression of wild-type or catalytically inactive USP15 in isogenic cell lines revealed a clear requirement for DUB activity. The elevation of CTLA4 at the plasma membrane exceeded that of the total cellular pool, consistent with a diversion from ubiquitin-driven lysosomal sorting towards recycling. This easily adaptable platform enables systematic testing of DUB-substrate combinations that informs rational ENhancement TArgeting Chimera (ENTAC) design for downstream drug discovery efforts and targeted protein rescue in therapeutic contexts. Significance statementBy showing that DUB proximity redirects CTLA4 from lysosomal degradation to the plasma membrane, this work defines the mechanistic foundation for Enhancement Targeting Chimeras (ENTACs) that stabilise and correctly reposition unstable proteins.

cell biology↗

bioRxiv2024

A long-lived pool of PINK1 imparts a molecular memory of depolarisation-induced activity.

The Parkinsons disease linked kinase, PINK1, is a short lived protein that undergoes cleavage upon mitochondrial import leading to its release to the cytosol and proteasomal degradation. Under mitochondria depolarising conditions, it accumulates on mitochondria where it becomes activated, phosphorylating both ubiquitin and the ubiquitin E3 ligase Parkin, at Ser65. Here we have used a ubiquitylation inhibitor TAK-243 to accumulate cleaved PINK1 (cPINK1) in a cell line that lacks Parkin. We show that cPINK1 phosphorylates free ubiquitin and can be released to the cytosol in an active form. We show that in RPE1 cells under mitochondria depolarising conditions (i) the majority of PINK1 cleavage proceeds unimpeded and (ii) accrued PINK1 cannot be accounted for by protein stabilisation alone. Accordingly, we suggest that translation of PINK1 mRNA must be mobilised under mitochondrial depolarisation. We have further discovered a pre-conditioning phenomenon, whereby an initial depolarising treatment leaves a residual pool of active PINK1, which remains competent for seeding the activation of nascent cPINK1, despite a 16 hour recuperation period.

cell biology↗

bioRxiv2024

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here we show that its lysosomal degradation is dependent on ubiquitylation at Lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells and in cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype, but shows distinct properties vis-a-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive, nor a localisation-compromised {Delta}MIT domain mutant can rescue delayed degradation of CTLA4, or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

cell biology↗

bioRxiv2023

The Parkinson's Disease related mutant VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress

The identification of multiple genes linked to Parkinsons Disease invites the question as to how they may cooperate. We have generated isogenic cell lines that inducibly express either wild-type or a mutant form of the retromer component VPS35 (D620N), which has been linked to Parkinsons Disease. This has enabled us to test proposed effects of this mutation in a setting where the relative expression reflects the physiological occurrence. We confirm that this mutation compromises VPS35 association with the WASH complex, but find no defect in WASH recruitment to endosomes, nor in the distribution of lysosomal receptors, cation-independent mannose-6-phosphate receptor and Sortilin. We show VPS35 (D620N) enhances the activity of the Parkinsons associated kinase LRRK2 towards RAB12 under basal conditions. Furthermore, VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress resulting in enhanced phosphorylation of RABs 10 and 12. By comparing different types of endolysosomal stresses such as the ionophore nigericin and the membranolytic agent LLOMe, we are able to dissociate phospho-RAB accumulation from membrane rupture.

cell biology↗

bioRxiv2023

Whole organism and tissue specific analysis of pexophagy in Drosophila

Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenic Drosophila melanogaster models expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. Using light sheet microscopy, we have been able to obtain a global overview of pexophagy levels, across the entire organism at different stages of development. Tissue specific control of pexophagy is exemplified by areas of peroxisome abundance but minimal pexophagy observed in clusters of oenocytes, which are the major site of long chain fatty acid synthesis. They are surrounded by epithelial cells where pexophagy is much more evident. Enhancement of pexophagy was achieved by feeding flies with the iron chelator deferiprone, in line with past results using mammalian cells. Specific drivers were used to visualise pexophagy in neurons, in which we tested the role of two proteins proposed to regulate pexophagy. Firstly, depletion of CG8814, the Drosophila homologue of the yeast protein Atg37, had no noticeable impact on pexophagy. In contrast, specific depletion in the larval central nervous system of Hsc70-5, the Drosophila homologue of the chaperone HSPA9/Mortalin, led to a substantial elevation in pexophagy.

cell biology↗

bioRxiv2023

Microtubule association of TRIM3 revealed by differential extraction proteomics

The microtubule network is formed from polymerised Tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from cells, whilst leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug Nocodazole or the microtubule stabilising drug, Taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of Tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule binding proteins, from which we have selected the ubiquitin E3 ligase TRIM3 (Tripartite motif-containing protein 3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated Tubulin, following treatment with Taxol. Furthermore, loss of TRIM3, partially recapitulates the reduction in Nocodazole-resistant microtubules characteristic of Alpha-Tubulin Acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription.

cell biology↗

bioRxiv2023

ISGylation-independent protection of cell growth by USP18 following interferon stimulation

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

cell biology↗

bioRxiv2022

Segregation of pathways leading to pexophagy

Peroxisomes are organelles with key roles in metabolism including long-chain fatty acid production. Their metabolic functions overlap and interconnect with mitochondria, with which they share an overlapping but distinct proteome. Both organelles are degraded by selective autophagy processes termed pexophagy and mitophagy. Whilst mitophagy has received intense attention, the pathways linked to pexophagy and associated tools are less well developed. We have identified the neddylation inhibitor, MLN4924, as a potent activator of pexophagy and show that this is mediated by the HIF1-dependent upregulation of BNIP3L/NIX, a known adaptor for mitophagy. We show that this pathway is distinct from pexophagy induced by the USP30 deubiquitylase inhibitor, CMPD-39, for which we identify the adaptor NBR1 as a central player. Our work suggests a level of complexity to the regulation of peroxisome turnover that includes the capacity to co-ordinate with mitophagy, via NIX, which acts as a rheostat for both processes.

cell biology↗

bioRxiv2022

FBXL4 deficiency promotes mitophagy by elevating NIX.

The selective autophagy of mitochondria is linked to mitochondrial quality control and is critical to a healthy organism. We have conducted a CRISPR/Cas9 screen of human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and following acute mitochondrial depolarisation. We identify two Cullin RING ligases, VHL and FBXL4 as the most profound negative regulators of basal mitophagy. We show that these converge through control of the mitophagy adaptors BNIP3 and BNIP3L/NIX through different mechanisms. FBXL4 suppression of BNIP3 and NIX levels is mediated via direct interaction and protein destabilisation rather than suppression of HIF1-mediated transcription. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study enables a full understanding of the aetiology of early onset mitochondrial encephalomyopathy that is supported by analysis of a disease associated mutation. We further show that the compound MLN4924, which globally interferes with Cullin RING ligase activity, is a strong inducer of mitophagy providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.

cell biology↗

bioRxiv2022

The deubiquitylase USP31 controls the Chromosomal Passenger Complex and spindle dynamics

We have identified USP31 as a microtubule and centrosome associated deubiquitylase, depletion of which leads to an increase in individual cell mass and defective proliferation. We have examined its dynamics and impact during mitosis. GFP-USP31 associates with the mitotic and central spindles, its levels are increased 2-3-fold in prometaphase compared to asynchronous cells and it is dynamically phosphorylated in a CDK1 dependent manner. We find that USP31 depleted cells display altered spindle morphology and chromosomal segregation errors, whilst stable expression of a catalytically inactive form of USP31 leads to polyploidy. At prometaphase, levels of multiple CPC components are destabilised, most prominently INCENP. Under anaphase conditions, depletion of USP31 impairs the translocation of both endogenous and ectopically expressed INCENP to the spindle mid-zone, whilst expression of catalytically inactive USP31 results in multiple ectopic cleavage furrows. In summary, our data indicate a multifaceted regulatory role for USP31 during mitosis, with a profound impact on chromosomal passenger complex abundance, dynamics and function.

cell biology↗

bioRxiv2021

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1/Parkin pathway leading to mitophagy. These include Parkinsons disease and pulmonary fibrosis. We provide a detailed cell biological characterisation of a benzenesulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition following mitochondrial depolarisation. This includes enhancement of TOM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinsons disease patients carrying loss of function Parkin mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1 independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U20S cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

cell biology↗

bioRxiv2020

USP28 deletion and small molecule inhibition destabilises c-Myc and elicitsregression of squamous cell lung carcinoma

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient 5-year survival rate is less than 5%. The ubiquitin specific protease 28 (USP28) has been implicated in tumorigenesis through its stabilization of the oncoprotein c-MYC. Here, we show that genetic inactivation of Usp28 induced regression of established murine LSCC lung tumors. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-Myc proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumors and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

cancer biology↗

bioRxiv2020

A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinsons Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

cell biology↗

bioRxiv2020

The deubiquitylase USP9X controls ribosomal stalling

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome. It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

cell biology↗

bioRxiv2018

Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin

Parkinsons disease factors, PINK1 and parkin, are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues including Parkinsons disease relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies we did not observe any substantial impact on basal mitophagy. As these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/Parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.\n\nSummaryPINK1/parkin are key mediators of stress-induced mitophagy in vitro but their impact on basal mitophagy in vivo is unclear. Novel Drosophila reporters lines reveal abundant mitophagy in many tissues including dopaminergic neurons but is unaffected by loss of PINK1/parkin.

cell biology↗