A5075455832- Ubiquitin and proteasome pathways
- interferon and immune responses
- Advanced Proteomics Techniques and Applications
- Peptidase Inhibition and Analysis
- Mitochondrial Function and Pathology
- Cancer-related Molecular Pathways
- Autophagy in Disease and Therapy
- Cancer, Hypoxia, and Metabolism
- RNA modifications and cancer
- Cell death mechanisms and regulation
- Cellular transport and secretion
- Retinoids in leukemia and cellular processes
- Glycosylation and Glycoproteins Research
- RNA Research and Splicing
- Immune Cell Function and Interaction
- NF-κB Signaling Pathways
- Endoplasmic Reticulum Stress and Disease
- DNA Repair Mechanisms
- Amyotrophic Lateral Sclerosis Research
- Histone Deacetylase Inhibitors Research
- PARP inhibition in cancer therapy
- Lysosomal Storage Disorders Research
- Calcium signaling and nucleotide metabolism
- Microtubule and mitosis dynamics
- Genetics and Neurodevelopmental Disorders
MRC Protein Phosphorylation and Ubiquitylation Unit
2022-2025
University of Dundee
2022-2025
Medical Research Council
2022-2025
Newcastle University
2017-2024
Wellcome Trust
2022-2024
Institute for Research in Immunology and Cancer
2013-2020
Université de Montréal
2014-2020
Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with ubiquitin-like protein UFM1 on 60S ribosomal subunit RPL26 (also known as uL24)
Abstract Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine interrelationship these modifications. Here, we report an optimized immunoaffinity method that permits study of both protein ubiquitylation SUMOylation from a single sample. This enables unprecedented identification 10,388 sites in HEK293 cells. The sequential use remnant purification facilitates dynamic profiling SUMOylated ubiquitylated proteins cells...
The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present characterisation an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. demonstrate that ubiquitylation TOM20, a component outer membrane import machinery, represents robust biomarker both loss and inhibition. A proteomics analysis, on SHSY5Y neuroblastoma cell line model, directly compares effects genetic chemical have thereby identified subset events...
Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 conjugated onto substrates. Here, use rebuilding approach to define the minimal requirements protein UFMylation. We find that reported cognate E3 ligase UFL1 inactive on own instead requires adaptor UFBP1 form an active complex. Structure predictions suggest...
An essential first step in the post-translational modification of proteins with UFM1, UFMylation, is proteolytic cleavage pro-UFM1 to expose a C-terminal glycine. Of two UFM1-specific proteases (UFSPs) identified humans, only UFSP2 reported be active, since annotated sequence UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs cells lacking UFSP2, suggesting presence another active protease. We herein identify translated from non-canonical start site this...
Abstract UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify characterize distinct within human consisting ‘hemiRING’ zinc finger, helical-rich UBR zinc-finger interacting (UZI) subdomain, an N-terminal region can serve as affinity factor for E2 conjugating enzyme (E2). structure E2–E3 complex provides atomic-level...
Abstract Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding branched function cell signaling has been stunted by the absence accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins devise approaches to probe K48–K63-branched function. We establish method monitor cleavage linkages within complex unveil ATXN3 MINDY as debranching enzymes. engineer K48–K63 branch-specific...
Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on single molecule modified with creating bifurcated architectures. Despite their abundance, we have poor understanding the cellular functions branched signals that stems from lack facile tools methods to study them. Here develop comprehensive pipeline define function, using K48-K63-branched as case study. We discover branch-specific binders and, by developing...
E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to attachment of ubiquitin substrate. This process, termed ubiquitylation, is required maintain cellular homeostasis and affects almost all process. By interacting with multiple E3 ligases, E2s dictate ubiquitylation landscape within cell. Since its discovery, has been regarded as posttranslational modification specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser...
The NEK1 kinase controls ciliogenesis, mitosis, and DNA repair, mutations cause human diseases including axial spondylometaphyseal dysplasia amyotrophic lateral sclerosis. C21ORF2 a similar pattern of diseases, suggesting close functional links with . Here, we report that endogenous form tight complex in cells. A interaction domain “CID” at the C-terminus is necessary for its association cells, pathogenic this region disrupt complex. AlphaFold modelling predicts an extended binding interface...
Abstract Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling content using tag-based immunoprecipitation (LysoTagIP) cell animal models allowed major discoveries the field, however studying dysfunction patients remains challenging. Here, we report development “tagless LysoIP method” to enable rapid enrichment lysosomes, via immunoprecipitation, endogenous integral membrane...
Rising temperatures and water scarcity caused by climate change are increasingly exposing our cells tissues to ionic stress, a consequence of elevated cytoplasmic strength that can disrupt protein, organelle, genome function. Here, we unveil single-protein mechanism for sensing mitigation in animal cells, one is notably different from the analogous high osmolarity glycerol kinase cascade yeast. The Rel family transcription factor NFAT5 directly senses intracellular using C-terminal...
Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping Lys residues mass spectrometry (MS) challenging because dynamic nature this modification, existence three functionally distinct human SUMO paralogs, large...
We report that interferon (IFN) α treatment at short and long periods increases the global cellular SUMOylation requires presence of SUMO E3 ligase promyelocytic leukemia protein (PML), organizer PML nuclear bodies (NBs). Several isoforms (PMLI-PMLVII) derived from a single gene by alternative splicing, share same N-terminal region but differ in their C-terminal sequences. Introducing each human isoform PML-negative cells revealed enhanced response to IFN is orchestrated PMLIII PMLIV....
Several regulators of SUMOylation have been previously linked to senescence but most targets this modification in senescent cells remain unidentified. Using a two-step purification modified SUMO3, we profiled the SUMO proteome site-specific manner. We identified 25 sites on 23 proteins that were significantly regulated during senescence. Of note, these PML nuclear body (PML-NB) associated, which correlates with increased number and size PML-NBs observed cells. Interestingly, sole E2 enzyme,...
ADP-heptose activates the protein kinase ALPK1 triggering TIFA phosphorylation at Thr9, recruitment of TRAF6 and subsequent production inflammatory mediators. Here, we demonstrate that also stimulates formation Lys63- Met1-linked ubiquitin chains to activate TAK1 canonical IKK complexes, respectively. We further show E3 ligases c-IAP1 operate redundantly generate Lys63-linked required for pathway activation, which are attached TRAF6, TRAF2 c-IAP1, is recruited by TRAF2. induces activation...
Ubiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect driven by the ability to form complex polyubiquitin chain architectures that can influence functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification Parallel Reaction Monitoring (Ub-AQUA-PRM). Using refined Ub-AQUA-PRM assay, were able quantify ubiquitin types 10-min LC-MS/MS runs. We used...
Abstract WNK family kinases are regulated by osmotic stress and control ion homeostasis activating SPAK OXSR1 kinases. Using a proximity ligation approach, we found that promotes the association of WNK1 with NRBP1 pseudokinase TSC22D2/4 adaptor proteins. is closely related to isoforms also contains RΦ-motif binding conserved C-terminal (CCT) domain, similar CCT domains in WNKs, OXSR1. Knockdown markedly inhibited sorbitol-induced activation downstream components. AlphaFold-3 modelling...
Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling content using tag-based immunoprecipitation (LysoTagIP) cell animal models has substantially moved the field forward, but studying dysfunction patients remains challenging. Here, we report development 'tagless LysoIP' method, designed to enable rapid enrichment lysosomes, via immunoprecipitation, endogenous integral...
Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated tight control TORC1 activity. Upon inhibition, protein degradation increased and synthesis reduced through inhibition translation initiation to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system, composed HSP70 Ssb its HSP40 co-chaperone Zuo1, required proteostasis viability under in Saccharomyces cerevisiae. In absence does not decrease response loss A...
The homohexameric p97 complex, composed of Cdc48 subunits in yeast, is a crucial component protein quality control pathways including ER-associated degradation. complex acts to segregate complexes an ATP-dependent manner, requiring the engagement cofactor proteins that determine substrate specificity. function different cofactors and how they are regulated remains relatively poorly understood. In this study, we assess phosphorylation adaptor proteins, revealing unique distinctive pattern...