ORCID Profile
0000-0003-0702-3259
Current Organisation
University of Nottingham
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Publisher: Wiley
Date: 19-10-2020
Publisher: Wiley
Date: 14-02-2023
Abstract: The site‐selective modification of peptides and proteins facilitates the preparation of targeted therapeutic agents and tools to interrogate biochemical pathways. Among the numerous bioconjugation techniques developed to install groups of interest, those that generate C(sp 3 )−C(sp 3 ) bonds are significantly underrepresented despite affording proteolytically stable, biogenic linkages. Herein, a visible‐light‐mediated reaction is described that enables the site‐selective modification of peptides and proteins via desulfurative C(sp 3 )−C(sp 3 ) bond formation. The reaction is rapid and high yielding in peptide systems, with comparable translation to proteins. Using this chemistry, a range of moieties is installed into model systems and an effective PTM‐mimic is successfully integrated into a recombinantly expressed histone.
Publisher: Informa UK Limited
Date: 04-2012
DOI: 10.4161/AUTO.19496
Publisher: The Endocrine Society
Date: 08-2013
DOI: 10.1210/ER.2012-1034
Abstract: Paget's disease of bone (PDB) is characterized by focal areas of aberrant and excessive bone turnover, specifically increased bone resorption and disorganized bone formation. Germline mutations in the sequestosome 1 62 (SQSTM1 62) gene are common in PDB patients, with most mutations affecting the ubiquitin-associated domain of the protein. In vitro, osteoclast precursor cells expressing PDB-mutant SQSTM1 62 protein are associated with increases in nuclear factor κB activation, osteoclast differentiation, and bone resorption. Although the precise mechanisms by which SQSTM1 62 mutations contribute to disease pathogenesis and progression are not well defined, it is apparent that as well as affecting nuclear factor κB signaling, SQSTM1 62 is a master regulator of ubiquitinated protein turnover via autophagy and the ubiquitin-proteasome system. Additional roles for SQSTM1 62 in the oxidative stress-induced Keap1/Nrf2 pathway and in caspase-mediated apoptosis that were recently reported are potentially relevant to the pathogenesis of PDB. Thus, SQSTM1 62 may serve as a molecular link or switch between autophagy, apoptosis, and cell survival signaling. The purpose of this review is to outline recent advances in understanding of the multiple pathophysiological roles of SQSTM1 62 protein, with particular emphasis on their relationship to PDB, including challenges associated with translating SQSTM1 62 research into clinical diagnosis and treatment.
Publisher: Wiley
Date: 03-12-2021
Abstract: Post‐translational modifications (PTMs) enhance the repertoire of protein function and mediate or influence the activity of many cellular processes. The preparation of site‐specifically and homogeneously modified proteins, to apply as tools to understand the biological role of PTMs, is a challenging task. Herein, we describe a visible‐light‐mediated desulfurative C(sp 3 )–C(sp 3 ) bond forming reaction that enables the site‐selective installation of N ϵ ‐modified sidechains into peptides and proteins of interest. Rapid, operationally simple, and tolerant to ambient atmosphere, we demonstrate the installation of a range of lysine (Lys) PTMs into model peptide systems and showcase the potential of this technology by site‐selectively installing an N ϵ Ac sidechain into recombinantly expressed ubiquitin (Ub).
Publisher: Oxford University Press (OUP)
Date: 21-04-2011
DOI: 10.1093/HMG/DDR172
Abstract: Mutations of SQSTM1 occur in about10% of patients with Paget's disease of bone (PDB), but it is unclear whether they play a causal role or regulate susceptibility to an environmental trigger. Here we show that mice with a proline to leucine mutation at codon 394 of mouse sqstm1 (P394L), equivalent to the P392L SQSTM1 mutation in humans, develop a bone disorder with remarkable similarity to PDB. The P394L mutant mice developed focal bone lesions with increasing age and by 12 months, 14/18 (77%) heterozygotes and 20/21 (95%) homozygotes had lesions, compared with 0/18 (0%) wild-type littermates (P< 0.001). Lesions predominantly affected the lower limbs in an asymmetric manner and were characterized by focal increases in bone turnover, with increased bone resorption and formation, disruption of the normal bone architecture and accumulation of woven bone. Osteoclasts within lesions were larger and more nucleated than normal and some contained nuclear inclusions similar to those observed in human PDB. Osteoclast precursors from P394L mutant mice had increased sensitivity to RANKL in vitro resulting in the generation of osteoclasts that were larger and more nucleated than those generated from wild-type littermates. There was increased expression of sqstm1, autophagy-related gene 5 (atg5) and light chain 3 gene (lc3) in osteoclast precursors and increased LC3-II protein levels in Bafilomycin-treated osteoclasts from P394L mutant mice compared with wild-type suggesting dysregulation of autophagy and enhanced autophagosome formation. These studies demonstrate that SQSTM1 mutations can cause a PDB-like skeletal disorder in the absence of an additional trigger and provide a new disease model for PDB.
Publisher: Wiley
Date: 03-12-2021
Abstract: Post‐translational modifications (PTMs) enhance the repertoire of protein function and mediate or influence the activity of many cellular processes. The preparation of site‐specifically and homogeneously modified proteins, to apply as tools to understand the biological role of PTMs, is a challenging task. Herein, we describe a visible‐light‐mediated desulfurative C(sp 3 )–C(sp 3 ) bond forming reaction that enables the site‐selective installation of N ϵ ‐modified sidechains into peptides and proteins of interest. Rapid, operationally simple, and tolerant to ambient atmosphere, we demonstrate the installation of a range of lysine (Lys) PTMs into model peptide systems and showcase the potential of this technology by site‐selectively installing an N ϵ Ac sidechain into recombinantly expressed ubiquitin (Ub).
Publisher: Wiley
Date: 07-2009
DOI: 10.1359/JBMR.090214
Abstract: Previously reported Sequestosome 1(SQSTM1) 62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype henotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-kappaB luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-kappaB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-kappaB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.
Publisher: Elsevier
Date: 2016
Publisher: American Physiological Society
Date: 09-2008
DOI: 10.1152/AJPENDO.90411.2008
Abstract: We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1- 13 C]leucine [i.e., muscle protein synthesis (MPS), P 0.01] without affecting the rate of leg protein breakdown (LPB appearance of d 5 -phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB ( P 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB Ser 473 and p70 S6k Thr 389 increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR Ser 2448 , 4E-BP1 Thr 37/46 , or GSK3β Ser 9 and decreased that of eEF2 Thr 56 , higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB.
Publisher: Springer Science and Business Media LLC
Date: 16-11-2016
DOI: 10.1038/NCOMMS13288
Abstract: Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex ex le, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.
Publisher: Wiley
Date: 18-09-2002
DOI: 10.1046/J.1471-4159.2002.01137.X
Abstract: Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins.
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.BONE.2012.10.023
Abstract: Mutations affecting the Sequestosome 1 (SQSTM1) gene commonly occur in patients with the skeletal disorder Paget's disease of bone (PDB), a condition characterised by defective osteoclast differentiation and function. Whilst most mutations cluster within the ubiquitin-associated (UBA) domain of the SQSTM1 protein, and are associated with dysregulated NFκB signalling, several non-UBA domain mutations have also been identified. Keap1 is a SQSTM1-interacting protein that regulates the levels and activity of the Nrf2 transcription factor. This in turn controls the expression of numerous cytoprotective genes that contribute to the cell's capacity to defend itself against chemical and oxidative stress, through binding to the antioxidant response element (ARE). The PDB-associated S349T mutation maps to the Keap1-interacting region (KIR) of SQSTM1, however the effects of PDB mutant SQSTM1 on Keap1 function have not been investigated. Here we show that unlike other SQSTM1 mutations, the S349T mutation results in neither impaired ubiquitin-binding function in pull-down assays, nor dysregulated NFκB signalling in luciferase reporter assays. Keap1 is expressed in differentiating osteoclast-like cells and the S349T mutation selectively impairs the SQSTM1-Keap1 interaction in co-immunoprecipitations, which molecular modelling indicates results from effects on critical hydrogen bonds required to stabilise the KIR-Keap1 complex. Further, S349T mutant SQSTM1, but not other PDB-associated mutants, showed reduced ability to activate Nrf2 signalling as assessed by ARE-luciferase reporter assays. Thus, SQSTM1-mediated dysregulation of the Keap1-Nrf2 axis, which could potentially lead to aberrant production of oxidative response genes, may contribute to disease aetiology in a subset of PDB patients.
Publisher: Elsevier BV
Date: 10-2016
Publisher: Informa UK Limited
Date: 02-01-2016
Publisher: Wiley
Date: 19-10-2020
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.BBRC.2006.12.179
Abstract: Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain s les, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.
Publisher: Elsevier BV
Date: 07-2014
Publisher: Elsevier BV
Date: 07-2014
DOI: 10.1016/J.YEXCR.2014.01.020
Abstract: Paget disease of bone (PDB) is a skeletal disorder common in Western Europe but extremely rare in the Indian subcontinent and Far East. The condition has a strong genetic element with mutations affecting the SQSTM1 gene, encoding the p62 protein, frequently identified. Recently SQSTM1 mutations have also been reported in a small number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), neurodegenerative disorders in which significant coexistence with PDB has not been previously recognized. Although several SQSTM1 mutations are common to both ALS/FTLD and PDB, many are ALS/FTLD-specific. The p62 protein regulates various cellular processes including NF-κB signaling and autophagy pathways. Here we consider how knowledge of the impact of PDB-associated SQSTM1 mutations (several of which are now known to be relevant for ALS/FTLD) on these pathways, as well as the locations of the mutations within the p62 primary sequence, may provide new insights into ALS/FTLD disease mechanisms.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Robert Layfield.