Publications by authors named "Rod Chalk"

15 Publications

  • Page 1 of 1

Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

Commun Biol 2021 08 3;4(1):934. Epub 2021 Aug 3.

Centre for Medicines Discovery, ORCRB, University of Oxford, Oxford, UK.

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.
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http://dx.doi.org/10.1038/s42003-021-02455-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8333269PMC
August 2021

Structural Insights into Notum Covalent Inhibition.

J Med Chem 2021 08 22;64(15):11354-11363. Epub 2021 Jul 22.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, U.K.

The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer's disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365597PMC
August 2021

An In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells.

Chem Sci 2020 Sep 10;11(33):8936-8944. Epub 2020 Aug 10.

Oxford Institute for Radiation Oncology , Department of Oncology , University of Oxford , Oxford , UK . Email:

Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics. Herein we report the synthesis of a mismatch DNA binding bis-ruthenium(ii) dipyridophenazine (dppz) complex that can be radiolabelled with the Auger electron emitting radionuclide indium-111 (In). Greater stabilisation accompanied by enhanced MLCT (metal to ligand charge-transfer) luminescence of both the bis-Ru(dppz) chelator and non-radioactive indium-loaded complex was observed in the presence of a TT mismatch-containing duplex compared to matched DNA. The radioactive construct [In]In-bisRu(dppz) ([In][In-]) targets cell nuclei and is radiotoxic towards MMR-deficient human colorectal cancer cells showing substantially less detrimental effects in a paired cell line with restored MMR function. Additional cell line studies revealed that [In][In-] is preferentially radiotoxic towards MMR-deficient colorectal cancer cells accompanied by increased DNA damage due to In decay. The biodistribution of [In][In-] in live mice was demonstrated using SPECT. These results illustrate how a Ru(ii) polypyridyl complex can incorporate mismatch DNA binding and radiometal chelation in a single molecule, generating a DNA-targeting AE radiopharmaceutical that displays selective radiotoxicity towards MMR-deficient cancer cells and is compatible with whole organism SPECT imaging.
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http://dx.doi.org/10.1039/d0sc02825hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989384PMC
September 2020

Glibenclamide reverses cardiovascular abnormalities of Cantu syndrome driven by KATP channel overactivity.

J Clin Invest 2020 03;130(3):1116-1121

Center for the Investigation of Membrane Excitability Diseases.

Cantu syndrome (CS) is a complex disorder caused by gain-of-function (GoF) mutations in ABCC9 and KCNJ8, which encode the SUR2 and Kir6.1 subunits, respectively, of vascular smooth muscle (VSM) KATP channels. CS includes dilated vasculature, marked cardiac hypertrophy, and other cardiovascular abnormalities. There is currently no targeted therapy, and it is unknown whether cardiovascular features can be reversed once manifest. Using combined transgenic and pharmacological approaches in a knockin mouse model of CS, we have shown that reversal of vascular and cardiac phenotypes can be achieved by genetic downregulation of KATP channel activity specifically in VSM, and by chronic administration of the clinically used KATP channel inhibitor, glibenclamide. These findings demonstrate that VSM KATP channel GoF underlies CS cardiac enlargement and that CS-associated abnormalities are reversible, and provide evidence of in vivo efficacy of glibenclamide as a therapeutic agent in CS.
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http://dx.doi.org/10.1172/JCI130571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269588PMC
March 2020

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Cell 2018 11;175(4):1045-1058.e16

Neurosciences Group, Nuffield Department of Clinical Neuroscience, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
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http://dx.doi.org/10.1016/j.cell.2018.10.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218659PMC
November 2018

Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition.

Nat Commun 2018 06 11;9(1):2261. Epub 2018 Jun 11.

Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, OX3 7DQ, UK.

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.
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http://dx.doi.org/10.1038/s41467-018-04735-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995969PMC
June 2018

Structural complexity in the KCTD family of Cullin3-dependent E3 ubiquitin ligases.

Biochem J 2017 11 1;474(22):3747-3761. Epub 2017 Nov 1.

Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.

Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.
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http://dx.doi.org/10.1042/BCJ20170527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664961PMC
November 2017

Mass Spectrometric Analysis of Proteins.

Authors:
Rod Chalk

Methods Mol Biol 2017 ;1586:373-395

Structural Genomics Consortium (SGC), Nuffield Department of Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK.

Mass spectrometry is a generic technique for the structural and functional analysis of purified proteins. Instrument capabilities and the possibilities of intact protein, peptide fragmentation and native analyses are discussed. Detailed experimental protocols are described for the most commonly applied techniques of protein identification, posttranslational modification (PTM) characterization, PTM mapping, native mass spectrometry, and analysis of membrane proteins using electrospray mass spectrometry.
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http://dx.doi.org/10.1007/978-1-4939-6887-9_25DOI Listing
February 2018

Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).

Nat Struct Mol Biol 2017 02 19;24(2):114-122. Epub 2016 Dec 19.

Structural Genomics Consortium, University of Oxford, Oxford, UK.

Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
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http://dx.doi.org/10.1038/nsmb.3343DOI Listing
February 2017

Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase.

Hum Mol Genet 2016 06 22;25(11):2234-2244. Epub 2016 Mar 22.

Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ , UK

Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.
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http://dx.doi.org/10.1093/hmg/ddw091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081055PMC
June 2016

Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking.

J Biol Chem 2015 Dec 19;290(49):29167-77. Epub 2015 Oct 19.

the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and

Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations.
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http://dx.doi.org/10.1074/jbc.M115.683268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705923PMC
December 2015

Serum indoleamine 2,3-dioxygenase activity is associated with reduced immunogenicity following vaccination with MVA85A.

BMC Infect Dis 2014 Dec 3;14:660. Epub 2014 Dec 3.

The Jenner Institute, University of Oxford, Oxford, UK.

Background: There is an urgent need for improved vaccines to protect against tuberculosis. The currently available vaccine Bacille Calmette-Guerin (BCG) has varying immunogenicity and efficacy across different populations for reasons not clearly understood. MVA85A is a modified vaccinia virus expressing antigen 85A from Mycobacterium tuberculosis which has been in clinical development since 2002 as a candidate vaccine to boost BCG-induced protection. A recent efficacy trial in South African infants failed to demonstrate enhancement of protection over BCG alone. The immunogenicity was lower than that seen in UK trials. The enzyme Indoleamine 2,3-dioxygenase (IDO) catalyses the first and rate-limiting step in the breakdown of the essential amino acid tryptophan. T cells are dependent on tryptophan and IDO activity suppresses T-cell proliferation and function.

Methods: Using samples collected during phase I trials with MVA85A across the UK and South Africa we have investigated the relationship between vaccine immunogenicity and IDO using IFN-γ ELISPOT, qPCR and liquid chromatography mass spectrometry.

Results: We demonstrate an IFN-γ dependent increase in IDO mRNA expression in peripheral blood mononuclear cells (PBMC) following MVA85A vaccination in UK subjects. IDO mRNA correlates positively with the IFN-γ ELISPOT response indicating that vaccine specific induction of IDO in PBMC is unlikely to limit the development of vaccine specific immunity. IDO activity in the serum of volunteers from the UK and South Africa was also assessed. There was no change in serum IDO activity following MVA85A vaccination. However, we observed higher baseline IDO activity in South African volunteers when compared to UK volunteers. In both UK and South African serum samples, baseline IDO activity negatively correlated with vaccine-specific IFN-γ responses, suggesting that IDO activity may impair the generation of a CD4+ T cell memory response.

Conclusions: Baseline IDO activity was higher in South African volunteers when compared to UK volunteers, which may represent a potential mechanism for the observed variation in vaccine immunogenicity in South African and UK populations and may have important implications for future vaccination strategies.

Trial Registration: Trials are registered at ClinicalTrials.gov; UK cohort NCT00427830, UK LTBI cohort NCT00456183, South African cohort NCT00460590, South African LTBI cohort NCT00480558.
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http://dx.doi.org/10.1186/s12879-014-0660-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265419PMC
December 2014

High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins.

Anal Biochem 2011 Mar 17;410(2):272-80. Epub 2010 Nov 17.

Structural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK.

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.
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http://dx.doi.org/10.1016/j.ab.2010.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041925PMC
March 2011

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

Proteomics 2002 Nov;2(11):1586-600

School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.
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http://dx.doi.org/10.1002/1615-9861(200211)2:11<1586::AID-PROT1586>3.0.CO;2-KDOI Listing
November 2002

Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry.

Nucleic Acids Res 2002 Mar;30(6):e27

CRC Chromosome Molecular Biology Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

We have established the use of a primer extension/mass spectrometry method (the PinPoint assay) for high-throughput SNP genotyping of the human Y chromosome. 118 markers were used to define 116 haplogroups and typing was organised in a hierarchical fashion. Twenty multiplex PCR/primer extension reactions were set up and each sample could be assigned to a haplogroup with only two to five of these multiplex analyses. A single aliquot of one enzyme was found to be sufficient for both PCR and primer extension. We observed 100% accuracy in blind validation tests. The technique thus provides a reliable, cost-effective and automated method for Y genotyping, and the advantages of using a hierarchical strategy can be applied to any DNA segment lacking recombination.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101371PMC
http://dx.doi.org/10.1093/nar/30.6.e27DOI Listing
March 2002
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