ORCID Profile
0000-0002-1330-9109
Current Organisation
University of Oxford
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Publisher: Proceedings of the National Academy of Sciences
Date: 04-01-2012
Abstract: N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a ( β / α ) 8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc 1/3 Man 9 GlcNAc 2 yields Glc 1/3 -Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.
Publisher: Elsevier BV
Date: 07-2012
Publisher: American Chemical Society (ACS)
Date: 05-12-0001
Publisher: Elsevier BV
Date: 04-2021
Publisher: Public Library of Science (PLoS)
Date: 14-03-2016
Publisher: Springer Science and Business Media LLC
Date: 16-03-2013
DOI: 10.1007/S00018-013-1304-6
Abstract: Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin roteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.
Publisher: American Society for Clinical Investigation
Date: 27-04-2015
DOI: 10.1172/JCI59987
Publisher: Cold Spring Harbor Laboratory
Date: 27-12-2019
DOI: 10.1101/2019.12.25.888438
Abstract: UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecule’s flexibility. Principal component analysis of the MD trajectories affords a description of UGGT’s overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and cl ing. These inter-domain motions modify the accessible surface area of the enzyme’s central saddle, likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name “Parodi limit” the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 Å. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a “one-size-fits-all adjustable spanner” substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzyme’s active site.
Publisher: Proceedings of the National Academy of Sciences
Date: 24-07-2017
Abstract: A dedicated endoplasmic reticulum quality control (ERQC) machinery ensures the correct fold of secreted proteins bearing N-linked glycans, which constitute around a fifth of the whole proteome and are essential for many important cellular processes such as signaling, immunity, adhesion, transport, and metabolism. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the sole checkpoint enzyme of ERQC, flagging incorrectly folded glycoproteins for ER retention. Here, we describe crystal structures of full-length UGGT. We show that enzymatic activity depends on interdomain conformational mobility, indicating that the intrinsic flexibility of UGGT may endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates.
Publisher: Cold Spring Harbor Laboratory
Date: 07-2020
DOI: 10.1101/2020.06.30.179523
Abstract: Mammalian protein N - linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition and function. N - linked glycans are synthesized from Glc 3 Man 9 GlcNAc 2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo -acting glycoside hydrolase involved in N - glycan trimming and unusually is located within the Golgi, where it allows ER escaped glycoproteins to bypass the classical N - glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N - glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation. The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
Publisher: American Chemical Society (ACS)
Date: 31-03-2020
Publisher: Proceedings of the National Academy of Sciences
Date: 26-07-2016
Abstract: Most pathogenic enveloped viruses crucially depend on the quality control (QC) machinery in the endoplasmic reticulum (ER) of the host cell. ERQC inhibitors therefore have the double potential benefit of targeting a wide variety of viruses (“broad-spectrum antivirals”) without the risk of losing efficacy due to escape mutations in the viral genome. Our recent work has proven that inhibition of the central enzyme of ERQC, α-glucosidase II (α-GluII), is sufficient for antiviral activity against dengue fever in vitro and in vivo. Here, we show how antiviral inhibitors bind to portions of α-GluII that are unique to this enzyme, and we open the way to the development of potent and selective antivirals against existing and emerging infectious disease.
Publisher: Elsevier BV
Date: 05-2016
Publisher: Springer Singapore
Date: 2018
DOI: 10.1007/978-981-10-8727-1_19
Abstract: Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and β-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.
Publisher: Proceedings of the National Academy of Sciences
Date: 05-11-2020
Abstract: The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play key roles in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, that holds great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase (MANEA) provides an alternative processing pathway to evade glucosidase inhibitors. We report the three-dimensional structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
Publisher: Cold Spring Harbor Laboratory
Date: 30-10-2020
DOI: 10.1101/2020.10.29.339317
Abstract: The COVID-19 pandemic was a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Pandemics could be prevented in principle by accessible, easily deployable broad-spectrum oral antivirals. Here we report the results of the COVID Moonshot , a fully open-science, crowd sourced, structure-enabled drug discovery c aign targeting the SARS-CoV-2 main protease. We discovered a novel chemical series that is differentiated from current Mpro inhibitors in that it maintains a new non-covalent, non-peptidic scaffold with nanomolar potency. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations and high-throughput chemistry. In the process, we generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery c aign, all compound designs ( ,000 designs), crystallographic data ( ligand-bound X-ray structures), assay data ( ,000 measurements), and synthesized molecules ( ,400 compounds) for this c aign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Dominic Alonzi.