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11 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Chemical toolkit for PARK7: potent, selective, and high-throughput Yuqing Jia , Robbert Q. Kim , Raymond Kooij , Huib Ovaa , Aysegul Sapmaz , Paul P. Geurink Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.2c01113 Diamond Proposal Number(s): [19800] Open Access Show Abstract ▼ Abstract: The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors.	Sep 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Manno-epi-cyclophellitols enable activity-based protein profiling of human α-mannosidases and discovery of new golgi mannosidase II inhibitors Zachary Armstrong , Chi-Lin Kuo , Daniël Lahav , Bing Liu , Rachel Johnson , Thomas J. M. Beenakker , Casper De Boer , Chung-Sing Wong , Erwin R. Van Rijssel , Marjoke F. Debets , Bogdan I. Florea , Colin Hissink , Rolf G. Boot , Paul P. Geurink , Huib Ovaa , Mario Van Der Stelt , Gijsbert M. Van Der Marel , Jeroen D. C. Codée , Johannes M. F. G. Aerts , Liang Wu , Herman S. Overkleeft , Gideon Davies Journal Of The American Chemical Society DOI: 10.1021/jacs.0c03880 Diamond Proposal Number(s): [18598] Show Abstract ▼ Abstract: Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.	Jul 2020
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Identification and characterization of diverse OTU deubiquitinases in bacteria Alexander F. Schubert , Justine V Nguyen , Tyler G. Franklin , Paul P. Geurink , Cameron G. Roberts , Daniel J. Sanderson , Lauren N. Miller , Huib Ovaa , Kay Hofmann , Jonathan N. Pruneda , David Komander The Embo Journal 81 DOI: 10.15252/embj.2020105127 Diamond Proposal Number(s): [8547, 11235] Open Access Show Abstract ▼ Abstract: Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co‐opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin‐bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross‐kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases.	Jun 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Rapid covalent-probe discovery by electrophile-fragment screening Efrat Resnick , Anthony Bradley , Jinrui Gan , Alice Douangamath , Tobias Krojer , Ritika Sethi , Paul P. Geurink , Anthony Aimon , Gabriel Amitai , Dom Bellini , James Bennett , Michael Fairhead , Oleg Fedorov , Ronen Gabizon , Jin Gan , Jingxu Guo , Alexander Plotnikov , Nava Reznik , Gian Filippo Ruda , Laura Diaz-Saez , Verena M. Straub , Tamas Szommer , Srikannathasan Velupillai , Daniel Zaidman , Yanling Zhang , Alun R. Coker , Christopher G. Dowson , Haim Barr , Chu Wang , Kilian V. M. Huber , Paul E. Brennan , Huib Ovaa , Frank Von Delft , Nir London Journal Of The American Chemical Society DOI: 10.1021/jacs.9b02822 Show Abstract ▼ Abstract: Covalent probes can display unmatched potency, selectivity and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.	May 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies Kirby N. Swatek , Martina Aumayr , Jonathan N. Pruneda , Linda J. Visser , Stephen Berryman , Anja F. Kueck , Paul P. Geurink , Huib Ovaa , Frank J. M. Van Kuppeveld , Tobias J. Tuthill , Tim Skern , David Komander Proceedings Of The National Academy Of Sciences 35 DOI: 10.1073/pnas.1710617115 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.	Feb 2018
I03-Macromolecular Crystallography	A fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human nonlysosomal glucosylceramidase Daniël Lahav , Bing Liu , Richard J. B. H. N. Van Den Berg , Adrianus M. C. H. Van Den Nieuwendijk , Tom Wennekes , Amar T. Ghisaidoobe , Imogen Breen , Maria J. Ferraz , Chi-Lin Kuo , Liang Wu , Paul P. Geurink , Huib Ovaa , Gijsbert A. Van Der Marel , Mario Van Der Stelt , Rolf G. Boot , Gideon J. Davies , Johannes M. F. G. Aerts , Herman S. Overkleeft Journal Of The American Chemical Society DOI: 10.1021/jacs.7b07352 Diamond Proposal Number(s): [13587] Open Access Show Abstract ▼ Abstract: Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.	Sep 2017
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis of the specificity of USP18 toward ISG15 Anja Basters , Paul P Geurink , Annika Röcker , Katharina F Witting , Roya Tadayon , Sandra Hess , Marta S Semrau , Paola Storici , Huib Ovaa , Klaus-Peter Knobeloch , Günter Fritz Nature Structural & Molecular Biology DOI: 10.1038/nsmb.3371 Diamond Proposal Number(s): [9694, 12090] Show Abstract ▼ Abstract: Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.	Feb 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne Tycho E. T. Mevissen , Yogesh Kulathu , Monique P. C. Mulder , Paul P. Geurink , Sarah L. Maslen , Malte Gersch , Paul R. Elliott , John E. Burke , Bianca D. M. Van Tol , Masato Akutsu , Farid El Oualid , Masato Kawasaki , Stefan M. V. Freund , Huib Ovaa , David Komander Nature 538, 402 - 405 DOI: 10.1038/nature19836 Diamond Proposal Number(s): [11235] Show Abstract ▼ Abstract: The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen–deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.	Oct 2016
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases Jonathan N. Pruneda , Charlotte H. Durkin , Paul P. Geurink , Huib Ovaa , Balaji Santhanam , David W. Holden , David Komander Molecular Cell 63, 261 - 276 DOI: 10.1016/j.molcel.2016.06.015 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins.	Jul 2016
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis Tycho E. T. Mevissen , Manuela K. Hospenthal , Paul P. Geurink , Paul R. Elliott , Masato Akutsu , Nadia Arnaudo , Reggy Ekkebus , Yogesh Kulathu , Tobias Wauer , Farid El oualid , Stefan Freund , Huib Ovaa , David Komander Cell 154 (1), 169 - 184 DOI: 10.1016/j.cell.2013.05.046 PMID: 23827681 Open Access Show Abstract ▼ Abstract: The 16 human OTU DUBs cleave distinct sets of ubiquitin chain types Five crystal structures of three human OTU DUBs reveal uncharacterized Ub-binding sites We reveal four distinct mechanisms of linkage specificity in OTU DUBs OTU DUBs can be used to identify the linkage types on a ubiquitinated substrate	Jul 2013

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