31 results on '"Yue, Wyatt W'
Search Results
2. Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate
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Thomas J. McCorvie, Paula M. Loria, Meihua Tu, Seungil Han, Leela Shrestha, D. Sean Froese, Igor M. Ferreira, Allison P. Berg, and Wyatt W. Yue
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Structural Biology ,Molecular Biology - Abstract
Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0–4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.
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- 2022
3. Snapshots of actin and tubulin folding inside the TRiC chaperonin
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John J. Kelly, Dale Tranter, Els Pardon, Gamma Chi, Holger Kramer, Lotta Happonen, Kelly M. Knee, Jay M. Janz, Jan Steyaert, Christine Bulawa, Ville O. Paavilainen, Juha T. Huiskonen, Wyatt W. Yue, Institute of Biotechnology, Helsinki Institute of Life Science HiLIFE, Biosciences, Molecular and Integrative Biosciences Research Programme, Department of Bio-engineering Sciences, and Structural Biology Brussels
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MECHANISM ,PROTEIN ,EUKARYOTIC CHAPERONIN ,ATP HYDROLYSIS ,CYTOSOLIC CHAPERONIN ,GROUP-II CHAPERONIN ,Structural Biology ,SUBUNIT ,CRYO-EM STRUCTURE ,1182 Biochemistry, cell and molecular biology ,CRYSTAL-STRUCTURE ,sense organs ,CCT CHAPERONIN ,Molecular Biology - Abstract
The integrity of a cell's proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC. Tagging of the endogenous type II chaperonin TRiC complex using CRISPR knock-in enables its purification for cryo-EM. A series of structures reveal the fate of substrates and co-chaperones inside the TRiC chamber to uncover its inner workings.
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- 2022
4. NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy
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Tetsushi Kataura, Elsje G Otten, Yoana Rabanal‐Ruiz, Elias Adriaenssens, Francesca Urselli, Filippo Scialo, Lanyu Fan, Graham R Smith, William M Dawson, Xingxiang Chen, Wyatt W Yue, Agnieszka K Bronowska, Bernadette Carroll, Sascha Martens, Michael Lazarou, Viktor I Korolchuk, Kataura, T., Otten, E. G., Rabanal-Ruiz, Y., Adriaenssens, E., Urselli, F., Scialo, F., Fan, L., Smith, G. R., Dawson, W. M., Chen, X., Yue, W. W., Bronowska, A. K., Carroll, B., Martens, S., Lazarou, M., and Korolchuk, V. I.
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autophagy ,mitophagy ,General Immunology and Microbiology ,General Neuroscience ,redox ,p62 ,NDP52 ,Molecular Biology ,General Biochemistry, Genetics and Molecular Biology - Abstract
Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.
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- 2022
5. A novel image-based high-throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease
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Peixiang Wang, Wyatt W. Yue, Or Kakhlon, Hanoch Senderowitz, Netaly Khazanov, Igor M. Ferreira, Leonardo J. Solmesky, Alexander Lossos, Miguel Weil, and Berge A. Minassian
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Adult ,Male ,0301 basic medicine ,High-throughput screening ,Cell ,Phosphatase ,Drug Evaluation, Preclinical ,Biology ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Humans ,Glycogen synthase ,Molecular Biology ,Glycogen ,Cell Biology ,Adult polyglucosan body disease ,Fibroblasts ,Glycogen Storage Disease ,Glycogen Synthase ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,biology.protein ,Female ,Nervous System Diseases ,030217 neurology & neurosurgery ,Image based ,Intracellular - Abstract
Glycogen storage disorders (GSDs) are caused by excessive accumulation of glycogen. Some GSDs [adult polyglucosan (PG) body disease (APBD), and Tarui and Lafora diseases] are caused by intracellular accumulation of insoluble inclusions, called PG bodies (PBs), which are chiefly composed of malconstructed glycogen. We developed an APBD patient skin fibroblast cell-based assay for PB identification, where the bodies are identified as amylase-resistant periodic acid–Schiff's-stained structures, and quantified. We screened the DIVERSet CL 10 084 compound library using this assay in high-throughput format and discovered 11 dose-dependent and 8 non-dose-dependent PB-reducing hits. Approximately 70% of the hits appear to act through reducing glycogen synthase (GS) activity, which can elongate glycogen chains and presumably promote PB generation. Some of these GS inhibiting hits were also computationally predicted to be similar to drugs interacting with the GS activator protein phosphatase 1. Our work paves the way to discovering medications for the treatment of PB-involving GSD, which are extremely severe or fatal disorders.
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- 2017
6. Human ISPD Is a Cytidyltransferase Required for Dystroglycan O-Mannosylation
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Wyatt W. Yue, Hans van Bokhoven, Walinka van Tol, Moniek Riemersma, Jolanta Kopec, D. Sean Froese, Hiroshi Manya, Ewa Swiezewska, Dirk Lefeber, Thijn R. Brummelkamp, T. Krojer, Monique van Scherpenzeel, Lucas T. Jae, Angel Ashikov, Frank von Delft, Tamao Endo, Anna Buczkowska, Udo F. H. Engelke, Marco Tessari, Klinische Genetica, and RS: CARIM - R2 - Cardiac function and failure
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Glycan ,Glycosylation ,Clinical Biochemistry ,Biology ,Crystallography, X-Ray ,Nucleotide sugar ,Ribitol ,Biochemistry ,Gene Knockout Techniques ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Ribose ,Drug Discovery ,Dystroglycan ,Humans ,Transferase ,Choline-Phosphate Cytidylyltransferase ,Disorders of movement Radboud Institute for Molecular Life Sciences [Radboudumc 3] ,Dystroglycans ,Molecular Biology ,Cells, Cultured ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7] ,ATP synthase ,General Medicine ,Fibroblasts ,Disorders of movement Donders Center for Medical Neuroscience [Radboudumc 3] ,Nucleotidyltransferases ,carbohydrates (lipids) ,chemistry ,biology.protein ,Molecular Medicine ,030217 neurology & neurosurgery - Abstract
Contains fulltext : 152310.pdf (Publisher’s version ) (Closed access) A unique, unsolved O-mannosyl glycan on alpha-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional alpha-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.
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- 2015
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7. Genetic, structural, and functional analysis of mutations causing methylmalonyl-CoA epimerase deficiency
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Matthias R. Baumgartner, Seraina Lutz, Patricie Burda, Brian Fowler, Wyatt W. Yue, Henry J. Bailey, Apirat Chaikuad, Terttu Suormala, Heuberger K, Céline Bürer, Krysztofinska E, D. S. Froese, and University of Zurich
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chemistry.chemical_classification ,0303 health sciences ,Mutation ,Functional analysis ,Globular protein ,Nonsense mutation ,610 Medicine & health ,medicine.disease_cause ,Molecular biology ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Enzyme ,Methylmalonic aciduria ,chemistry ,law ,10036 Medical Clinic ,medicine ,Recombinant DNA ,Missense mutation ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of D-methylmalonyl-CoA and L-methylmalonyl-CoA in propionate catabolism. Autosomal recessive mutations inMCEEreportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with mutations inMCEE. Nine patients were homozygous for the known nonsense mutation p.Arg47* (c.139C>T), and one for the novel missense mutation p.Ile53Arg (c.158T>G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described patient mutations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Å structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this mutation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. We solved the structure of MCEE-Arg143Cys to 1.9 Å and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.
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- 2018
8. Tryptophan-mediated interactions between tristetraprolin and the CNOT9 subunit are required for CCR4-NOT deadenylase complex recruitment
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D. Bulbrook, Sarah Picaud, M. Kliszczak, Wyatt W. Yue, Andrew R. Clark, Panagis Filippakopoulos, Anna Aubareda, Rod Chalk, Oleg Fedorov, N.A. Burgess-Brown, Claire Strain-Damerell, H. Brazier, Pravin Mahajan, Jonathan L.E. Dean, Opher Gileadi, and Francesco P. Marchese
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0301 basic medicine ,Untranslated region ,Receptors, CCR4 ,RNA Stability ,Protein subunit ,Tristetraprolin ,Catabolite repression ,Repressor ,Autoantigens ,03 medical and health sciences ,Structural Biology ,hemic and lymphatic diseases ,Humans ,Protein Interaction Domains and Motifs ,RNA, Messenger ,3' Untranslated Regions ,Molecular Biology ,AU-rich element ,Messenger RNA ,030102 biochemistry & molecular biology ,Chemistry ,Mutagenesis ,Tryptophan ,RNA-Binding Proteins ,respiratory system ,Cell biology ,030104 developmental biology ,Exoribonucleases ,Mutagenesis, Site-Directed ,HeLa Cells ,Transcription Factors - Abstract
The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3' untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex "carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT)," which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3'-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.
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- 2017
9. A homozygous variant in mitochondrial RNase P subunit PRORP is associated with Perrault syndrome characterized by hearing loss and primary ovarian insufficiency
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Jill E. Urquhart, Zeev Blumenfeld, Emma M. Jenkinson, Johannes Zschocke, Albert Amberger, Walter Rossmanith, James O'Sullivan, William G. Newman, Thomas B. Friedman, Kyle Thompson, Irit Hochberg, Wyatt W. Yue, Leigh Ann Mary Demain, Andrea J. Deutschmann, Kevin J. Munro, Stephanie Oerum, Sergey Yalonetsky, Nada Al-Sheqaih, Sandra Demetz, Raymond T. O'Keefe, Robert W. Taylor, Sanjeev S. Bhaskar, Inna A. Belyantseva, Melanie Barzik, and Simon G. Williams
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Genetics ,0303 health sciences ,Messenger RNA ,Mitochondrial translation ,RNase P ,Wild type ,TRNA processing ,Biology ,medicine.disease ,Molecular biology ,03 medical and health sciences ,0302 clinical medicine ,Transfer RNA ,medicine ,Missense mutation ,Sensorineural hearing loss ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46 XX, females. It is genetically heterogeneous with biallelic variants in six genes identified to date (HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1). Most genes possessing variants associated with Perrault syndrome are involved in mitochondrial translation. We describe a consanguineous family with three affected individuals homozygous for a novel missense variant c.1454C>T; p.(Ala485Val) in KIAA0391, encoding proteinaceous RNase P (PRORP), the metallonuclease subunit of the mitochondrial RNase P complex, responsible for the 5’-end processing of mitochondrial precursor tRNAs. In RNase P activity assays, RNase P complexes containing the PRORP disease variant produced ~35-45% less 5’-processed tRNA than wild type PRORP. Consistently, the accumulation of unprocessed polycistronic mitochondrial transcripts was observed in patient dermal fibroblasts, leading to an observable loss of steady-state levels of mitochondrial oxidative phosphorylation components. Expression of wild type KIAA0391 in patient fibroblasts rescued tRNA processing. Immunohistochemistry analyses of the auditory sensory epithelium from postnatal and adult mouse inner ear showed a high level of PRORP in the efferent synapses and nerve fibres of hair cells, indicating a possible mechanism for the sensorineural hearing loss observed in affected individuals. We have identified a variant in an additional gene associated with Perrault syndrome. With the identification of this disease-causing variant in KIAA0391, reduced function of each of the three subunits of mitochondrial RNase P have now been associated with distinct clinical presentations.Author SummaryPerrault syndrome is a rare genetic condition which results in hearing loss in both sexes and ovarian dysfunction in females. Perrault syndrome may also cause neurological symptoms in some patients. Here, we present the features and genetic basis of the condition in three sisters affected by Perrault syndrome. The sisters did not have pathogenic variants in any of the genes previously associated with Perrault syndrome. We identified a change in the gene KIAA0391, encoding PRORP, a subunit of the mitochondrial RNase P complex. Mitochondrial RNase P is a key enzyme in RNA processing in mitochondria. Impaired RNA processing reduces protein production in mitochondria, which we observed in patient cells along with high levels of unprocessed RNA. When we expressed wild type PRORP in patient cells, the RNA processing improved. We also investigated PRORP localisation in the mouse inner ear and found high levels in the synapses and nerve fibers that transmit sound. It may be that disruption of RNA processing in the mitochondria of these cells causes hearing loss in this family.
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- 2017
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10. The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats
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Ana Gonzalez, Camila Cristina Pasquali, Ricardo Diogo Righeto, Wyatt W. Yue, Igor M. Ferreira, Andre Luis Berteli Ambrosio, Zeyaul Islam, Sandra Martha Gomes Dias, Jefferson Bettini, Rodrigo Villares Portugal, and Douglas Adamoski
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0301 basic medicine ,Gene isoform ,Models, Molecular ,Retrotransposon ,Biology ,Crystallography, X-Ray ,Biochemistry ,Evolution, Molecular ,03 medical and health sciences ,0302 clinical medicine ,Glutaminase ,Protein Domains ,Phylogenetics ,Ankyrin ,Humans ,Protein Structure, Quaternary ,Molecular Biology ,chemistry.chemical_classification ,Genetics ,Phylogenetic tree ,Models, Genetic ,Cell Biology ,Ankyrin Repeat ,Isoenzymes ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Protein Structure and Folding ,Human genome ,Ankyrin repeat - Abstract
On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C-termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.
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- 2017
11. Novel patient missense mutations in the HSD17B10 gene affect dehydrogenase and mitochondrial tRNA modification functions of the encoded protein
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Martine Roovers, Udo Oppermann, Louis Droogmans, Corinne Gemperle-Britschgi, Henry J. Bailey, Michael Leichsenring, Wyatt W. Yue, Frauke Beermann, Alain Fouilhoux, Stephanie Oerum, Joern Oliver Sass, Anne Korwitz-Reichelt, and Cecile Acquaviva-Bourdain
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0301 basic medicine ,Male ,Models, Molecular ,Protein Conformation ,Mutant ,Mutation, Missense ,TRNA processing ,Gene Expression ,Biology ,Methylation ,Ribonuclease P ,HSD17B10 ,Mitochondrial Proteins ,03 medical and health sciences ,RNA, Transfer ,Humans ,Molecular Biology ,Gene ,HSPA9 ,Genetics ,030102 biochemistry & molecular biology ,Wild type ,Mitochondrial tRNA modification ,3-Hydroxyacyl CoA Dehydrogenases ,Infant ,Methyltransferases ,Recombinant Proteins ,Mitochondria ,030104 developmental biology ,Biochemistry ,Transfer RNA ,Molecular Medicine ,Female - Abstract
MRPP2 (also known as HSD10/SDR5C1) is a multifunctional protein that harbours both catalytic and non-catalytic functions. The protein belongs to the short-chain dehydrogenase/reductases (SDR) family and is involved in the catabolism of isoleucine in vivo and steroid metabolism in vitro. MRPP2 also moonlights in a complex with the MRPP1 (also known as TRMT10C) protein for N1-methylation of purines at position 9 of mitochondrial tRNA, and in a complex with MRPP1 and MRPP3 (also known as PRORP) proteins for 5'-end processing of mitochondrial precursor tRNA. Inherited mutations in the HSD17B10 gene encoding MRPP2 protein lead to a childhood disorder characterised by progressive neurodegeneration, cardiomyopathy or both. Here we report two patients with novel missense mutations in the HSD17B10 gene (c.34G>C and c.526G>A), resulting in the p.V12L and p.V176M substitutions. Val12 and Val176 are highly conserved residues located at different regions of the MRPP2 structure. Recombinant mutant proteins were expressed and characterised biochemically to investigate their effects towards the functions of MRPP2 and associated complexes in vitro. Both mutant proteins showed significant reduction in the dehydrogenase, methyltransferase and tRNA processing activities compared to wildtype, associated with reduced stability for protein with p.V12L, whereas the protein carrying p.V176M showed impaired kinetics and complex formation. This study therefore identified two distinctive molecular mechanisms to explain the biochemical defects for the novel missense patient mutations.
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- 2017
12. Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA
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Sylvestre Grizot, Susan K. Buchanan, and Wyatt W. Yue
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Models, Molecular ,Siderophore ,Protein Conformation ,Iron ,Molecular Sequence Data ,Receptors, Cell Surface ,Plasma protein binding ,Crystallography, X-Ray ,Ligands ,Ferric Compounds ,Citric Acid ,Protein structure ,Bacterial Proteins ,Structural Biology ,medicine ,Escherichia coli ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Binding Sites ,Chemistry ,Escherichia coli Proteins ,Membrane Proteins ,Biological Transport ,Periplasmic space ,Ligand (biochemistry) ,Biochemistry ,Ferric ,Bacterial outer membrane ,Carrier Proteins ,Sequence Alignment ,medicine.drug ,Protein Binding - Abstract
Escherichia coli possesses a TonB-dependent transport system, which exploits the iron-binding capacity of citrate and its natural abundance. Here, we describe three structures of the outer membrane ferric citrate transporter FecA: unliganded and complexed with iron-free or diferric dicitrate. We show the structural mechanism for discrimination between the iron-free and ferric siderophore: the binding of diferric dicitrate, but not iron-free dicitrate alone, causes major conformational rearrangements in the transporter. The structure of FecA bound with iron-free dicitrate represents the first structure of a TonB-dependent transporter bound with an iron-free siderophore. Binding of diferric dicitrate to FecA results in changes in the orientation of the two citrate ions relative to each other and in their interactions with FecA, compared to the binding of iron-free dicitrate. The changes in ligand binding are accompanied by conformational changes in three areas of FecA: two extracellular loops, one plug domain loop and the periplasmic TonB-box motif. The positional and conformational changes in the siderophore and transporter initiate two independent events: ferric citrate transport into the periplasm and transcription induction of the fecABCDE transport genes. From these data, we propose a two-step ligand recognition event: FecA binds iron-free dicitrate in the non-productive state or first step, followed by siderophore displacement to form the transport-competent, diferric dicitrate-bound state in the second step.
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- 2016
13. Crystal structure of the PHF8 Jumonji domain, an Nepsilon-methyl lysine demethylase
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Viktorija Hozjan, Wyatt W. Yue, Michael A. McDonough, Christopher D.O. Cooper, Udo Oppermann, Christopher J. Schofield, Christoph Loenarz, Wei Ge, and Kathryn L. Kavanagh
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Models, Molecular ,Jumonji Domain-Containing Histone Demethylases ,Protein Conformation ,Iron ,Lysine ,Molecular Sequence Data ,Biophysics ,chemistry ,Crystallography, X-Ray ,Biochemistry ,Protein structure ,Transcriptional regulation ,Structural Biology ,Humans ,genetics ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Histone Demethylases ,Epigenetic modification ,Binding Sites ,biology ,Sequence Homology, Amino Acid ,PHF8 ,Histone lysine demethylase ,2-Oxoglutarate oxygenase ,Cell Biology ,Protein Structure, Tertiary ,JmjC ,PHD finger ,Mutation ,biology.protein ,Demethylase ,Ketoglutaric Acids ,JARID1B ,metabolism ,Protein Binding ,Transcription Factors - Abstract
Crystallographic analysis of the catalytic domain of PHD finger protein 8 (PHF8), an N(epsilon)-methyl lysine histone demethylase associated with mental retardation and cleft lip/palate, reveals a double-stranded beta-helix fold with conserved Fe(II) and cosubstrate binding sites typical of the 2-oxoglutarate dependent oxygenases. The PHF8 active site is highly conserved with those of the FBXL10/11demethylases, which are also selective for the di-/mono-methylated lysine states, but differs from that of the JMJD2 demethylases which are selective for tri-/di-methylated states. The results rationalize the lack of activity for the clinically observed F279S PHF8 variant and they will help to identify inhibitors selective for specific N(epsilon)-methyl lysine demethylase subfamilies.
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- 2016
14. Dynamic protein methylation in chromatin biology
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S.S. Ng, Wyatt W. Yue, Udo Oppermann, and Robert J. Klose
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Models, Molecular ,Protein Conformation ,Review Article ,histone ,Computational biology ,Biology ,Arginine ,Methylation ,Chromatin remodeling ,Epigenesis, Genetic ,Histones ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Neoplasms ,Histone H2A ,Histone methylation ,Animals ,Histone code ,Protein Methyltransferases ,Epigenetics ,Cancer epigenetics ,Molecular Biology ,Embryonic Stem Cells ,030304 developmental biology ,Epigenomics ,Genetics ,Pharmacology ,0303 health sciences ,Molecular Structure ,epigenetics ,Lysine ,demethylation ,Oxidoreductases, N-Demethylating ,Histone-Lysine N-Methyltransferase ,Cell Biology ,Chromatin ,Germ Cells ,Gene Expression Regulation ,Histone methyltransferase ,Histone Methyltransferases ,Molecular Medicine ,Tumor Suppressor Protein p53 ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,DNA Damage - Abstract
Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.
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- 2016
15. Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design
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Andrew P. Turnbull, Wyatt W. Yue, Naeem Shafqat, Udo Oppermann, and Johannes Zschocke
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Gene isoform ,Hydroxymethylglutaryl-CoA Synthase ,Models, Molecular ,Coenzyme A ,Mutation, Missense ,chemistry ,Crystallography, X-Ray ,chemistry.chemical_compound ,Structural Biology ,Ketogenesis ,Transferase ,Humans ,Protein Isoforms ,genetics ,Protein Structure, Quaternary ,Molecular Biology ,antagonists and inhibitors ,ATP synthase ,biology ,deficiency ,Ketones ,Cytosol ,Biochemistry ,HMG-CoA reductase ,biology.protein ,Mevalonate pathway ,metabolism ,Dimerization - Abstract
3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.
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- 2016
16. Use of methylmalonyl‐CoA epimerase in enhancing crotonase stereoselectivity
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Wyatt W. Yue, Refaat B. Hamed, D. Sean Froese, E. Krysztofinska, J. Ruben Gomez-Castellanos, and Christopher J. Schofield
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0301 basic medicine ,Stereochemistry ,Organic Chemistry ,Racemases and Epimerases ,Stereoisomerism ,Reductase ,Enoyl-CoA hydratase ,010402 general chemistry ,Methylmalonyl CoA epimerase ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Pyruvate carboxylase ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Biosynthesis ,chemistry ,Biocatalysis ,Molecular Medicine ,Stereoselectivity ,Molecular Biology ,Enoyl-CoA Hydratase - Abstract
The use of methylmalonyl‐CoA epimerase (MCEE) to improve stereoselectivity in crotonase‐mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl‐CoA carboxylase reductase and carboxymethylproline synthase in a three‐enzyme one‐pot sequential synthesis of functionalised C‐5 carboxyalkylprolines starting from crotonyl‐CoA and carbon dioxide.
- Published
- 2015
17. Structures of the Human GTPase MMAA and Vitamin B12-dependent Methylmalonyl-CoA Mutase and Insight into Their Complex Formation
- Author
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João R. C. Muniz, Udo Oppermann, E. Krysztofinska, Emelie Ugochukwu, Roy A. Gravel, Grazyna Kochan, D. Sean Froese, Xuchu Wu, Wyatt W. Yue, and Carina Gileadi
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Protein domain ,Mutation, Missense ,GTPase ,chemistry ,Crystallography, X-Ray ,Mitochondrial Membrane Transport Proteins ,Guanosine Diphosphate ,Biochemistry ,Cofactor ,Mitochondrial Proteins ,Cytosol ,Protein structure ,Mutase ,Humans ,genetics ,Methionine synthase ,Child ,Protein Structure, Quaternary ,Molecular Biology ,biology ,Membrane transport protein ,Methylmalonyl-CoA mutase ,Membrane Transport Proteins ,Methylmalonyl-CoA Mutase ,Cell Biology ,Mitochondria ,Child, Preschool ,Multiprotein Complexes ,biology.protein ,Guanosine Triphosphate ,Cobamides ,metabolism ,Metabolism, Inborn Errors - Abstract
Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.
- Published
- 2010
18. Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation
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Wyatt W. Yue, He Huang, Lunzhi Dai, Jeongsoon Park, Zhongyi Cheng, Olga Pougovkina, Yingming Zhao, Xiaojing Liu, Xuelian Wan, Ronald J.A. Wanders, Vincent C. J. de Boer, Heleen te Brinke, Jason W. Locasale, David B. Lombard, Minjia Tan, Gozde Colak, Other departments, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, and Laboratory Genetic Metabolic Diseases
- Subjects
Cell physiology ,Male ,Models, Molecular ,fatty acid oxydation ,Carboxy-Lyases ,macromolecular substances ,Biochemistry ,Analytical Chemistry ,Cell Line ,Mice ,mitochondrial function ,medicine ,lysine malonylation ,Animals ,Humans ,Sirtuins ,Fibroblast ,Molecular Biology ,Beta oxidation ,Mice, Knockout ,biology ,Lysine ,Research ,Fatty Acids ,Proteomic ,nutritional and metabolic diseases ,Metabolism ,Fibroblasts ,Pathophysiology ,Malonates ,Mitochondria ,Malonyl Coenzyme A ,medicine.anatomical_structure ,Liver ,Cell culture ,Sirtuin ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Oxidation-Reduction ,Function (biology) ,Metabolism, Inborn Errors ,Methylmalonic Acid - Abstract
The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD−/− fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases.
- Published
- 2015
19. Recognition of iron-free siderophores by TonB-dependent iron transporters
- Author
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Wyatt W. Yue, Isabelle J. Schalk, and Susan K. Buchanan
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0303 health sciences ,Siderophore ,Inner membrane complex ,030306 microbiology ,Chemiosmosis ,Transporter ,Periplasmic space ,Biology ,Microbiology ,03 medical and health sciences ,Biochemistry ,Sigma factor ,Transcription (biology) ,Biophysics ,bacteria ,Inner membrane ,Molecular Biology ,030304 developmental biology - Abstract
TonB-dependent iron transporters reside in the outer membranes of Gram-negative bacteria, transporting ferric-complexes into the periplasm by a mechanism requiring proton motive force and an integral inner membrane complex, TonB-ExbB-ExbD. Certain TonB-dependent transporters contain an additional domain at the N-terminus, which interacts with an inner membrane regulatory protein and a cytoplasmic sigma factor to induce transcription of iron transport genes when a ferric-ligand is bound at the extracellular surface of the transporter. Transport of the ferric-ligand is apparently not necessary for transcription induction. Recent biophysical and crystallographic experiments have shown that this subclass of TonB-dependent iron transporters can bind iron-free ligands, whereas only the ferric-ligands are transported into the periplasm. This review focuses on the ligand binding properties of these transporters and includes a discussion of the biological function of the additional domain, the mechanism of transcription induction and the mechanism of ferric-ligand transport.
- Published
- 2004
20. Mutation or knock-down of 17β-hydroxysteroid dehydrogenase type 10 cause loss of MRPP1 and impaired processing of mitochondrial heavy strand transcripts
- Author
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Stephanie Oerum, Johannes Zschocke, Renée G. Feichtinger, Claudia Zavadil, Wyatt W. Yue, Johannes A. Mayr, Albert Amberger, Herbert Steinbeisser, and Andrea J. Deutschmann
- Subjects
Mutation ,RNase P ,3-Hydroxyacyl CoA Dehydrogenases ,General Medicine ,Methyltransferases ,Biology ,Mitochondrion ,Fibroblasts ,medicine.disease_cause ,Molecular biology ,Ribonuclease P ,HSD17B10 ,Heavy strand ,RNA, Transfer ,Transfer RNA ,Genetics ,medicine ,Humans ,Ectopic expression ,Mitochondrial tRNA processing ,Molecular Biology ,Genetics (clinical) - Abstract
17β-Hydroxysteroid dehydrogenase type 10 (HSD10) is multifunctional protein coded by the X-chromosomal HSD17B10 gene. Mutations in this gene cause HSD10 disease characterized by progressive neurological abnormalities and cardiomyopathy. Disease progression and severity of symptoms is unrelated to the protein's dehydrogenase activity. Recently, it was shown that HSD10 is an essential component of mitochondrial Ribonuclease P (RNase P), an enzyme required for mitochondrial tRNA processing, but little is known about the role of HSD10 in RNase P function. RNase P consists of three different proteins MRPP1, MRPP2 (HSD10) and MRPP3, each of which is essential for RNase P function. Here, we show that HSD10 protein levels are significantly reduced in fibroblasts from patients carrying the HSD17B10 mutation p.R130C. A reduction in HSD10 levels was accompanied by a reduction in MRPP1 protein but not MRPP3 protein. In HSD10 knock-down cells, MRPP1 protein content was also reduced, indicating that HSD10 is important for the maintenance of normal MRPP1 protein levels. Ectopic expression of HSD10 partially restored RNA processing in HSD10 knock-down cells and fibroblasts, and also expression of MRPP1 protein was restored to values comparable to controls. In both, patient fibroblasts and HSD10 knock-down cells, there was evidence of impaired processing of precursor tRNA transcripts of the mitochondrial heavy strand but not the light strand compared with controls. Our findings indicate that HSD10 is important for the maintenance of the MRPP1-HSD10 subcomplex of RNase P and that loss of HSD10 causes impaired mitochondrial precursor transcript processing which may explain mitochondrial dysfunction observed in HSD10 disease.
- Published
- 2014
21. Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations
- Author
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Froese, D. Sean, Forouhar, Farhad, Tran, Timothy H., Vollmar, Melanie, Kim, Yi Seul, Lew, Scott, Neely, Helen, Seetharaman, Jayaraman, Shen, Yang, Xiao, Rong, Acton, Thomas B., Everett, John K., Cannone, Giuseppe, Puranik, Sriharsha, Savitsky, Pavel, Krojer, Tobias, Pilka, Ewa S., Kiyani, Wasim, Lee, Wen Hwa, Marsden, Brian D., von Delft, Frank, Allerston, Charles K., Spagnolo, Laura, Gileadi, Opher, Montelione, Gaetano T., Oppermann, Udo, Yue, Wyatt W., and Tong, Liang
- Subjects
Models, Molecular ,Carboxy-Lyases ,Molecular Sequence Data ,Mutation, Missense ,Hydrogen Bonding ,Crystallography, X-Ray ,Article ,Protein Structure, Secondary ,Kinetics ,Bacterial Proteins ,Structural Homology, Protein ,Structural Biology ,Catalytic Domain ,Enzyme Stability ,Humans ,Amino Acid Sequence ,Deficiency Diseases ,Protein Structure, Quaternary ,Molecular Biology - Abstract
Summary Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design., Highlights • Structures of human and bacterial MCDs were determined at up to 2.3 Å resolution • Distinct tetrameric and dimeric MCD oligomerizations were observed • Unexpected homology to the GNAT superfamily gives insights into catalytic mechanism • The structures provide the molecular basis for the disease-causing mutations in MCD, Malonyl-CoA decarboxylase (MCD) is important in fatty acid metabolism. Froese et al. report structures of several MCDs and show that the MCD catalytic domain shares structural homology with GNAT superfamily. The structures further our understanding of catalysis, pathogenic mutations, and drug design.
- Published
- 2013
22. Crystal structure of the secretory isozyme of mammalian carbonic anhydrases CA VI: implications for biological assembly and inhibitor development
- Author
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Udo Oppermann, Grazyna Kochan, Wyatt W. Yue, and Ewa S. Pilka
- Subjects
Gene isoform ,Protein Conformation ,Bicarbonate ,Biophysics ,Crystallography, X-Ray ,Biochemistry ,Isozyme ,chemistry.chemical_compound ,Protein structure ,Carbonic anhydrase ,Catalytic Domain ,Drug Discovery ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Carbonic Anhydrases ,biology ,Active site ,Cell Biology ,Lyase ,Isoenzymes ,Zinc ,Secretory protein ,chemistry ,biology.protein ,Protein Multimerization - Abstract
Zn(2+)-dependent carbonic anhydrases (CA) catalyse the reversible hydration of carbon dioxide to bicarbonate and participate in diverse physiological processes, hence having manifold therapeutic potentials. Among the 15 human CAs with wide-ranging sub-cellular localisation and kinetic properties, CA VI is the only secretory isoform. The 1.9Å crystal structure of the human CA VI catalytic domain reveals a prototypical mammalian CA fold, and a novel dimeric arrangement as compared to previously-reported CA structures. The active site cavity contains a cluster of non-conserved residues that may be involved in ligand binding and have significant implications for developing the next-generation of isoform-specific inhibitors.
- Published
- 2012
23. Structural and evolutionary basis for the dual substrate selectivity of human KDM4 histone demethylase family
- Author
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Carina Gileadi, Wyatt W. Yue, James E. Bray, Udo Oppermann, S.S. Ng, Simon H. Bello, Lars Hillringhaus, Nathan R. Rose, Christopher J. Schofield, and Christoph Loenarz
- Subjects
Jumonji Domain-Containing Histone Demethylases ,DNA and Chromosomes ,Crystallography, X-Ray ,Jumonji-C Demethylases ,Biochemistry ,Evolution, Molecular ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Histone H1 ,Structural Biology ,Histone methylation ,Animals ,Humans ,Histone code ,Molecular Biology ,030304 developmental biology ,Enzyme Kinetics ,0303 health sciences ,biology ,Cell Biology ,Enzyme structure ,Mutagenesis in Vitro ,Histone ,2-Oxoglutarate ,Mutagenesis ,Structural Homology, Protein ,030220 oncology & carcinogenesis ,Histone methyltransferase ,Enzyme Structure ,Oxygenases ,biology.protein ,Demethylase ,Histone Methylation ,Histone Demethylases - Abstract
Background: Lysine demethylases reverse Nϵ-methylation in a sequence- and methylation-selective manner. Results: Enzyme-histone interactions away from the conserved oxygenase active site are important in determining sequence selectivity in the JMJD2 (KDM4) subfamily. Conclusion: The catalytic JmjC domain determines sequence selectivity for at least some JmjC demethylases. Significance: This work might be a basis for the development of selective inhibitors., Nϵ-Methylations of histone lysine residues play critical roles in cell biology by “marking” chromatin for transcriptional activation or repression. Lysine demethylases reverse Nϵ-methylation in a sequence- and methylation-selective manner. The determinants of sequence selectivity for histone demethylases have been unclear. The human JMJD2 (KDM4) H3K9 and H3K36 demethylases can be divided into members that act on both H3K9 and H3K36 and H3K9 alone. Kinetic, crystallographic, and mutagenetic studies in vitro and in cells on KDM4A–E reveal that selectivity is determined by multiple interactions within the catalytic domain but outside the active site. Structurally informed phylogenetic analyses reveal that KDM4A–C orthologues exist in all genome-sequenced vertebrates with earlier animals containing only a single KDM4 enzyme. KDM4D orthologues only exist in eutherians (placental mammals) where they are conserved, including proposed substrate sequence-determining residues. The results will be useful for the identification of inhibitors for specific histone demethylases.
- Published
- 2011
24. Interactive JIMD articles using the iSee concept: turning a new page on structural biology data
- Author
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Eugene Raush, Brian D. Marsden, Ruben Abagyan, Wyatt W. Yue, Wen Hwa Lee, Udo Oppermann, and Maxim Totrov
- Subjects
Models, Molecular ,Protein Conformation ,Computer science ,Text annotation ,Bioinformatics ,Public domain ,Data type ,Molecular graphics ,Structural genomics ,User-Computer Interface ,Software ,Genetics ,Humans ,Databases, Protein ,Molecular Biology ,Dissemination ,Genetics (clinical) ,Publishing ,business.industry ,Data science ,High-Throughput Screening Assays ,Structural biology ,Periodicals as Topic ,business ,Algorithms - Abstract
Since its inception, researchers in structural biology have reported detailed molecular models of macromolecules involved in the functioning of the human body. This wealth of information has provided a better understanding of the molecular mechanisms by which the proteins and enzymes perform their biochemical and physiological functions. Several scientific fields have benefited from these advancements, including clinicians and those studying inherited metabolic disorders caused by mutational defects in proteins. Furnished with structural data, scientists are able to make sense of the different mutations observed in patients and understand their effects at the protein level (Yue and Oppermann 2011). In recent years, advances in high-throughput methods and concerted structural genomics efforts have yielded a large increase in the number of publically available solved structures. The Structural Genomics Consortium (SGC) was created in 2004 to tackle the challenging task of solving structures of medically relevant human proteins in a high-throughput manner (Gileadi et al. 2007; Weigelt 2010). While contributing to over 20% of novel protein structures in the public domain, the SGC recognised that dissemination of its results and data beyond the boundaries of structural biology is fundamental in promoting tangible advancements in human health and medicine. This has been the main motivation behind the development of the iSee concept by the SGC, in collaboration with MolSoft L.L.C., as a means to enable flexible, instructive and intuitive data dissemination (Abagyan et al. 2006). The iSee concept provides a flexible and intuitive tool to deliver text annotation and easy-to-use interactive molecular graphics to authors and readers alike. In one single file, the authors can include many different data types, including full atomic coordinates of protein structures or chemical compounds, DNA and amino acid sequences, images, tables, text and molecular graphics animations. These files can be created, viewed and edited using the free ICM Browser software provided by Communicated by: Verena Peters
- Published
- 2011
25. Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS
- Author
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Jan Frayne, Apirat Chaikuad, Gus Cameron, Anthony R. Clarke, Wyatt W. Yue, R Al-Mokhtar, Udo Oppermann, Naeem Shafqat, and R L Brady
- Subjects
Male ,Models, Molecular ,Stereochemistry ,Dehydrogenase ,Protonation ,Models, Biological ,Biochemistry ,Catalysis ,Protein Structure, Secondary ,Phosphates ,Oxidoreductase ,Animals ,Humans ,Spermatogenesis ,Molecular Biology ,Glyceraldehyde 3-phosphate dehydrogenase ,chemistry.chemical_classification ,Binding Sites ,biology ,Ligand ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Cell Biology ,Spermatozoa ,Rats ,Amino acid ,Isoenzymes ,Kinetics ,Enzyme ,chemistry ,Organ Specificity ,biology.protein ,NAD+ kinase ,Protein Binding - Abstract
hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDS ΔN ), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDS ΔN –NAD + –phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved P s site and the newly recognized P i site identified in other organisms. The hGAPDS ΔN –NAD + –glycerol structure shows serendipitous binding of glycerol at the P s and new P i sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDS ΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDS ΔN . This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.
- Published
- 2011
26. Structural impact of human and Escherichia coli biotin carboxyl carrier proteins on biotin attachment
- Author
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Xuchu Wu, Wyatt W. Yue, Udo Oppermann, Shannon Healy, Megan K. McDonald, Grazyna Kochan, and Roy A. Gravel
- Subjects
Methylmalonyl-CoA Decarboxylase ,Biology ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Biotin ,Consensus sequence ,medicine ,Fatty Acid Synthase, Type II ,Humans ,Carbon-Nitrogen Ligases ,Escherichia coli ,chemistry.chemical_classification ,DNA ligase ,Escherichia coli Proteins ,Substrate (chemistry) ,Molecular biology ,Peptide Fragments ,Pyruvate carboxylase ,Protein Structure, Tertiary ,Repressor Proteins ,Protein Subunits ,chemistry ,Biotinylation ,Holocarboxylase synthetase ,Carrier Proteins ,Acetyl-CoA Carboxylase ,Protein Binding - Abstract
Holocarboxylase synthetase (HCS, human) and BirA (Escherichia coli) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases. Biotin attachment occurs on a highly conserved lysine residue within a consensus sequence (Ala/Val-Met-Lys-Met) that is found in carboxylases in most organisms. Numerous studies have indicated that HCS and BirA, as well as biotin protein ligases from other organisms, can attach biotin to apocarboxylases from different organisms, indicating that the mechanism of biotin attachment is well conserved. In this study, we examined the cross-reactivity of biotin attachment between human and bacterial biotin ligases by comparing biotinylation of p-67 and BCCP87, the biotin-attachment domain fragments from human propionyl-CoA carboxylase and E. coli acetyl-CoA carboxylase, respectively. While BirA has similar biotinylation activity toward the two substrates, HCS has reduced activity toward bacterial BCCP87 relative to its native substrate, p-67. The crystal structure of a digested form of p-67, spanning a sequence that contains a seven-residue protruding thumb loop in BCCP87, revealed the absence of a similar structure in the human peptide. Significantly, an engineered "thumbless" bacterial BCCP87 could be biotinylated by HCS, with substrate affinity restored to near normal. This study suggests that the thumb loop found in bacterial carboxylases interferes with optimal interaction with the mammalian biotin protein ligase. While the function of the thumb loop remains unknown, these results indicate a constraint on specificity of the bacterial substrate for biotin attachment that is not itself a feature of BirA.
- Published
- 2010
27. Structural snapshots for the conformation-dependent catalysis by human medium-chain acyl-coenzyme A synthetase ACSM2A
- Author
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Udo Oppermann, Grazyna Kochan, Wyatt W. Yue, Ewa S. Pilka, and Frank von Delft
- Subjects
Models, Molecular ,Stereochemistry ,Protein Conformation ,Coenzyme A ,Molecular Sequence Data ,Molecular Conformation ,chemistry ,Crystallography, X-Ray ,Cofactor ,Catalysis ,Substrate Specificity ,chemistry.chemical_compound ,Protein structure ,Adenosine Triphosphate ,Structural Biology ,Coenzyme A Ligases ,Humans ,genetics ,Amino Acid Sequence ,Molecular Biology ,Magnesium ion ,Binding Sites ,biology ,Fatty Acids ,Active site ,Adenosine Monophosphate ,Isoenzymes ,biology.protein ,Salt bridge ,Adenosine triphosphate ,metabolism ,Sequence Alignment - Abstract
Acyl-CoA synthetases belong to the superfamily of adenylate-forming enzymes, and catalyze the two-step activation of fatty acids or carboxylate-containing xenobiotics. The carboxylate substrate first reacts with ATP to form an acyl-adenylate intermediate, which then reacts with CoA to produce an acyl-CoA ester. Here, we report the first crystal structure of a medium-chain acyl-CoA synthetase ACSM2A, in a series of substrate/product/cofactor complexes central to the catalytic mechanism. We observed a substantial rearrangement between the N- and C-terminal domains, driven purely by the identity of the bound ligand in the active site. Our structures allowed us to identify the presence or absence of the ATP pyrophosphates as the conformational switch, and elucidated new mechanistic details, including the role of invariant Lys557 and a divalent magnesium ion in coordinating the ATP pyrophosphates, as well as the involvement of a Gly-rich P-loop and the conserved Arg472-Glu365 salt bridge in the domain rearrangement.
- Published
- 2009
28. Crystal structure of human carbonic anhydrase-related protein VIII reveals the basis for catalytic silencing
- Author
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Ewa S. Pilka, Wyatt W. Yue, Udo Oppermann, Sarah Picaud, Grazyna Kochan, Anneke Kramm, and João R. C. Muniz
- Subjects
Gene isoform ,Models, Molecular ,biology ,Chemistry ,Protein Conformation ,Active site ,chemistry ,Lyase ,Crystallography, X-Ray ,Biochemistry ,Isozyme ,Catalysis ,Cytosol ,Protein structure ,Tumor Markers, Biological ,Structural Biology ,Carbonic anhydrase ,Catalytic Domain ,biology.protein ,Biomarkers, Tumor ,Humans ,metabolism ,Molecular Biology ,Histidine - Abstract
Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton.1 They are ubiquitous in prokaryotes and eukaryotes, and are encoded by four evolutionarily-unrelated gene classes (a, b, g, d). The human CA family belongs to the a class and contains 12 catalytic isozymes with different tissue distribution, subcellular localization and kinetic properties. These include the cytosolic (CA I, CA II, CA III, CA VII, CA XIII), membrane-bound (CA IV, CA IX, CA XII, CA XIV), mitochondrial (CA VA, CA VB) and secreted (CA VI) isoforms. They share an absolute requirement for a catalytic Zn ion in the active site, coordinated by an hydroxide ion and three invariant histidine residues H94CAII, H96CAII and H119CAII (human CA II numbering) that are in turn hydrogen bonded to conserved partners Q92CAII, N244CAII and E117CAII, respectively. CAs participate in physiological processes such as respiration, metabolite biosynthesis and pH regulation, and are interesting pharmaceutical targets.2 Sulphonamide-based CA inhibitors are established diuretics and anti-glaucoma drugs, and may have further therapeutic potentials in anti-obesity and anti-cancer treatment.3 The human CA family includes a subclass of three noncatalytic isoforms (CA VIII, CA X, CA XI), also known as CA-related proteins (CA-RPs), based on sequence homology with the catalytic isozymes. CA-RPs lack one or more of the essential Zn-coordinating histidines and are devoid of CO2 hydration activity.4 To date, the biological functions of CA-RPs remain undefined. The first identified CA-RP, CA VIII, replaces the Zn-coordinating H94CAII and its hydrogen-bonding partner Q92CAII with R116 and E114, respectively (human CA VIII numbering).5 CA VIII is highly expressed in the cerebellum,6 and a mouse gene deletion causes a motor coordination defect.7 Relevant to this, CA VIII has been identified as a binding partner for the inositol 1,4,5 triphosphate (IP3) receptor type 1 which is abundant in the cerebellum.8 To provide insights into the biological properties of CA-RPs, we have determined the 1.6 A crystal structure of human CA VIII (hCA VIII). This work represents the first structural characterization of a CA-RP and offers a structural basis for its catalytic silencing effect.
- Published
- 2009
29. Crystal structure of the Retinoblastoma protein N-domain provides insight into tumor suppression, ligand interaction and holoprotein architecture
- Author
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Rachid Lakbir, Wyatt W. Yue, Sibylle Mittnacht, Maciej T. Luczynski, Laurence H. Pearl, Thomas Bader, Francisco Sánchez-Sánchez, Shradha Singh, and Markus Hassler
- Subjects
Models, Molecular ,Molecular Sequence Data ,Cell Cycle Proteins ,Crystallography, X-Ray ,Ligands ,Retinoblastoma Protein ,Article ,Protein structure ,Protein Interaction Mapping ,medicine ,Humans ,Amino Acid Sequence ,Binding site ,Nuclear protein ,Peptide sequence ,Molecular Biology ,Genetics ,Binding Sites ,biology ,Retinoblastoma ,Retinoblastoma protein ,Nuclear Proteins ,Cell Biology ,medicine.disease ,Ligand (biochemistry) ,Cell biology ,Protein Structure, Tertiary ,Repressor Proteins ,Structural biology ,biology.protein - Abstract
The retinoblastoma susceptibility protein, Rb, has a key role in regulating cell cycle progression via interactions involving the central 'pocket' and C-terminal regions. While the N-terminal domain of Rb is dispensable for this function, it is nonetheless strongly conserved, and harbours many missense mutations found in hereditary retinoblastoma, indicating that disruption of its function is oncogenic. The crystal structure of the Rb N-terminal domain (RbN), reveals a single globular entity formed by two cyclin-like folds. The intrinsic similarity of RbN to the A and B boxes of the Rb pocket domain suggests that Rb evolved through domain duplication. Structural and functional analysis provides insight into oncogenicity of mutations in RbN and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation.
- Published
- 2007
30. Insights into histone code syntax from structural and biochemical studies of CARM1 methyltransferase
- Author
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Laurence H. Pearl, S. Mark Roe, Markus Hassler, Vivienne Thompson-Vale, and Wyatt W. Yue
- Subjects
Protein-Arginine N-Methyltransferases ,Molecular Sequence Data ,Biology ,Arginine ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Protein Structure, Secondary ,Article ,Histones ,Histone H3 ,Mice ,Histone methylation ,Histone H2A ,Histone code ,Animals ,Histone octamer ,Amino Acid Sequence ,Molecular Biology ,Binding Sites ,General Immunology and Microbiology ,Sequence Homology, Amino Acid ,General Neuroscience ,EZH2 ,Methylation ,Protein Structure, Tertiary ,Rats ,Biochemistry ,Histone methyltransferase ,Crystallization ,Protein Binding - Abstract
Coactivator-associated arginine methyltransferase (CARM1) is a transcriptional coactivator that methylates Arg17 and Arg26 in histone H3. CARM1 contains a conserved protein arginine methyltransferase (PRMT) catalytic core flanked by unique pre- and post-core regions. The crystal structures of the CARM1 catalytic core in the apo and holo states reveal cofactor-dependent formation of a substrate-binding groove providing a specific access channel for arginine to the active site. The groove is supported by the first eight residues of the post-core region (C-extension), not present in other PRMTs. In vitro methylation assays show that the C-extension is essential for all histone H3 methylation activity, whereas the pre-core region is required for methylation of Arg26, but not Arg17. Kinetic analysis shows Arg17 methylation is potentiated by pre-acetylation of Lys18, and this is reflected in k(cat) rather than K(m). Together with the absence of specificity subsites in the structure, this suggests an electrostatic sensing mechanism for communicating the modification status of vicinal residues as part of the syntax of the 'histone code.'
- Published
- 2007
31. Expanding the clinical and molecular spectrum of thiamine pyrophosphokinase deficiency: A treatable neurological disorder caused by TPK1 mutations.
- Author
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Banka, Siddharth, de Goede, Christian, Yue, Wyatt W., Morris, Andrew A.M., von Bremen, Beate, Chandler, Kate E., Feichtinger, René G., Hart, Claire, Khan, Nasaim, Lunzer, Verena, Mataković, Lavinija, Marquardt, Thorsten, Makowski, Christine, Prokisch, Holger, Debus, Otfried, Nosaka, Kazuto, Sonwalkar, Hemant, Zimmermann, Franz A., Sperl, Wolfgang, and Mayr, Johannes A.
- Subjects
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NEUROLOGICAL disorders , *THIAMIN pyrophosphate , *ENZYME deficiency , *GENETIC mutation , *KETOGLUTARATE dehydrogenase , *BIOMARKERS , *MOLECULAR biology - Abstract
Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458 ) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood. These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting. Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence suggesting that thiamine supplementation may rescue TPK enzyme activity. Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK dimerization, which may be a novel mechanism for the disease. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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