42 results on '"Aleš Hnízda"'
Search Results
2. Conserved roles of C. elegans and human MANFs in sulfatide binding and cytoprotection
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Meirong Bai, Roman Vozdek, Aleš Hnízda, Chenxiao Jiang, Bingying Wang, Ladislav Kuchar, Tiejun Li, Yuefan Zhang, Chase Wood, Liang Feng, Yongjun Dang, and Dengke K. Ma
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Science - Abstract
MANF is a secreted ER stress-inducible protein that protects neurons, pancreatic β cells and cardiomyocytes from cell death under oxidative stress, hypoxic or ischemic conditions. Here the authors show that MANF confers cytoprotection through direct binding to sulfatide followed by cellular uptake in both C. elegans and mammalian cells.
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- 2018
- Full Text
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3. Data from Mechanisms of NT5C2-Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia
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Jun J. Yang, William L. Carroll, Aleš Hnízda, Richard Heath, Youming Shao, Wentao Yang, Xujie Zhao, Julia Meyer, Jing Li, Shuguang Liu, and Takaya Moriyama
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Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5′-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.
- Published
- 2023
4. Supplementary Table 1 from Mechanisms of NT5C2-Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia
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Jun J. Yang, William L. Carroll, Aleš Hnízda, Richard Heath, Youming Shao, Wentao Yang, Xujie Zhao, Julia Meyer, Jing Li, Shuguang Liu, and Takaya Moriyama
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This supplementary table describes metabolite levels and statistics from metabolite experiments
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- 2023
5. Supplementary Figures 1-5 from Mechanisms of NT5C2-Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia
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Jun J. Yang, William L. Carroll, Aleš Hnízda, Richard Heath, Youming Shao, Wentao Yang, Xujie Zhao, Julia Meyer, Jing Li, Shuguang Liu, and Takaya Moriyama
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Supplementary figure 1 and 2 describe protein purity and expression. Supplementary Figure 3 shows cell-based cytotoxic assays. Supplementary Figure 4 shows protein activity against several substrates. Supplementary Figure 5 describes levels of individual metabolites.
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- 2023
6. AGAL misprocessing-induced ER stress and the unfolded protein response: lysosomal storage-independent mechanism of Fabry disease pathogenesis?
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Martina Živná, Gabriela Dostálová, Veronika Barešová, Dita Mušálková, Ladislav Kuchař, Befekadu Asfaw, Helena Poupětová, Hana Vlášková, Tereza Kmochová, Petr Vyletal, Hana Hartmannová, Kateřina Hodaňová, Viktor Stránecký, Lenka Steiner-Mrázová, Aleš Hnízda, Martin Radina, Miroslav Votruba, Jana Sovová, Helena Trešlová, Larisa Stolnaja, Petra Reková, Lenka Roblová, Eva Honsová, Helena Hůlková, Ivan Rychlík, Anthony J. Bleyer, Aleš Linhart, Jakub Sikora, and Stanislav Kmoch
- Abstract
BackgroundClassic Fabry disease (FD) is caused by GLA mutations that result in enzymatic deficiency of alpha-galactosidase A (AGAL), lysosomal storage of globotriaosylceramide, and a resulting multisystemic disease. In non-classic later-onset FD, patients have some preserved AGAL activity and a milder disease course, though female carriers may also be affected. While FD pathogenesis has been mostly attributed to catalytic deficiency of mutated AGAL, lysosomal storage and impairment of lysosomal functions, other pathogenic factors may be important, especially in non-classic later-onset FD.MethodsWe characterized the clinical, biochemical, genetic, molecular, cellular and organ pathology correlates of the p.L394P AGAL variant that was identified in six individuals with end-stage kidney disease by the Czech national screening program for FD and by further screening of 25 family members.ResultsClinical findings revealed a milder clinical course with ~15% residual AGAL activity. Laboratory investigations documented intracellular retention of mutated AGAL with resulting ER stress and the unfolded protein response (UPR). Kidney biopsies did not show lysosomal storage. We observed similar findings of ER stress and UPR with several other classic and non-classic FD missense GLA variants.ConclusionsWe identified defective proteostasis of mutated AGAL resulting in chronic ER stress and UPR of AGAL expressing cells (hereafter referred to as AGALopathy) as an important contributor to FD pathogenesis. These findings provide insight into non-classic later-onset FD and may better explain clinical manifestations with implications for pathogenesis, clinical characterization and treatment of all FD forms.Significance statementCatalytic deficiency of mutated AGAL is responsible for classicFabry disease (FD) pathogenesis but does not fully explain the findings in non-classic later-onset FD, in which affected individuals and female carriers develop clinical manifestations despite some AGAL activity and variably mitigated lysosomal storage. In this investigation of individuals with the p.L394P AGAL variant, we identified defective proteostasis of mutated AGAL resulting in chronic endoplasmic reticulum stress and the unfolded protein response as significant contributors to pathogenesis of non-classic later-onset FD. Similar effects were documented also in other AGAL variants identified in classic and non-classicFD. Endoplasmic reticulum stress and the unfolded protein response therefore play an important role in FD.
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- 2022
7. Stages, scaffolds and strings in the spatial organisation of non-homologous end joining: Insights from X-ray diffraction and Cryo-EM
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Shikang Liang, Tom L. Blundell, Amanda K. Chaplin, Aleš Hnízda, Antonia Kefala Stavridi, Robert Appleby, Liang, Shikang [0000-0002-6930-7882], Blundell, Tom [0000-0002-2708-8992], and Apollo - University of Cambridge Repository
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Materials science ,DNA End-Joining Repair ,Spatial organisation ,DNA Repair ,Cryo-electron microscopy ,DNA repair ,Double-strand breaks ,030303 biophysics ,Biophysics ,KBM, Ku-binding motif ,Computational biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,DNA-PK, DNA-dependent protein kinase ,PAXX, Paralog of XRCC4 and XLF ,XLF, XRCC4-Like Factor ,Stages ,Strings ,Humans ,DSB, double-strand break ,DNA Breaks, Double-Stranded ,Orchestration (computing) ,vWA, von Willebrand factor type A ,cryo-EM, cryo-electron microscopy ,PTIP, Pax transcription-activation-domain interacting protein ,Molecular Biology ,NHEJ ,Cryo-EM ,IR, ionising radiation ,Scaffolds ,0303 health sciences ,53BP1, p53-binding protein 1 ,Cryoelectron Microscopy ,X-ray diffraction ,Non-homologous end joining ,enzymes and coenzymes (carbohydrates) ,chemistry ,DNA-PKcs, DNA-PK catalytic subunit ,NHEJ, non-homologous end joining ,X-ray crystallography ,PNKP, polynucleotide kinase ,APLF, aprataxin and PNKP-like factor ,BRCT, BRCA1 C-Terminus domain ,lncRNA, long noncoding RNA ,HR, homologous recombination ,DNA ,CTR, C-terminal region - Abstract
Non-homologous end joining (NHEJ) is the preferred pathway for the repair of DNA double-strand breaks in humans. Here we describe three structural aspects of the repair pathway: stages, scaffolds and strings. We discuss the orchestration of DNA repair to guarantee robust and efficient NHEJ. We focus on structural studies over the past two decades, not only using X-ray diffraction, but also increasingly exploiting cryo-EM to investigate the macromolecular assemblies.
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- 2021
8. SAP domain forms a flexible part of DNA aperture in Ku70/80
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Roland Beckmann, Thanh Binh Nguyen, Zdeněk Kukačka, Amanda K. Chaplin, Aleš Hnízda, Petr Novák, Petr Tesina, Lukas Kater, Tom L. Blundell, David B. Ascher, Hnízda, Aleš [0000-0002-1521-4453], and Apollo - University of Cambridge Repository
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0301 basic medicine ,DNA double‐strand break ,DNA End-Joining Repair ,Protein Conformation ,Protein domain ,Protein Data Bank (RCSB PDB) ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Protein Domains ,Humans ,A-DNA ,DNA Breaks, Double-Stranded ,Molecular Biology ,Ku Autoantigen ,integrative structural biology ,Physics ,Original Articles ,Cell Biology ,computer.file_format ,DNA ,Protein Data Bank ,SAP domain ,nonhomologous end joining ,Non-homologous end joining ,Ku70/80 ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,DNA double-strand break ,Biophysics ,Original Article ,computer - Abstract
Funder: Victorian Government, Funder: BBSRC, Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-bound state. The second position, which was observed in both apo and DNA-bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. DATABASES: EM maps have been deposited in EMDB (EMD-11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.
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- 2021
9. Mechanisms of NT5C2-Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia
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William L. Carroll, Jing Li, Richard J. Heath, Xujie Zhao, Takaya Moriyama, Shuguang Liu, Youming Shao, Aleš Hnízda, Julia Meyer, Jun J. Yang, and Wentao Yang
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0301 basic medicine ,Purine ,Cancer Research ,Nucleotidase activity ,Thiopurine methyltransferase ,HEK 293 cells ,Mutant ,Endogeny ,Drug resistance ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Metabolomics ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein - Abstract
Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5′-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.
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- 2019
10. Thiopurine intolerance-causing mutations in NUDT15 induce temperature-dependent destabilization of the catalytic site
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Milan Fábry, Aleš Hnízda, Irena Sieglová, Petr Novák, Daniel Kavan, and Petr Man
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0301 basic medicine ,Mutant ,Thermolysin ,Biophysics ,Context (language use) ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Germline mutation ,Protein structure ,Catalytic Domain ,Pyrophosphatases ,Molecular Biology ,chemistry.chemical_classification ,Thiopurine methyltransferase ,biology ,Protein Stability ,Chemistry ,Temperature ,Deoxyguanine Nucleotides ,030104 developmental biology ,Enzyme ,Protein destabilization ,030220 oncology & carcinogenesis ,Mutation ,Mutagenesis, Site-Directed ,biology.protein - Abstract
Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.
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- 2019
11. Multicomponent assemblies in DNA-double-strand break repair by NHEJ
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Aleš Hnízda and Tom L. Blundell
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0303 health sciences ,DNA Repair ,Macromolecular Substances ,Chemistry ,DNA repair ,Protein subunit ,Synapsis ,DNA ,DNA-Activated Protein Kinase ,Double Strand Break Repair ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Structural Biology ,Humans ,DNA Breaks, Double-Stranded ,Kinase activity ,Protein kinase A ,Ligation ,Ku Autoantigen ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Non-homologous end joining (NHEJ), a process for repair of DNA-breaks that does not require a DNA-template, involves synapsis, end-processing and ligation. Synapsis is initiated by assembly of the Ku-heterodimer on DNA broken ends, followed by the formation of DNA-dependent protein kinase (DNA-PK) - an assembly of the catalytic subunit (DNA-PKcs), the Ku-heterodimer and DNA. Recent progress in understanding the structural architecture of DNA-PK, achieved by X-ray crystallography and cryo-electron microscopy, has revealed a stage of DNA-PKcs on which other components from the pathway assemble and mediate kinase activity allosterically. This review provides a comparative overview of recently published structures of DNA-PK, together with a discussion of other complexes mediated by the Ku heterodimer. It also shows that some binders are specific to particular patho-physiological conditions.
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- 2019
12. Author response for 'SAP domain forms a flexible part of DNA aperture in Ku70/80'
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Tom L. Blundell, Petr Novák, Roland Beckmann, Zdeněk Kukačka, Thanh Binh Nguyen, Lukas Kater, David B. Ascher, Amanda K. Chaplin, Petr Tesina, and Aleš Hnízda
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Physics ,Optics ,Aperture ,business.industry ,business ,Domain (software engineering) - Published
- 2021
13. Dimers of DNA-PK create a stage for DNA double-strand break repair
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Taiana Maia de Oliveira, Lee R Cooper, Antonia Kefala Stavridi, Amanda K. Chaplin, Tom L. Blundell, Shikang Liang, Aleš Hnízda, Dimitri Y. Chirgadze, Steven W. Hardwick, Blundell, Tom L [0000-0002-2708-8992], and Apollo - University of Cambridge Repository
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DNA End-Joining Repair ,Ku Autoantigen ,Molecular Conformation ,DNA-Activated Protein Kinase ,Crystallography, X-Ray ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Dna genetics ,Structural Biology ,Humans ,DNA Breaks, Double-Stranded ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,0303 health sciences ,Cryoelectron Microscopy ,DNA ,Molecular biology ,Double Strand Break Repair ,chemistry ,Dna breaks ,Double stranded ,Dimerization ,030217 neurology & neurosurgery - Abstract
DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.
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- 2021
14. Mechanism of efficient double-strand break repair by a long non-coding RNA
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Shujuan Fang, Ruiqiong Ye, Aleš Hnízda, Roopa Thapar, Isaac Forrester, Chunru Lin, Linda Lee, Susan P. Lees-Miller, Heather Villarreal, Su S. Maw, Terence R. Strick, Miaw Sheue Tsai, Anthony J. Davis, Michal Hammel, Cheng-Cao Sun, Jing L. Wang, John A. Tainer, Shikang Liang, Tom L. Blundell, Ke Liang, Liang, Shikang [0000-0002-6930-7882], Blundell, Tom [0000-0002-2708-8992], and Apollo - University of Cambridge Repository
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Ku80 ,DNA End-Joining Repair ,DNA repair ,AcademicSubjects/SCI00010 ,Biology ,Genome Integrity, Repair and Replication ,chemistry.chemical_compound ,03 medical and health sciences ,Double-Stranded ,Information and Computing Sciences ,Genetics ,Humans ,DNA Breaks, Double-Stranded ,Ku Autoantigen ,030304 developmental biology ,Cancer ,Ku70 ,0303 health sciences ,030302 biochemistry & molecular biology ,DNA Breaks ,RNA ,Biological Sciences ,Double Strand Break Repair ,Long non-coding RNA ,Cell biology ,DNA-Binding Proteins ,chemistry ,Hela Cells ,RNA, Long Noncoding ,Long Noncoding ,Protein Multimerization ,Corrigendum ,DNA ,Environmental Sciences ,HeLa Cells ,Protein Binding ,Developmental Biology - Abstract
Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1’s ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA–RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.
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- 2021
15. An international cohort study of autosomal dominant tubulointerstitial kidney disease due to REN mutations identifies distinct clinical subtypes
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Veronika Baresova, Miroslav Votruba, Kálmán Tory, Aleš Hnízda, Jakub Sikora, Matthias T.F. Wolf, Marisa Santostefano, Neila Belghith, Lídia Balogh, Jan Živný, Tal Kopel, Robert M. Haws, Bertrand Knebelmann, Andrea Wenzel, Bodo B. Beck, Lawrence R. Shoemaker, Laurent Mesnard, Anna Jakubowska, Kendrah Kidd, Charles Shaw-Smith, Christoforos Stavrou, Mayssa Abdelwahed, Constantinos Deltas, John A. Sayer, Claudio Graziano, Rhian L Clissold, Petr Vyleťal, Stanislav Kmoch, Victoria Robins, Howard Trachtman, Michael E. Bleyer, Marie Matignon, Anthony J. Bleyer, Kathleen Claes, Jana Sovová, Irene Capelli, Philippe Grimbert, Sharon M. Moe, Luca Rampoldi, Ivana Jedličková, Karsten Häeffner, Stéphane Decramer, Kateřina Hodaňová, Helena Trešlová, Matthew R. Sinclair, Raj Munshi, Gregory Papagregoriou, Hana Hartmannová, Albert C.M. Ong, Mohamad Zaidan, Agnieszka Łaszkiewicz, Amy N. Sussman, Claudia Izzi, Martina Živná, Helena Hůlková, Francesco Scolari, Živná, M, Kidd, K, Zaidan, M, Vyleťal, P, Barešová, V, Hodaňová, K, Sovová, J, Hartmannová, H, Votruba, M, Trešlová, H, Jedličková, I, Sikora, J, Hůlková, H, Robins, V, Hnízda, A, Živný, J, Papagregoriou, G, Mesnard, L, Beck, Bb, Wenzel, A, Tory, K, Häeffner, K, Wolf, Mtf, Bleyer, Me, Sayer, Ja, Ong, Acm, Balogh, L, Jakubowska, A, Łaszkiewicz, A, Clissold, R, Shaw-Smith, C, Munshi, R, Haws, Rm, Izzi, C, Capelli, I, Santostefano, M, Graziano, C, Scolari, F, Sussman, A, Trachtman, H, Decramer, S, Matignon, M, Grimbert, P, Shoemaker, Lr, Stavrou, C, Abdelwahed, M, Belghith, N, Sinclair, M, Claes, K, Kopel, T, Moe, S, Deltas, C, Knebelmann, B, Rampoldi, L, Kmoch, S, and Bleyer, Aj
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0301 basic medicine ,Signal peptide ,Adult ,Male ,medicine.medical_specialty ,Mutant ,030232 urology & nephrology ,Chromosomal translocation ,autosomal dominant tubulointerstitial kidney disease ,characterization ,mutation ,prosegment ,renin ,signal peptide ,medicine.disease_cause ,Cohort Studies ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Internal medicine ,Renin–angiotensin system ,Renin ,medicine ,Humans ,Secretion ,Child ,Mutation ,Polycystic Kidney Diseases ,business.industry ,Endoplasmic reticulum ,Anemia ,medicine.disease ,030104 developmental biology ,Endocrinology ,Nephrology ,Female ,business ,Kidney disease - Abstract
There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.
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- 2020
16. Chronic tubulointerstitial kidney disease in untreated adenine phosphoribosyl transferase (APRT) deficiency: A case report
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Kendrah Kidd, Benjamin Cochran, Angela G Niehaus, Stanislav Kmoch, Alex Bonnecaze, Anthony J. Bleyer, Tereza Kovačíková, Gowrie Balasubraminiam, Amret T. Hawfield, Martina Živná, Aleš Hnízda, Kateřina Hodaňová, and Irene Ceballos-Picot
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Male ,medicine.medical_specialty ,Antimetabolites ,Allopurinol ,medicine.medical_treatment ,Adenine Phosphoribosyltransferase ,030232 urology & nephrology ,Adenine phosphoribosyltransferase ,Urology ,Renal function ,030204 cardiovascular system & hematology ,Nephropathy ,Kidney Calculi ,03 medical and health sciences ,0302 clinical medicine ,Urolithiasis ,medicine ,Humans ,Renal Insufficiency, Chronic ,Dialysis ,Kidney ,business.industry ,Phosphoribosyl transferase ,General Medicine ,Middle Aged ,medicine.disease ,medicine.anatomical_structure ,Nephrology ,Nephritis, Interstitial ,business ,Metabolism, Inborn Errors ,Glomerular Filtration Rate ,medicine.drug ,Kidney disease - Abstract
Adenine phosphoribosyltransferase (APRT) deficiency (OMIM #614723) is a rare autosomal recessive defect in the purine salvage pathway that causes excessive production of 2,8-dihydroxyadenine, leading to nephrolithiasis and chronic kidney disease (CKD). This case report describes the natural history of CKD in untreated APRT deficiency. We describe a novel APRT mutation (chr16:88877985 G / C; c.195 C>/G; p.His54Asp) presenting with CKD without nephrolithiasis. The patient initially required dialysis, but kidney function improved with allopurinol. We reviewed APRT deficiency reported in the literature to determine the loss of kidney function in individuals with untreated APRT deficiency and its relationship to nephrolithiasis. We identified 95 individuals in whom kidney function was assessed prior to treatment. There was a bimodal distribution of kidney failure. AKI occurred frequently in childhood due to obstructing nephrolithiasis or crystalline nephropathy and was usually reversible. CKD developed after age 20 in all patients irrespective of nephrolithiasis history, with 36/42 patients > 40 years of age having at least stage 3 CKD, and 24/42 having an eGFR > 10 mL/min/1.73m2 or being on dialysis. There were 13 adults without nephrolithiasis and 50 adults with nephrolithiasis. The mean age of end-stage renal diesease (ESRD) was 50.52 ± 13.9 for those without nephrolithiasis and 43.4 ± 15.8 years for those with nephrolithiasis (p = 0.24). APRT deficiency is associated with slowly progressive CKD that occurs independently of nephrolithiasis. Diagnosis should be considered in all individuals with chronic tubulointerstitial kidney disease, with or without the presence of nephrolithiasis. In our patient, allopurinol 300 mg/day resulted in improvement of kidney function. .
- Published
- 2018
17. Clinical manifestations and molecular aspects of phosphoribosylpyrophosphate synthetase superactivity in females
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Marie Zikanova, Stanislav Kmoch, Aleš Hnízda, Martina Živná, Charles Pitts, Arielle Hay, Kateřina Hodaňová, Veronika Baresova, Viktor Stránecký, Blanka Stibůrková, Vaclava Skopova, Dita Musalkova, Anthony J. Bleyer, Hana Hartmannová, Dawn M. Wahezi, and Olga Souckova
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Adult ,Male ,musculoskeletal diseases ,0301 basic medicine ,Purine-Pyrimidine Metabolism, Inborn Errors ,medicine.medical_specialty ,Adolescent ,030105 genetics & heredity ,Nephrolithiasis ,03 medical and health sciences ,Rheumatology ,Internal medicine ,Ribose-Phosphate Pyrophosphokinase ,medicine ,OMIM : Online Mendelian Inheritance in Man ,Humans ,Pharmacology (medical) ,Hyperuricemia ,Family history ,Exome sequencing ,Molecular Structure ,Whole Genome Sequencing ,Arthritis, Gouty ,business.industry ,Genetic Diseases, X-Linked ,Clinical Science ,medicine.disease ,Purine/pyrimidine metabolism ,Penetrance ,Pedigree ,Gout ,030104 developmental biology ,Mutation ,Female ,business ,Kidney disease - Abstract
Objectives Phosphoribosylpyrophosphate synthetase (PRPS1) superactivity is an X-linked disorder characterized by urate overproduction Online Mendelian Inheritance in Man (OMIM) gene reference 300661. This condition is thought to rarely affect women, and when it does, the clinical presentation is mild. We describe a 16-year-old African American female who developed progressive tophi, nephrolithiasis and acute kidney failure due to urate overproduction. Family history included a mother with tophaceous gout who developed end-stage kidney disease due to nephrolithiasis and an affected sister with polyarticular gout. The main aim of this study was to describe the clinical manifestations of PRPS1 superactivity in women. Methods Whole exome sequencing was performed in affected females and their fathers. Results Mutational analysis revealed a new c.520 G > A (p.G174R) mutation in the PRPS1 gene. The mutation resulted in decreased PRPS1 inhibition by ADP. Conclusion Clinical findings in previously reported females with PRPS1 superactivity showed a high clinical penetrance of this disorder with a mean serum urate level of 8.5 (4.1) mg/dl [506 (247) μmol/l] and a high prevalence of gout. These findings indicate that all women in families with PRPS1 superactivity should be genetically screened for a mutation (for clinical management and genetic counselling). In addition, women with tophaceous gout, gout presenting in childhood, or a strong family history of severe gout should be considered for PRPS1 mutational analysis.
- Published
- 2018
18. Towards personalized chemotherapy of acute lymphoblastic leukemia
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Aleš Hnízda, Tai Yang, Hnízda, Aleš [0000-0002-1521-4453], and Apollo - University of Cambridge Repository
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Oncology ,personalized therapy ,medicine.medical_specialty ,Chemotherapy ,chemotherapy resistance ,Thiopurine methyltransferase ,biology ,business.industry ,Lymphoblastic Leukemia ,medicine.medical_treatment ,purine metabolism ,Editorial ,Internal medicine ,thiopurine ,medicine ,biology.protein ,Personalized therapy ,Purine metabolism ,business ,relapsed leukemia ,Chemotherapy resistance - Published
- 2018
19. Mechanisms of
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Takaya, Moriyama, Shuguang, Liu, Jing, Li, Julia, Meyer, Xujie, Zhao, Wentao, Yang, Youming, Shao, Richard, Heath, Aleš, Hnízda, William L, Carroll, and Jun J, Yang
- Subjects
Kinetics ,HEK293 Cells ,Drug Resistance, Neoplasm ,Mercaptopurine ,Cell Line, Tumor ,Mutation ,Humans ,Metabolomics ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,5'-Nucleotidase ,Models, Biological ,Article - Abstract
Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared to wildtype NT5C2, mutant proteins showed elevated 5’-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of MP metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant vs wildtype NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influenced endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.
- Published
- 2018
20. Conserved Roles of C. elegans and Human MANFs in Sulfatide Binding and Cytoprotection
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Chase Wood, Chenxiao Jiang, Yuefan Zhang, Roman Vozdek, Liang Feng, Bingying Wang, Ladislav Kuchar, Dengke K. Ma, Aleš Hnízda, Tiejun Li, Meirong Bai, Yongjun Dang, Ma, Dengke K [0000-0002-5619-7485], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Programmed cell death ,1.1 Normal biological development and functioning ,Science ,Mutant ,General Physics and Astronomy ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Underpinning research ,Neurotrophic factors ,Genetics ,2.1 Biological and endogenous factors ,Animals ,Humans ,Nerve Growth Factors ,Aetiology ,lcsh:Science ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Cerebral dopamine neurotrophic factor ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Sulfoglycosphingolipids ,biology ,Chemistry ,Endoplasmic reticulum ,HEK 293 cells ,Neurosciences ,General Chemistry ,Lipid signaling ,biology.organism_classification ,Lipid Metabolism ,Endoplasmic Reticulum Stress ,Cytoprotection ,3. Good health ,Cell biology ,030104 developmental biology ,HEK293 Cells ,Unfolded protein response ,lcsh:Q ,Generic health relevance ,030217 neurology & neurosurgery ,Genetic screen - Abstract
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) protein that can be secreted and protects dopamine neurons and cardiomyocytes from ER stress and apoptosis. The mechanism of action of extracellular MANF has long been elusive. From a genetic screen for mutants with abnormal ER stress response, we identified the gene Y54G2A.23 as the evolutionarily conserved C. elegans MANF orthologue. We find that MANF binds to the lipid sulfatide, also known as 3-O-sulfogalactosylceramide present in serum and outer-cell membrane leaflets, directly in isolated forms and in reconstituted lipid micelles. Sulfatide binding promotes cellular MANF uptake and cytoprotection from hypoxia-induced cell death. Heightened ER stress responses of MANF-null C. elegans mutants and mammalian cells are alleviated by human MANF in a sulfatide-dependent manner. Our results demonstrate conserved roles of MANF in sulfatide binding and ER stress response, supporting sulfatide as a long-sought lipid mediator of MANF’s cytoprotection., MANF is a secreted ER stress-inducible protein that protects neurons, pancreatic β cells and cardiomyocytes from cell death under oxidative stress, hypoxic or ischemic conditions. Here the authors show that MANF confers cytoprotection through direct binding to sulfatide followed by cellular uptake in both C. elegans and mammalian cells.
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- 2018
21. Chaperone therapy for homocystinuria: the rescue of CBS mutations by heme arginate
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Petra Melenovská, Jana Kopecka, Viktor Kožich, Jakub Krijt, Aleš Hnízda, Miroslav Janosik, Kateřina Raková, and Bridget Wilcken
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Models, Molecular ,Protein Folding ,Protein Conformation ,Mutant ,Cystathionine beta-Synthase ,CBS domain ,Homocystinuria ,CHO Cells ,Heme ,Arginine ,Transfection ,Substrate Specificity ,Structure-Activity Relationship ,chemistry.chemical_compound ,Cricetulus ,Catalytic Domain ,Genetics ,medicine ,Animals ,Humans ,Genetic Predisposition to Disease ,Proteostasis Deficiencies ,Genetics (clinical) ,biology ,Homozygote ,Heme arginate ,Fibroblasts ,medicine.disease ,Cystathionine beta synthase ,Phenotype ,Biochemistry ,chemistry ,Chaperone (protein) ,Mutation ,biology.protein ,Female ,Chemical chaperone ,Molecular Chaperones - Abstract
Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.
- Published
- 2014
22. Structural Basis for Inhibition of Mycobacterial and Human Adenosine Kinase by 7-Substituted 7-(Het)aryl-7-deazaadenine Ribonucleosides
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Jindřich Fanfrlík, Radek Pohl, Jiřina Stolaříková, Petr Džubák, Petr Nauš, Petr Konečný, Jiří Dostál, Hana Dvořáková, Ivan Votruba, Marian Hajduch, Aleš Hnízda, Jiří Brynda, Michal Hocek, Helena Zábranská, Pavlína Řezáčová, Vaclav Veverka, Aurelie Bourderioux, Iva Pichová, Michal Dušek, and Jan Snášel
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Protein Conformation ,medicine.drug_class ,Stereochemistry ,Antitubercular Agents ,Drug Evaluation, Preclinical ,Microbial Sensitivity Tests ,Adenosine kinase ,Crystallography, X-Ray ,Antimycobacterial ,Structure-Activity Relationship ,chemistry.chemical_compound ,Adenosine Triphosphate ,Drug Discovery ,medicine ,Humans ,Transferase ,Structure–activity relationship ,Binding site ,Adenosine Kinase ,Nuclear Magnetic Resonance, Biomolecular ,Binding Sites ,biology ,Chemistry ,Adenine ,Mycobacterium tuberculosis ,respiratory system ,bacterial infections and mycoses ,Adenosine ,3. Good health ,ADK ,Biochemistry ,biology.protein ,Molecular Medicine ,Ribonucleosides ,Adenosine triphosphate ,medicine.drug - Abstract
Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.
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- 2014
23. Biochemical properties of nematode O-acetylserine(thiol)lyase paralogs imply their distinct roles in hydrogen sulfide homeostasis
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Aleš Hnízda, Leona Šerá, Roman Vozdek, Viktor Kožich, and Jakub Krijt
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Sulfide ,Hydrogen sulfide ,Biophysics ,Biology ,Cysteine synthase ,Biochemistry ,Substrate Specificity ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Sulfur assimilation ,Catalytic Domain ,Serine ,Animals ,Homeostasis ,Hydrogen Sulfide ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,030304 developmental biology ,Cell Nucleus ,chemistry.chemical_classification ,Cysteine Synthase ,0303 health sciences ,Cyanides ,Active site ,biology.organism_classification ,Lyase ,chemistry ,biology.protein ,O-Acetylserine ,030217 neurology & neurosurgery - Abstract
O-Acetylserine(thiol)lyases (OAS-TLs) play a pivotal role in a sulfur assimilation pathway incorporating sulfide into amino acids in microorganisms and plants, however, these enzymes have not been found in the animal kingdom. Interestingly, the genome of the roundworm Caenorhabditis elegans contains three expressed genes predicted to encode OAS-TL orthologs (cysl-1–cysl-3), and a related pseudogene (cysl-4); these genes play different roles in resistance to hypoxia, hydrogen sulfide and cyanide. To get an insight into the underlying molecular mechanisms we purified the three recombinant worm OAS-TL proteins, and we determined their enzymatic activities, substrate binding affinities, quaternary structures and the conformations of their active site shapes. We show that the nematode OAS-TL orthologs can bind O-acetylserine and catalyze the canonical reaction although this ligand may more likely serve as a competitive inhibitor to natural substrates instead of being a substrate for sulfur assimilation. In addition, we propose that S-sulfocysteine may be a novel endogenous substrate for these proteins. However, we observed that the three OAS-TL proteins are conformationally different and exhibit distinct substrate specificity. Based on the available evidences we propose the following model: CYSL-1 interacts with EGL-9 and activates HIF-1 that upregulates expression of genes detoxifying sulfide and cyanide, the CYSL-2 acts as a cyanoalanine synthase in the cyanide detoxification pathway and simultaneously produces hydrogen sulfide, while the role of CYSL-3 remains unclear although it exhibits sulfhydrylase activity in vitro. All these data indicate that C. elegans OAS-TL paralogs have distinct cellular functions and may play different roles in maintaining hydrogen sulfide homeostasis.
- Published
- 2013
24. Backbone resonance assignments of human cytosolic dNT-1 nucleotidase
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Aleš Hnízda, Petr Pachl, Milan Fábry, Zdeněk Tošner, Radka Skleničková, Vaclav Veverka, and Jiří Brynda
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chemistry.chemical_classification ,Nucleotidase activity ,Chemistry ,Biochemistry ,Small molecule ,Deoxyribonucleotides ,5'-nucleotidase ,Dephosphorylation ,Nucleotidases ,Structural Biology ,Drug Design ,Nucleotidase ,Humans ,Nucleotide ,Enzyme Inhibitors ,Nuclear Magnetic Resonance, Biomolecular ,Nucleoside - Abstract
Cytosolic dNT-1 nucleotidase plays a key role in the homeostasis of pyrimidine deoxyribonucleotides in mammalian cells. The enzyme is responsible for the dephosphorylation of physiological substrates as well as nucleoside analogues that are used in antiviral and anticancer therapies, therefore selective inhibition of the dNT-1 nucleotidase activity may lead to an increase in efficacy of this type of therapeutic compounds. Here, we report the backbone ¹H, ¹³C and ¹⁵N assignments for the 47 kDa dNT-1 dimer, which will be used for structural characterisation of dNT-1 complexes with small molecule inhibitors obtained through modification of pyrimidine nucleotide scaffolds or optimisation of successful binders obtained from the screening of fragment libraries.
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- 2013
25. Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia
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Milan Fábry, Petr Pachl, Aleš Hnízda, Lukáš Vrzal, Martina Šinalová, Jana Škerlová, Vaclav Veverka, Petr Man, Pavlína Řezáčová, and Petr Novák
- Subjects
0301 basic medicine ,Physiology ,Purine metabolism ,Protein Conformation ,medicine.medical_treatment ,Allosteric regulation ,Mutant ,Cancer mutations ,Plant Science ,Biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Targeted therapy ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Recurrence ,Nucleotidase ,medicine ,Humans ,Enzyme kinetics ,Allele ,Cloning, Molecular ,Gene ,5'-Nucleotidase ,Ecology, Evolution, Behavior and Systematics ,Alleles ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Gene Expression Regulation, Leukemic ,Cell Biology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,medicine.disease ,Relapsed ALL ,Leukemia ,Cytosol ,030104 developmental biology ,Biochemistry ,030220 oncology & carcinogenesis ,Mutation ,Cancer research ,General Agricultural and Biological Sciences ,Developmental Biology ,Biotechnology ,Research Article - Abstract
Background Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experience relapsed ALL (R367Q, R238W and L375F). Results Enzyme kinetics measurements revealed that the mutants are consitutively active without need for allosteric activators. This shows that hyperactivity is not caused by a direct catalytic effect but rather by misregulation of cN-II. X-ray crystallography combined with mass spectrometry-based techniques demonstrated that this misregulation is driven by structural modulation of the oligomeric interface within the cN-II homotetrameric assembly. These specific conformational changes are shared between the studied variants, despite the relatively random spatial distribution of the mutations. Conclusions These findings define a common molecular mechanism for cN-II hyperactivity, which provides a solid basis for targeted therapy of leukemia. Our study highlights the cN-II oligomerization interface as an attractive pharmacological target. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0313-y) contains supplementary material, which is available to authorized users.
- Published
- 2016
26. Additional file 11: of Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia
- Author
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Aleš Hnízda, Škerlová, Jana, Fábry, Milan, Pachl, Petr, Šinalová, Martina, Lukáš Vrzal, Man, Petr, Novák, Petr, Řezáčová, Pavlína, and Veverka, Václav
- Abstract
Cross-linking of the wild-type enzyme and R367Q mutant: modified residues identified and quantified using mass spectrometry. Relative abundance refers to a quantitative comparison between products obtained from wild-type and R367Q. The type of crosslinking (i.e., intermolecular, intramolecular or hanging) was inferred from the mass spectrometric data or from the crystal structure. Each number represents the mean value and standard deviation from three experiments. (DOCX 18 kb)
- Published
- 2016
- Full Text
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27. Heterozygous Loss-of-Function SEC61A1 Mutations Cause Autosomal-Dominant Tubulo-Interstitial and Glomerulocystic Kidney Disease with Anemia
- Author
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Hana Hartmannová, Christelle Golzio, Nicholas Katsanis, Lut Van Laer, Geert Vandeweyer, Nikhita Ajit Bolar, Igor Pediaditakis, Christine Van Hemelrijk, Bart Loeys, Jana Sovová, Kendrah Kidd, Martina Živná, Geert Mortier, Guy Van Camp, Han G. Brunner, Kateřina Hodaňová, Richard Spong, Alexander Hoischen, Jeroen R. Huyghe, Gaëlle Hayot, Anna Přistoupilová, Myriam Azou, Erve Matthys, Viktor Stránecký, Ann Raes, Stanislav Kmoch, Marie Claire Gubler, Emiel Sys, Veronika Baresova, Dorien Schepers, Ivana Jedličková, Marleen Praet, Aleš Hnízda, Johan Vande Walle, Petr Vyleťal, Anthony J. Bleyer, Helena Hůlková, and Kelsey McFadden
- Subjects
Male ,Models, Molecular ,0301 basic medicine ,Pathology ,Biopsy ,medicine.medical_treatment ,PRONEPHROS ,030232 urology & nephrology ,Golgi Apparatus ,Endoplasmic Reticulum ,0302 clinical medicine ,ENDOPLASMIC-RETICULUM STRESS ,Medicine and Health Sciences ,Exome ,Genetics(clinical) ,Child ,SIGNAL SEQUENCE ,Zebrafish ,Genetics (clinical) ,Kidney transplantation ,Genes, Dominant ,Genetics ,Kidney ,Fetal Growth Retardation ,medicine.diagnostic_test ,Anemia ,Syndrome ,Middle Aged ,ER STRESS ,Pedigree ,TRANSLOCATION ,3. Good health ,GENOME ,Phenotype ,medicine.anatomical_structure ,Disease Progression ,Female ,Kidney Diseases ,Renal biopsy ,Adult ,Heterozygote ,medicine.medical_specialty ,Neutropenia ,NEPHROPATHY ,Tubular atrophy ,Mutation, Missense ,DNA-SEQUENCING DATA ,Other Research Radboud Institute for Molecular Life Sciences [Radboudumc 0] ,Biology ,Article ,Nephropathy ,Young Adult ,03 medical and health sciences ,GENE MUTATION ,medicine ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Alleles ,Dialysis ,Aged ,Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7] ,LINKAGE ANALYSIS ,Infant, Newborn ,Biology and Life Sciences ,Glomerulosclerosis ,medicine.disease ,030104 developmental biology ,Chronic Disease ,Mutation ,Human medicine ,SEC Translocation Channels ,Kidney disease - Abstract
Contains fulltext : 167296.pdf (Publisher’s version ) (Open Access) Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.
- Published
- 2016
28. Additional file 6: of Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia
- Author
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Aleš Hnízda, Škerlová, Jana, Fábry, Milan, Pachl, Petr, Šinalová, Martina, Lukáš Vrzal, Man, Petr, Novák, Petr, Řezáčová, Pavlína, and Veverka, Václav
- Abstract
Kinetic parameters of the C-terminally truncated enzymes in the presence and absence of ATP. The n value refers to the calculated Hill coefficient. (DOCX 15 kb)
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- 2016
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29. Additional file 7: of Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia
- Author
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Aleš Hnízda, Škerlová, Jana, Fábry, Milan, Pachl, Petr, Šinalová, Martina, Lukáš Vrzal, Man, Petr, Novák, Petr, Řezáčová, Pavlína, and Veverka, Václav
- Abstract
Differential scanning fluorimetry of the C-terminally truncated cN-II variants in the presence or absence of 3 mM ATP. Truncated proteins are stabilized upon ATP binding, as described for full-length variants. Mean values and standard deviations from duplicates of two independent measurements are listed. (DOCX 14 kb)
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- 2016
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30. Additional file 9: of Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia
- Author
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Aleš Hnízda, Škerlová, Jana, Fábry, Milan, Pachl, Petr, Šinalová, Martina, Lukáš Vrzal, Man, Petr, Novák, Petr, Řezáčová, Pavlína, and Veverka, Václav
- Abstract
Oligomeric interface of cN-II crystals analyzed by PISA. NHB and NSB represent the number of identified hydrogen bonds and salt bridges, respectively. ∆i G is the energy of solvation. (DOCX 15 kb)
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- 2016
- Full Text
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31. Conformational Properties of Nine Purified Cystathionine β-Synthase Mutants
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John F. Carpenter, Viktor Kožich, Tomas Majtan, Angel L. Pey, Milan Kodíček, Lu Liu, Aleš Hnízda, and Jan P. Kraus
- Subjects
chemistry.chemical_classification ,medicine.diagnostic_test ,biology ,Proteolysis ,Mutant ,Wild type ,Biochemistry ,Cystathionine beta synthase ,Molecular biology ,Protein structure ,Enzyme ,chemistry ,Thermolysin ,medicine ,biology.protein ,Protein folding - Abstract
Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS.
- Published
- 2012
32. Novel structural arrangement of nematode cystathionine β-synthases: characterization of Caenorhabditis elegans CBS-1
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Marta Kostrouchová, Viktor Kožich, Aleš Hnízda, Roman Vozdek, and Jakub Krijt
- Subjects
Models, Molecular ,Cytoplasm ,AdoMet, S-adenosylmethionine ,domain architecture ,Mutant ,hydrogen sulfide ,BN, blue native ,Biochemistry ,0302 clinical medicine ,RNA interference ,Homeostasis ,LC–MS/MS, liquid chromatography–tandem MS ,Homocysteine ,Conserved Sequence ,Caenorhabditis elegans ,GFP, green fluorescent protein ,chemistry.chemical_classification ,0303 health sciences ,RT, reverse transcription ,biology ,cystathionine β-synthase (CBS) ,knockdown ,UTR, untranslated region ,RNAi, RNA interference ,Organ Specificity ,EST, expressed sequence tag ,Research Article ,inorganic chemicals ,congenital, hereditary, and neonatal diseases and abnormalities ,SEC, size-exclusion chromatography ,Molecular Sequence Data ,BS3, bis(sulfosuccinimidyl) suberate ,Cystathionine beta-Synthase ,Cofactor ,03 medical and health sciences ,Tandem repeat ,Animals ,Humans ,Amino Acid Sequence ,Protein Structure, Quaternary ,CBS, cystathionine β-synthase ,Molecular Biology ,Gene ,030304 developmental biology ,organic chemicals ,nutritional and metabolic diseases ,Cell Biology ,biology.organism_classification ,WT, wild-type ,Cystathionine beta synthase ,Protein Structure, Tertiary ,CGL, cystathionine γ-lyase ,Enzyme ,chemistry ,DTT, dithiothreitol ,Biocatalysis ,biology.protein ,Sequence Alignment ,PLP, pyridoxal 5*-phosphate ,030217 neurology & neurosurgery - Abstract
CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.
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- 2012
33. Cystathionine beta‐synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts
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Aleš Hnízda, Viktor Kožich, Kateřina Raková, and Vojtěch Jurga
- Subjects
Protein Denaturation ,Protein Folding ,Time Factors ,Protein Conformation ,Proteolysis ,Mutant ,Thermolysin ,Cystathionine beta-Synthase ,CBS domain ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Escherichia coli ,Genetics ,medicine ,Humans ,Urea ,Genetics(clinical) ,Genetics (clinical) ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,medicine.diagnostic_test ,Chemistry ,Cystathionine beta synthase ,Protein Structure, Tertiary ,Kinetics ,Enzyme ,Biochemistry ,Mutation ,Solvents ,biology.protein ,Original Article ,Protein folding ,Dimerization ,030217 neurology & neurosurgery - Abstract
Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism. Electronic supplementary material The online version of this article (doi:10.1007/s10545-011-9407-4) contains supplementary material, which is available to authorized users.
- Published
- 2011
34. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency
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Stuart J. Moat, Viktor Kožich, Jakub Krijt, Jana Kopecka, Leo A. J. Kluijtmans, Aleš Hnízda, and Philip Mayne
- Subjects
inorganic chemicals ,S-Adenosylmethionine ,congenital, hereditary, and neonatal diseases and abnormalities ,Cystathionine beta-Synthase ,Homocystinuria ,Genomic disorders and inherited multi-system disorders [IGMD 3] ,Immunoenzyme Techniques ,Serine ,Plasma ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Enzyme Stability ,medicine ,Genetics ,Humans ,natural sciences ,Genetics(clinical) ,Pyridoxal phosphate ,Pyridoxal ,Genetics (clinical) ,chemistry.chemical_classification ,Homocysteine and B-Vitamin Metabolism ,biology ,organic chemicals ,nutritional and metabolic diseases ,Pyridoxine ,medicine.disease ,Cystathionine beta synthase ,Enzyme assay ,Enzyme ,chemistry ,Biochemistry ,Case-Control Studies ,Pyridoxal Phosphate ,Calibration ,biology.protein ,Blood Chemical Analysis ,Chromatography, Liquid ,medicine.drug - Abstract
Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-2H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-2H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66–1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0–9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0–358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5′-phosphate, and by configuration of the detected reaction product, 3,3-2H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency. Electronic supplementary material The online version of this article (doi:10.1007/s10545-010-9178-3) contains supplementary material, which is available to authorized users.
- Published
- 2011
35. Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling†
- Author
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Viktor Kozich, Vojtech Jurga, Vojtech Spiwok, Milan Kodíček, Aleš Hnízda, and Jan P. Kraus
- Subjects
Stereochemistry ,Protein Conformation ,Allosteric regulation ,CBS domain ,Cystathionine beta-Synthase ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Article ,Serine ,03 medical and health sciences ,Protein structure ,Thermolysin ,Catalytic Domain ,Humans ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,010401 analytical chemistry ,Computational Biology ,Receptor Cross-Talk ,Cystathionine beta synthase ,0104 chemical sciences ,Protein Structure, Tertiary ,Enzyme ,chemistry ,biology.protein ,Linker ,Hydrophobic and Hydrophilic Interactions ,Allosteric Site - Abstract
Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms.
- Published
- 2010
36. Biochemical and structural analysis of 14 mutant adsl enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency
- Author
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Stanislav Kmoch, Vaclava Skopova, Jakub Krijt, Marie Zikanova, and Aleš Hnízda
- Subjects
Mutant ,Biology ,Compound heterozygosity ,Protein Structure, Secondary ,Genetic Heterogeneity ,Structure-Activity Relationship ,Multienzyme Complexes ,Enzyme Stability ,Genetics ,medicine ,Humans ,Adenylosuccinate lyase ,Genetics (clinical) ,Adenylosuccinate lyase deficiency ,Genetic heterogeneity ,Adenylosuccinate Lyase ,Infant, Newborn ,medicine.disease ,Phenotype ,Complementation ,Biochemistry ,Mutation ,Electrophoresis, Polyacrylamide Gel ,Mutant Proteins ,Sample collection - Abstract
Adenylosuccinate lyase (ADSL) deficiency is neurometabolic disease characterized by accumulation of dephosphorylated enzyme substrates SAICA-riboside (SAICAr) and succinyladenosine (S-Ado) in body fluids of affected individuals. The phenotypic severity differs considerably among patients: neonatal fatal, severe childhood, and moderate phenotypic forms correlating with different values for the ratio between S-Ado and SAICAr concentrations in cerebrospinal fluid have been distinguished. To reveal the biochemical and structural basis for this phenotypic heterogeneity, we expressed and characterized 19 ADSL mutant proteins identified in 16 patients representing clinically distinct subgroups. Respecting compound heterozygosity and considering the homotetrameric structure of ADSL, we used intersubunit complementation and prepared and characterized genotype-specific heteromeric mutant ADSL complexes. We correlated clinical phenotypes with biochemical properties of the mutant proteins and predicted structural impacts of the mutations. We found that phenotypic severity in ADSL deficiency is correlated with residual enzymatic activity and structural stability of the corresponding mutant ADSL complexes and does not seem to result from genotype-specific disproportional catalytic activities toward one of the enzyme substrates. This suggests that the S-Ado/SAICAr ratio is probably not predictive of phenotype severity; rather, it may be secondary to the degree of the patient's development (i.e., to the age of the patient at the time of sample collection). Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc.
- Published
- 2010
37. Conformationally constrained nucleoside phosphonic acids--potent inhibitors of human mitochondrial and cytosolic 5'(3')-nucleotidases
- Author
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Pavlína Řezáčová, Milan Fábry, Ondřej Šimák, Miloš Buděšínský, Aleš Hnízda, Ivan Rosenberg, Jiří Brynda, Magdalena Petrová, Petr Pachl, Vaclav Veverka, Tomáš Jandušík, and Radka Skleničková
- Subjects
biology ,Dose-Response Relationship, Drug ,Stereochemistry ,Organic Chemistry ,Molecular Conformation ,Organophosphonates ,Active site ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Thymine ,Mitochondria ,chemistry.chemical_compound ,Nucleotidases ,Structure-Activity Relationship ,Cytosol ,chemistry ,biology.protein ,Moiety ,Structure–activity relationship ,Humans ,Physical and Theoretical Chemistry ,Enzyme Inhibitors ,Nucleoside ,Lead compound ,5'-Nucleotidase - Abstract
This work describes novel in vitro inhibitors of human mitochondrial (mdN) and cytosolic (cdN) 5′(3′)-deoxynucleotidases. We designed a series of derivatives of the lead compound (S)-1-[2-deoxy-3,5-O-(phosphonobenzylidene)-β-D-threo-pentofuranosyl]thymine bearing various substituents in the para position of the benzylidene moiety. Detailed kinetic study revealed that certain para substituents increase the inhibitory potency (iodo derivative; KmdNi = 2.71 μM) and some induce a shift in selectivity toward cdN (carboxy derivative, KcdNi = 11.60 μM; iodoxy derivative, KcdNi = 6.60 μM). Crystal structures of mdN in complex with three of these compounds revealed that various para substituents lead to two alternative inhibitor binding modes within the enzyme active site. Two binding modes were also identified for cdN complexes by heteronuclear NMR spectroscopy.
- Published
- 2014
38. Correction to Conformational Properties of Nine Purified Cystathionine β-Synthase Mutants
- Author
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John F. Carpenter, Tomas Majtan, Viktor Kožich, Aleš Hnízda, Jan P. Kraus, Angel L. Pey, Lu Liu, and Milan Kodíček
- Subjects
Models, Molecular ,Protein Folding ,S-Adenosylmethionine ,ATP synthase ,biology ,Chemistry ,Protein Conformation ,Circular Dichroism ,Mutant ,Mutation, Missense ,Cystathionine beta-Synthase ,Biochemistry ,Cystathionine beta synthase ,Article ,Proteolysis ,biology.protein ,Escherichia coli ,Humans ,Spectrophotometry, Ultraviolet - Abstract
Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS.
- Published
- 2012
39. Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: a cellular model of adenylosuccinate lyase deficiency
- Author
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Terry G. Wilkinson, Christine Graham, Stanislav Kmoch, Vaclava Skopova, Nathan Duval, Marie Zikanova, Guido N. Vacano, Veronika Baresova, Lydia K. Vliet, Aleš Hnízda, and David Patterson
- Subjects
Purine ,Purine-Pyrimidine Metabolism, Inborn Errors ,Transcription, Genetic ,Endocrinology, Diabetes and Metabolism ,Mutant ,Molecular Sequence Data ,Mutation, Missense ,CHO Cells ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,chemistry.chemical_compound ,Mice ,Endocrinology ,Cricetulus ,Catalytic Domain ,Cricetinae ,Genetics ,medicine ,Animals ,Humans ,RNA, Messenger ,Autistic Disorder ,Adenylosuccinate lyase ,Protein Structure, Quaternary ,Molecular Biology ,Adenylosuccinate lyase deficiency ,Enzyme Assays ,Alanine ,Mutation ,Chinese hamster ovary cell ,Adenylosuccinate Lyase ,medicine.disease ,chemistry ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,Cellular model - Abstract
Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells ( AdeI ) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency.
- Published
- 2010
40. Biochemical‐structural‐phenotypic correlation of adenylosuccinate lyase deficiency based on analysis of mutant enzyme complexes
- Author
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Marie Zikanova, Jakub Krijt, Aleš Hnízda, Vaclava Skopova, and Stanislav Kmoch
- Subjects
Biochemistry ,Chemistry ,Genetics ,medicine ,medicine.disease ,Adenylosuccinate lyase ,Molecular Biology ,Mutant enzyme ,Phenotype ,Biotechnology ,Adenylosuccinate lyase deficiency - Published
- 2010
41. Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins
- Author
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Miloslav Sanda, Jiří Šantrůček, Milan Kodíček, Aleš Hnízda, and Martin Strohalm
- Subjects
Surface Properties ,Lysine ,Biophysics ,Peptide ,Biochemistry ,Protein structure ,Diethyl Pyrocarbonate ,medicine ,Animals ,Humans ,Histidine ,Horses ,chemistry.chemical_classification ,Molecular Structure ,Chemistry ,Chemical modification ,Proteins ,Human serum albumin ,Angiotensin II ,Amino acid ,Spectrophotometry ,Chickens ,medicine.drug - Abstract
The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.
- Published
- 2007
42. Towards personalized chemotherapy of acute lymphoblastic leukemia.
- Author
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Hnízda A and Yang T
- Published
- 2018
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