176 results on '"Lars C. Pedersen"'
Search Results
2. A genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase
- Author
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Da-Yun Jin, Xuejie Chen, Yizhou Liu, Craig M. Williams, Lars C. Pedersen, Darrel W. Stafford, and Jian-Ke Tie
- Subjects
Science - Abstract
The authors develop a Vitamin K-dependent apoptotic reporter cell line for large-scale screening of enzymes associated with Vitamin K-dependent carboxylation and identify ferroptosis suppressor protein 1 (FSP1) as responsible for warfarin-resistant vitamin K reduction.
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- 2023
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3. Targeting heparan sulfate-protein interactions with oligosaccharides and monoclonal antibodies
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Miaomiao Li, Lars C. Pedersen, and Ding Xu
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heparan sulfate binding proteins ,drug target ,oligosaccharide ,monoclonal antibody ,crystallization ,specificity ,Biology (General) ,QH301-705.5 - Abstract
Heparan sulfate-binding proteins (HSBPs) are structurally diverse extracellular and membrane attached proteins that interact with HS under normal physiological conditions. Interactions with HS offer an additional level of control over the localization and function of HSBPs, which enables them to behave in a more refined manner. Because all cell signaling events start at the cell membrane, and cell-cell communication relies on translocation of soluble factors across the extracellular matrix, HS occupies an apical position in cellular signal transduction by interacting with hundreds of growth factors, cytokines, chemokines, enzymes, enzyme inhibitors, receptors and adhesion molecules. These extracellular and membrane proteins can play important roles in physiological and pathological conditions. For most HS-binding proteins, the interaction with HS represents an essential element in regulating their normal physiological functions. Such dependence on HS suggests that manipulating HS-protein interactions could be explored as a therapeutic strategy to selectively antagonize/activate HS-binding proteins. In this review, we will discuss current understanding of the diverse nature of HS-HSBP interactions, and the latest advancements in targeting the HS-binding site of HSBPs using structurally-defined HS oligosaccharides and monoclonal antibodies.
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- 2023
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4. Targeting the Structural Maturation Pathway of HIV-1 Reverse Transcriptase
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Thomas W. Kirby, Scott A. Gabel, Eugene F. DeRose, Lalith Perera, Juno M. Krahn, Lars C. Pedersen, and Robert E. London
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HIV-1 reverse transcriptase ,RT structural maturation ,maturation inhibitors ,RT polymerase domain ,ground state stabilization ,RT dimerization inhibitor ,Microbiology ,QR1-502 - Abstract
Formation of active HIV-1 reverse transcriptase (RT) proceeds via a structural maturation process that involves subdomain rearrangements and formation of an asymmetric p66/p66′ homodimer. These studies were undertaken to evaluate whether the information about this maturation process can be used to identify small molecule ligands that retard or interfere with the steps involved. We utilized the isolated polymerase domain, p51, rather than p66, since the initial subdomain rearrangements are largely limited to this domain. Target sites at subdomain interfaces were identified and computational analysis used to obtain an initial set of ligands for screening. Chromatographic evaluations of the p51 homodimer/monomer ratio support the feasibility of this approach. Ligands that bind near the interfaces and a ligand that binds directly to a region of the fingers subdomain involved in subunit interface formation were identified, and the interactions were further characterized by NMR spectroscopy and X-ray crystallography. Although these ligands were found to reduce dimer formation, further efforts will be required to obtain ligands with higher binding affinity. In contrast with previous ligand identification studies performed on the RT heterodimer, subunit interface surfaces are solvent-accessible in the p51 and p66 monomers, making these constructs preferable for identification of ligands that directly interfere with dimerization.
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- 2023
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5. Analysis of diverse double-strand break synapsis with Polλ reveals basis for unique substrate specificity in nonhomologous end-joining
- Author
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Andrea M. Kaminski, Kishore K. Chiruvella, Dale A. Ramsden, Katarzyna Bebenek, Thomas A. Kunkel, and Lars C. Pedersen
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Science - Abstract
Using X-ray crystallography and nonhomologous end-joining assays, this study reveals structural features within Polλ that provide it with the ability to bridge and stabilize tenuous DNA double-strand break ends, allowing for religation.
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- 2022
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6. Structural and ligand binding analysis of the pet allergens Can f 1 and Fel d 7
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Jungki Min, Alexander C. Y. Foo, Scott A. Gabel, Lalith Perera, Eugene F. DeRose, Anna Pomés, Lars C. Pedersen, and Geoffrey A. Mueller
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allergen ,dog ,cat ,lipocalin ,structure ,Immunologic diseases. Allergy ,RC581-607 - Abstract
IntroductionPet lipocalins are respiratory allergens with a central hydrophobic ligand-binding cavity called a calyx. Molecules carried in the calyx by allergens are suggested to influence allergenicity, but little is known about the native ligands.MethodsTo provide more information on prospective ligands, we report crystal structures, NMR, molecular dynamics, and florescence studies of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7.ResultsStructural comparisons with reported lipocalins revealed that Can f 1 and Fel d 7 calyxes are open and positively charged while other dog lipocalin allergens are closed and negatively charged. We screened fatty acids as surrogate ligands, and found that Can f 1 and Fel d 7 bind multiple ligands with preferences for palmitic acid (16:0) among saturated fatty acids and oleic acid (18:1 cis-9) among unsaturated ones. NMR analysis of methyl probes reveals that conformational changes occur upon binding of pinolenic acid inside the calyx. Molecular dynamics simulation shows that the carboxylic group of fatty acids shuttles between two positively charged amino acids inside the Can f 1 and Fel d 7 calyx. Consistent with simulations, the stoichiometry of oleic acid-binding is 2:1 (fatty acid: protein) for Can f 1 and Fel d 7.DiscussionThe results provide valuable insights into the determinants of selectivity and candidate ligands for pet lipocalin allergens Can f 1 and Fel d 7.
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- 2023
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7. Immunotherapy-induced neutralizing antibodies disrupt allergen binding and sustain allergen tolerance in peanut allergy
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Nicole A. LaHood, Jungki Min, Tarun Keswani, Crystal M. Richardson, Kwasi Amoako, Jingjia Zhou, Orlee Marini-Rapoport, Hervé Bernard, Stéphane Hazebrouck, Wayne G. Shreffler, J. Christopher Love, Anna Pomes, Lars C. Pedersen, Geoffrey A. Mueller, and Sarita U. Patil
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Immunology ,Medicine - Abstract
In IgE-mediated food allergies, exposure to the allergen activates systemic allergic responses. Oral immunotherapy (OIT) treats food allergies through incremental increases in oral allergen exposure. However, OIT only induces sustained clinical tolerance and decreased basophil sensitivity in a subset of individuals despite increases in circulating allergen-specific IgG in all treated individuals. Therefore, we examined the allergen-specific antibodies from 2 OIT cohorts of patients with sustained and transient responses. Here, we compared antibodies from individuals with sustained or transient responses and discovered specific tolerance-associated conformational epitopes of the immunodominant allergen Ara h 2 recognized by neutralizing antibodies. First, we identified what we believe to be previously unknown conformational, intrahelical epitopes using x-ray crystallography with recombinant antibodies. We then identified epitopes only recognized in sustained tolerance. Finally, antibodies recognizing tolerance-associated epitopes effectively neutralized allergen to suppress IgE-mediated effector cell activation. Our results demonstrate the molecular basis of antibody-mediated protection in IgE-mediated food allergy, by defining how these antibodies disrupt IgE-allergen interactions to prevent allergic reactions. Our approach to studying the structural and functional basis for neutralizing antibodies demonstrates the clinical relevance of specific antibody clones in antibody-mediated tolerance. We anticipate that our findings will form the foundation for treatments of peanut allergy using neutralizing antibodies and hypoallergens.
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- 2023
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8. Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break
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Andrea M. Kaminski, John M. Pryor, Dale A. Ramsden, Thomas A. Kunkel, Lars C. Pedersen, and Katarzyna Bebenek
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Science - Abstract
Polymerase μ (Polμ) participates in the repair of DNA double-strand breaks (DSBs) via the nonhomologous end-joining (NHEJ) pathway. Here, the authors determine the crystal structure of a pre-catalytic ternary complex of human Polμ with a bound DSB substrate and they obtain further mechanistic insights by allowing the insertion reaction to proceed in crystallo, which enabled them to determine a Polμ structure with incomplete incorporation and the structure of the post-catalytic nicked state.
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- 2020
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9. Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis
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Andrea M. Kaminski, Percy P. Tumbale, Matthew J. Schellenberg, R. Scott Williams, Jason G. Williams, Thomas A. Kunkel, Lars C. Pedersen, and Katarzyna Bebenek
- Subjects
Science - Abstract
DNA Ligase IV (LigIV) catalyzes nick sealing of DNA double-strand break substrates during non-homologous end-joining. Here the authors present the crystal structures of two human LigIV DNA-bound catalytic states, which provide insights into its catalytic mechanism and the molecular basis of LIG4 syndrome causing disease mutations.
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- 2018
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10. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action
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Joonas A. Jamsen, William A. Beard, Lars C. Pedersen, David D. Shock, Andrea F. Moon, Juno M. Krahn, Katarzyna Bebenek, Thomas A. Kunkel, and Samuel H. Wilson
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Science - Abstract
DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.
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- 2017
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11. Increased 3- O -sulfated heparan sulfate in Alzheimer’s disease brain is associated with genetic risk gene HS3ST1
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Zhangjie Wang, Vaishali N. Patel, Xuehong Song, Yongmei Xu, Andrea M. Kaminski, Vivien Uyen Doan, Guowei Su, Yien Liao, Dylan Mah, Fuming Zhang, Vijayakanth Pagadala, Chunyu Wang, Lars C. Pedersen, Lianchun Wang, Matthew P. Hoffman, Marla Gearing, and Jian Liu
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Multidisciplinary - Abstract
HS3ST1 is a genetic risk gene associated with Alzheimer’s disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3- O -sulfated HS displayed sevenfold increase in the AD group ( n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3- O -sulfated HS is made by 3- O -sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3- O -sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3- O -sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.
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- 2023
12. Emerging chemical and biochemical tools for studying 3-O-sulfated heparan sulfate
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Jian Liu and Lars C. Pedersen
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carbohydrates (lipids) ,Sulfates ,Tandem Mass Spectrometry ,Physiology ,Protein Isoforms ,Review ,Heparitin Sulfate ,Cell Biology ,Sulfotransferases ,Chromatography, Liquid - Abstract
Heparan sulfate is a widely expressed polysaccharide in the extracellular matrix and on the cell surface. 3- O-sulfated heparan sulfate represents only a small percentage of heparan sulfate from biological sources. However, this subpopulation is closely associated with biological functions of heparan sulfate. The 3- O-sulfated heparan sulfate is biosynthesized by heparan sulfate 3- O-sulfotransferase, which exists in seven different isoforms. This review article summarizes the recent progress in the substrate specificity studies of different 3- O-sulfotransferase isoforms involving the use of homogeneous oligosaccharide substrates and crystal structural analysis. The article also reviews a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to analyze the level of 3- O-sulfated heparan sulfate with high sensitivity and quantitative capability. This newly emerged technology will provide new tools to study the structure and function relationship of heparan sulfate.
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- 2022
13. Structural and Substrate Specificity Analysis of 3-O-Sulfotransferase Isoform 5 to Synthesize Heparan Sulfate
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Eduardo Stancanelli, Vijayakanth Pagadala, Truong Quang Pham, Juno M. Krahn, Andrea M. Kaminski, Rylee Wander, Yongmei Xu, Lars C. Pedersen, Jian Liu, Zhangjie Wang, and Jine Li
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Gene isoform ,chemistry.chemical_classification ,Sulfotransferase ,Chemistry ,General Chemistry ,Heparan sulfate ,Oligosaccharide ,Article ,Catalysis ,carbohydrates (lipids) ,chemistry.chemical_compound ,Biochemistry ,Glucosamine ,Substrate specificity ,Heparan Sulfate Biosynthesis - Abstract
Heparan sulfate 3-O-sulfotransferase (3-OST) transfers a sulfo group to the 3-OH position of a glucosamine saccharide unit to form 3-O-sulfated heparan sulfate. 3-O-sulfation is known to be critically important for bestowing anticoagulant activity and other biological functions of heparan sulfate. Here, we report two ternary crystal structures of 3-OST-5 with PAP (3’-phosphoadenosine 5’-phosphate) and two octasaccharide substrates. We also used 3-OST-5 to synthesize six 3-O-sulfated 8-mers. Results from the structural analysis of the six 3-O-sulfated 8-mers revealed the substrate specificity of 3-OST-5. The enzyme prefers to sulfate a 6-O-sulfo glucosamine saccharide that is surrounded by glucuronic acid over a 6-O-sulfo glucosamine saccharide that is surrounded by 2-O-sulfated iduronic acid. 3-OST-5 modified 8-mers display a broad range of anti-factor Xa activity, depending on the structure of the 8-mer. We also discovered that the substrate specificity of 3-OST-5 is not governed solely by the side chains from amino acid residues in the active site. The conformational flexibility of the 2-O-sulfated iduronic acid in the saccharide substrates also contributes to the substrate specificity. These findings advance our understanding for how to control the biosynthesis of 3-O-sulfated heparan sulfate with desired biological activities.
- Published
- 2021
14. A genome-wide CRISPR-Cas9 knockout screen reveals FSP1 as warfarin-resistant vitamin K reductase
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Da-Yun Jin, Xuejie Chen, Yizhou Liu, Craig M. Williams, Lars C. Pedersen, Darrel W. Stafford, and Jian-Ke Tie
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Vitamin K is a vital micronutrient implicated in a variety of human diseases1. Warfarin, a vitamin K antagonist, is the most commonly prescribed oral anticoagulant2. Warfarin exerts its effect by inhibiting vitamin K epoxide reductase (VKOR) leading to the depletion of reduced vitamin K, a cofactor of gamma-glutamyl carboxylase (GGCX) required for the post-translational modification of proteins with diverse functions. Patients overdosed on warfarin can be rescued by administering high doses of vitamin K because of the existence of a warfarin-resistant vitamin K reductase (VKR)3,4. Despite the functional discovery of VKR over eight decades ago, the identity of VKR remained elusive. Here, we report the identification of warfarin-resistant VKR using a genome-wide CRISPR-Cas9 knockout screening with a novel vitamin K-dependent (VKD) apoptotic reporter cell line. We found that ferroptosis suppressor protein 1 (FSP1) is responsible for vitamin K reduction in a warfarin-resistant manner. Knocking out FSP1 in HEK293 cells dramatically decreased VKR activity, while HEK293 cells overexpressing FSP1 exhibited robust VKR activity that is resistant to warfarin inhibition. Inhibitor of FSP1 that inhibited ubiquinone reduction and thus triggering cancer cell ferroptosis, displayed strong inhibition of VKD carboxylation. Intriguingly, dihydroorotate dehydrogenase (DHODH), another ubiquinone-associated ferroptosis suppressor protein parallel to the function of FSP15, does not support VKD carboxylation and its inhibitors have no effect on carboxylation. These findings provide new insights into selectively controlling the physiological and pathological processes involving electron transfers mediated by vitamin K and ubiquinone.
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- 2022
15. Deciphering the substrate recognition mechanisms of the heparan sulfate 3-O-sulfotransferase-3
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Andrea M. Kaminski, Juno M. Krahn, Lars C. Pedersen, Vijayakanth Pagadala, Jian Liu, Rylee Wander, Truong Quang Pham, and Yongmei Xu
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0303 health sciences ,Sulfotransferase ,biology ,010405 organic chemistry ,Substrate (chemistry) ,Heparan sulfate ,Plasma protein binding ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,Enzyme assay ,0104 chemical sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Sulfation ,chemistry ,Chemistry (miscellaneous) ,Product inhibition ,Glucosamine ,biology.protein ,Molecular Biology ,030304 developmental biology - Abstract
The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.
- Published
- 2021
16. The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase
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Lars C. Pedersen, Robert E. London, Elizabeth E. Howell, Scott A. Gabel, Juno M. Krahn, Michael R. Duff, and Eugene F. DeRose
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Models, Molecular ,Drug ,medicine.drug_class ,media_common.quotation_subject ,Diflunisal ,Pharmacology ,Crystallography, X-Ray ,01 natural sciences ,Article ,Anti-inflammatory ,03 medical and health sciences ,Folic Acid ,Drug Discovery ,Dihydrofolate reductase ,medicine ,Humans ,Moiety ,Potency ,030304 developmental biology ,media_common ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,Chemistry ,Anti-Inflammatory Agents, Non-Steroidal ,0104 chemical sciences ,Tetrahydrofolate Dehydrogenase ,010404 medicinal & biomolecular chemistry ,Enzyme ,Drug Design ,biology.protein ,Folic Acid Antagonists ,Molecular Medicine ,Cyclooxygenase ,medicine.drug - Abstract
Although nonsteroidal anti-inflammatory drugs (NSAIDs) target primarily cyclooxygenase enzymes, a subset of NSAIDs containing carboxylate groups also has been reported to competitively inhibit dihydrofolate reductase (DHFR). In this study, we have characterized NSAID interactions with human DHFR based on kinetic, NMR, and X-ray crystallographic methods. The NSAIDs target a region of the folate binding site that interacts with the p-aminobenzoyl-l-glutamate (pABG) moiety of folate and inhibit cooperatively with ligands that target the adjacent pteridine-recognition subsite. NSAIDs containing benzoate or salicylate groups were identified as having the highest potency. Among those tested, diflunisal, a salicylate derivative not previously identified to have anti-folate activity, was found to have a Ki of 34 μM, well below peak plasma diflunisal levels reached at typical dosage levels. The potential of these drugs to interfere with the inflammatory process by multiple pathways introduces the possibility of further optimization to design dual-targeted analogs.
- Published
- 2020
17. Using engineered 6-O-sulfotransferase to improve the synthesis of anticoagulant heparin
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Genmin Lu, Andrea M Kaminski, Maurice Horton, Zhangjie Wang, Yongmei Xu, Vijayakanth Pagadala, Xiaobing Chang, Lars C. Pedersen, Lin Yi, Pamela Conley, Zhenqing Zhang, Guowei Su, and Jian Liu
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chemistry.chemical_classification ,0303 health sciences ,Sulfotransferase ,Anticoagulant drug ,medicine.drug_class ,Organic Chemistry ,Anticoagulant ,Heparan sulfate ,Heparin ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,03 medical and health sciences ,Residue (chemistry) ,chemistry.chemical_compound ,Enzyme ,chemistry ,Glucosamine ,medicine ,Physical and Theoretical Chemistry ,030304 developmental biology ,medicine.drug - Abstract
Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of glucosamine to synthesize HS with desired biological activities. Chemoenzymatic synthesis is a widely adopted method to obtain HS oligosaccharides to support biological studies. However, this method is unable to synthesize all possible structures due to the specificity of natural enzymes. Here, we report the use of an engineered 6-OST to achieve fine control of the 6-O-sulfation. Unlike wild type enzyme, the engineered 6-OST only sulfates the non-reducing end glucosamine residue. Utilizing the engineered enzyme and wild type enzyme, we successfully completed the synthesis of five hexasaccharides and one octasaccharide differing in 6-O-sulfation patterns. We also identified a hexasaccharide construct as a new anticoagulant drug candidate. Our results demonstrate the feasibility of using an engineered HS biosynthetic enzyme to prepare HS-based therapeutics.
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- 2020
18. Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Family X DNA Polymerases
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Andrea M. Kaminski, Thomas A. Kunkel, Lars C. Pedersen, and Katarzyna Bebenek
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DNA Replication ,8-oxo-guanine (8OG) ,DNA Repair ,Family X polymerases ,Review ,DNA ,DNA-Directed DNA Polymerase ,QH426-470 ,base excision repair (BER) ,nonhomologous end-joining (NHEJ) ,oxidized base damage ,8-Hydroxy-2'-Deoxyguanosine ,Catalytic Domain ,Genetics ,Humans ,Genetics (clinical) - Abstract
8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols β, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles—the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.
- Published
- 2021
19. Analysis of diverse double-strand break synapsis with Polλ reveals basis for unique substrate specificity in nonhomologous end-joining
- Author
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Andrea M. Kaminski, Kishore K. Chiruvella, Dale A. Ramsden, Katarzyna Bebenek, Thomas A. Kunkel, and Lars C. Pedersen
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Chromosome Pairing ,Multidisciplinary ,DNA End-Joining Repair ,Synapses ,General Physics and Astronomy ,DNA Breaks, Double-Stranded ,General Chemistry ,Nucleotidyltransferases ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity - Abstract
DNA double-strand breaks (DSBs) threaten genomic stability, since their persistence can lead to loss of critical genetic information, chromosomal translocations or rearrangements, and cell death. DSBs can be repaired through the nonhomologous end-joining pathway (NHEJ), which processes and ligates DNA ends efficiently to prevent or minimize sequence loss. Polymerase λ (Polλ), one of the Family X polymerases, fills sequence gaps of DSB substrates with a strict specificity for a base-paired primer terminus. There is little information regarding Polλ’s approach to engaging such substrates. We used in vitro polymerization and cell-based NHEJ assays to explore the contributions of conserved loop regions toward DSB substrate specificity and utilization. In addition, we present multiple crystal structures of Polλ in synapsis with varying biologically relevant DSB end configurations, revealing how key structural features and hydrogen bonding networks work in concert to stabilize these tenuous, potentially cytotoxic DNA lesions during NHEJ.
- Published
- 2021
20. Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine
- Author
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Katarzyna Bebenek, Andrea M. Kaminski, Kishore K. Chiruvella, Lars C. Pedersen, Thomas A. Kunkel, and Dale A. Ramsden
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DNA Replication ,DNA End-Joining Repair ,Ultraviolet Rays ,Stereochemistry ,Guanosine ,DNA-Directed DNA Polymerase ,DNA Ligase ATP ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Structural Biology ,Radiation, Ionizing ,Genetics ,Humans ,Transferase ,DNA Breaks, Double-Stranded ,Nucleotide ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,DNA ligase ,biology ,030302 biochemistry & molecular biology ,Active site ,chemistry ,Mutagenesis ,biology.protein ,Reactive Oxygen Species ,DNA polymerase mu ,DNA ,DNA Damage - Abstract
DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.
- Published
- 2019
21. Deciphering the substrate recognition mechanisms of the heparan sulfate 3
- Author
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Rylee, Wander, Andrea M, Kaminski, Yongmei, Xu, Vijayakanth, Pagadala, Juno M, Krahn, Truong Quang, Pham, Jian, Liu, and Lars C, Pedersen
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Chemistry - Abstract
The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3′-phosphoadenosine 5′-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes., Co-crystallization and biochemical analyses with structurally defined oligosaccharides show the low reactivity of HS 3-OST-3 toward 6-O-sulfated substrates is due to inhibition of enzyme activity by 6-O-sulfated oligosaccharides.
- Published
- 2021
22. The mosquito protein AEG12 displays both cytolytic and antiviral properties via a common lipid transfer mechanism
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Eugene F. DeRose, Simrat Arora, Peter M. Thompson, Lars C. Pedersen, Brianna Lupo, Negin P. Martin, Victoria C. Placentra, Geoffrey A. Mueller, Lalith Perera, Alexander C. Y. Foo, Shih Heng Chen, Ramesh Jadi, and Lakshmanane Premkumar
- Subjects
0301 basic medicine ,Erythrocytes ,viruses ,Ligands ,Antiviral Agents ,Virus ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Viral envelope ,Animals ,Humans ,Lipid bilayer ,Unsaturated fatty acid ,Binding selectivity ,Multidisciplinary ,biology ,Chemistry ,Hemolytic Agents ,Cell Membrane ,Biological Sciences ,biology.organism_classification ,Lipids ,Cell biology ,Protein Structure, Tertiary ,Flavivirus ,030104 developmental biology ,Culicidae ,Viral Envelope ,Lentivirus ,Viruses ,Fatty Acids, Unsaturated ,Insect Proteins ,Hydrophobic and Hydrophilic Interactions ,030217 neurology & neurosurgery ,Function (biology) ,Protein Binding - Abstract
The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.
- Published
- 2021
23. DNA polymerase mu: An inflexible scaffold for substrate flexibility
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Lars C. Pedersen, Katarzyna Bebenek, Andrea M. Kaminski, and Thomas A. Kunkel
- Subjects
DNA End-Joining Repair ,DNA polymerase ,DNA-Directed DNA Polymerase ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,DNA Ligase ATP ,0302 clinical medicine ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,DNA ligase ,biology ,fungi ,Substrate (chemistry) ,Cell Biology ,DNA ,Non-homologous end joining ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Biophysics ,DNA polymerase mu ,Recombination - Abstract
DNA polymerase μ is a Family X member that participates in repair of DNA double strand breaks (DSBs) by non-homologous end joining. Its role is to fill short gaps arising as intermediates in the process of V(D)J recombination and during processing of accidental double strand breaks. Pol μ is the only known template-dependent polymerase that can repair non-complementary DSBs with unpaired 3´primer termini. Here we review the unique properties of Pol μ that allow it to productively engage such a highly unstable substrate to generate a nick that can be sealed by DNA Ligase IV.
- Published
- 2020
24. Using engineered 6
- Author
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Lin, Yi, Yongmei, Xu, Andrea M, Kaminski, Xiaobing, Chang, Vijayakanth, Pagadala, Maurice, Horton, Guowei, Su, Zhangjie, Wang, Genmin, Lu, Pamela, Conley, Zhenqing, Zhang, Lars C, Pedersen, and Jian, Liu
- Subjects
Sulfotransferases ,Article - Abstract
Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of glucosamine to synthesize HS with desired biological activities. Chemoenzymatic synthesis is a widely adopted method to obtain HS oligosaccharides to support biological studies. However, this method is unable to synthesize all possible structures due to the specificity of natural enzymes. Here, we report the use of an engineered 6-OST to achieve fine control of the 6-O-sulfation. Unlike wild type enzyme, the engineered 6-OST only sulfates the non-reducing end glucosamine residue. Utilizing the engineered enzyme and wild type enzyme, we successfully completed the synthesis of five hexasaccharides and one octasaccharide differing in 6-O-sulfation patterns. We also identified a hexasaccharide construct as a new anticoagulant drug candidate. Our results demonstrate the feasibility of using an engineered HS biosynthetic enzyme to prepare HS-based therapeutics.
- Published
- 2020
25. Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break
- Author
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Katarzyna Bebenek, Andrea M. Kaminski, John M. Pryor, Thomas A. Kunkel, Dale A. Ramsden, and Lars C. Pedersen
- Subjects
Models, Molecular ,0301 basic medicine ,DNA End-Joining Repair ,DNA Repair ,Protein Conformation ,DNA damage ,DNA repair ,Science ,General Physics and Astronomy ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,Article ,General Biochemistry, Genetics and Molecular Biology ,DNA Ligase ATP ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Humans ,DNA Breaks, Double-Stranded ,lcsh:Science ,Polymerase ,X-ray crystallography ,chemistry.chemical_classification ,DNA ligase ,Multidisciplinary ,biology ,Synapsis ,Hydrogen Bonding ,DNA ,General Chemistry ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,chemistry ,Coding strand ,biology.protein ,lcsh:Q ,030217 neurology & neurosurgery ,DNA Damage - Abstract
Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks., Polymerase μ (Polμ) participates in the repair of DNA double-strand breaks (DSBs) via the nonhomologous end-joining (NHEJ) pathway. Here, the authors determine the crystal structure of a pre-catalytic ternary complex of human Polμ with a bound DSB substrate and they obtain further mechanistic insights by allowing the insertion reaction to proceed in crystallo, which enabled them to determine a Polμ structure with incomplete incorporation and the structure of the post-catalytic nicked state.
- Published
- 2020
26. Variations in nuclear localization strategies among pol X family enzymes
- Author
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Natalie R. Gassman, Lars C. Pedersen, Thomas W. Kirby, Robert E. London, and Scott A. Gabel
- Subjects
0301 basic medicine ,030102 biochemistry & molecular biology ,biology ,Chemistry ,DNA repair ,DNA polymerase ,viruses ,Cell Biology ,Importin ,Biochemistry ,Article ,DNA polymerase lambda ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Structural Biology ,Genetics ,biology.protein ,NLS ,Molecular Biology ,DNA polymerase mu ,Polymerase ,Nuclear localization sequence ,Biotechnology - Abstract
Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol μ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.
- Published
- 2018
27. Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications
- Author
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Lars C. Pedersen, Thomas W. Kirby, Robert E. London, Kyungmin Kim, and Eugene F. DeRose
- Subjects
0301 basic medicine ,Models, Molecular ,Phosphopeptides ,Plasma protein binding ,Biology ,Genome Integrity, Repair and Replication ,03 medical and health sciences ,XRCC1 ,chemistry.chemical_compound ,Genetics ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Protein Interaction Domains and Motifs ,Binding site ,Casein Kinase II ,Poly-ADP-Ribose Binding Proteins ,Aprataxin ,Binding Sites ,030102 biochemistry & molecular biology ,Phosphopeptide ,DNA repair protein XRCC4 ,Hydrogen-Ion Concentration ,030104 developmental biology ,Phosphothreonine ,X-ray Repair Cross Complementing Protein 1 ,chemistry ,Phosphoserine ,Biophysics ,Protein Binding - Abstract
Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA–FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors.
- Published
- 2017
28. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action
- Author
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Lars C. Pedersen, Joonas A. Jamsen, David D. Shock, Andrea F. Moon, Samuel H. Wilson, William A. Beard, Katarzyna Bebenek, Juno M. Krahn, and Thomas A. Kunkel
- Subjects
0301 basic medicine ,DNA Replication ,Models, Molecular ,DNA Repair ,DNA polymerase ,DNA repair ,Science ,General Physics and Astronomy ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Catalytic Domain ,DNA Breaks, Double-Stranded ,Polymerase ,Multidisciplinary ,DNA clamp ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Nucleotides ,DNA replication ,General Chemistry ,Processivity ,DNA ,Double Strand Break Repair ,Crystallography ,Kinetics ,030104 developmental biology ,biology.protein - Abstract
DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5′-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance., DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.
- Published
- 2017
29. Activation-induced deoxycytidine deaminase: Structural basis for favoring WRC hot motif specificities unique among APOBEC family members
- Author
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Kazuhiko Maeda, Phuong Pham, Samir A. Afif, Nobuo Sakaguchi, Myron F. Goodman, Mayuko Shimoda, and Lars C. Pedersen
- Subjects
Models, Molecular ,0301 basic medicine ,Genetics ,Spectrum Analysis ,Antibody Diversity ,Cell Biology ,Biology ,Biochemistry ,Article ,Deoxycytidine deaminase ,Structure-Activity Relationship ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Multigene Family ,030220 oncology & carcinogenesis ,Mutation ,Animals ,Humans ,Protein Interaction Domains and Motifs ,APOBEC Family ,APOBEC Deaminases ,Motif (music) ,Molecular Biology - Published
- 2017
30. Structure Based Substrate Specificity Analysis of Heparan Sulfate 6-O-Sulfotransferases
- Author
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Yongmei Xu, Jian Liu, Juno M. Krahn, Andrea F. Moon, Lars C. Pedersen, and Shuqin Xu
- Subjects
Models, Molecular ,0301 basic medicine ,Protein Conformation ,Stereochemistry ,Oligosaccharides ,Sequence alignment ,Crystallography, X-Ray ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Sulfation ,Protein structure ,Glucosamine ,Catalytic Domain ,Animals ,Humans ,Protein Isoforms ,Transferase ,Amino Acid Sequence ,Ternary complex ,Peptide sequence ,Zebrafish ,030102 biochemistry & molecular biology ,fungi ,General Medicine ,Heparan sulfate ,Zebrafish Proteins ,Adenosine Diphosphate ,030104 developmental biology ,chemistry ,Molecular Medicine ,Sulfotransferases ,Sequence Alignment - Abstract
Heparan sulfate (HS) is a sulfated polysaccharide exhibiting essential physiological functions. HS 6-O-sulfotransferase (6-OST) transfers a sulfo group to the 6-OH position of glucosamine units to confer a variety of HS biological activities. There are three different isoforms of 6-OST in the human genome. Here, we report crystal structures of the ternary complex of 6-OST with the sulfo donor analog 3'-phosphoadenosine 5'-phosphate and three different oligosaccharide substrates at 1.95 to 2.1 Å resolutions. Structural and mutational analyses reveal amino acid residues that contribute to catalysis and substrate recognition of 6-OST. Unexpectedly, the structures reveal 6-OST engages HS in a completely different orientation than other HS sulfotransferases and sheds light on the basic HS requirements for specificity. These findings also contribute structural information to understand mutations in human 6-OST isoform 1 associated with the human genetic disease idiopathic hypogonadotropic hypogonadism characterized by incomplete or lack of puberty.
- Published
- 2016
31. Ligand binding characteristics of the Ku80 von Willebrand domain
- Author
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Jungki Min, Kyungmin Kim, Robert E. London, Lars C. Pedersen, Thomas W. Kirby, and Scott A. Gabel
- Subjects
Models, Molecular ,Ku80 ,Werner Syndrome Helicase ,DNA repair ,Protein Conformation ,Xenopus ,Peptide ,Xenopus Proteins ,Ligands ,Biochemistry ,Article ,03 medical and health sciences ,Xenopus laevis ,0302 clinical medicine ,Retrovirus ,Protein Domains ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Animals ,Binding site ,Poly-ADP-Ribose Binding Proteins ,Molecular Biology ,Ku Autoantigen ,Conserved Sequence ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,Synapsis ,Cell Biology ,biology.organism_classification ,Cell biology ,chemistry ,030220 oncology & carcinogenesis ,Fluorescence anisotropy ,Protein Binding - Abstract
The N-terminal von Willebrand domain of Ku80 supports interactions with a Ku binding motif (KBM) that has been identified in at least three other DNA repair proteins: the non-homologous end joining (NHEJ) scaffold APLF, the modulator of retrovirus infection, MRI, and the Werner syndrome protein (WRN). A second, more recently identified Ku binding motif present in XLF and several other proteins (KBMX) has also been reported to interact with this domain. The isolated Ku80 von Willebrand antigen domain (vWA) from Xenopus laevis has a sequence that is 60% identical with the human domain, is readily expressed and has been used to investigate these interactions. Structural characterization of the complexes formed with the KBM motifs in human APLF, MRI, and WRN identify a conserved binding site that is consistent with previously-reported mutational studies. In contrast with the KBM binding site, structural studies indicate that the KBMX site is occluded by a distorted helix. Fluorescence polarization and 19F NMR studies of a fluorinated XLF C-terminal peptide failed to indicate any interaction with the frog vWA. It was hypothesized that availability of this binding site is conditional, i.e., dependent on specific experimental conditions or other repair factors to make the site available for binding. Modulating the fraction of KBMX-accessible binding site mutationally demonstrated that the more open site is capable of binding the KBMXXLF motif peptide. It is suggested that the conditional nature of KBMX binding limits formation of non-productive complexes so that activation-dependent site availability can more optimally support advancing the synapsis process.
- Published
- 2019
32. A ubiquitin-like domain is required for stabilizing the N-terminal ATPase module of human SMCHD1
- Author
-
Lars C. Pedersen, Susan Kim, Lalith Perera, Natalie D. Shaw, and Kaoru Inoue
- Subjects
Chromosomal Proteins, Non-Histone ,Hydrolases ,ATPase ,Dimer ,Mutant ,Medicine (miscellaneous) ,Repressor ,Epigenetic Repression ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Protein Structure, Secondary ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Adenosine Triphosphate ,Ubiquitin ,Protein Domains ,Catalytic Domain ,Hydrolase ,medicine ,Facioscapulohumeral muscular dystrophy ,Humans ,lcsh:QH301-705.5 ,030304 developmental biology ,X-ray crystallography ,Adenosine Triphosphatases ,0303 health sciences ,biology ,Chemistry ,Neuromuscular disease ,medicine.disease ,Chromatin ,Muscular Dystrophy, Facioscapulohumeral ,Cell biology ,Kinetics ,lcsh:Biology (General) ,Mutation ,biology.protein ,Protein Multimerization ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery - Abstract
Variants in the gene SMCHD1, which encodes an epigenetic repressor, have been linked to both congenital arhinia and a late-onset form of muscular dystrophy called facioscapulohumeral muscular dystrophy type 2 (FSHD2). This suggests that SMCHD1 has a diversity of functions in both developmental time and space. The C-terminal end of SMCHD1 contains an SMC-hinge domain which mediates homodimerization and chromatin association, whereas the molecular architecture of the N-terminal region, which harbors the GHKL-ATPase domain, is not well understood. We present the crystal structure of the human SMCHD1 N-terminal ATPase module bound to ATP as a functional dimer. The dimer is stabilized by a novel N-terminal ubiquitin-like fold and by a downstream transducer domain. While disease variants map to what appear to be critical interdomain/intermolecular interfaces, only the FSHD2-specific mutant constructs we tested consistently abolish ATPase activity and/or dimerization. These data suggest that the full functional profile of SMCHD1 has yet to be determined., Lars Pedersen, Kaoru Inoue et al. present the crystal structure of the N-terminal ATPase module of human SMCHD1 bound to ATP. They show that the dimer is stabilized by a ubiquitin-like fold at the N-terminal, as well as by a downstream transducer domain.
- Published
- 2019
33. Structural and functional consequences of SMCHD1 mutations associated with arhinia and muscular dystrophy
- Author
-
Susan Ok Kim, Natalie D. Shaw, Lalith Perera, Lars C. Pedersen, and Kaoru Inoue
- Subjects
Pathology ,medicine.medical_specialty ,business.industry ,Genetics ,medicine ,Muscular dystrophy ,medicine.disease ,business ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2019
34. Structural Analysis of Recent Allergen-Antibody Complexes and Future Directions
- Author
-
Alexander C. Y. Foo, Geoffrey A. Mueller, Lars C. Pedersen, Jungki Min, and Anna Pomés
- Subjects
Pulmonary and Respiratory Medicine ,biology ,Immunology ,Antigen-Antibody Complex ,Computational biology ,Allergens ,Immunoglobulin E ,Protein Structure, Secondary ,Article ,Long Variable ,Epitope ,Epitopes ,03 medical and health sciences ,0302 clinical medicine ,030228 respiratory system ,biology.protein ,Humans ,Immunology and Allergy ,Amino Acid Sequence ,Antibody ,030223 otorhinolaryngology ,Function (biology) - Abstract
PURPOSE OF REVIEW: Allergen-antibody complexes are extremely valuable in describing the detailed molecular features of epitopes. This review summarizes insights gained from recently published co-structures and what obstacles impede the acquisition of further data. RECENT FINDINGS: Structural epitope data helped define the epitopes of two anti-Fel d 1 antibodies undergoing phase I clinical trials, providing a greater level of detail than was possible through hydrogen exchange protection studies. Separately, a human camelid-like antibody structure with lysozyme described several unique features in a long variable loop that interacted with the active site cleft of Gal d 4. Finally, a co-structure conclusively demonstrated that Phl p 7 could function as a superantigen, and that an antibody could simultaneously recognize two epitopes. These remarkable assertions would not have been possible without visualization of the complex. Only 3 new complexes have appeared in the last few years, suggesting there are major impediments to traditional production and crystallization. SUMMARY: The structural data was extremely valuable in describing epitopes. New techniques like cryo-EM may provide an alternative to crystallography.
- Published
- 2019
35. Structural characterization of the virulence factor Sda1 nuclease fromStreptococcus pyogenes
- Author
-
Andrea F. Moon, Lars C. Pedersen, Xun Lu, Matthew J. Cuneo, and Juno M. Krahn
- Subjects
Models, Molecular ,0301 basic medicine ,Streptococcus pyogenes ,Virulence Factors ,Virulence ,Biology ,medicine.disease_cause ,Virulence factor ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Bacterial Proteins ,Protein Domains ,Structural Biology ,Genetics ,medicine ,Deoxyribonuclease I ,Nuclease ,Innate immune system ,Streptococcus ,Virology ,3. Good health ,030104 developmental biology ,biology.protein ,Protein Multimerization ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
Infection by Group A Streptococcus pyogenes (GAS) is a leading cause of severe invasive disease in humans, including streptococcal toxic shock syndrome and necrotizing fasciitis. GAS infections lead to nearly 163,000 annual deaths worldwide. Hypervirulent strains of S. pyogenes have evolved a plethora of virulence factors that aid in disease-by promoting bacterial adhesion to host cells, subsequent invasion of deeper tissues and blocking the immune system's attempts to eradicate the infection. Expression and secretion of the extracellular nuclease Sda1 is advantageous for promoting bacterial dissemination throughout the host organism, and evasion of the host's innate immune response. Here we present two crystal structures of Sda1, as well as biochemical studies to address key structural features and surface residues involved in DNA binding and catalysis. In the active site, Asn211 is observed to directly chelate a hydrated divalent metal ion and Arg124, on the putative substrate binding loop, likely stabilizes the transition state during phosphodiester bond cleavage. These structures provide a foundation for rational drug design of small molecule inhibitors to be used in prevention of invasive streptococcal disease.
- Published
- 2016
36. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war
- Author
-
Robert E. London, Eugene F. DeRose, Geoffrey A. Mueller, Lars C. Pedersen, Xunhai Zheng, and Scott A. Gabel
- Subjects
0301 basic medicine ,Protein Conformation ,Dimer ,Protein subunit ,Ribonuclease H ,Cleavage (embryo) ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Catalytic Domain ,Hydrolase ,Genetics ,Humans ,Binding site ,RNase H ,Polymerase ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Nucleic Acid Enzymes ,Molecular biology ,HIV Reverse Transcriptase ,Protein Structure, Tertiary ,3. Good health ,030104 developmental biology ,chemistry ,HIV-1 ,biology.protein ,Biophysics ,Reverse Transcriptase Inhibitors ,Dimerization - Abstract
Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. This study demonstrates the feasibility of directly targeting RT maturation with therapeutics.
- Published
- 2016
37. Erratum: 'Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity'
- Author
-
John E. French, Daniel M. Gatti, Daniel L. Morgan, Grace E. Kissling, Keith R. Shockley, Gabriel A. Knudsen, Kim G. Shepard, Herman C. Price, Deborah King, Kristine L. Witt, Lars C. Pedersen, Steven C. Munger, Karen L. Svenson, and Gary A. Churchill
- Subjects
Inhalation Exposure ,Micronucleus Tests ,Reticulocytes ,Dose-Response Relationship, Drug ,Genetic Linkage ,Health, Toxicology and Mutagenesis ,Public Health, Environmental and Occupational Health ,Benzene ,Bone Marrow Cells ,Risk Assessment ,Hazardous Substances ,Mice ,Animals, Outbred Strains ,Animals ,Erratum ,Sulfotransferases ,DNA Damage - Abstract
Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers.We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity.We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis.We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity.The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.
- Published
- 2018
38. Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis
- Author
-
Katarzyna Bebenek, Thomas A. Kunkel, Matthew J. Schellenberg, Percy P. Tumbale, R. Scott Williams, Lars C. Pedersen, Jason Williams, and Andrea M. Kaminski
- Subjects
0301 basic medicine ,Immunoglobulin gene ,Science ,LIG4 syndrome ,General Physics and Astronomy ,General Biochemistry, Genetics and Molecular Biology ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,DNA Ligase ATP ,Catalytic Domain ,medicine ,Humans ,lcsh:Science ,chemistry.chemical_classification ,DNA ligase ,Multidisciplinary ,Polymorphism, Genetic ,030102 biochemistry & molecular biology ,biology ,Base Sequence ,Chemistry ,Adenine ,Lysine ,Mutagenesis ,Active site ,General Chemistry ,DNA ,medicine.disease ,Cell biology ,030104 developmental biology ,Mutation ,biology.protein ,Nucleic acid ,Biocatalysis ,lcsh:Q ,Function (biology) ,Protein Binding - Abstract
DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct states—an open lysyl-AMP intermediate, and a closed DNA–adenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome., DNA Ligase IV (LigIV) catalyzes nick sealing of DNA double-strand break substrates during non-homologous end-joining. Here the authors present the crystal structures of two human LigIV DNA-bound catalytic states, which provide insights into its catalytic mechanism and the molecular basis of LIG4 syndrome causing disease mutations.
- Published
- 2017
39. Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation
- Author
-
Masahiko Negishi, Mack Sobhany, Lars C. Pedersen, Shingo Mutoh, Ryota Shizu, and Jungki Min
- Subjects
0301 basic medicine ,Receptors, Cytoplasmic and Nuclear ,Retinoid X receptor ,Ligands ,Biochemistry ,Protein–protein interaction ,Cell Line ,Dephosphorylation ,03 medical and health sciences ,Protein Domains ,Humans ,Protein Phosphatase 2 ,Nuclear protein ,Phosphorylation ,Molecular Biology ,Constitutive Androstane Receptor ,Retinoid X Receptor alpha ,Chemistry ,DNA-binding domain ,Cell Biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,030104 developmental biology ,Retinoid X Receptors ,Nuclear receptor ,Biophysics ,human activities ,Dimerization ,hormones, hormone substitutes, and hormone antagonists ,Binding domain ,Protein Binding - Abstract
The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer–heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD–LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D–LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD–LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.
- Published
- 2017
40. Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate
- Author
-
Vijayakanth Pagadala, Jian Liu, Wenfang Dou, Lars C. Pedersen, and Yongmei Xu
- Subjects
chemistry.chemical_classification ,Sulfotransferase ,biology ,Chemistry ,Molecular Sequence Data ,Glycobiology and Extracellular Matrices ,Cell Biology ,Heparan sulfate ,Biochemistry ,Substrate Specificity ,carbohydrates (lipids) ,chemistry.chemical_compound ,Sulfation ,Enzyme ,Carbohydrate Sequence ,Tandem Mass Spectrometry ,Glucosamine ,Glycosyltransferase ,biology.protein ,Phosphofructokinase 2 ,Heparitin Sulfate ,Sulfotransferases ,Molecular Biology ,Deacetylase activity - Abstract
Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.
- Published
- 2015
41. Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity
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Gabriel A. Knudsen, Keith R. Shockley, John E. French, Herman C. Price, Kristine L. Witt, Gary A. Churchill, Karen L. Svenson, Deborah King, Grace E. Kissling, Steven C. Munger, Lars C. Pedersen, Daniel L. Morgan, Kim G. Shepard, and Daniel M. Gatti
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Inhalation exposure ,0303 health sciences ,Extramural ,Health, Toxicology and Mutagenesis ,Research ,Public Health, Environmental and Occupational Health ,Population based ,Biology ,medicine.disease_cause ,3. Good health ,03 medical and health sciences ,chemistry.chemical_compound ,Dose–response relationship ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Immunology ,Micronucleus test ,Benzene toxicity ,medicine ,Benzene ,human activities ,Genotoxicity ,030304 developmental biology - Abstract
Background Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. Objective We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. Methods We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. Results We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. Conclusions The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity. Citation French JE, Gatti DM, Morgan DL, Kissling GE, Shockley KR, Knudsen GA, Shepard KG, Price HC, King D, Witt KL, Pedersen LC, Munger SC, Svenson KL, Churchill GA. 2015. Diversity Outbred mice identify population-based exposure thresholds and genetic factors that influence benzene-induced genotoxicity. Environ Health Perspect 123:237–245; http://dx.doi.org/10.1289/ehp.1408202
- Published
- 2014
42. Probing Dominant Negative Behavior of Glucocorticoid Receptor β through a Hybrid Structural and Biochemical Approach
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John A. Cidlowski, Andrea F. Moon, Christine M. Jewell, Lalith Perera, Lars C. Pedersen, Juno M. Krahn, and Jungki Min
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0301 basic medicine ,chemistry.chemical_classification ,Antagonist ,Peptide ,Cell Biology ,Biology ,Cell biology ,Amino acid ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Glucocorticoid receptor ,Receptors, Glucocorticoid ,chemistry ,Transcription (biology) ,Hormone receptor ,030220 oncology & carcinogenesis ,Escherichia coli ,Humans ,Amino Acid Sequence ,Molecular Biology ,Corepressor ,Glucocorticoids ,Transrepression ,Research Article - Abstract
Glucocorticoid receptor β (GRβ) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRβ functions as a dominant negative inhibitor of GRα at a molecular level, we determined the crystal structure of the ligand binding domain of GRβ complexed with the antagonist RU-486. The structure reveals that GRβ binds RU-486 in the same ligand binding pocket as GRα, and the unique C-terminal amino acids of GRβ are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRβ do not contribute to RU-486 binding. Intriguingly, the GRβ/RU-486 complex binds corepressor peptide with affinity similar to that of a GRα/RU-486 complex, despite the lack of helix 12. Our biophysical and biochemical analyses reveal that in the presence of RU-486, GRβ is found in a conformation that favors corepressor binding, potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRβ could repress transcription.
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- 2017
43. A Structural Basis for Biguanide Activity
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Michael R. Duff, Eugene F. DeRose, Lars C. Pedersen, Scott A. Gabel, Elizabeth E. Howell, Robert E. London, and Juno M. Krahn
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0301 basic medicine ,Models, Molecular ,medicine.drug_class ,Protein Conformation ,Biguanides ,Pharmacology ,Phenformin ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,0302 clinical medicine ,Oxidoreductase ,Dihydrofolate reductase ,medicine ,Escherichia coli ,Structure–activity relationship ,Hypoglycemic Agents ,Binding site ,Buformin ,chemistry.chemical_classification ,Binding Sites ,biology ,Molecular Structure ,Chemistry ,Biguanide ,Metformin ,Tetrahydrofolate Dehydrogenase ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,Folic Acid Antagonists ,Crystallization ,medicine.drug - Abstract
Metformin is the most commonly prescribed treatment for type II diabetes and related disorders; however, molecular insights into its mode(s) of action have been limited by an absence of structural data. Structural considerations along with a growing body of literature demonstrating its effects on one-carbon metabolism suggest the possibility of folate mimicry and anti-folate activity. Motivated by the growing recognition that anti-diabetic biguanides may act directly upon the gut microbiome, we have determined structures of the complexes formed between the anti-diabetic biguanides (phenformin, buformin, and metformin) and Escherichia coli dihydrofolate reductase (ecDHFR) based on nuclear magnetic resonance, crystallographic, and molecular modeling studies. Interligand Overhauser effects indicate that metformin can form ternary complexes with p-aminobenzoyl-l-glutamate (pABG) as well as other ligands that occupy the region of the folate-binding site that interacts with pABG; however, DHFR inhibition is not cooperative. The biguanides competitively inhibit the activity of ecDHFR, with the phenformin inhibition constant being 100-fold lower than that of metformin. This inhibition may be significant at concentrations present in the gut of treated individuals, and inhibition of DHFR in intestinal mucosal cells may also occur if accumulation levels are sufficient. Perturbation of folate homeostasis can alter the pyridine nucleotide redox ratios that are important regulators of cellular metabolism.
- Published
- 2017
44. Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase Mu
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Thomas A. Kunkel, Katarzyna Bebenek, Andrea F. Moon, John M. Pryor, Lars C. Pedersen, and Dale A. Ramsden
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Models, Molecular ,Protein Conformation, alpha-Helical ,0301 basic medicine ,DNA End-Joining Repair ,Ribonucleotide ,DNA polymerase ,DNA repair ,DNA polymerase II ,Amino Acid Motifs ,Deoxyribonucleotides ,Gene Expression ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,DNA polymerase delta ,Substrate Specificity ,03 medical and health sciences ,RNTP ,Structural Biology ,Catalytic Domain ,Escherichia coli ,Genetics ,Humans ,Protein Interaction Domains and Motifs ,Cloning, Molecular ,DNA clamp ,Base Sequence ,biology ,DNA ,Ribonucleotides ,Recombinant Proteins ,Kinetics ,030104 developmental biology ,Biochemistry ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,Thermodynamics ,Protein Conformation, beta-Strand ,DNA polymerase mu ,Protein Binding - Abstract
While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol μ with a ribonucleotide bound at the active site. These structures reveal that Pol μ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol μ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol μ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.
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- 2017
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45. Characterization of an anti-Bla g 1 scFv: Epitope mapping and cross-reactivity
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Lori L. Edwards, Lalith Perera, Jill Glesner, Jay E. Slater, Anna Pomés, Robert E. London, Geoffrey A. Mueller, Lars C. Pedersen, John A. Ankney, and Taruna Khurana
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Models, Molecular ,Immunology ,Cockroaches ,chemical and pharmacologic phenomena ,Cross Reactions ,Crystallography, X-Ray ,medicine.disease_cause ,Cross-reactivity ,Article ,Epitope ,Epitopes ,medicine ,Animals ,Humans ,Molecular Biology ,Alanine ,biology ,Wild type ,Environmental exposure ,Allergens ,Immunoglobulin E ,respiratory system ,Alanine scanning ,biology.organism_classification ,Molecular biology ,Epitope mapping ,Mutation ,Binding Sites, Antibody ,American cockroach ,Epitope Mapping ,Single-Chain Antibodies - Abstract
Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE–Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.
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- 2014
46. Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse
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Sylvia C. Hewitt, Günther Schütz, Rainer B. Lanz, Wipawee Winuthayanon, David C. Fargo, Leping Li, Kenneth S. Korach, Lars C. Pedersen, Sara A. Grimm, Francesco J. DeMayo, Katherine J. Hamilton, Brianna Pockette, and Cory A. Rubel
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Transcriptional Activation ,Mice, 129 Strain ,Mutant ,Kruppel-Like Transcription Factors ,Mutation, Missense ,Estrogen receptor ,Biology ,Response Elements ,Kruppel-Like Factor 4 ,Endocrinology ,Transcription (biology) ,Consensus Sequence ,Animals ,Molecular Biology ,Transcription factor ,Original Research ,Mice, Knockout ,Hormone response element ,Reporter gene ,Base Sequence ,Estradiol ,Uterus ,Estrogen Receptor alpha ,Nuclear Proteins ,General Medicine ,Molecular biology ,Chromatin ,Mice, Inbred C57BL ,Phenotype ,Female ,Estrogen receptor alpha ,Protein Binding - Abstract
Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.
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- 2014
47. Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation
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Robert E. London, Xunhai Zheng, Matthew J. Cuneo, Geoffrey A. Mueller, Juno M. Krahn, Scott A. Gabel, Lars C. Pedersen, and Eugene F. DeRose
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Models, Molecular ,Stereochemistry ,Protein subunit ,Ribonuclease H ,010402 general chemistry ,01 natural sciences ,03 medical and health sciences ,Structural Biology ,Genetics ,Transferase ,RNase H ,Polymerase ,030304 developmental biology ,Protein Unfolding ,0303 health sciences ,biology ,Nuclear magnetic resonance spectroscopy ,Reverse transcriptase ,Random coil ,HIV Reverse Transcriptase ,0104 chemical sciences ,3. Good health ,Protein Structure, Tertiary ,Folding (chemistry) ,Biochemistry ,biology.protein ,Protein Multimerization - Abstract
HIV-1 reverse transcriptase (RT), a critical enzyme 10 of the HIV life cycle and an important drug target, undergoes complex and largely uncharacterized conformational rearrangements that underlie its asymmetric folding, dimerization and subunit-selective ribonuclease H domain (RH) proteolysis. In the 15 present article we have used a combination of NMR spectroscopy, small angle X-ray scattering and X-ray crystallography to characterize the p51 and p66 monomers and the conformational maturation of the p66/p66 0 homodimer. The p66 monomer exists as a 20 loosely structured molecule in which the fingers/ palm/connection, thumb and RH substructures are connected by flexible (disordered) linking segments. The initially observed homodimer is asymmetric and includes two fully folded RH domains, while exhibiting 25 other conformational features similar to that of the RT heterodimer. The RH 0 domain of the p66 0 subunit undergoes selective unfolding with time constant ! 6.5 h, consistent with destabilization due to residue transfer to the polymerase 0 domain on the 30 p66 0 subunit. A simultaneous increase in the intensity of resonances near the random coil positions is characterized by a similar time constant. Consistent with the residue transfer hypothesis, a construct of the isolated RH domain lacking the two N-terminal 35 residues is shown to exhibit reduced stability. These results demonstrate that RH 0 unfolding is coupled to homodimer formation.
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- 2014
48. Evaluation of the allergenic activity of the Glutathione Transferase from Blomia tropicalis (Blo t 8) in a mouse model of airway inflammation
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Sandra M. Coronado, Ronald Regino, Josefina Zakzuk, Luis Caraballo, Juana Bustillo, Ines Benedetti, Geoffrey A. Mueller, and Lars C. Pedersen
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Glutathione transferase ,Blomia tropicalis ,Immunology ,Airway inflammation ,Immunology and Allergy ,Biology - Published
- 2019
49. Mimicking of Estradiol Binding by Flame Retardants and Their Metabolites: A Crystallographic Analysis
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Rajendrakumar A. Gosavi, Linda S. Birnbaum, Lars C. Pedersen, and Gabriel A. Knudsen
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0303 health sciences ,03 medical and health sciences ,Chemistry ,Health, Toxicology and Mutagenesis ,Research ,Public Health, Environmental and Occupational Health ,Organic chemistry ,Polybrominated Biphenyls ,010501 environmental sciences ,Estradiol binding ,01 natural sciences ,030304 developmental biology ,0105 earth and related environmental sciences - Abstract
Background: Brominated flame retardants (BFRs), used in many types of consumer goods, are being studied because of concerns about possible health effects related to endocrine disruption, immunotoxicity, reproductive toxicity, and neurotoxicity. Tetrabromobisphenol A (TBBPA), the most widely used BFR, and human metabolites of certain congeners of polybrominated diphenyl ether (e.g., 3-OH-BDE-47) have been suggested to inhibit estrogen sulfotransferase, potentially affecting estrogen metabolism. Objectives: Our primary goal was to understand the structural mechanism for inhibition of the hormone-metabolizing enzyme estrogen sulfotransferase by certain BFRs. We also sought to understand various factors that facilitate the binding of flame retardants in the enzyme binding pocket. Methods: We used X-ray crystallography to obtain atomic detail of the binding modes of TBBPA and 3-OH-BDE-47 to estrogen sulfotransferase for comparison with binding of the endogenous substrate estradiol. Results: The crystal structures reveal how BFRs mimic estradiol binding as well as the various interactions between the compounds and protein residues that facilitate its binding. In addition, the structures provide insights into the ability of the sulfotransferase substrate binding pocket to accommodate a range of halogenated compounds that satisfy minimal structural criteria. Conclusions: Our results show how BFRs or their metabolites can bind to and inhibit a key hormone-metabolizing enzyme, potentially causing endocrine disruption. Citation: Gosavi RA, Knudsen GA, Birnbaum LS, Pedersen LC. 2013. Mimicking of estradiol binding by flame retardants and their metabolites: a crystallographic analysis. Environ Health Perspect 121:1194–1199; http://dx.doi.org/10.1289/ehp.1306902
- Published
- 2013
50. Amino Acid Substitution in the Active Site of DNA Polymerase β Explains the Energy Barrier of the Nucleotidyl Transfer Reaction
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Lars C. Pedersen, Lee G. Pedersen, David D. Shock, Vinod K. Batra, Lalith Perera, Samuel H. Wilson, William A. Beard, and Ping Lin
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Models, Molecular ,DNA clamp ,biology ,Chemistry ,Stereochemistry ,DNA polymerase ,Base pair ,Active site ,General Chemistry ,Processivity ,Crystallography, X-Ray ,Biochemistry ,DNA polymerase delta ,Article ,Catalysis ,Colloid and Surface Chemistry ,Amino Acid Substitution ,Catalytic Domain ,biology.protein ,DNA polymerase I ,DNA Polymerase beta ,Polymerase - Abstract
DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3′ group, thus activating O3′ as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1,000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3′ proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3′ proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3′. The theoretical results with the wild-type enzyme indicate early charge reorganization associated with the O3′ proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.
- Published
- 2013
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