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Principal Investigator: Lars Pedersen
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2020
Award: $1,453,130
Funding agency: National Institute of Environmental Health Sciences
Our lab works on a variety of different biological systems including heparan/chondroitin sulfate biosynthesis, DNA replication/repair, nuclear receptors, folate metabolism, and antibody/antigen interactions. Listed below are a few of our main projects form last year:
1) Work on glycosylaminoglycan (GAG) biosynthesis (in collaboration with Dr. Jian Liu at UNC) was centered around GAG biosynthesis enzymes that are utilized for the production of potential therapeutics dealing with coagulation, inflammation, and viral infection. By understanding how these enzymes interact with their substrates, we hope to modify them to improve their effectiveness in generating novel therapeutics with specific lengths, sequences, and sulfation patterns. CHST15 is a sulfotransferase that transfers a sulfate to the C-6 hydroxyl of GalNAc4S of chondroitin sulfate A to produce chondroitin sulfate E (CS-E). We have been able to obtain soluble protein from insect cells and partially deglycosylate it, finally giving us good material for ongoing crystallization studies. In addition, we have focused on structures of sulfotransferases involved in heparan sulfate biosynthesis. To this end, we have solved a co-crystal structure of 3-O-sulfotransferase (3-OST) isoform 5 in the presence of an oligosaccharide to mid-resolution. This structure allows for comparisons of substrate binding to isoforms 1 and 3 for which we have already determined structures, to help us better understand the specificity differences between these enzymes. Finally, our structures have directed research demonstrating that a triple mutant of 6-O-sulfotransferase, generated in our lab, only sulfates the non-reducing end of HS chains. This mutant provides a critical tool for the synthesis of homogenous HS oligosaccharides with specific 6-O-sulfation.
2) Double-strand breaks in DNA can result from exposure to ionizing radiation, reactive oxygen species, and other DNA damaging agents. Such damage can lead to cell death or cancer. Non-homologous End Joining (NHEJ) is a favored form of double-strand break (DSB) repair and is critical in non-replicating cells. NHEJ is also utilized in immunoglobin gene rearrangement and class-switch recombination. In support of Dr. Thomas Kunkels Replication Fidelity Group at NIEHS, our work this year was focused on understanding how X family polymerase Mu (pol mu) engages and repairs DSBs. Andrea Kaminski in our group was able to obtain pre-, intermediate and post-catalytic structures of pol mu bound to a DNA DSB. These represent the first structures from either pol mu or lambda bound to a DSB. Synapsis is mediated solely by Pol, 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 complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Pol may address complementary DSB substrates during NHEJ in a manner nearly indistinguishable from single-strand breaks, with subtle conformational changes in N457 and H459 near the break. This supports the notion that the rigid Pol mu scaffold provides for great flexibility in the types of substrates it can support for catalysis to repair damaged DNA. In addition, we worked in support of Dr. Samuel Wilsons group at NIEHS to critically refine and validate an array of time-lapse crystallography structures of Pol lambda (also an X-family polymerase) to investigate the process of correct nucleotide incorporation compared to incorporation of a mispair. These results demonstrate that misincorporation proceeds through a mostly open state of the enzyme that more closely resembles the binary rather than the ternary complex.
3) SMCHD1 (structural maintenance of chromosome flexible hinge domain containing 1) is a 2005 amino acid protein involved in gene silencing, with reported roles in X inactivation, genomic imprinting, and non-homologous end joining (NHEJ). Missense mutations in SMCHD1 are associated with arhinia, and Facioscapulohumeral Muscular Dystrophy Type 2 (FSHD2). In supporting the research by Dr. Natalie Shaw at NIEHS, we are trying to understand how these mutations lead to disease. Having previously solved the crystal structure of the ATPase module of SMCHD1, this year we focused on solving crystal structures of mutants and started CryoEM experiments to address how various mutations may affect the conformational landscape of SMCHD1 function. The mutations we investigated have surprisingly provided no changes in the global conformation of the protein in the dimeric state.
This project involves research on human coronavirus, novel coronavirus, COVID-19, Severe Acute Respiratory Syndrome coronavirus disease, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.
Terms: <2019 novel coronavirus><2019-nCoV><ATP phosphohydrolase><ATPase><Active Oxygen><Active Sites><Address><Adenosine Triphosphatase><Adenosinetriphosphatase><Affect><Amino Acids><Anabolism><Antibodies><Antigens><Behavior><Binding><COVID-19><COVID19><Cancers><Catalysis><Cell Body><Cell Death><Cells><Chondroitin 4 Sulfate><Chondroitin Sulfate A><Chondroitin Sulfates><Chromosomal Synapsis><Chromosome Pairing><Chromosomes><Class Switching><Class Switchings><Clotting><CoV emergence><Coagulation><Coagulation Process><Collaborations><Communities><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallization><Crystallographies><Crystallography><DNA><DNA Damage><DNA Damage Repair><DNA Double Strand Break><DNA Injury><DNA Rearrangement><DNA Repair><DNA Replication><DNA Synthesis><DNA biosynthesis><DNA polymerase mu><Deoxyribonucleic Acid><Disease><Disorder><Double Strand Break Repair><Effectiveness><Electron Cryomicroscopy><Enzyme Gene><Enzymes><Exposure to><FSHD><Facioscapulohumeral Atrophy><Facioscapulohumeral Muscular Dystrophy><Facioscapulohumeral Type Progressive Muscular Dystrophy><Family><Fasioscapulohumeral Muscular Dystrophy><Folate Metabolism><Folic Acid Metabolosm><Gene Inactivation><Gene Rearrangement><Gene Silencing><Genetic Alteration><Genetic Change><Genetic Imprinting><Genetic defect><Genomic Imprinting><Goals><H-bond><HCoV><Health><Heparan Sulfate Biosynthesis><Human><Hydrogen Bonding><Hydroxyl><Hydroxyl Radical><Immunoglobulin Class Switching><Immunoglobulin Class Switchings><Immunoglobulin Isotype-Switch Recombination><Immunoglobulin Switch Recombination><Inflammation><Insect Proteins><Ionizing Electromagnetic Radiation><Ionizing radiation><Isoforms><Isotype Switching><Isotype Switchings><Landouzy Dejerine muscular dystrophy><Landouzy-Dejerine Dystrophy><Lead><Length><Lyonization><Macromolecular Complexes><Macromolecular Structure><Maintenance><Malignant Neoplasms><Malignant Tumor><Mediating><Missense Mutation><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Molecular Structure><Mutation><NHEJ><NIEHS><National Institute of Environmental Health Sciences><Non-Homologous End Joining><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nuclear Receptors><Nucleotides><Oligosaccharides><Oxygen Radicals><Parental Imprinting><Pattern><Pb element><Pol mu><Polymerase><Pro-Oxidants><Process><Production><Protein Conformation><Protein Isoforms><Proteins><Radiation-Ionizing Total><Reactive Oxygen Species><Reporting><Research><Research Support><Resolution><Roentgen Rays><Role><SARS><SARS Virus><SARS corona virus><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe acute respiratory syndrome coronavirus 2><Site><Specificity><Structural Protein><Structure><Sulfate><Switch Recombination><Synapsis><Techniques><Therapeutic><Time><Unscheduled DNA Synthesis><Viral Diseases><Virus Diseases><Work><Wuhan coronavirus><X Inactivation><X-Chromosome Inactivation><X-Radiation><X-Ray Radiation><X-ray><Xray><aminoacid><biological systems><biosynthesis><conformation><conformational state><corona virus disease 2019><coronavirus disease 2019><coronavirus emergence><cryo-EM><cryoEM><dimer><emergent CoV><emergent coronavirus><emerging CoV><emerging coronavirus><enzyme biosynthesis><experiment><experimental research><experimental study><flexibility><flexible><folic acid metabolism><genome mutation><heavy metal Pb><heavy metal lead><human CoV><human corona virus><human coronavirus><hydrogen sulfate Chondroitin><immunogen><improved><insight><interest><ionizing output><malignancy><mu opioid receptors><mutant><nCoV><necrocytosis><neoplasm/cancer><new CoV><new coronavirus><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel CoV><novel coronavirus><novel drug treatments><novel drugs><novel therapeutics><novel therapy><protein complex><protein function><repair><repaired><scaffold><scaffolding><severe acute respiratory syndrome-CoV><social role><sulfotransferase><tool><transcriptional silencing><viral infection><virus infection><virus-induced disease><μ opioid receptors><μ-OR><μOR>