43 results on '"Juno M. Krahn"'
Search Results
2. 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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3. Predicting tumor response to drugs based on gene-expression biomarkers of sensitivity learned from cancer cell lines
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Yuanyuan Li, David M. Umbach, Juno M. Krahn, Igor Shats, Xiaoling Li, and Leping Li
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Drug sensitivity ,RNA-seq ,Cancer cell line ,GDSC ,GA/KNN ,TCGA ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Human cancer cell line profiling and drug sensitivity studies provide valuable information about the therapeutic potential of drugs and their possible mechanisms of action. The goal of those studies is to translate the findings from in vitro studies of cancer cell lines into in vivo therapeutic relevance and, eventually, patients’ care. Tremendous progress has been made. Results In this work, we built predictive models for 453 drugs using data on gene expression and drug sensitivity (IC50) from cancer cell lines. We identified many known drug-gene interactions and uncovered several potentially novel drug-gene associations. Importantly, we further applied these predictive models to ~ 17,000 bulk RNA-seq samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database to predict drug sensitivity for both normal and tumor tissues. We created a web site for users to visualize and download our predicted data ( https://manticore.niehs.nih.gov/cancerRxTissue ). Using trametinib as an example, we showed that our approach can faithfully recapitulate the known tumor specificity of the drug. Conclusions We demonstrated that our approach can predict drugs that 1) are tumor-type specific; 2) elicit higher sensitivity from tumor compared to corresponding normal tissue; 3) elicit differential sensitivity across breast cancer subtypes. If validated, our prediction could have relevance for preclinical drug testing and in phase I clinical design.
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- 2021
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4. Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics
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Monica C. Pillon, Meredith N. Frazier, Lucas B. Dillard, Jason G. Williams, Seda Kocaman, Juno M. Krahn, Lalith Perera, Cassandra K. Hayne, Jacob Gordon, Zachary D. Stewart, Mack Sobhany, Leesa J. Deterding, Allen L. Hsu, Venkata P. Dandey, Mario J. Borgnia, and Robin E. Stanley
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Science - Abstract
Nsp15 is a uridine specific endoribonuclease present in all coronaviruses. Here, the authors determine the cryo-EM structures of SARS-CoV-2 Nsp15 in the apo and UTP-bound states, which together with biochemical experiments, mass spectrometry and molecular dynamics simulations provide insights into the catalytic mechanism of Nsp15 and its conformational dynamics.
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- 2021
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5. Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7
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Yu-Hua Lo, Mack Sobhany, Allen L. Hsu, Brittany L. Ford, Juno M. Krahn, Mario J. Borgnia, and Robin E. Stanley
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Science - Abstract
Rix7 is a type II AAA-ATPase that is required for the assembly of the large ribosomal subunit. Here the authors present the 4.5 Å cryo-EM structure of the Rix7 homohexamer with a polypeptide fragment bound in its central channel and provide insights into the function of Rix7 as a molecular unfoldase.
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- 2019
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6. 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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7. A comprehensive genomic pan-cancer classification using The Cancer Genome Atlas gene expression data
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Yuanyuan Li, Kai Kang, Juno M. Krahn, Nicole Croutwater, Kevin Lee, David M. Umbach, and Leping Li
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Pan-cancer ,Classification ,Ga/KNN ,RNA-seq ,TCGA ,And sex dimorphism ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background The Cancer Genome Atlas (TCGA) has generated comprehensive molecular profiles. We aim to identify a set of genes whose expression patterns can distinguish diverse tumor types. Those features may serve as biomarkers for tumor diagnosis and drug development. Methods Using RNA-seq expression data, we undertook a pan-cancer classification of 9,096 TCGA tumor samples representing 31 tumor types. We randomly assigned 75% of samples into training and 25% into testing, proportionally allocating samples from each tumor type. Results We could correctly classify more than 90% of the test set samples. Accuracies were high for all but three of the 31 tumor types, in particular, for READ (rectum adenocarcinoma) which was largely indistinguishable from COAD (colon adenocarcinoma). We also carried out pan-cancer classification, separately for males and females, on 23 sex non-specific tumor types (those unrelated to reproductive organs). Results from these gender-specific analyses largely recapitulated results when gender was ignored. Remarkably, more than 80% of the 100 most discriminative genes selected from each gender separately overlapped. Genes that were differentially expressed between genders included BNC1, FAT2, FOXA1, and HOXA11. FOXA1 has been shown to play a role for sexual dimorphism in liver cancer. The differentially discriminative genes we identified might be important for the gender differences in tumor incidence and survival. Conclusions We were able to identify many sets of 20 genes that could correctly classify more than 90% of the samples from 31 different tumor types using TCGA RNA-seq data. This accuracy is remarkable given the number of the tumor types and the total number of samples involved. We achieved similar results when we analyzed 23 non-sex-specific tumor types separately for males and females. We regard the frequency with which a gene appeared in those sets as measuring its importance for tumor classification. One third of the 50 most frequently appearing genes were pseudogenes; the degree of enrichment may be indicative of their importance in tumor classification. Lastly, we identified a few genes that might play a role in sexual dimorphism in certain cancers.
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- 2017
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8. GADGETS: a genetic algorithm for detecting epistasis using nuclear families.
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Michael Nodzenski, Min Shi 0002, Juno M. Krahn, Alison S. Wise, Yuanyuan Li, Leping Li, David M. Umbach, and Clarice R. Weinberg
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- 2022
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9. Erratum to: GADGETS: a genetic algorithm for detecting epistasis using nuclear families.
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Michael Nodzenski, Min Shi 0002, Juno M. Krahn, Alison S. Wise, Yuanyuan Li, Leping Li, David M. Umbach, and Clarice R. Weinberg
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- 2022
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10. Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM
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Amanda A. Riccio, Jonathan Bouvette, Matthew J. Longley, Juno M. Krahn, Mario J. Borgnia, and William C. Copeland
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DNA Replication ,Mitochondrial Proteins ,Cryoelectron Microscopy ,DNA Helicases ,DNA, Mitochondrial ,Molecular Biology ,General Biochemistry, Genetics and Molecular Biology ,Mitochondria - Abstract
The mitochondrial replisome replicates the 16.6 kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.
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- 2022
11. Mechanism by which T7 bacteriophage protein Gp1.2 inhibits
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Bradley P, Klemm, Deepa, Singh, Cassandra E, Smith, Allen L, Hsu, Lucas B, Dillard, Juno M, Krahn, Robert E, London, Geoffrey A, Mueller, Mario J, Borgnia, and Roel M, Schaaper
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DNA Replication ,Viral Proteins ,Protein Conformation ,Bacteriophage T7 ,Escherichia coli Proteins ,Cryoelectron Microscopy ,DNA, Viral ,Escherichia coli ,Guanosine Triphosphate ,Virus Replication ,GTP Phosphohydrolases - Abstract
Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses.
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- 2023
12. 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.
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- 2021
13. Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase
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Bradley P. Klemm, Deepa Singh, Cassandra E. Smith, Allen L. Hsu, Lucas B. Dillard, Juno M. Krahn, Robert E. London, Geoffrey A. Mueller, Mario J. Borgnia, and Roel M. Schaaper
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Multidisciplinary - Abstract
Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2′-deoxyguanosine-5′-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host “arms race.” We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt–Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt–Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.
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- 2022
14. Structural Basis for pre-tRNA Recognition and Processing by the Human tRNA Splicing Endonuclease Complex
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Cassandra K. Hayne, Kevin John U. Butay, Zachary D. Stewart, Juno M. Krahn, Lalith Perera, Jason G. Williams, Robert M. Petrovitch, Leesa J. Deterding, A. Gregory Matera, Mario J. Borgnia, and Robin E. Stanley
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Structural Biology ,Molecular Biology - Abstract
Across all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN•pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.
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- 2022
15. Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease
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Meredith N. Frazier, Isha M. Wilson, Juno M. Krahn, Kevin John Butay, Lucas B. Dillard, Mario J. Borgnia, and Robin E. Stanley
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Structural Biology ,SARS-CoV-2 ,viruses ,fungi ,Endoribonucleases ,Genetics ,COVID-19 ,Humans ,Viral Nonstructural Proteins ,Uridine ,RNA, Double-Stranded - Abstract
Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.
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- 2022
16. 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.
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- 2021
17. 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.
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- 2020
18. GADGETS: a genetic algorithm for detecting epistasis using nuclear families
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Alison S Wise, David M. Umbach, Min Shi, Juno M. Krahn, Clarice R. Weinberg, Leping Li, Yuanyuan Li, and Michael Nodzenski
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Statistics and Probability ,Computer science ,business.industry ,Machine learning ,computer.software_genre ,Biochemistry ,Original Papers ,Computer Science Applications ,Visualization ,Computational Mathematics ,Identification (information) ,Computational Theory and Mathematics ,Genetic algorithm ,Disease risk ,Epistasis ,Relevance (information retrieval) ,Artificial intelligence ,business ,Molecular Biology ,computer ,Nuclear family ,Selection (genetic algorithm) - Abstract
Motivation Epistasis may play an etiologic role in complex diseases, but research has been hindered because identification of interactions among sets of single nucleotide polymorphisms (SNPs) requires exploration of immense search spaces. Current approaches using nuclear families accommodate at most several hundred candidate SNPs. Results GADGETS detects epistatic SNP-sets by applying a genetic algorithm to case-parent or case-sibling data. To allow for multiple epistatic sets, island subpopulations of SNP-sets evolve separately under selection for evident joint relevance to disease risk. The software evaluates the identified SNP-sets via permutation testing and provides graphical visualization. GADGETS correctly identified epistatic SNP-sets in realistically simulated case-parent triads with 10 000 candidate SNPs, far more SNPs than competitors can handle, and it outperformed competitors in simulations with many fewer SNPs. Applying GADGETS to family-based oral-clefting data from dbGaP identified SNP-sets with possible epistatic effects on risk. Availability and implementation GADGETS is part of the epistasisGA package at https://github.com/mnodzenski/epistasisGA. Supplementary information Supplementary data are available at Bioinformatics online.
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- 2021
19. Characterization of SARS2 Nsp15 Nuclease Activity Reveals it’s Mad About U
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Mario J. Borgnia, Leesa J. Deterding, Robin E. Stanley, Lucas B. Dillard, Juno M. Krahn, Monica C. Pillon, Lalith Perera, Isha M Wilson, Jason Williams, Zachary D. Stewart, and Meredith N Frazier
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Nuclease ,biology ,RNase P ,Chemistry ,Endoribonuclease ,RNA ,Active site ,biology.organism_classification ,Uridine ,chemistry.chemical_compound ,Biochemistry ,Cleave ,biology.protein ,Coronaviridae - Abstract
Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3’ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3’ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.
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- 2021
20. Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex
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Monica C. Pillon, Mack Sobhany, Mario J. Borgnia, Juno M. Krahn, Allen L. Hsu, Jason Williams, Robin E. Stanley, and Kevin H Goslen
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Protein Conformation ,RNase P ,Endoribonuclease ,02 engineering and technology ,Chaetomium ,010403 inorganic & nuclear chemistry ,01 natural sciences ,Biochemistry ,Ribosome ,Article ,Ribosome assembly ,Fungal Proteins ,Inorganic Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Multienzyme Complexes ,Structural Biology ,Catalytic Domain ,RNA Precursors ,General Materials Science ,Physical and Theoretical Chemistry ,RRNA processing ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Nuclease ,biology ,Chemistry ,Cryoelectron Microscopy ,RNA ,Ribosomal RNA ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,0104 chemical sciences ,biology.protein ,0210 nano-technology ,030217 neurology & neurosurgery - Abstract
Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the poly-nucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA. RNase PNK belongs to the functionally-diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-electron microscopy structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.
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- 2019
21. Deciphering the substrate recognition mechanisms of the heparan sulfate 3
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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.
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- 2021
22. Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics
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Lucas B. Dillard, Cassandra K. Hayne, Meredith N Frazier, Lalith Perera, Robin E. Stanley, Allen L. Hsu, Leesa J. Deterding, Jason Williams, Zachary D. Stewart, Mario J. Borgnia, Jacob Gordon, Venkata P. Dandey, Monica C. Pillon, Mack Sobhany, Seda Kocaman, Juno M. Krahn, Pillon, Monica C [0000-0001-8571-6873], Perera, Lalith [0000-0003-0823-1631], Hayne, Cassandra K [0000-0002-2485-5949], Gordon, Jacob [0000-0001-7199-7416], Hsu, Allen L [0000-0003-2065-3802], Borgnia, Mario J [0000-0001-9159-1413], Stanley, Robin E [0000-0002-2106-3102], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Models, Molecular ,viruses ,Science ,Endoribonuclease ,General Physics and Astronomy ,Uridine Triphosphate ,Computational biology ,Viral Nonstructural Proteins ,Cleavage (embryo) ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Catalytic Domain ,Endoribonucleases ,Amino Acid Sequence ,Peptide sequence ,Nuclease ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,Chemistry ,SARS-CoV-2 ,Cryoelectron Microscopy ,RNA ,Active site ,General Chemistry ,Uridine ,030104 developmental biology ,Models, Chemical ,Phosphodiester bond ,biology.protein - Abstract
Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.
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- 2021
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23. Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15
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Monica C. Pillon, Meredith N. Frazier, Lucas B. Dillard, Jason G. Williams, Seda Kocaman, Juno M. Krahn, Lalith Perera, Cassandra K. Hayne, Jacob Gordon, Zachary D. Stewart, Mack Sobhany, Leesa J. Deterding, Allen L. Hsu, Venkata P. Dandey, Mario J. Borgnia, and Robin E. Stanley
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Nuclease ,biology ,Mechanism (biology) ,viruses ,Endoribonuclease ,Active site ,Computational biology ,Random hexamer ,Biochemistry ,RNA decay ,Uridine ,Virus ,Article ,chemistry.chemical_compound ,chemistry ,Cryoelectron microscopy ,Viral infection ,Phosphodiester bond ,biology.protein - Abstract
Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics., Nsp15 is a uridine specific endoribonuclease present in all coronaviruses. Here, the authors determine the cryo-EM structures of SARS-CoV-2 Nsp15 in the apo and UTP-bound states, which together with biochemical experiments, mass spectrometry and molecular dynamics simulations provide insights into the catalytic mechanism of Nsp15 and its conformational dynamics.
- Published
- 2020
24. Predicting Tumor Response to Drugs Based on Gene-expression Biomarkers of Sensitivity Learned From Cancer Cell Lines
- Author
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David M. Umbach, Juno M. Krahn, Yuanyuan Li, Xiaoling Li, Igor Shats, and Leping Li
- Subjects
Drug ,GDSC ,media_common.quotation_subject ,Gene Expression ,Breast Neoplasms ,RNA-Seq ,Computational biology ,Biology ,QH426-470 ,Proteomics ,03 medical and health sciences ,Cancer cell line ,0302 clinical medicine ,Breast cancer ,In vivo ,Cell Line, Tumor ,Gene expression ,Biomarkers, Tumor ,Genetics ,Humans ,Medicine ,Clinical significance ,030304 developmental biology ,media_common ,Trametinib ,0303 health sciences ,business.industry ,Gene Expression Profiling ,TCGA ,medicine.disease ,Pharmaceutical Preparations ,Cell culture ,030220 oncology & carcinogenesis ,GA/KNN ,And CCLE ,GTEx ,DNA microarray ,RNA-seq ,Cancer cell lines ,business ,Biomarkers ,TP248.13-248.65 ,Research Article ,Drug sensitivity ,Biotechnology - Abstract
Background Human cancer cell line profiling and drug sensitivity studies provide valuable information about the therapeutic potential of drugs and their possible mechanisms of action. The goal of those studies is to translate the findings from in vitro studies of cancer cell lines into in vivo therapeutic relevance and, eventually, patients’ care. Tremendous progress has been made. Results In this work, we built predictive models for 453 drugs using data on gene expression and drug sensitivity (IC50) from cancer cell lines. We identified many known drug-gene interactions and uncovered several potentially novel drug-gene associations. Importantly, we further applied these predictive models to ~ 17,000 bulk RNA-seq samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database to predict drug sensitivity for both normal and tumor tissues. We created a web site for users to visualize and download our predicted data (https://manticore.niehs.nih.gov/cancerRxTissue). Using trametinib as an example, we showed that our approach can faithfully recapitulate the known tumor specificity of the drug. Conclusions We demonstrated that our approach can predict drugs that 1) are tumor-type specific; 2) elicit higher sensitivity from tumor compared to corresponding normal tissue; 3) elicit differential sensitivity across breast cancer subtypes. If validated, our prediction could have relevance for preclinical drug testing and in phase I clinical design.
- Published
- 2020
25. Ubiquitin stimulated reversal of topoisomerase 2 DNA-protein crosslinks by TDP2
- Author
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C. Denise Appel, Logan R. Butler, Felipe Cortés-Ledesma, Juno M. Krahn, A.A. Riccio, Jenna A Liebermann, Matthew J. Schellenberg, R. Scott Williams, National Institutes of Health (US), National Institute of Environmental Health Sciences (US), Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), European Commission, European Research Council, Universidad de Sevilla, Department of Energy (US), United States of Department of Health & Human Services, and NIH - National Institute of Environmental Health Sciences (NIEHS) (Estados Unidos)
- Subjects
Models, Molecular ,European Regional Development Fund ,Protein dna ,Library science ,Advanced Photon Source ,Biology ,Crystallography, X-Ray ,Substrate Specificity ,03 medical and health sciences ,Light source ,Structural Biology ,Catalytic Domain ,Genetics ,Humans ,User Facility ,Polyubiquitin ,030304 developmental biology ,0303 health sciences ,Government ,Binding Sites ,Phosphoric Diester Hydrolases ,Ubiquitin ,030302 biochemistry & molecular biology ,Sumoylation ,DNA ,DNA-Binding Proteins ,DNA Topoisomerases, Type II ,Mutation ,Small Ubiquitin-Related Modifier Proteins ,Topoisomerase 2 ,National laboratory ,Protein Binding - Abstract
Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA–protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA–protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface., Intramural research program of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) [1Z01ES102765 to R.S.W.]; Work in the F.C.L. lab is supported by Ministerio de Economía y Competitividad, Gobierno de España [SAF2017-89619-R, European Regional Development Fund]; European Research Council [ERC-CoG-2014-647359]; University of Seville Predoctoral Studentship [PIF-2011 to J.A.L.]; M.J.S. is supported by Mayo Clinic start-up funds and the Center for Biomedical Discovery new investigator funds; Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory; Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]; SAXS data were collected at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE [P30 GM124169]; High-End Instrumentation Grant [S10OD018483]. Funding for open access charge: US government, Intramural NIH.
- Published
- 2020
26. Erratum to: GADGETS: a genetic algorithm for detecting epistasis using nuclear families
- Author
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Michael Nodzenski, Min Shi, Juno M Krahn, Alison S Wise, Yuanyuan Li, Leping Li, David M Umbach, and Clarice R Weinberg
- Subjects
Statistics and Probability ,Computational Mathematics ,Computational Theory and Mathematics ,Molecular Biology ,Biochemistry ,Computer Science Applications - Published
- 2021
27. 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
28. 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
29. Structural characterization of the virulence factor Sda1 nuclease fromStreptococcus pyogenes
- Author
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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
30. Glypican 6 is a putative biomarker for metastatic progression of cutaneous melanoma
- Author
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Igor Shats, David M. Umbach, Gordon P. Flake, Xiaoling Li, Melissa Li, Juno M. Krahn, Leping Li, and Yuanyuan Li
- Subjects
0301 basic medicine ,Male ,Melanomas ,Glypican ,Skin Neoplasms ,Fibroblast Growth Factor ,Physiology ,Gene Expression ,Fibroblast growth factor ,Epithelium ,Metastasis ,0302 clinical medicine ,Endocrinology ,Cell Signaling ,Animal Cells ,Medicine and Health Sciences ,RNA, Neoplasm ,Neoplasm Metastasis ,Melanoma ,Cultured Tumor Cells ,Multidisciplinary ,Wnt signaling pathway ,3. Good health ,Neoplasm Proteins ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Oncology ,030220 oncology & carcinogenesis ,Cutaneous Melanoma ,Biomarker (medicine) ,Medicine ,Melanoma Cells ,Melanocytes ,Female ,Biological Cultures ,Cellular Types ,Anatomy ,Research Article ,Signal Transduction ,Science ,Malignant Skin Neoplasms ,Dermatology ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Glypicans ,Cell Line, Tumor ,Growth Factors ,medicine ,Biomarkers, Tumor ,Genetics ,Cell Adhesion ,Humans ,Chromatophores ,Endocrine Physiology ,Cancers and Neoplasms ,Biology and Life Sciences ,Epithelial Cells ,Cell Biology ,Cell Cultures ,medicine.disease ,MicroRNAs ,030104 developmental biology ,Biological Tissue ,Cutaneous melanoma ,Cancer research ,Hedgehog Signaling ,Ovarian cancer - Abstract
Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.
- Published
- 2018
31. Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease
- Author
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Emma E. Fairweather, R.S. Williams, Matthew J. Schellenberg, Ian D. Waddell, J Little, Percy P. Tumbale, Mandy Watson, Geoffrey A. Mueller, Robert E. London, and Juno M. Krahn
- Subjects
0301 basic medicine ,Models, Molecular ,Magnetic Resonance Spectroscopy ,DNA repair ,Protein Conformation ,Ribonucleotide excision repair ,DNA Mutational Analysis ,aptX ,DNA Ligases ,Ataxia Oculomotor Apraxia 1 ,X‐ray crystallography ,medicine.disease_cause ,Crystallography, X-Ray ,APTX ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Catalytic Domain ,medicine ,Humans ,Molecular Biology of Disease ,DNA Breaks, Single-Stranded ,Molecular Biology ,chemistry.chemical_classification ,Mutation ,DNA ligase ,General Immunology and Microbiology ,biology ,Protein Stability ,General Neuroscience ,missense mutation ,Active site ,Nuclear Proteins ,DNA Replication, Repair & Recombination ,Neurodegenerative Diseases ,DNA ,Articles ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,chemistry ,biology.protein ,RNA ,Protein Binding - Abstract
The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA‐DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X‐ray structures of APTX engaging nicked RNA‐DNA substrates that provide direct evidence for a wedge‐pivot‐cut strategy for 5′‐AMP resolution shared with the alternate 5′‐AMP processing enzymes POLβ and FEN1. Our results uncover a DNA‐induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X‐ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.
- Published
- 2017
32. Characterization of the Redox Transition of the XRCC1 N-terminal Domain
- Author
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Matthew J. Cuneo, Geoffrey A. Mueller, Scott A. Gabel, Robert E. London, Thomas W. Kirby, Juno M. Krahn, Cassandra E. Smith, and Eugene F. DeRose
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Protein Folding ,DNA Repair ,biology ,DNA polymerase ,DNA repair ,Redox ,Article ,Cell Line ,Adduct ,DNA-Binding Proteins ,Residue (chemistry) ,Crystallography ,X-ray Repair Cross Complementing Protein 1 ,Enzyme ,chemistry ,Structural Biology ,Escherichia coli ,biology.protein ,Protein disulfide-isomerase ,Oxidation-Reduction ,Molecular Biology ,Polymerase ,Protein Binding - Abstract
Summary XRCC1, a scaffold protein involved in DNA repair, contains an N-terminal domain (X1NTD) that interacts specifically with DNA polymerase β. It was recently discovered that X1NTD contains a disulfide switch that allows it to adopt either of two metamorphic structures. In the present study, we demonstrate that formation of an N-terminal proline carbimate adduct resulting from the nonenzymatic reaction of Pro2 with CO 2 is essential for stabilizing the oxidized structure, X1NTDox. The kinetic response of X1NTDred to H 2 O 2 , monitored by NMR, was determined to be very slow, consistent with involvement of the buried, kinetically trapped Cys12 residue, but was significantly accelerated by addition of protein disulfide isomerase or by Cu 2+ . NMR analysis of a sample containing the pol β polymerase domain, and both the reduced and oxidized forms of X1NTD, indicates that the oxidized form binds to the enzyme 25-fold more tightly than the reduced form.
- Published
- 2014
33. Probing Dominant Negative Behavior of Glucocorticoid Receptor β through a Hybrid Structural and Biochemical Approach
- Author
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John A. Cidlowski, Andrea F. Moon, Christine M. Jewell, Lalith Perera, Lars C. Pedersen, Juno M. Krahn, and Jungki Min
- Subjects
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.
- Published
- 2017
34. A Structural Basis for Biguanide Activity
- Author
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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
- Subjects
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
35. A comprehensive genomic pan-cancer classification using The Cancer Genome Atlas gene expression data
- Author
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Kevin Lee, Nicole Croutwater, Kai Kang, David M. Umbach, Leping Li, Juno M. Krahn, and Yuanyuan Li
- Subjects
0301 basic medicine ,Male ,lcsh:QH426-470 ,lcsh:Biotechnology ,Pseudogene ,RNA-Seq ,Pan-cancer ,Biology ,Proteomics ,03 medical and health sciences ,Ga/KNN ,lcsh:TP248.13-248.65 ,Neoplasms ,Databases, Genetic ,Genetics ,medicine ,Humans ,Gene ,Sequence Analysis, RNA ,Gene Expression Profiling ,Genomics ,TCGA ,medicine.disease ,Classification ,3. Good health ,Sexual dimorphism ,Gene Expression Regulation, Neoplastic ,lcsh:Genetics ,030104 developmental biology ,Adenocarcinoma ,Female ,DNA microarray ,RNA-seq ,Liver cancer ,And sex dimorphism ,Biotechnology ,Genes, Neoplasm ,Research Article - Abstract
Background The Cancer Genome Atlas (TCGA) has generated comprehensive molecular profiles. We aim to identify a set of genes whose expression patterns can distinguish diverse tumor types. Those features may serve as biomarkers for tumor diagnosis and drug development. Methods Using RNA-seq expression data, we undertook a pan-cancer classification of 9,096 TCGA tumor samples representing 31 tumor types. We randomly assigned 75% of samples into training and 25% into testing, proportionally allocating samples from each tumor type. Results We could correctly classify more than 90% of the test set samples. Accuracies were high for all but three of the 31 tumor types, in particular, for READ (rectum adenocarcinoma) which was largely indistinguishable from COAD (colon adenocarcinoma). We also carried out pan-cancer classification, separately for males and females, on 23 sex non-specific tumor types (those unrelated to reproductive organs). Results from these gender-specific analyses largely recapitulated results when gender was ignored. Remarkably, more than 80% of the 100 most discriminative genes selected from each gender separately overlapped. Genes that were differentially expressed between genders included BNC1, FAT2, FOXA1, and HOXA11. FOXA1 has been shown to play a role for sexual dimorphism in liver cancer. The differentially discriminative genes we identified might be important for the gender differences in tumor incidence and survival. Conclusions We were able to identify many sets of 20 genes that could correctly classify more than 90% of the samples from 31 different tumor types using TCGA RNA-seq data. This accuracy is remarkable given the number of the tumor types and the total number of samples involved. We achieved similar results when we analyzed 23 non-sex-specific tumor types separately for males and females. We regard the frequency with which a gene appeared in those sets as measuring its importance for tumor classification. One third of the 50 most frequently appearing genes were pseudogenes; the degree of enrichment may be indicative of their importance in tumor classification. Lastly, we identified a few genes that might play a role in sexual dimorphism in certain cancers. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3906-0) contains supplementary material, which is available to authorized users.
- Published
- 2017
36. Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation
- Author
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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
- Subjects
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.
- Published
- 2014
37. Structure of an aprataxin–DNA complex with insights into AOA1 neurodegenerative disease
- Author
-
R.S. Williams, Patrick D. Robertson, C.D. Appel, Juno M. Krahn, Ivan Ahel, Percy P. Tumbale, Jessica S. Williams, and Rolf Kraehenbuehl
- Subjects
Models, Molecular ,Cerebellar Ataxia ,DNA Repair ,Apraxias ,DNA damage ,DNA repair ,Amino Acid Motifs ,Molecular Sequence Data ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Article ,Ataxia Telangiectasia ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Structural Biology ,medicine ,Humans ,DNA Breaks, Single-Stranded ,Molecular Biology ,030304 developmental biology ,Aprataxin ,Genetics ,0303 health sciences ,Mutation ,Binding Sites ,Mutagenesis ,DNA replication ,Nuclear Proteins ,Zinc Fingers ,DNA ,biology.organism_classification ,Protein Structure, Tertiary ,DNA-Binding Proteins ,chemistry ,Schizosaccharomyces pombe ,Nucleic Acid Conformation ,Hypoalbuminemia ,030217 neurology & neurosurgery ,DNA Damage - Abstract
DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5'-adenylation DNA damage. Aprataxin (Aptx) catalyzes direct reversal of 5'-adenylate adducts to protect genome integrity. Here the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn(2+) complex reveals active site and DNA interaction clefts formed by fusing a histidine triad (HIT) nucleotide hydrolase with a DNA minor groove-binding C(2)HE zinc finger (Znf). An Aptx helical 'wedge' interrogates the base stack for sensing DNA ends or DNA nicks. The HIT-Znf, the wedge and an '[F/Y]PK' pivot motif cooperate to distort terminal DNA base-pairing and direct 5'-adenylate into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5'-adenylate adduct recognition and removal and suggest that mutations affecting protein folding, the active site pocket and the pivot motif underlie Aptx dysfunction in the neurodegenerative disorder ataxia with oculomotor apraxia 1 (AOA1).
- Published
- 2011
38. Toward predicting metastatic progression of melanoma based on gene expression data
- Author
-
Leping Li, Yuanyuan Li, Gordon P. Flake, David M. Umbach, and Juno M. Krahn
- Subjects
Skin Neoplasms ,Cell ,Dermatology ,Biology ,Bioinformatics ,General Biochemistry, Genetics and Molecular Biology ,Article ,Metastasis ,microRNA ,Gene expression ,medicine ,Humans ,Neoplasm Metastasis ,Gene ,Melanoma ,Sequence Analysis, RNA ,medicine.disease ,Prognosis ,Gene Expression Regulation, Neoplastic ,MicroRNAs ,medicine.anatomical_structure ,Gene Ontology ,Oncology ,Cutaneous melanoma ,Cancer research ,Disease Progression ,Biomarker (medicine) ,Genes, Neoplasm - Abstract
Primary and metastatic melanoma tumors share the same cell origin, making it challenging to identify genomic biomarkers that can differentiate them. Primary tumors themselves can be heterogeneous, reflecting ongoing genomic changes as they progress toward metastasizing. We developed a computational method to explore this heterogeneity and to predict metastatic progression of the primary tumors. We applied our method separately to gene expression and to microRNA (miRNA) expression data from ~450 primary and metastatic skin cutaneous melanoma (SKCM) samples from the Cancer Genome Atlas (TCGA). Metastatic progression scores from RNA-seq data were significantly associated with clinical staging of patients' lymph nodes, whereas scores from miRNA-seq data were significantly associated with Clark's level. The loss of expression of many characteristic epithelial lineage genes in primary SKCM tumor samples was highly correlated with predicted progression scores. We suggest that those genes/miRNAs might serve as putative biomarkers for SKCM metastatic progression.
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- 2015
39. Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase
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Yongmei Xu, Lars C. Pedersen, Wenfang Dou, Jian Liu, Chunhui Liu, Juno M. Krahn, Lalith Perera, Juzheng Sheng, and Po Hung Hsieh
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Sulfotransferase ,Stereochemistry ,Glycobiology and Extracellular Matrices ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Structure-Activity Relationship ,Sulfation ,Catalytic Domain ,Structure–activity relationship ,Animals ,Molecular Biology ,Ternary complex ,chemistry.chemical_classification ,biology ,Active site ,Substrate (chemistry) ,Cell Biology ,Heparan sulfate ,carbohydrates (lipids) ,Enzyme ,chemistry ,biology.protein ,Sulfotransferases ,Chickens - Abstract
Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.
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- 2014
40. Stable RAGE-heparan sulfate complexes are essential for signal transduction
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Walter J. Chazin, Danyin Song, Jeffrey H. Young, Jeffrey D. Esko, Juno M. Krahn, Kevin D. Corbett, Ding Xu, and Lars C. Pedersen
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Models, Molecular ,endocrine system diseases ,Receptor for Advanced Glycation End Products ,Random hexamer ,Biochemistry ,Article ,RAGE (receptor) ,chemistry.chemical_compound ,Drug Stability ,X-Ray Diffraction ,Glycation ,Coordination Complexes ,Humans ,Receptor ,Chemistry ,Endothelial Cells ,General Medicine ,Heparan sulfate ,Cell biology ,Molecular Medicine ,Phosphorylation ,Heparan sulfate binding ,Heparitin Sulfate ,Signal transduction ,Dimerization ,Signal Transduction - Abstract
RAGE (Receptor for Advanced Glycation End-Products) has emerged as a major receptor that mediates vascular inflammation. Signaling through RAGE by damage-associated molecular pattern molecules often leads to uncontrolled inflammation that exacerbates the impact of the underlying disease. Oligomerization of RAGE is believed to play an essential role in signal transduction, but the molecular mechanism of oligomerization remains elusive. Here we report that RAGE activation of Erk1/2 phosphorylation on endothelial cells in response to a number of ligands depends on a mechanism that involves heparan sulfate-induced hexamerization of the RAGE extracellular domain. Structural studies of the extracellular V-C1 domain-dodecasaccharide complex by X-ray diffraction and small-angle X-ray scattering revealed that the hexamer consists of a trimer of dimers, with a stoichiometry of 2:1 RAGE:dodecasaccharide. Mutagenesis studies mapped the heparan sulfate binding site and the interfacial surface between the monomers, and demonstrated that electrostatic interactions with heparan sulfate and inter-monomer hydrophobic interactions work in concert to stabilize the dimer. The importance of oligomerization was demonstrated by inhibition of signaling with a new epitope-defined monoclonal antibody that specifically targets oligomerization. These findings indicate that RAGE-heparan sulfate oligomeric complexes are essential for signaling and that interfering with RAGE oligomerization might be of therapeutic value.
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- 2013
41. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease
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Ronald T. Hay, Richard C. Trembath, Rosa Morra, Ege Ozkan, Juno M. Krahn, C. Denise Appel, Reza Sharifi, Ivan Matic, Marianna Nicoletta Rossi, Andreas G. Ladurner, Ria Weston, Jason Williams, R. Scott Williams, Hamid Galehdari, Dragana Ahel, Michael Tallis, Matthew J. Schellenberg, Barry A. Chioza, Andrew H. Crosby, Gyula Timinszky, Michael A. Simpson, Y. B. Alexander Wan, Ivan Ahel, Gytis Jankevicius, and Barbara Golia
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chemistry.chemical_classification ,0303 health sciences ,PARG ,Mutation ,General Immunology and Microbiology ,DNA repair ,General Neuroscience ,Poly ADP ribose polymerase ,030302 biochemistry & molecular biology ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Chromatin ,03 medical and health sciences ,Adenosine diphosphate ,chemistry.chemical_compound ,Enzyme ,Biochemistry ,chemistry ,medicine ,Molecular Biology ,Peptide sequence ,030304 developmental biology - Abstract
Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.
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- 2013
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42. Abstract A17: Assessing the similarity and dissimilarity between primary and metastatic melanoma using gene expression data
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Juno M. Krahn, Yuanyuan Li, and Leping Li
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Cancer Research ,Metastatic melanoma ,Melanoma ,Equal probability ,Epidermal cell differentiation ,Computational biology ,Biology ,medicine.disease ,Bioinformatics ,Tumor Status ,Oncology ,Cutaneous melanoma ,Gene expression ,medicine ,Gene - Abstract
The Cancer Genome Atlas (TCGA) consortium measured genome-wide gene expression using RNA-seq for 336 skin cutaneous melanoma (SKCM) samples among which 272 were clinically classified as metastatic SKCM tumors and the remaining 64 as primary SKCM tumors. We aimed to identify gene signatures that separate the primary SKCM from the metastatic SKCM samples. Our initial analysis showed that the primary and metastatic SKCM samples shared enough similarity at the gene expression level so that misclassification rates were unacceptably high. We reasoned that some of the primary SKCM tumors plausibly might have evolved to resemble the metastatic tumors in their gene expression. This idea led us to propose an alternative computational method to find a gene signature for accurate classification but does so while making explicit allowance for allegiance switching moving samples from one group to the other, e.g., primary to metastatic or vice versa. Based on an iterative stochastic search algorithm that delivers nearly optimal gene signatures for classification, our alternative algorithm is rooted in the groups defined by clinical classification but allows for switching between groups when a sample is clearly discordant with other group members based on its gene expression profile. We began by seeking such near-optimal partitioning of the 336 samples into the primary and metastatic groups based on the gene expression data using the clinical classification as the guide/basis. Specifically, our algorithm gives each of the 336 samples a small but equal probability to be switched to the other group at each iteration (e.g., from metastatic to primary, or vice versa). We carried out a massive computational search for gene signatures (a set of 20 genes) that provide a near optimal partitioning of the groups while keeping the clinical classification for most of the 336 samples but reassigning a few to the other group. Distinguishing between the newly re-assigned primary and metastatic partitioning now possible based on gene expression data. The search carried out 5,000 independent runs of our alternative stochastic search algorithm to generate 5,000 near-optimal gene signatures and 5,000 sets of near-optimal partitioning of the groups. By examining how often a sample was assigned to the primary and metastatic groups, we could estimate the proportion of runs where the sample was classified as a primary or metastatic SKCM tumor. We found that nearly all the clinically classified metastatic tumors were consistently assigned to the metastatic tumor group in 90-100% of the runs whereas the clinically classified primary SKCM tumors were often reassigned to the metastatic tumor group in proportions ranging from 2% to 80%. This result suggests that the gene expression profiles of many primary tumors resemble those of metastatic tumors to various degrees. Gene ontology analysis of the 500 most frequently selected genes (those appearing most frequently in the 5,000 gene signatures) suggested that the top-ranked genes are enriched in ectoderm and epidermis development, epithelia and epidermal cell differentiation, kerationization, and regulation of inflammatory and defense response. In summary, we have developed a unique computational method that not only assesses the relevance of genes in sample classification but also classifies each sample probabilistically to uncover the true tumor status. Our analysis may provide useful information for treatment and disease management. Citation Format: Yuanyuan Li, Juno Krahn, Leping Li. Assessing the similarity and dissimilarity between primary and metastatic melanoma using gene expression data. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A17.
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- 2015
43. Glypican 6 is a putative biomarker for metastatic progression of cutaneous melanoma.
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Yuanyuan Li, Melissa Li, Igor Shats, Juno M Krahn, Gordon P Flake, David M Umbach, Xiaoling Li, and Leping Li
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Medicine ,Science - Abstract
Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.
- Published
- 2019
- Full Text
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