82 results on '"D. Sohl"'
Search Results
2. Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen–Deuterium Exchange Mass Spectrometry
- Author
-
Kaitlyn A. Sabo, Elene Albekioni, Danielle Caliger, Nalani J. Coleman, Ella Thornberg, Diego Avellaneda Matteo, Elizabeth A. Komives, Steve Silletti, and Christal D. Sohl
- Subjects
Biochemistry ,Article - Abstract
Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP(+), and its catalytic metal cation. Here, we used hydrogen–deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.
- Published
- 2023
3. Supplementary Figures 1-2, Supplementary Tables 1-3 from The FGFR1 V561M Gatekeeper Mutation Drives AZD4547 Resistance through STAT3 Activation and EMT
- Author
-
Karen S. Anderson, BeiBei Luo, Christal D. Sohl, and Molly R. Ryan
- Abstract
Figure S1: qPCR of FGF2 mRNA expression in H1581 and L6 cells Figure S2: STAT3 knockdown sensitizes H1581-V561M cells to AZD4547 treatment. Table S1 and S2: Proliferation after various timepoints Table S3: AZD4547 sensitivity of L6 shRNA-treated cells
- Published
- 2023
4. Multiple chiral doublet bands in 104Rh
- Author
-
A. Krakó, D. Sohler, J. Timár, I. Kuti, Q.B. Chen, S.Q. Zhang, J. Meng, K. Starosta, T. Koike, E.S. Paul, D.B. Fossan, and C. Vaman
- Subjects
Physics ,QC1-999 - Abstract
Two new negative-parity bands have been identified in the odd-odd 104Rh nucleus. According to their experimentally observed properties, they have the same π(1g9/2)−1⊗ν(1h11/2)1 high-j configuration as the previously known negative-parity chiral doublet bands. This observation raises the possibility of the existence of multiple chiral doublet bands, MχD, in this nucleus. Comparing the properties of the observed bands with results of detailed theoretical calculations, one can conclude that the lower energy parts of bands 1 and 2 are chiral partner bands with the π(1g9/2)−1⊗ν(1h11/2)1 two-quasiparticle configuration, while bands 3 and 4 are chiral partner bands with the π(1g9/2)−1⊗ν(1g7/2)−2(1h11/2)1 four-quasiparticle configuration. Thus, MχD based on different configurations is observed in 104Rh.
- Published
- 2024
- Full Text
- View/download PDF
5. Spectroscopy of deeply bound orbitals in neutron-rich Ca isotopes
- Author
-
P.J. Li, J. Lee, P. Doornenbal, S. Chen, S. Wang, A. Obertelli, Y. Chazono, J.D. Holt, B.S. Hu, K. Ogata, Y. Utsuno, K. Yoshida, N.L. Achouri, H. Baba, F. Browne, D. Calvet, F. Château, N. Chiga, A. Corsi, M.L. Cortés, A. Delbart, J.-M. Gheller, A. Giganon, A. Gillibert, C. Hilaire, T. Isobe, T. Kobayashi, Y. Kubota, V. Lapoux, H.N. Liu, T. Motobayashi, I. Murray, H. Otsu, V. Panin, N. Paul, W. Rodriguez, H. Sakurai, M. Sasano, D. Steppenbeck, L. Stuhl, Y.L. Sun, Y. Togano, T. Uesaka, K. Wimmer, K. Yoneda, O. Aktas, T. Aumann, K. Boretzky, C. Caesar, L.X. Chung, F. Flavigny, S. Franchoo, I. Gasparic, R.-B. Gerst, J. Gibelin, K.I. Hahn, J. Kahlbow, D. Kim, T. Koiwai, Y. Kondo, D. Körper, P. Koseoglou, C. Lehr, B.D. Linh, T. Lokotko, M. MacCormick, K. Miki, K. Moschner, T. Nakamura, S.Y. Park, D. Rossi, E. Sahin, F. Schindler, H. Simon, P.-A. Söderström, D. Sohler, S. Takeuchi, H. Toernqvist, J. Tscheuschner, V. Vaquero, V. Wagner, V. Werner, X. Xu, H. Yamada, D. Yan, Z. Yang, M. Yasuda, and L. Zanetti
- Subjects
Unbound states ,Knockout reaction ,Single-particle strength ,Shell evolution ,Physics ,QC1-999 - Abstract
The calcium isotopes are an ideal system to investigate the evolution of shell structure and magic numbers. Although the properties of surface nucleons in calcium have been well studied, probing the structure of deeply bound nucleons remains a challenge. Here, we report on the first measurement of unbound states in 53Ca and 55Ca, populated from 54,56Ca(p,pn) reactions at a beam energy of around 216 MeV/nucleon at the RIKEN Radioactive Isotopes Beam Factory. The resonance properties, partial cross sections, and momentum distributions of these unbound states were analyzed. Orbital angular momentum l assignments were extracted from momentum distributions based on calculations using the distorted wave impulse approximation (DWIA) reaction model. The resonances at excitation energies of 5516(41)keV in 53Ca and 6000(250)keV in 55Ca indicate a significant l =3 component, providing the first experimental evidence for the ν0f7/2 single-particle strength of unbound hole states in the neutron-rich Ca isotopes. The observed excitation energies and cross-sections point towards extremely localized and well separated strength distributions, with some fragmentation for the ν0f7/2 orbital in 55Ca. These results are in good agreement with predictions from shell-model calculations using the effective GXPF1Bs interaction and ab initio calculations and diverge markedly from the experimental distributions in the nickel isotones at Z=28.
- Published
- 2024
- Full Text
- View/download PDF
6. An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity
- Author
-
Zachary Lesecq, David Scott, Katharine A. White, Diane L. Barber, Christal D. Sohl, Lucas A. Luna, Jamie M. Schiffer, Olga Zagnitko, Andrey A. Bobkov, Daniel G. Isom, and An Hoang
- Subjects
Isocitrates ,Biochemistry & Molecular Biology ,Protein Conformation ,Dimer ,Protonation ,Medical and Health Sciences ,Biochemistry ,Article ,Catalysis ,Substrate Specificity ,Glutarates ,Residue (chemistry) ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Catalytic Domain ,enzyme kinetics ,Lipid biosynthesis ,Humans ,cancer ,Enzyme kinetics ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Active site ,Cell Biology ,Hydrogen-Ion Concentration ,Biological Sciences ,Isocitrate Dehydrogenase ,post translational modification ,Kinetics ,Cytosol ,Isocitrate dehydrogenase ,Enzyme ,post-translational modification ,chemistry ,pH regulation ,buried ionizable residues ,030220 oncology & carcinogenesis ,Mutation ,Chemical Sciences ,biology.protein ,Biophysics ,tumor metabolism - Abstract
Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (α-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasingkcatvalues for α-KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKavalues. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKavalue upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.
- Published
- 2020
7. Probing altered enzyme activity in the biochemical characterization of cancer
- Author
-
Mowaffaq Adam Ahmed Adam and Christal D. Sohl
- Subjects
Kinetics ,Neoplasms ,Mutation ,Biophysics ,Tumor Microenvironment ,Humans ,Cell Biology ,Molecular Biology ,Biochemistry ,Catalysis - Abstract
Enzymes have evolved to catalyze their precise reactions at the necessary rates, locations, and time to facilitate our development, to respond to a variety of insults and challenges, and to maintain a healthy, balanced state. Enzymes achieve this extraordinary feat through their unique kinetic parameters, myriad regulatory strategies, and their sensitivity to their surroundings, including substrate concentration and pH. The Cancer Genome Atlas (TCGA) highlights the extraordinary number of ways in which the finely tuned activities of enzymes can be disrupted, contributing to cancer development and progression often due to somatic and/or inherited genetic alterations. Rather than being limited to the domain of enzymologists, kinetic constants such as kcat, Km, and kcat/Km are highly informative parameters that can impact a cancer patient in tangible ways—these parameters can be used to sort tumor driver mutations from passenger mutations, to establish the pathways that cancer cells rely on to drive patients’ tumors, to evaluate the selectivity and efficacy of anti-cancer drugs, to identify mechanisms of resistance to treatment, and more. In this review, we will discuss how changes in enzyme activity, primarily through somatic mutation, can lead to altered kinetic parameters, new activities, or changes in conformation and oligomerization. We will also address how changes in the tumor microenvironment can affect enzymatic activity, and briefly describe how enzymology, when combined with additional powerful tools, and can provide us with tremendous insight into the chemical and molecular mechanisms of cancer.
- Published
- 2021
8. Evaluating Mechanisms of IDH1 Regulation through Site-Specific Acetylation Mimics
- Author
-
Alexandra I Strom, Joi Weeks, Christal D. Sohl, Sati Alexander, Vinnie Widjaja, and Dahra K Pucher
- Subjects
0301 basic medicine ,IDH1 ,Kinetics ,Mutant ,Lysine ,Proteomics ,Biochemistry ,Microbiology ,Article ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Computer Simulation ,Molecular Biology ,acetylation ,Brain Neoplasms ,Chemistry ,Glioma ,Isocitrate Dehydrogenase ,Recombinant Proteins ,QR1-502 ,Glutamine ,030104 developmental biology ,Isocitrate dehydrogenase ,post-translational modification ,Acetylation ,kinetics ,Mutation ,Mutagenesis, Site-Directed ,Glioblastoma ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery - Abstract
Isocitrate dehydrogenase (IDH1) catalyzes the reversible NADP+-dependent oxidation of isocitrate to α-ketoglutarate (αKG). IDH1 mutations, primarily R132H, drive >, 80% of low-grade gliomas and secondary glioblastomas and facilitate the NADPH-dependent reduction of αKG to the oncometabolite D-2-hydroxyglutarate (D2HG). While the biochemical features of human WT and mutant IDH1 catalysis have been well-established, considerably less is known about mechanisms of regulation. Proteomics studies have identified lysine acetylation in WT IDH1, indicating post-translational regulation. Here, we generated lysine to glutamine acetylation mimic mutants in IDH1 to evaluate the effects on activity. We show that mimicking lysine acetylation decreased the catalytic efficiency of WT IDH1, with less severe catalytic consequences for R132H IDH1.
- Published
- 2021
9. The FGFR1 V561M Gatekeeper Mutation Drives AZD4547 Resistance through STAT3 Activation and EMT
- Author
-
Molly R. Ryan, Christal D. Sohl, BeiBei Luo, and Karen S. Anderson
- Subjects
STAT3 Transcription Factor ,0301 basic medicine ,Cancer Research ,Epithelial-Mesenchymal Transition ,Lung Neoplasms ,Antineoplastic Agents ,Drug resistance ,medicine.disease_cause ,Article ,Piperazines ,03 medical and health sciences ,0302 clinical medicine ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,medicine ,Humans ,Receptor, Fibroblast Growth Factor, Type 1 ,Epithelial–mesenchymal transition ,STAT3 ,Molecular Biology ,Mutation ,Gene knockdown ,biology ,Kinase ,Cancer ,medicine.disease ,stomatognathic diseases ,030104 developmental biology ,Oncology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Benzamides ,Cancer cell ,Cancer research ,biology.protein ,Pyrazoles - Abstract
FGFR1 has been implicated in numerous cancer types including squamous cell lung cancer, a subset of non–small cell lung cancer with a dismal 5-year survival rate. Small-molecule inhibitors targeting FGFR1 are currently in clinical trials, with AZD4547 being one of the furthest along; however, the development of drug resistance is a major challenge for targeted therapies. A prevalent mechanism of drug resistance in kinases occurs through mutation of the gatekeeper residue, V561M in FGFR1; however, mechanisms underlying V561M resistance to AZD4547 are not fully understood. Here, the cellular consequences of the V561M gatekeeper mutation were characterized, and it was found that although AZD4547 maintains nanomolar affinity for V561M FGFR1, based on in vitro binding assays, cells expressing V561M demonstrate dramatic resistance to AZD4547 driven by increased STAT3 activation downstream of V561M FGFR1. The data reveal that the V561M mutation biases cells toward a more mesenchymal phenotype, including increased levels of proliferation, migration, invasion, and anchorage-independent growth, which was confirmed using CyTOF, a novel single-cell analysis tool. Using shRNA knockdown, loss of STAT3 restored sensitivity of cancer cells expressing V561M FGFR1 to AZD4547. Thus, the data demonstrate that combination therapies including FGFR and STAT3 may overcome V561M FGFR1–driven drug resistance in the clinic. Implications: The V561M FGFR1 gatekeeper mutation leads to devastating drug resistance through activation of STAT3 and the epithelial–mesenchymal transition; this study demonstrates that FGFR1 inhibitor sensitivity can be restored upon STAT3 knockdown.
- Published
- 2019
10. Readying students for careers in industry: A guided inquiry activity to prepare students for success in biotechnology and pharmaceutical industry positions
- Author
-
Stefan Gross and Christal D. Sohl
- Subjects
Drug Industry ,media_common.quotation_subject ,Context (language use) ,Biochemistry ,03 medical and health sciences ,Presentation ,ComputingMilieux_COMPUTERSANDEDUCATION ,Humans ,Sociology ,Students ,Molecular Biology ,030304 developmental biology ,media_common ,0303 health sciences ,Career Choice ,business.industry ,05 social sciences ,050301 education ,Due diligence ,Biotechnology ,Framing (social sciences) ,Critical thinking ,Instructional scaffolding ,Coursework ,Curriculum ,business ,0503 education ,Career development - Abstract
While science students are well prepared for careers in biotechnology and pharmaceutical sciences in terms of technical expertise and critical thinking, they rarely have an opportunity to practice the due diligence required for success in industry in their coursework. This includes framing their expertise as solutions to challenges a company may be experiencing, an important skill for the interview process. As most academics have not applied for positions in industry, they may feel ill equipped to help students practice the important skills of framing their expertise within company goals and to discuss the business and financial concepts relevant to careers in scientific industry. Here, we describe an educational activity first developed by a leader in the biotech/pharmaceutical industry that was modified and given educational context by an academic in a class of upper-level undergraduate and graduate students. In this guided inquiry activity, students were instructed to select a start-up company in their field-ideally one to which they intended to apply for a job. Students were empowered by scaffolded hands-on exercises to research the company's scientific focus and finances, and to frame how their expertise could help companies achieve stated goals. Students compiled and delivered their research as an in-class presentation.
- Published
- 2020
11. Water Networks and Correlated Motions in Mutant Isocitrate Dehydrogenase 1 (IDH1) Are Critical for Allosteric Inhibitor Binding and Activity
- Author
-
Wade Miller, Jennifer M. Chambers, Christal D. Sohl, Jamie M. Schiffer, Andrey A. Bobkov, Viraj Upadhye, Diego Avellaneda Matteo, and Giovanni Quichocho
- Subjects
Isocitrates ,IDH1 ,Allosteric regulation ,Mutant ,Molecular Dynamics Simulation ,Biochemistry ,Article ,Biophysical Phenomena ,Catalysis ,Glutarates ,Catalytic Domain ,Humans ,Binding site ,chemistry.chemical_classification ,Binding Sites ,Chemistry ,Wild type ,Water ,Small molecule ,Isocitrate Dehydrogenase ,Kinetics ,Isocitrate dehydrogenase ,Enzyme ,Mutation ,Biophysics ,Ketoglutaric Acids - Abstract
Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP(+)-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP(+) binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.
- Published
- 2020
12. Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells
- Author
-
Lucas A. Luna, Christal D. Sohl, Michael Coste, Alex E. Clark, Sungjun Beck, Grace Wells, David Hecht, Jean A. Bernatchez, Jeremy N. Rich, Jair L. Siqueira-Neto, Byron W. Purse, and Zhe Zhu
- Subjects
0301 basic medicine ,Sofosbuvir ,NS5 ,Hepatitis C virus ,viruses ,030106 microbiology ,lcsh:QR1-502 ,Protide ,medicine.disease_cause ,RNA-dependent RNA polymerase ,lcsh:Microbiology ,Article ,Zika virus ,Small Molecule Libraries ,03 medical and health sciences ,immune system diseases ,prodrugs ,Virology ,Drug Discovery ,antiviral agents ,medicine ,Humans ,reproductive and urinary physiology ,neural stem cells ,Nucleoside analogue ,Chemistry ,virus diseases ,Phosphoramidate ,Nucleosides ,ProTides ,Prodrug ,Ribonucleoside ,3. Good health ,nervous system diseases ,030104 developmental biology ,Infectious Diseases ,nucleoside analogues ,Nucleoside ,medicine.drug - Abstract
Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré, syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2&prime, C-methyluridine and 2&prime, C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2&prime, C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2&prime, C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound&rsquo, s triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.
- Published
- 2019
- Full Text
- View/download PDF
13. Post Translational Modifications in MDH1 May Affect Lung Cancer Survival
- Author
-
Joi Weeks, Sati Alexander, An Hoang, and Christal D. Sohl
- Subjects
business.industry ,Genetics ,Cancer research ,Posttranslational modification ,Medicine ,business ,Lung cancer ,medicine.disease ,Affect (psychology) ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2019
14. Molecular Mechanisms of Isocitrate Dehydrogenase 1 Mutants in Driving the Oncogenic Neomorphic Reaction
- Author
-
Giovanni Quichocho, Diego Avellaneda Matteo, Ruth Xu, Michelle Scott, Lucas A. Luna, Christal D. Sohl, and Jeon Erik Fonbon
- Subjects
Isocitrate dehydrogenase ,Biochemistry ,Chemistry ,Mutant ,Genetics ,Molecular Biology ,Biotechnology - Published
- 2019
15. Ocean Color Instrument Integration and Testing
- Author
-
George Hilton, Mellina Espiritu, Susanna Petro, and D. Sohl
- Subjects
Spacecraft ,business.industry ,Integration testing ,Electromagnetic compatibility ,law.invention ,Telescope ,Ocean color ,Observatory ,law ,Environmental science ,System integration ,Satellite ,Aerospace engineering ,business ,Physics::Atmospheric and Oceanic Physics - Abstract
This paper describes the plans, flows, key facilities, components and equipment necessary to fully integrate, functionally test, qualify and calibrate the Ocean Color Instrument (OCI) on the Plankton, Aerosols, Clouds, and oceans Ecosystem (PACE) observatory. PACE is currently in the design phase of mission development. It is scheduled to launch in 2022, extending and improving NASA's twenty-year record of satellite observations of global ocean biology, aerosols and clouds. PACE will advance the assessment of ocean health by measuring the distribution of phytoplankton, which are small plants and algae that sustain the marine food web. It will also continue systematic records of key atmospheric variables associated with air quality and the Earth's climate. PACE's primary sensor, the OCI, is a highly advanced optical spectrometer that will be used to measure properties of light over portions of the electromagnetic spectrum. It will enable continuous measurement of light at finer wavelength resolution than previous NASA satellite sensors, extending key system ocean color data records for climate studies. The color of the ocean is determined by the interaction of sunlight with substances or particles present in seawater such as chlorophyll. By monitoring global phytoplankton distribution and abundance with unprecedented detail, the OCI will contribute to a better understanding of the complex systems that drive ocean ecology and it's impacts on global fisheries. This paper will focus on the Integration and Test (I&T) activities for OCI while it is at the NASA Goddard Space Flight Center. The OCI integration consists of assembly and alignment of the rotating telescope, electronics box integration, fixed deck assembly integration, thermal systems integration and the final assembly and testing. This I&T phase will be followed by the OCI calibration and characterization, environmental tests which include electromagnetic interference (EMI)/electromagnetic compatibility (EMC), vibration with sine sweep, acoustics, shock, thermal balance, thermal vacuum, mass properties and center of gravity. This paper will briefly discuss OCI shipment and delivery to the spacecraft vendor for observatory level I&T as well as some launch preparation activities
- Published
- 2019
16. Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells
- Author
-
Michael Coste, Lucas A. Luna, Zhe Zhu, Byron W. Purse, Grace Wells, Sungjun Beck, Jair L. Siqueira-Neto, Jean A. Bernatchez, Christal D. Sohl, and Alex E. Clark
- Subjects
0303 health sciences ,Sofosbuvir ,Nucleoside analogue ,030306 microbiology ,Chemistry ,Hepatitis C virus ,Protide ,Phosphoramidate ,Prodrug ,Ribonucleoside ,medicine.disease_cause ,Virology ,3. Good health ,03 medical and health sciences ,medicine ,Nucleoside ,030304 developmental biology ,medicine.drug - Abstract
Zika virus (ZIKV), an emerging flavivirus which causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome, continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2′-C-methyluridine and 2′-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Conversion of the aryloxyl phosphoramidate ProTide group of 2′-C-methyluridine to a 2-(methylthio)ethyl phosphoramidate completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2′-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound’s triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. Molecular superpositioning revealed different orientations of residues opposite the 2′-fluoro group of sofosbuvir. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.
- Published
- 2019
- Full Text
- View/download PDF
17. A Tie2 kinase mutation causing venous malformations increases phosphorylation rates and enhances cooperativity
- Author
-
Zeqing Xu, Christal D. Sohl, Yunjin Wu, and Madison A. Kennedy
- Subjects
0301 basic medicine ,Vascular Malformations ,Biophysics ,Cooperativity ,Plasma protein binding ,medicine.disease_cause ,Biochemistry ,Receptor tyrosine kinase ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Phosphorylation ,Molecular Biology ,Mutation ,biology ,Chemistry ,Kinase ,Wild type ,Cooperative binding ,Cell Biology ,Molecular biology ,Receptor, TIE-2 ,Kinetics ,030104 developmental biology ,030220 oncology & carcinogenesis ,embryonic structures ,biology.protein ,cardiovascular system ,Biocatalysis ,Endothelium, Vascular ,Protein Binding - Abstract
The endothelial receptor tyrosine kinase Tie2 plays an important role in vascular formation and maintenance. Mutations in Tie2 lead to vascular malformations, which are painful vascular lesions that cause disfigurement, bleeding, and thrombosis. R849W Tie2 is the most common mutation implicated in an inherited form of vascular malformations and has been shown to be activating, though little is known about the kinetic features of catalysis. Here we undertake a steady-state kinetic analysis of heterologously expressed and purified wild type (WT) and R849W Tie2. While the catalytic efficiencies of the two forms are not significantly different, the observed maximal rate of phosphorylation, k(cat,obs), is > 3-fold higher for R849W Tie2 compared to WT. Notably, steady-state catalysis by R849W Tie2 has more striking sigmoidal features compared to WT, suggesting enhanced positive cooperativity. We propose that activating catalytic features are one important consequence of the R849W mutation, though likely other factors such as increased protein binding affinity also contribute to the phenotypes observed in patients.
- Published
- 2018
18. Abstract 3732: Characterization of tumor relevant isocitrate dehydrogenase 1 (IDH1)
- Author
-
Christal D. Sohl, Lucas A. Luna, Nalani J. Coleman, Danielle Caliger, and Ella Thornberg
- Subjects
Cancer Research ,IDH1 ,Isocitrate dehydrogenase ,Oncology ,Biochemistry ,Chemistry - Abstract
Isocitrate dehydrogenase 1 (IDH1) is found commonly mutated in grade II and III gliomas and secondary glioblastomas. Normally, IDH1 catalyzes the conversion of isocitrate and NADP+ to α-ketoglutarate and NADPH. However, in mutated form, IDH1 catalyzes α-ketoglutarate and NADPH into NADP+ and D-2-hydroxyglutarate, an oncometabolite. D-2-hydroxyglutarate is toxic to cells and drives a number of pro-tumorigenic pathways. To understand the molecular mechanisms of how mutated IDH1 can lead to brain cancer, we created point mutations in IDH1 to understand the effects on enzyme activity. Site-directed mutagenesis was used to generate mutations at residue 132, the most common site of mutations in cancer patients. Wild type (WT) IDH1 and mutant IDH1 were heterologously expressed in bacteria . Following purification using affinity column chromatography, steady-state kinetic assays were used to compare the catalytic activity of WT versus mutant IDH1. By elucidating the types of mutations at R132 that facilitate D2HG production, we can have a better understanding of how IDH1 mutations affect prognosis and therapeutic response. This work was funded by a Research Scholar Grant, RSG-19-075-01-TBE, from the American Cancer Society (CDS), National Institutes of Health R00 CA187594 (CDS), National Institutes of Health U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), and the California Metabolic Research Foundation (SDSU). Citation Format: Nalani J. Coleman, Ella Thornberg, Danielle Caliger, Lucas Luna, Christal D. Sohl. Characterization of tumor relevant isocitrate dehydrogenase 1 (IDH1) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3732.
- Published
- 2020
19. The Catalytic Features of IDH1 Mutations Can Drive Neomorphic Activity, Phenotype Severity, and Inhibitor Binding
- Author
-
Lucas A. Luna, Jennifer M. Chambers, Diego Avellaneda Matteo, Jamie M. Schiffer, Christal D. Sohl, Wade Miller, David Scott, Grace Wells, Viraj Upadhye, and Giovanni Quichocho
- Subjects
IDH1 ,Chemistry ,Genetics ,Molecular Biology ,Biochemistry ,Phenotype ,Biotechnology ,Catalysis ,Cell biology - Published
- 2020
20. Catalytic Characterization of Human Malate Dehydrogenase 1 (MDH1)
- Author
-
An Hoang, Christal D. Sohl, and Ngoc H. Huynh
- Subjects
Biochemistry ,Chemistry ,Genetics ,Malate dehydrogenase 1 ,Molecular Biology ,Biotechnology ,Catalysis - Published
- 2020
21. The effect of acetylation on isocitrate dehydrogenase 1 regulation
- Author
-
Joi Weeks, Alexandra I Strom, Diego Avellaneda Matteo, Sati Alexander, Lucas A. Luna, An Hoang, Tin Duc Nguyen, Vinnie Widjaja, Dahra K Pucher, and Christal D. Sohl
- Subjects
Isocitrate dehydrogenase ,Biochemistry ,Acetylation ,Chemistry ,Genetics ,Molecular Biology ,Biotechnology - Published
- 2020
22. Characterization of Amplified Malate Dehydrogenase 1 (MDH1) in the Context of Squamous Cell Lung Cancer
- Author
-
Sati Alexander, An Hoang, Joi Weeks, and Christal D. Sohl
- Subjects
Genetics ,Cancer research ,Context (language use) ,Malate dehydrogenase 1 ,Biology ,Molecular Biology ,Biochemistry ,Squamous cell lung cancer ,Biotechnology - Published
- 2020
23. Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants
- Author
-
Olga Zagnitko, An Hoang, Amit Luthra, Manal A. Swairjo, Christal D. Sohl, Lucas A. Luna, Adam J. Grunseth, Jamie M. Schiffer, Grace Wells, David Scott, and Diego Avellaneda Matteo
- Subjects
0301 basic medicine ,IDH1 ,Carcinogenesis ,Mutant ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Molecular Biology ,Mutation ,Binding Sites ,biology ,Chemistry ,Myeloid leukemia ,Cell Biology ,Resistance mutation ,Molecular biology ,Isocitrate Dehydrogenase ,Protein Structure, Tertiary ,030104 developmental biology ,Isocitrate dehydrogenase ,HEK293 Cells ,Enzyme inhibitor ,030220 oncology & carcinogenesis ,biology.protein ,HeLa Cells - Abstract
Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient's therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.
- Published
- 2018
24. Development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting the Zika virus
- Author
-
Jean A. Bernatchez, Jerry Li, Zunhua Yang, Christal D. Sohl, Lucas A. Luna, Rongshi Li, Yan Liu, Alex E. Clark, Michael Coste, Sungjun Beck, Jair L. Siqueira-Neto, Zhe Zhu, and Byron W. Purse
- Subjects
Microcephaly ,biology ,medicine.diagnostic_test ,medicine.disease ,biology.organism_classification ,Immunofluorescence ,Virology ,Virus ,Zika virus ,Cell culture ,Vero cell ,medicine ,Viability assay ,Cytopathic effect - Abstract
Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat infection, and accordingly there is an unmet medical need for discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay used to detect cells affected by the viral-induced cytopathic effect (CPE) using automated imaging and analysis. Protection from CPE correlates with a decrease in viral antigen production as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues against ZIKV; the previously reported antiviral activities of 2’-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. Profiling of a novel library of 24 natural product derivatives using our assay revealed compound 1 as an inhibitor of ZIKV-induced cytopathic effect; activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling large compound libraries against ZIKV, and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.
- Published
- 2018
25. Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus
- Author
-
Jerry Li, Jair L. Siqueira-Neto, Alex E. Clark, Zhe Zhu, Zunhua Yang, Yan Liu, Lucas A. Luna, Sungjun Beck, Rongshi Li, Christal D. Sohl, Byron W. Purse, Michael Coste, and Jean A. Bernatchez
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,High-throughput screening ,030106 microbiology ,Biology ,Immunofluorescence ,Antiviral Agents ,Virus ,Zika virus ,03 medical and health sciences ,Chlorocebus aethiops ,medicine ,Animals ,Pharmacology (medical) ,Viability assay ,Vero Cells ,Cytopathic effect ,Pharmacology ,medicine.diagnostic_test ,Zika Virus Infection ,Zika Virus ,biology.organism_classification ,Virology ,030104 developmental biology ,Infectious Diseases ,Cell culture ,Vero cell - Abstract
Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barre syndrome. There are currently no drugs available to treat ZIKV infection, and accordingly, there is an unmet medical need for the discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity against ZIKV; the previously reported antiviral activities of 2'-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKV-induced cytopathic effect; the activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.
- Published
- 2018
26. Expression and Characterization of the Receptor Tyrosine Kinase Tie2
- Author
-
Adam J. Grunseth, Christal D. Sohl, Madison A. Kennedy, and Yunjin Wu
- Subjects
biology ,Chemistry ,Genetics ,biology.protein ,Molecular Biology ,Biochemistry ,Angiopoietin receptor ,Receptor tyrosine kinase ,Biotechnology ,Cell biology - Published
- 2018
27. Understanding the Structure‐Function Relationship of IDH1 R132 mutants and the Effect of Allosteric Inhibitors
- Author
-
Grace Wells, Adam J. Grunseth, Jamie M. Schiffer, Diego Avellaneda Matteo, and Christal D. Sohl
- Subjects
IDH1 ,Biochemistry ,Chemistry ,Structure function ,Allosteric regulation ,Mutant ,Genetics ,Molecular Biology ,Biotechnology - Published
- 2018
28. Illuminating the Molecular Mechanisms of Tyrosine Kinase Inhibitor Resistance for the FGFR1 Gatekeeper Mutation: The Achilles’ Heel of Targeted Therapy
- Author
-
BeiBei Luo, Karen S. Anderson, Kathleen M. Frey, Molly R. Ryan, and Christal D. Sohl
- Subjects
Models, Molecular ,Protein Conformation ,medicine.drug_class ,Mutant ,medicine.disease_cause ,Biochemistry ,Article ,Receptor tyrosine kinase ,Tyrosine-kinase inhibitor ,medicine ,Humans ,Receptor, Fibroblast Growth Factor, Type 1 ,Phosphorylation ,Protein Kinase Inhibitors ,Mutation ,biology ,Autophosphorylation ,Wild type ,General Medicine ,Protein-Tyrosine Kinases ,Kinetics ,Drug Resistance, Neoplasm ,Fibroblast growth factor receptor ,biology.protein ,Cancer research ,Molecular Medicine ,Tyrosine kinase - Abstract
Human fibroblast growth factor receptors (FGFRs) 1–4 are a family of receptor tyrosine kinases that can serve as drivers of tumorigenesis. In particular, FGFR1 gene amplification has been implicated in squamous cell lung and breast cancers. Tyrosine kinase inhibitors (TKIs) targeting FGFR1, including AZD4547 and E3810 (Lucitanib), are currently in early phase clinical trials. Unfortunately, drug resistance limits the long-term success of TKIs, with mutations at the “gatekeeper” residue leading to tumor progression. Here we show the first structural and kinetic characterization of the FGFR1 gatekeeper mutation, V561M FGFR1. The V561M mutation confers a 38-fold increase in autophosphorylation achieved at least in part by a network of interacting residues forming a hydrophobic spine to stabilize the active conformation. Moreover, kinetic assays established that the V561M mutation confers significant resistance to E3810, while retaining affinity for AZD4547. Structural analyses of these TKIs with wild type (WT) and gatekeeper mutant forms of FGFR1 offer clues to developing inhibitors that maintain potency against gatekeeper mutations. We show that AZD4547 affinity is preserved by V561M FGFR1 due to a flexible linker that allows multiple inhibitor binding modes. This is the first example of a TKI binding in distinct conformations to WT and gatekeeper mutant forms of FGFR, highlighting adaptable regions in both the inhibitor and binding pocket crucial for drug design. Exploiting inhibitor flexibility to overcome drug resistance has been a successful strategy for combatting diseases such as AIDS and may be an important approach for designing inhibitors effective against kinase gatekeeper mutations.
- Published
- 2015
29. Level structures of 56,58Ca cast doubt on a doubly magic 60Ca
- Author
-
S. Chen, F. Browne, P. Doornenbal, J. Lee, A. Obertelli, Y. Tsunoda, T. Otsuka, Y. Chazono, G. Hagen, J.D. Holt, G.R. Jansen, K. Ogata, N. Shimizu, Y. Utsuno, K. Yoshida, N.L. Achouri, H. Baba, D. Calvet, F. Château, N. Chiga, A. Corsi, M.L. Cortés, A. Delbart, J.-M. Gheller, A. Giganon, A. Gillibert, C. Hilaire, T. Isobe, T. Kobayashi, Y. Kubota, V. Lapoux, H.N. Liu, T. Motobayashi, I. Murray, H. Otsu, V. Panin, N. Paul, W. Rodriguez, H. Sakurai, M. Sasano, D. Steppenbeck, L. Stuhl, Y.L. Sun, Y. Togano, T. Uesaka, K. Wimmer, K. Yoneda, O. Aktas, T. Aumann, L.X. Chung, F. Flavigny, S. Franchoo, I. Gasparic, R.-B. Gerst, J. Gibelin, K.I. Hahn, D. Kim, T. Koiwai, Y. Kondo, P. Koseoglou, C. Lehr, B.D. Linh, T. Lokotko, M. MacCormick, K. Moschner, T. Nakamura, S.Y. Park, D. Rossi, E. Sahin, P.-A. Söderström, D. Sohler, S. Takeuchi, H. Törnqvist, V. Vaquero, V. Wagner, S. Wang, V. Werner, X. Xu, H. Yamada, D. Yan, Z. Yang, M. Yasuda, and L. Zanetti
- Subjects
Shell evolution ,γ Ray spectroscopy ,Physics ,QC1-999 - Abstract
Gamma decays were observed in 56Ca and 58Ca following quasi-free one-proton knockout reactions from 57,59Sc beams at ≈200 MeV/nucleon. For 56Ca, a γ ray transition was measured to be 1456(12) keV, while for 58Ca an indication for a transition was observed at 1115(34) keV. Both transitions were tentatively assigned as the 21+→0gs+ decays, and were compared to results from ab initio and conventional shell-model approaches. A shell-model calculation in a wide model space with a marginally modified effective nucleon-nucleon interaction depicts excellent agreement with experiment for 21+ level energies, two-neutron separation energies, and reaction cross sections, corroborating the formation of a new nuclear shell above the N = 34 shell. Its constituents, the 0f5/2 and 0g9/2 orbitals, are almost degenerate. This degeneracy precludes the possibility for a doubly magic 60Ca and potentially drives the dripline of Ca isotopes to 70Ca or even beyond.
- Published
- 2023
- Full Text
- View/download PDF
30. Intruder configurations in 29Ne at the transition into the island of inversion: Detailed structure study of 28Ne
- Author
-
H. Wang, M. Yasuda, Y. Kondo, T. Nakamura, J.A. Tostevin, K. Ogata, T. Otsuka, A. Poves, N. Shimizu, K. Yoshida, N.L. Achouri, H. Al Falou, L. Atar, T. Aumann, H. Baba, K. Boretzky, C. Caesar, D. Calvet, H. Chae, N. Chiga, A. Corsi, H.L. Crawford, F. Delaunay, A. Delbart, Q. Deshayes, Zs. Dombrádi, C. Douma, Z. Elekes, P. Fallon, I. Gašparić, J.-M. Gheller, J. Gibelin, A. Gillibert, M.N. Harakeh, A. Hirayama, C.R. Hoffman, M. Holl, A. Horvat, Á. Horváth, J.W. Hwang, T. Isobe, J. Kahlbow, N. Kalantar-Nayestanaki, S. Kawase, S. Kim, K. Kisamori, T. Kobayashi, D. Körper, S. Koyama, I. Kuti, V. Lapoux, S. Lindberg, F.M. Marqués, S. Masuoka, J. Mayer, K. Miki, T. Murakami, M.A. Najafi, K. Nakano, N. Nakatsuka, T. Nilsson, A. Obertelli, N.A. Orr, H. Otsu, T. Ozaki, V. Panin, S. Paschalis, A. Revel, D. Rossi, A.T. Saito, T. Saito, M. Sasano, H. Sato, Y. Satou, H. Scheit, F. Schindler, P. Schrock, M. Shikata, Y. Shimizu, H. Simon, D. Sohler, O. Sorlin, L. Stuhl, S. Takeuchi, M. Tanaka, M. Thoennessen, H. Törnqvist, Y. Togano, T. Tomai, J. Tscheuschner, J. Tsubota, T. Uesaka, Z. Yang, and K. Yoneda
- Subjects
In-beam γ-ray spectroscopy ,Island of inversion ,Shell evolution ,Physics ,QC1-999 - Abstract
Detailed γ-ray spectroscopy of the exotic neon isotope 28Ne has been performed for the first time using the one-neutron removal reaction from 29Ne on a liquid hydrogen target at 240 MeV/nucleon. Based on an analysis of parallel momentum distributions, a level scheme with spin-parity assignments has been constructed for 28Ne and the negative-parity states are identified for the first time. The measured partial cross sections and momentum distributions reveal a significant intruder p-wave strength providing evidence of the breakdown of the N=20 and N=28 shell gaps. Only a weak, possible f-wave strength was observed to bound final states. Large-scale shell-model calculations with different effective interactions do not reproduce the large p-wave and small f-wave strength observed experimentally, indicating an ongoing challenge for a complete theoretical description of the transition into the island of inversion along the Ne isotopic chain.
- Published
- 2023
- Full Text
- View/download PDF
31. Molecular mechanisms of isocitrate dehydrogenase 1 (IDH1) mutations identified in tumors: The role of size and hydrophobicity at residue 132 on catalytic efficiency
- Author
-
Diego Avellaneda Matteo, Madison A. Kennedy, Precious Moman, An Hoang, Christal D. Sohl, Eric R. Gonzalez, David Scott, Stacy L. Anselmo, and Adam J. Grunseth
- Subjects
0301 basic medicine ,IDH1 ,Mutant ,Protein Engineering ,Biochemistry ,Catalysis ,Gas Chromatography-Mass Spectrometry ,03 medical and health sciences ,Catalytic Domain ,Neoplasms ,Humans ,Enzyme kinetics ,Molecular Biology ,chemistry.chemical_classification ,biology ,Dose-Response Relationship, Drug ,Circular Dichroism ,Temperature ,Active site ,Cell Biology ,Glioma ,Peroxisome ,Isocitrate Dehydrogenase ,Oxygen ,Cytosol ,030104 developmental biology ,Enzyme ,Isocitrate dehydrogenase ,chemistry ,Mutation ,biology.protein ,Enzymology ,Protein Multimerization ,Hydrophobic and Hydrophilic Interactions ,NADP ,Software - Abstract
Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate (ICT) to α-ketoglutarate (αKG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of αKG to d-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit αKG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ∼640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas.
- Published
- 2017
32. Abstract 4381: Investigating the reversible MDH1 catalytic reaction in squamous non-small cell lung cancer
- Author
-
Christian M. Metallo, Joi Weeks, Sati Alexander, Grace Wells, and Christal D. Sohl
- Subjects
chemistry.chemical_classification ,Cancer Research ,Cell growth ,Cell ,Cancer ,medicine.disease ,Malate dehydrogenase ,Cytosol ,Enzyme ,medicine.anatomical_structure ,Oncology ,chemistry ,Cancer research ,medicine ,Glycolysis ,NAD+ kinase - Abstract
Non-small-cell lung cancer (NSCLC) is a leading cause of death in men and women worldwide. NSCLC relies on glycolysis to support cell proliferation and to synthesize proteins, nucleic acids and lipids. Malate dehydrogenase (MDH) is an enzyme critical for replenishing intermediates in the tricarboxylic acid (TCA) cycle and for helping to drive glycolysis through its reversible coenzyme system NAD+/NADH by interconverting oxaloacetate and malate. The cytosolic form of MDH (MDH1) has been reported to be amplified in squamous cell NSCLC, which has high glycolytic activity. Elucidation of the role of MDH1 in altering cellular metabolism to drive tumorigenic progression has yet to be determined. Thus, our interest lies in identifying the chemical and cellular characteristics required to drive the reversible MDH1 catalytic reaction in squamous cell NSCLC. Previously, we have shown that metabolic dehydrogenases are regulated by microenvironmental changes in pH and oxidation levels, and we are currently extending this work to MDH1. We are also evaluating various cell lines to gain a better understanding of how the MDH1 reaction can be altered to decrease the tumorigenic capacity of squamous cell NSCLC. Experimental knowledge obtained has the potential to increase our understanding of the MDH1 reaction and educate the design of future anti-tumor therapeutics. Citation Format: Joi L. Weeks, Grace Wells, Sati Alexander, Christian Metallo, Christal D. Sohl. Investigating the reversible MDH1 catalytic reaction in squamous non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4381.
- Published
- 2019
33. On spaceflight instrument adaptive electrical and electronics subsystem functional framework
- Author
-
A. M. Parsons, Renee M. Reynolds, Jeffrey S. Smith, D. Sohl, and Semion Kizhner
- Subjects
Engineering ,Spacecraft ,business.industry ,Astrophysics::Instrumentation and Methods for Astrophysics ,NASA Deep Space Network ,01 natural sciences ,Space exploration ,Space launch ,law.invention ,010309 optics ,Telescope ,Orbiter ,law ,Observatory ,Physics::Space Physics ,0103 physical sciences ,Satellite ,Astrophysics::Earth and Planetary Astrophysics ,Aerospace engineering ,business ,010303 astronomy & astrophysics - Abstract
Near-earth heritage spaceflight missions and contemporary large observatories such as the NASA Hubble Space Telescope (HST), future James Web Space Telescope (JWST), or planetary fly-by (Cassini with its 12 instruments) and orbiting deep space observatories like the Mars Reconnaissance Orbiter (MRO) have carried large electronics subsystems. The Kepler cosmology observatory 95 mega pixels focal plane servicing electronics comprises some 50 electronic boards. The future cosmology mission Wide Field Infra-Red Survey Telescope (WFIRST with 2 instruments) envisions a focal plane with 18 large 4Kx4K sensors totaling 4.8318e+09 bits also serviced by a multitude of electronic boards. On the other hand, a new class of NASA Earth small satellite missions (SmallSat), the Department of Defense Operationally Responsive Space small satellites (ORS) and planetary surface mission instruments require smaller scale electrical and electronics subsystems. These are challenged with unique external space launch technology requirements for ever-smaller mass and volume, constraints on power and communications bandwidth, in addition to the requirements of space extreme environment of temperature variations and cosmic radiation. Within this wide range of space exploration and earth remote-sensing missions there is a need to revisit these external and internal spaceflight instrument science requirements from the point of view of developing the future spaceflight instrument scalable and adaptive electrical and electronics subsystem (IAEES) conceptual framework. We consider these instrument requirements and conceptual functionality framework on the precedent of the two future Decadal missions — the flagship cosmology observatory WFIRST pre-cursor study and proposal DESTINY [1] and the proposed Mars-2020 mission instrument — the Pulsed Neutron Generator and Gamma Ray Spectrometer (PING). Both proposals are now history with the first materializing as the winning WFIRST [2] and the PING destined to fly on some other than Mars-2020 mission. The purpose of this paper is to delineate the IAEES framework in proposal phase broad enough to be scalable and adaptive for future implementation. Representative top-level requirements — each originating in science definition and the instrument's other subsystems and the spacecraft needs constitute the basis of such a framework. A representative IAEES conceptual framework is elaborated on the precedent of the two future mission instruments' proposals and is analyzed as a reference scalable adaptive IAEES and its simulators.
- Published
- 2016
34. Correction for Sohl et al., Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ
- Author
-
Christie K. Shumate, Karen S. Anderson, Sheida Amiralaei, Yuhui Yin, Andrea C. Mislak, Raymond F. Schinazi, Christal D. Sohl, and Michal R. Szymanski
- Subjects
chemistry.chemical_classification ,Genetics ,Multidisciplinary ,chemistry ,biology.protein ,Nucleotide ,Biology ,Selectivity ,Human mitochondrial genetics ,Polymerase - Published
- 2016
35. Multi-step oxidations catalyzed by cytochrome P450 enzymes: Processive vs. distributive kinetics and the issue of carbonyl oxidation in chemical mechanisms
- Author
-
Goutam Chowdhury, Christal D. Sohl, and F. Peter Guengerich
- Subjects
Kinetics ,Biophysics ,Biochemistry ,Redox ,Aldehyde ,Article ,Substrate Specificity ,Catalysis ,chemistry.chemical_compound ,Aromatase ,Cytochrome P-450 Enzyme System ,Animals ,Humans ,Organic chemistry ,Molecular Biology ,chemistry.chemical_classification ,Aldehydes ,biology ,Chemistry ,Substrate (chemistry) ,Cytochrome P450 ,Combinatorial chemistry ,Enzyme ,biology.protein ,Xenobiotic ,Oxidation-Reduction - Abstract
Catalysis of sequential oxidation reactions is not unusual in cytochrome P450 (P450) reactions, not only in steroid metabolism but also with many xenobiotics. One issue is how processive/distributive these reactions are, i.e., how much do the "intermediate" products dissociate. Our work with human P450s 2E1, 2A6, and 19A1 on this subject has revealed a mixture of systems, surprisingly with a more distributive mechanism with an endogenous substrate (P450 19A1) than for some xenobiotics (P450s 2E1, 2A6). One aspect of this research involves carbonyl intermediates, and the choice of catalytic mechanism is linked to the hydration state of the aldehyde. The non-enzymatic rates of hydration and dehydration of carbonyls are not rapid and whether P450s catalyze the reversible hydration is unknown. If carbonyl hydration and dehydration are slow, the mechanism may be set by the carbonyl hydration status.
- Published
- 2011
36. High-throughput fluorescence assay of cytochrome P450 3A4
- Author
-
F. Peter Guengerich, Christal D. Sohl, and Qian Cheng
- Subjects
chemistry.chemical_classification ,biology ,CYP3A4 ,CYP1A2 ,Cytochrome P450 ,Oxidative phosphorylation ,Article ,General Biochemistry, Genetics and Molecular Biology ,Inhibitory Concentration 50 ,chemistry.chemical_compound ,Ketoconazole ,Spectrometry, Fluorescence ,Enzyme ,chemistry ,Biochemistry ,Coumarins ,biology.protein ,Cytochrome P-450 CYP3A ,Humans ,CYP2A6 ,Xenobiotic ,Oxidation-Reduction ,Software ,Drug metabolism - Abstract
Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less.
- Published
- 2009
37. Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2
- Author
-
Donald F. Stec, Glenn A. Marsch, Emre M. Isin, Christal D. Sohl, F. Peter Guengerich, and Robert L. Eoff
- Subjects
biology ,Stereochemistry ,Kinetics ,Active site ,Cytochrome P450 ,Cooperativity ,Cell Biology ,Photochemistry ,Biochemistry ,Redox ,Hydroxylation ,chemistry.chemical_compound ,chemistry ,biology.protein ,Pyrene ,Molecular Biology ,Heme - Abstract
Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of α-naphthoflavone (αNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for αNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (αNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-αNF complex show active site space for only one αNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.
- Published
- 2008
38. Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ
- Author
-
Sheida Amiralaei, Andrea C. Mislak, Y. Whitney Yin, Christie K. Shumate, Karen S. Anderson, Raymond F. Schinazi, Michal R. Szymanski, and Christal D. Sohl
- Subjects
0301 basic medicine ,Protein Conformation ,DNA-Directed DNA Polymerase ,Emtricitabine ,Crystallography, X-Ray ,Human mitochondrial genetics ,Corrections ,Nucleoside Reverse Transcriptase Inhibitor ,Substrate Specificity ,03 medical and health sciences ,immune system diseases ,medicine ,Humans ,Polymerase ,Nucleic Acid Synthesis Inhibitors ,Multidisciplinary ,biology ,virus diseases ,Biological Sciences ,medicine.disease ,Molecular biology ,Reverse transcriptase ,DNA Polymerase gamma ,Mitochondria ,Mitochondrial toxicity ,Kinetics ,030104 developmental biology ,Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors ,Molecular Probes ,biology.protein ,Reverse Transcriptase Inhibitors ,Nucleoside ,medicine.drug - Abstract
Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3′-dideoxy-5-fluoro-3′-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3′-dideoxy-3′-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.
- Published
- 2015
39. Recombinant Enzymes Overexpressed in Bacteria Show Broad Catalytic Specificity of Human Cytochrome P450 2W1 and Limited Activity of Human Cytochrome P450 2S1
- Author
-
Christal D. Sohl, Zhong-Liu Wu, Tsutomu Shimada, and F. Peter Guengerich
- Subjects
Pharmacology ,Base Sequence ,biology ,CYP2B6 ,Cytochrome c ,CYP1B1 ,Molecular Sequence Data ,25-Hydroxyvitamin D3 1-alpha-hydroxylase ,CYP1A2 ,Cytochrome P450 ,Cytochrome P450 reductase ,Molecular biology ,Catalysis ,Recombinant Proteins ,Mixed Function Oxygenases ,Cytochrome P-450 Enzyme System ,Biochemistry ,Escherichia coli ,biology.protein ,Humans ,Molecular Medicine ,Cytochrome P450, family 1, member A1 ,Amino Acid Sequence ,Cytochrome P450 Family 2 - Abstract
Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k(cat), 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (approximately 0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process.
- Published
- 2006
40. Synthesis and solid-state molecular structures of nitrosoalkane complexes of iron porphyrins containing methanol, pyridine, and 1-methylimidazole ligands
- Author
-
Christal D. Sohl, Susan S. Alguindigue, Jonghyuk Lee, George B. Richter-Addo, and Masood A. Khan
- Subjects
Hemeprotein ,Molecular Structure ,Pyridines ,Chemistry ,Ligand ,Stereochemistry ,Hydrogen bond ,Methanol ,Imidazoles ,Heme ,Nitroso ,Ligands ,Biochemistry ,Porphyrin ,Inorganic Chemistry ,chemistry.chemical_compound ,Myoglobin ,Pyridine ,Nitroso Compounds - Abstract
Nitrosoalkanes belong to the family of C-nitroso compounds and are known to bind to the iron center in heme proteins. We haveprepared and characterized a series of new nitrosoalkane heme model complexes of the form (por)Fe(RNO)(L) (por¼porphyrinatodianion; R¼isopropyl; L¼MeOH, pyridine, 1-methylimidazole) by infrared and 1 H NMR spectroscopy and X-ray crystallogra-phy. Within the set of octaethylporphyrinato (OEP) compounds, the infrared stretching frequencies of the NO groups decrease inthe order (OEP)Fe( i PrNO)(MeOH)"MeOH (1433 cm #1 )>(OEP)Fe( i PrNO)(py) (1429 cm )>(OEP)Fe( i PrNO)(1-MeIm) (1423cm #1 ), reflecting the increased backdonation of electron density in the 1-methylimidazole derivative. The molecular structures of thecompounds as determined by crystallography reveal N-binding of the nitrosoalkane ligands to the formally ferrous metal centers.! 2004 Elsevier Inc. All rights reserved. Keywords: Iron; Nitroso; Porphyrin; X-ray; Nitrosoalkane 1. IntroductionThe nitroso group of nitrosoalkanes (RNO; R¼al-kyl) is isoelectronic with dioxygen. Nitrosoalkanes be-long to the general class of C-nitroso compounds, andthe binding of nitrosoalkanes and/or nitrosoarenes toheme iron has been demonstrated in heme proteins suchas myoglobin (Mb), hemoglobin (Hb), leghemoglobin,soluble guanylyl cyclase (sGC), NO synthase, catalase,prostaglandin H synthase, microperoxidase, and cyto-chrome P450 [1]. Importantly, heme–RNO bond for-mation has been implicated in the severe inhibition ofthe catalytic functions of hepatic microsomal cyto-chrome P450. Such heme–RNO compounds can beproduced from the oxidative metabolism of amine-containing drugs or by reduction of nitroorganics.Excellent work by Mansuy and coworkers [2,3] hasshown that nitrosoalkane complexes of Mb can beprepared from treatment of met Mb with nitroalkanes inthe presence of dithionite. Related nitrosoarene Mbcomplexes have been reported [4–6]. We recently deter-mined the molecular structure of Mb(EtNO) by X-raycrystallography [7]. We showed that the nitrosoethaneligand was attached to the iron center through its N-atom (Fig. 1), although distal pocket residues alsohelped orient the nitrosoethane ligand to its final ori-entation with respect to the normal to the heme planevia a hydrogen bond between the nitroso O-atom andthe distal His64 residue. This was the first reportedstructure of a nitrosoalkane complex of a heme protein.Also, it is surprising that to date there is only one re-ported crystal structure of a nitrosoalkane complex of aheme model, namely that of (TPP)Fe(
- Published
- 2004
41. Temporal resolution of protein signaling (473.1)
- Author
-
Joseph Schlessinger, Christal D. Sohl, Cristina M. Furdui, Youngjoo Kim, BeiBei Luo, Karen S. Anderson, Shunyan Mo, Mihaela Apetri, and Erin Lew
- Subjects
Physics ,Temporal resolution ,Genetics ,Molecular Biology ,Biochemistry ,Neuroscience ,Biotechnology - Published
- 2014
42. Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics
- Author
-
Yagmur Muftuoglu, Stefan G. Sarafianos, Hiroaki Mitsuya, Christal D. Sohl, Andrea C. Mislak, and Karen S. Anderson
- Subjects
Anti-HIV Agents ,Article ,chemistry.chemical_compound ,Deoxyadenosine ,Virology ,medicine ,Nucleotide ,Enzyme kinetics ,Polymerase ,Pharmacology ,chemistry.chemical_classification ,Reverse-transcriptase inhibitor ,biology ,Deoxyadenosines ,Molecular biology ,Reverse transcriptase ,HIV Reverse Transcriptase ,Kinetics ,chemistry ,biology.protein ,HIV-1 ,Reverse Transcriptase Inhibitors ,Primer (molecular biology) ,Nucleoside ,medicine.drug - Abstract
The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed.
- Published
- 2013
43. Relationship between graded degrees of anemia and amniotic fluid volume in the ovine fetus
- Author
-
Bryan D. Sohl and Robert A. Brace
- Subjects
medicine.medical_specialty ,Polyhydramnios ,Time Factors ,Amniotic fluid ,Anemia ,Diuresis ,Hematocrit ,Hypoxemia ,Andrology ,Pregnancy ,Internal medicine ,medicine ,Animals ,Fetus ,Sheep ,medicine.diagnostic_test ,business.industry ,Obstetrics and Gynecology ,Amniotic Fluid ,Fetal Blood ,medicine.disease ,Oxygen ,Fetal Diseases ,Endocrinology ,Multivariate Analysis ,Lactates ,Gestation ,Female ,medicine.symptom ,business - Abstract
Severe fetal anemia is associated with polyhydramnios in both human and ovine fetuses. This study examined the relationship between varying degrees of anemia and amniotic fluid volume in fetal sheep.Eleven long-term catheterized ovine fetuses at 126 +/- 1 days' gestation (mean +/- SE) were subjected to hemorrhage of 20 to 80 mL daily for 9 consecutive days to produce varying degrees of fetal anemia. Five additional animals served as time control animals. Statistical analysis was by least squares regression and 3-factor analysis of variance.Amniotic fluid volume was 793 +/- 147 mL and did not change with time in the control fetuses. In the fetuses that were subjected to hemorrhage the amniotic fluid volume changed little through the hematocrit range of 40% to 25%. As fetal hematocrit fell below approximately 25%, amniotic fluid volume began to increase. With greater degrees of anemia the amniotic fluid volume increased as an exponential function of hematocrit and approached 2000 mL excess fluid as hematocrit dropped to15%. According to bivariate regression the increase in amniotic fluid volume was related to fetal hematocrit, PaO(2), and urinary flow rate as well as to plasma and amniotic fluid lactate concentrations. According to multivariate regression only fetal PO(2) and urinary flow rate were significantly related to the increase in amniotic fluid volume.Although mild anemia was not associated with increased amniotic fluid volume, moderate to severe fetal anemia was associated with an exponential rise in amniotic fluid volume. This rise may have been mediated by a hypoxemia-induced diuresis, by a diuresis related to a lactate-induced osmotic accumulation of fetal fluid, or by both mechanisms.
- Published
- 1999
44. Pools and Pols: Mechanism of a mutator phenotype
- Author
-
Joann B. Sweasy, Sreerupa Ray, and Christal D. Sohl
- Subjects
Genetics ,Multidisciplinary ,Deoxyribonucleoside triphosphate ,biology ,DNA synthesis ,DNA polymerase ,biology.protein ,DNA replication ,Proofreading ,DNA mismatch repair ,Processivity ,Polymerase - Abstract
The maintenance of the human genome is dependent upon several cellular processes including DNA replication. Ordinarily, DNA replication is an exceptionally faithful process, with approximately one error occurring for every 109–1010 nucleotides (1, 2). High-fidelity replicative DNA polymerases with exonucleolytic proofreading activity, along with DNA mismatch repair machinery, are responsible for accurate DNA synthesis. DNA polymerase δ (pol δ) and polymerase e (pol e) are two essential replicative lagging and leading strand polymerases, respectively, that ensure efficient and high-fidelity genome replication (3–5). Replicative polymerase variants have recently been identified in human tumors that harbor enormous numbers of mutations. In recent studies reported in PNAS, Mertz et al. and Williams et al. provide evidence that this hypermutator phenotype results from expansion of deoxyribonucleoside triphosphate (dNTP) pools (6, 7).
- Published
- 2015
45. Mutations in human DNA polymerase γ confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients
- Author
-
Karen S. Anderson, Rajesh Kasiviswanathan, William C. Copeland, and Christal D. Sohl
- Subjects
Mitochondrial DNA ,Mitochondrial Diseases ,Mitochondrial disease ,Mutant ,Mutation, Missense ,DNA-Directed DNA Polymerase ,Mitochondrion ,Biology ,medicine.disease_cause ,Human mitochondrial genetics ,DNA, Mitochondrial ,chemistry.chemical_compound ,Genetics ,medicine ,Humans ,Molecular Biology ,Genetics (clinical) ,Polymerase ,Mutation ,General Medicine ,Articles ,medicine.disease ,Molecular biology ,DNA Polymerase gamma ,Mitochondria ,chemistry ,biology.protein ,Biocatalysis ,DNA - Abstract
Human mitochondrial DNA polymerase γ (pol γ) is solely responsible for the replication and repair of the mitochondrial genome. Unsurprisingly, alterations in pol γ activity have been associated with mitochondrial diseases such as Alpers syndrome and progressive external ophthalmoplegia. Thus far, predicting the severity of mitochondrial disease based the magnitude of deficiency in pol γ activity has been difficult. In order to understand the relationship between disease severity in patients and enzymatic defects in vitro, we characterized the molecular mechanisms of four pol γ mutations, A957P, A957S, R1096C and R1096H, which have been found in patients suffering from aggressive Alpers syndrome to mild progressive external ophthalmoplegia. The A957P mutant showed the most striking deficiencies in the incorporation efficiency of a correct deoxyribonucleotide triphosphate (dNTP) relative to wild-type pol γ, with less, but still significant incorporation efficiency defects seen in R1096H and R1096C, and only a small decrease in incorporation efficiency observed for A957S. Importantly, this trend matches the disease severity observed in patients very well (approximated as A957P ≫ R1096C ≥ R1096H ≫ A957S, from most severe disease to least severe). Further, the A957P mutation conferred a two orders of magnitude loss of fidelity relative to wild-type pol γ, indicating that a buildup of mitochondrial genomic mutations may contribute to the death in infancy seen with these patients. We conclude that characterizing the unique molecular mechanisms of pol γ deficiency for physiologically important mutant enzymes is important for understanding mitochondrial disease and for predicting disease severity.
- Published
- 2012
46. Oxidation of dihydrotestosterone by human cytochromes P450 19A1 and 3A4
- Author
-
Francis K. Yoshimoto, Christal D. Sohl, F. Peter Guengerich, and Qian Cheng
- Subjects
Male ,medicine.drug_class ,medicine.medical_treatment ,Estrone ,Hydroxylation ,Biochemistry ,Steroid ,chemistry.chemical_compound ,Aromatase ,medicine ,Cytochrome P-450 CYP3A ,Humans ,Molecular Biology ,Testosterone ,biology ,Cytochrome P450 ,Dihydrotestosterone ,Cell Biology ,Androgen ,chemistry ,biology.protein ,Microsome ,Microsomes, Liver ,Enzymology ,Oxidation-Reduction ,medicine.drug - Abstract
Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy- and 19-oxo derivatives and the resulting Δ(1,10)-, Δ(5,10)-, and Δ(9,10)-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ~10-fold higher than reported for 3α-reduction by 3α-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5β-, and 9β-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the Δ(1,10)-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the Δ(4,5) bond is remarkable in shifting the course of hydroxylation from the 6β-, 2β-, 1β-, and 15β-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure.
- Published
- 2012
47. Balancing antiviral potency and host toxicity: identifying a nucleotide inhibitor with an optimal kinetic phenotype for HIV-1 reverse transcriptase
- Author
-
Masanori Baba, Jiae Kim, Raymond F. Schinazi, Karen S. Anderson, William C. Copeland, Christal D. Sohl, Hiroaki Mitsuya, Ugo Pradere, and Rajesh Kasiviswanathan
- Subjects
Anti-HIV Agents ,HIV Infections ,DNA-Directed DNA Polymerase ,DNA, Mitochondrial ,Nucleoside Reverse Transcriptase Inhibitor ,chemistry.chemical_compound ,medicine ,Humans ,Nucleotide ,Polymerase ,Pharmacology ,chemistry.chemical_classification ,biology ,Nucleotides ,Articles ,medicine.disease ,Molecular biology ,Reverse transcriptase ,Dideoxynucleosides ,HIV Reverse Transcriptase ,DNA Polymerase gamma ,Mitochondrial toxicity ,Kinetics ,Stavudine ,Enzyme ,chemistry ,Toxicity ,biology.protein ,HIV-1 ,Molecular Medicine ,Reverse Transcriptase Inhibitors ,Thymidine - Abstract
Two novel thymidine analogs, 3′-fluoro-3′-deoxythymidine (FLT) and 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre–steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol γ, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2′,3′-didehydro-2′,3′-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4′-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2′,3′-didehydro-2′,3′-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.
- Published
- 2012
48. Mechanism of interaction of human mitochondrial DNA polymerase γ with the novel nucleoside reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine indicates a low potential for host toxicity
- Author
-
Karen S. Anderson, Kamlendra Singh, Stefan G. Sarafianos, Hiroaki Mitsuya, William C. Copeland, Rajesh Kasiviswanathan, and Christal D. Sohl
- Subjects
Models, Molecular ,DNA polymerase ,Molecular Sequence Data ,DNA-Directed DNA Polymerase ,Mitochondrion ,Human mitochondrial genetics ,Antiviral Agents ,Nucleoside Reverse Transcriptase Inhibitor ,Mitochondrial Proteins ,chemistry.chemical_compound ,Deoxyadenosine ,Humans ,Pharmacology (medical) ,Polymerase ,Pharmacology ,biology ,Base Sequence ,Deoxyadenosines ,Processivity ,Molecular biology ,Reverse transcriptase ,HIV Reverse Transcriptase ,DNA Polymerase gamma ,Mitochondria ,Kinetics ,Infectious Diseases ,chemistry ,biology.protein ,HIV-1 ,Reverse Transcriptase Inhibitors - Abstract
The potent antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a promising experimental agent for treating HIV infection. Pre-steady-state kinetics were used to characterize the interaction of EFdA-triphosphate (EFdA-TP) with human mitochondrial DNA polymerase γ (Pol γ) to assess the potential for toxicity. Pol γ incorporated EFdA-TP 4,300-fold less efficiently than dATP, with an excision rate similar to ddATP. This strongly indicates EFdA is a poor Pol γ substrate, suggesting minimal Pol γ-mediated toxicity, although this should be examined under clinical settings.
- Published
- 2011
49. A first glimpse at the shell structure beyond 54Ca: Spectroscopy of 55K, 55Ca, and 57Ca
- Author
-
T. Koiwai, K. Wimmer, P. Doornenbal, A. Obertelli, C. Barbieri, T. Duguet, J.D. Holt, T. Miyagi, P. Navrátil, K. Ogata, N. Shimizu, V. Somà, Y. Utsuno, K. Yoshida, N.L. Achouri, H. Baba, F. Browne, D. Calvet, F. Château, S. Chen, N. Chiga, A. Corsi, M.L. Cortés, A. Delbart, J.-M. Gheller, A. Giganon, A. Gillibert, C. Hilaire, T. Isobe, T. Kobayashi, Y. Kubota, V. Lapoux, H.N. Liu, T. Motobayashi, I. Murray, H. Otsu, V. Panin, N. Paul, W. Rodriguez, H. Sakurai, M. Sasano, D. Steppenbeck, L. Stuhl, Y.L. Sun, Y. Togano, T. Uesaka, K. Yoneda, O. Aktas, T. Aumann, L.X. Chung, F. Flavigny, S. Franchoo, I. Gasparic, R.-B. Gerst, J. Gibelin, K.I. Hahn, D. Kim, Y. Kondo, P. Koseoglou, J. Lee, C. Lehr, B.D. Linh, T. Lokotko, M. MacCormick, K. Moschner, T. Nakamura, S.Y. Park, D. Rossi, E. Sahin, P.-A. Söderström, D. Sohler, S. Takeuchi, H. Toernqvist, V. Vaquero, V. Wagner, S. Wang, V. Werner, X. Xu, H. Yamada, D. Yan, Z. Yang, M. Yasuda, and L. Zanetti
- Subjects
Radioactive beams ,γ-ray spectroscopy ,Shell evolution ,Physics ,QC1-999 - Abstract
States in the N=35 and 37 isotopes 55,57Ca have been populated by direct proton-induced nucleon removal reactions from 56,58Sc and 56Ca beams at the RIBF. In addition, the (p,2p) quasi-free single-proton removal reaction from 56Ca was studied. Excited states in 55K, 55Ca, and 57Ca were established for the first time via in-beam γ-ray spectroscopy. Results for the proton and neutron removal reactions from 56Ca to states in 55K and 55Ca for the level energies, excited state lifetimes, and exclusive cross sections agree well with state-of-the-art theoretical calculations using different approaches. The observation of a short-lived state in 57Ca suggests a transition in the calcium isotopic chain from single-particle dominated states at N=35 to collective excitations at N=37.
- Published
- 2022
- Full Text
- View/download PDF
50. Kinetic analysis of steps involved in oxidation by cytochrome P450 19A1
- Author
-
F. Peter Guengerich and Christal D. Sohl
- Subjects
biology ,Computational chemistry ,Chemistry ,Kinetic analysis ,Genetics ,biology.protein ,Cytochrome P450 ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2010
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.