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2. An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Author

Luna, Lucas A, Lesecq, Zachary, White, Katharine A, Hoang, An, Scott, David A, Zagnitko, Olga, Bobkov, Andrey A, Barber, Diane L, Schiffer, Jamie M, Isom, Daniel G, and Sohl, Christal D

Subjects
Catalysis, Catalytic Domain, Glutarates, Humans, Hydrogen-Ion Concentration, Isocitrate Dehydrogenase, Isocitrates, Kinetics, Mutation, Protein Conformation, Substrate Specificity, buried ionizable residues, cancer, enzyme kinetics, pH regulation, post-translational modification, tumor metabolism, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology
Abstract

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) 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 increasing kcat values for αKG 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 pKa values. 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 pKa value 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

3. Readying Students for Careers in Industry: A Guided Inquiry Activity to Prepare Students for Success in Biotechnology and Pharmaceutical Industry Positions

Author

Gross, Stefan and Sohl, Christal D.

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
2021
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5. Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms

Author

Mealka, Matthew, primary, Sierra, Nicole A., additional, Matteo, Diego Avellaneda, additional, Albekioni, Elene, additional, Khoury, Rachel, additional, Mai, Timothy, additional, Conley, Brittany M., additional, Coleman, Nalani J., additional, Sabo, Kaitlyn A., additional, Komives, Elizabeth A., additional, Bobkov, Andrey A., additional, Cooksy, Andrew L., additional, Silletti, Steve, additional, Schiffer, Jamie M., additional, Huxford, Tom, additional, and Sohl, Christal D., additional

Published
2024
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9. Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen–Deuterium Exchange Mass Spectrometry

Author

Sabo, Kaitlyn A., primary, Albekioni, Elene, additional, Caliger, Danielle, additional, Coleman, Nalani J., additional, Thornberg, Ella, additional, Avellaneda Matteo, Diego, additional, Komives, Elizabeth A., additional, Silletti, Steve, additional, and Sohl, Christal D., additional

Published
2023
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19. An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Author

Luna, Lucas A., primary, Lesecq, Zachary, additional, White, Katharine A., additional, Hoang, An, additional, Scott, David A., additional, Zagnitko, Olga, additional, Bobkov, Andrey A., additional, Barber, Diane L., additional, Schiffer, Jamie M., additional, Isom, Daniel G., additional, and Sohl, Christal D., additional

Published
2020
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31. Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus

Author

Bernatchez, Jean A., primary, Yang, Zunhua, additional, Coste, Michael, additional, Li, Jerry, additional, Beck, Sungjun, additional, Liu, Yan, additional, Clark, Alex E., additional, Zhu, Zhe, additional, Luna, Lucas A., additional, Sohl, Christal D., additional, Purse, Byron W., additional, Li, Rongshi, additional, and Siqueira-Neto, Jair L., additional

Published
2018
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32. Development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting the Zika virus

Author

Bernatchez, Jean A., primary, Yang, Zunhua, additional, Coste, Michael, additional, Li, Jerry, additional, Beck, Sungjun, additional, Liu, Yan, additional, Clark, Alex E., additional, Zhu, Zhe, additional, Luna, Lucas A., additional, Sohl, Christal D., additional, Purse, Byron W., additional, Li, Rongshi, additional, and de Siqueira-Neto, Jair L., additional

Published
2018
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35. Water Networks and Correlated Motions in Mutant Isocitrate Dehydrogenase 1 (IDH1) Are Critical for Allosteric Inhibitor Binding and Activity

Author

Chambers, Jennifer M., Miller, Wade, Quichocho, Giovanni, Upadhye, Viraj, Matteo, Diego Avellaneda, Bobkov, Andrey A., Sohl, Christal D., and Schiffer, Jamie M.

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
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36. Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β: THE CRITICAL ROLE OF FINGERS DOMAIN MOVEMENTS AND A NOVEL NON-COVALENT STEP DURING NUCLEOTIDE SELECTION*

Author

Towle-Weicksel, Jamie B., Dalal, Shibani, Sohl, Christal D., Doublié, Sylvie, Anderson, Karen S., and Sweasy, Joann B.

Subjects
enzymes and coenzymes (carbohydrates), Base Sequence, Nucleotides, Fluorescence Resonance Energy Transfer, Humans, heterocyclic compounds, DNA and Chromosomes, DNA Polymerase beta, DNA Primers
Abstract

During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.

Published
2014

37. Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants.

Author

Matteo, Diego Avellaneda, Wells, Grace A., Luna, Lucas A., Grunseth, Adam J., Zagnitko, Olga, Scott, David A., An Hoang, Luthra, Amit, Swairjo, Manal A., Schiffer, Jamie M., and Sohl, Christal D.

Subjects
ISOCITRATE dehydrogenase, NEOPLASTIC cell transformation, KETOGLUTARATE dehydrogenase, NICOTINAMIDE adenine dinucleotide phosphate, BINDING sites
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. [ABSTRACT FROM AUTHOR]

Published
2018
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