Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.
Introductory-level laboratory courses provide students with hands-on experience using the discipline's tools and theories. These courses often rely on recipebased experiments due to the constraints of large enrollments, short lab periods, and the desire to minimize complexity. In addition, covering a breadth of topics can lead to a fragmented curriculum with little carryover in learning from week to week. Herein, we describe an overhaul of an introductory organic chemistry laboratory curriculum, informed by the strategies of meaningful learning and a desire to make the course experience mimic a research lab. This new course, primarily taught to first-year undergraduate students at the University of Michigan, is framed with three interconnected modules. We present herein the first module, which focuses on thinlayer chromatography (TLC). In the first week, students learn how to perform TLC using a variety of compounds and solvent mixtures, gaining an understanding of how intermolecular interactions affect their retention. In the second week, they practice using TLC to distinguish reagents and reaction byproducts and in the third week apply TLC to monitor reaction progress and test their hypothesis. We assessed student learning through a writing assignment at the end of the three-week module. We also assessed how the overall course affects student comprehension of TLC concepts and confidence. Our findings suggest that this learn, practice, apply approach toward teaching introductory organic chemistry laboratory concepts leads to learning gains and increased confidence.
Infographics are increasingly used to communicate complex topics to the public. By coupling visually appealing graphics with compelling narratives, infographics can both engage and inform the audience. We designed an assignment wherein students create infographics with the dual purpose of helping them connect course concepts to their daily lives and practice science communication. Most students focused on compounds found in food, beverages, cosmetics, and medicine. The infographics were assessed based on the effectiveness of the layout, the quality and clarity of the graphical and textual content, and the reliability of the cited sources. Student reflections on the assignment revealed strong connections between their course learning and their infographic topics. Although this assignment was part of a college laboratory course, it can be adapted for alternative settings.
Histone deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depends on the identity of the active site metal ion. Varied deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using a high-throughput Self-Assembled Monolayers for MALDI-TOF Mass Spectrometry (SAMDI) mass spectrometry screen. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the ratio of Fe(II)/Zn(II) HDAC8 activity varies widely (2 to 150) among the peptides tested. These data provide support for the hypothesis that HDAC8 may undergo metal switching in vivo which, in turn, may regulate its activity. However future studies are needed to explore the identity of the metal ion bound to HDAC8 in cells under varied conditions.
Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39 which leads to decreased catalytic activity.HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion (Zn(II) vs Fe(II)) with the value of kcat/KM for the mutant decreasing 9-to >200-fold compared to wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ~15fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency and substrate selectivity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.