User:Asif Hossain/Sandbox 1: Difference between revisions

Histone deacetylase 8 (HDAC8) is an enzyme that plays a role in controlling gene expression. Specifically, HDAC8 removes an acetyl group off of the ε-amino-Lys 382 of Histone 4's N-terminal core.<ref name="Vannini, A., Volpari, C., Gallinari, P.">Vannini, A., Volpari, C., Gallinari, P., Jones, P., Mattu, M., Carfí, A., ... & Di Marco, S. (2007). Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex. EMBO reports, 8(9), 879-884. https://doi.org/10.1038/sj.embor.7401047 </ref> [https://en.wikipedia.org/wiki/Histone Histones] consist of eight monomers to form an octomer complex. Each histone has a positive charge which allows interaction with negatively-charged DNA. This prevents transcription factors from accessing DNA, thus, decreasing gene expression. [https://en.wikipedia.org/wiki/Chromatin_remodeling Chromatin remodeling] by the addition or removal of an acetyl group on a histone is an example of [https://en.wikipedia.org/wiki/Epigenetics epigenetic regulation]. [https://en.wikipedia.org/wiki/Histone_acetyltransferase Histone Acetylase 1] (HAT1) catalyzes the addition of an acetyl group onto a histone. The lack of charge on the acetyl group weakens the interaction between DNA and histones which allows transcription factors to access the DNA to increase gene expression. HDAC8 reverses this reaction by catalyzing the removal of these acetyl groups from the Lys to reclaim the positive charge of the histone. This allows the histone to interact with the negative charge on the DNA. As a result, DNA binds more tightly to the histone protein, repressing transcription and gene expression.

There are four major classes of HDAC proteins (I,II, III, and IV). Other than the Class III “[https://en.wikipedia.org/wiki/Sirtuin sirtuins]” that utilize a [https://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2016/bichaw.2016.55.issue-11/acs.biochem.5b01210/20160316/images/medium/bi-2015-01210h_0006.gif NAD⁺ cofactor-dependent mechanism], all other HDAC classes use Zn²⁺-assisted catalysis through mechanisms (Figure 3) reminiscent of a typical [https://en.wikipedia.org/wiki/Serine_protease serine protease].<ref name="DesJarlais, R., & Tummino, P. J.">DesJarlais, R., & Tummino, P. J. (2016). Role of histone-modifying enzymes and their complexes in regulation of chromatin biology. Biochemistry, 55(11), 1584-1599. https://doi.org/10.1021/acs.biochem.5b01210 </ref> While Classes I, II, and IV do have some major distinctions such as size of the protein, in general, they share homology at the catalytic site. HDAC 8 is classified as a Class I HDAC alongside HDACs 1-3. In fact, within Class I HDACs,  there are many invariant residues involved in the catalytic site (such as His-Asp dyads), Zn-binding, and ligand binding pocket (such as Asp101) (Figure 1). <ref name="Vannini, A., Volpari, C., Gallinari, P.">Vannini, A., Volpari, C., Gallinari, P., Jones, P., Mattu, M., Carfí, A., ... & Di Marco, S. (2007). Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex. EMBO reports, 8(9), 879-884. https://doi.org/10.1038/sj.embor.7401047 </ref>  

[[Image:Conserved residues.PNG|600px||center||thumb|Figure 1: Weblogo representation comparing conservation of residues (143-182 in HDAC8) to homologous sequences in all class I HDACs. Nearly all active site residues (asterisk), zinc binding (dollar), and binding pocket residues (caret) are conserved across all class I HDACs. Other conserved residues not shown include active site residue Tyr306, zinc binding residue Asp267, and binding pocket residue Asp101. Nonconserved residues from 158 to l70 are part of an α-helix that moves outward from the active side before looping back around to the active site.]]

The crystal structure of human HDAC8 was determined using x-ray crystallography at a 2.0Å resolution. <ref name="Vannini, A., Volpari, C., Gallinari, P.">Vannini, A., Volpari, C., Gallinari, P., Jones, P., Mattu, M., Carfí, A., ... & Di Marco, S. (2007). Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex. EMBO reports, 8(9), 879-884. https://doi.org/10.1038/sj.embor.7401047 </ref> The structure includes two structural K⁺ ion and one catalytic Zn²⁺ ion. HDAC8 is bound to a [https://en.wikipedia.org/wiki/P53 p-53] <scene name='81/811084/Ligand/9'>derived diacetylated peptide substrate</scene> as opposed to the natural histone substrate. This peptide includes a <scene name='81/811084/Coumarin/2'>fluorescent coumarin ring</scene> likely used in past kinetic assays. The HDAC8 is made up of a <scene name='81/811084/Beta_sheets/6'>β-sheet</scene> with eight parallel β-strands located between 13 <scene name='81/811084/Alpha_helicesv2/4'>α-helices</scene>. The HDAC8 consists of 377 amino acids.  <ref name="Somoza"> Somoza J, Skene R. Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases. Structure, 12(7), 1325-1334.2004. https://doi.org/10.1016/j.str.2004.04.012 </ref>

The pentacoordinated Zn²⁺ ion involved in the metalloenzyme catalysis is tethered to the protein through interactions with <scene name='81/811085/Zinc_binding/2'>Asp178, His180, and Asp267</scene> . This positions the metal ion to favorably interact with the catalytic water and acetylated lysine substrate. <ref name="Vannini, A., Volpari, C., Gallinari, P.">Vannini, A., Volpari, C., Gallinari, P., Jones, P., Mattu, M., Carfí, A., ... & Di Marco, S. (2007). Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex. EMBO reports, 8(9), 879-884. https://doi.org/10.1038/sj.embor.7401047 </ref> The Zn²⁺ ion lowers the pKa of a water molecule that activates it as a nucleophile. By binding to both the nucleophile and the substrate simultaneously, the Zn²⁺ ion also assists the deacetylation process by lowering the entropy of the reaction. This polarizes the carbonyl of the acetyl-lysine and stabilizes the transition state.<ref name="Somoza">Somoza J, Skene R. Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases. Structure, 12(7), 1325-1334.2004. https://doi.org/10.1016/j.str.2004.04.012 </ref>

Once the substrate is bound to the binding pocket through interactions with <scene name='81/811087/Ligand_interaction/4'>Asp101,Phe152 and Phe208</scene>, the water molecule attacks the carbonyl carbon of the ε-amino-lysine sidechain of N-terminal core of histone proteins (Figure 2). This water molecule is recruited and stabilized by <scene name='81/811085/Dyads/5'>two catalytic dyads</scene>. The first dyad consists of His143 and Asp183. Asp183 interacts with His143 to shift electron density so that His143 may act as a general base to remove a proton from water. The second catalytic dyad consists of His142 and Asp176 and stabilizes the now deprotonated water molecule. A Zn²⁺ ion also makes the water more acidic making it a better nucleophile. The tetrahedral intermediate is stabilized by the Zn²⁺ ion as well as Tyr306. The amine group of the substrate's lysine acts as a general base and deprotonates His143. This drives the tetrahedral intermediate to collapse and expel the acetyl group to produce an acetate ion and a deacetylated lysine residue. <ref name="Seto, E., & Yoshida, M."> Seto, E., & Yoshida, M. (2014). Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harbor perspectives in biology, 6(4), a018713. https://doi.org/10.1101/cshperspect.a018713 </ref>

Besides controlling gene regulation through deacetylation of histones, HDAC8 also regulates other non-histone proteins such as chaperones, hormone receptors and signaling molecules. <ref name="Eckschlager">Eckschlager T, Plch, J, Stiborova M, Hrabeta J.Histone deacetylase inhibitors as anticancer drugs. International journal of molecular sciences, 18(7), 1414. 2017. https://dx.doi.org/10.3390%2Fijms18071414</ref> Thus, it has influences on protein stability and interactions between other proteins and DNA. HDAC8 can therefore affect the regulation of cell proliferation and cell death. These processes are typically being altered in cancer cells and that makes HDAC enzymes an interesting potential target for cancer drugs. HDAC inhibitors have been shown to be promising cancer drug agent as the HDAC inhibitors (HDACi) cease tumor growth in cancer cells by either making them differentiate, undergo apoptosis or upregulate cell cycle arrest proteins. <ref name="Eckschlager">Eckschlager T, Plch, J, Stiborova M, Hrabeta J.Histone deacetylase inhibitors as anticancer drugs. International journal of molecular sciences, 18(7), 1414. 2017. https://dx.doi.org/10.3390%2Fijms18071414</ref> One way the HDAC inhibitors cease tumor growth is by the reactivation of the transcription factor, [https://en.wikipedia.org/wiki/RUNX3 RUNX3], a known tumor suppressor. HDACi increases the acetylation of the protein and as the stability of RUNX3 is dependent on the acetylation status of the protein, the increased acetylation or HDAC inhibition will enhance the protein stability. A number of HDAC inhibitors have been purified from natural sources or synthesized and at least four structurally different inhibitor classes have been characterized: hydroxamates, cyclic peptides, aliphatic acids and benzamides. The Vorinostat (within the hydroxamate class) has been FDA-approved for treatment of cancer. A HDAC class 1 hydroxamic acid, compound 1 (Figure 3), <scene name='81/811087/Inhibitor_and_zinc_binding/13'>binds to the zinc ion</scene> in a bidendate fashion while making hydrogen bonds to important residues as His142, His143 and Tyr306 at the active site of HDAC8. Thus, the HDAC inhibitors can be used as antagonists to prevent the functioning of HDAC8 in cancer treatment. <ref name="Vannini, A., Volpari, C., Filocamo, G.">Vannini, A., Volpari, C., Filocamo, G., Casavola, E. C., Brunetti, M., Renzoni, D., ... & Steinkühler, C. (2004). Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor. Proceedings of the National Academy of Sciences, 101(42), 15064-15069. https://dx.doi.org/10.1073%2Fpnas.0404603101</ref>