Sandbox Reserved 1084

Chromatin, in eukaryotes, is a very compact structure mainly due to the electrostatic interaction between DNA and histones ^[1]. This interaction happens because DNA carries an overall negative charge and the histones carry a positive charge when deacetylated. In order to carry out the basic functions, such as transcription and replication, the chromatin has to be decondensed so that enzymes and transcription factors can access the DNA. The decondensation is mainly carried out by the acetylation of a lysine residue on the histone tail by Histone Acetyl Transferases (HATs). This then neutralizes its positive charge. The removal of the acetyl group is carried out by different histone deacetylases (HDACs).

Function

Histone Deacetylase 8 (HDAC8) has several features that make it different from other HDAC isozymes. HDAC8 contains a nuclear localization signal (NLS) at the center of the catalytic domain, and because of this tag it is found in the nucleus, however, it has been reported that HDAC8 has a cytosolic localization in muscle cells ^[2]. It has been reported that the global deletion of HDAC8 in mice leads to prenatal death due to instability of skull ^[3]. HDACs have been found to regulate learning, memory and cognition in human ^[4].

Disease

Class I HDAC isozymes are reportedly involved in expression p21, a cyclin-dependent kinase inhibitor ^[5]. The p21 proteins main job is to inhibit uncontrolled cell proliferation. A mutation in p53 gene has been widely reported in cancer patients ^[6]. Recently, HDAC8 has been shown to regulate the expression of both the wild type and the mutant form of p53 ^[6].Inhibition of HDACs has been shown to provide an anticancer ^[7]. However, a pan-HDAC inhibitor usually shows a considerable side effect in a clinical setting, primarily because of an indiscriminate inhibition of the multiple HDAC isozymes involved in several vital cellular processes. Isozyme selective inhibitors are likely to have fewer side effects as compared to a pan-inhibitor. They induce growth inhibition, dedifferentiation, and even cell death in cancer cells ^[8]. It is widely known that a pan-HDAC inhibitor significantly affects the acetylation status of its histone as well as several non-histone proteins, such as HSP90, p53 and others.The therapeutic potential of an HDAC8 selective activator for the treatment of various forms of cancers has recently emerged. In the above case the expression of the tumor suppressor protein (p53) is reportedly suppressed by HDAC8 ^[6].

In some cases of Cornelia de Lange syndrome (CLdS), the cohesion acetylation cycle has been reported to be impaired due to mutations in the HDAC8 gene ^[9]. The enzyme activity of Class I HDACs including HDAC8 has been found to be reduced in Chronic Obstructive Pulmonary Disease (COPD) ^[10]. Therapeutic potential of HDAC activators have not been well understood so far. However, there are several human diseases where an HDAC activator could be of great therapeutic benefits. In COPD and CLdS where the HDAC enzyme activity has been reported to be reduced, HDAC activators have a potential to lessen the disease conditions ^[9].

Structural highlights

HDAC8 was the first among the HDAC isozymes whose crystal structure became available. The image shows the ribbon structure of HDAC8 bound with a canonical hydroxamate inhibitor, SAHA (Suberoylanilide Hydroxamic Acid) [pdb 1T69]. HDAC8 contains a single α/β-deacetylase domain consisting of 13 α-helices and an eight-stranded parallel β-sheet. Multiple loops, which emanate from the protein core, play a significant role in maintaining the appropriate geometry of the catalytic pocket. The α/β-fold of the above kind was first observed in a metalloenzyme, [Arginase]. The active site of HDAC8 contains a tubular cavity leading to catalytic machinery at the end. The catalytic machinery is comprised of a Zn2+ ion penta-coordinated with a square pyramidal geometry. The residues His 180, Asp 267 and Asp 176 occupy the three co-ordination sites, whereas the hydroxamate moiety of SAHA occupies the remaining two sites. In addition, the carbonyl oxygen of the hydroxamate moiety forms a hydrogen bond with Tyr 306. In the absence of any ligand (substrate/inhibitors), two water molecules are bound to the Zn2+ ion. The linker domain of the inhibitor interacts with the residues: Phe 152, Phe 208, His 180, Gly 151, and Met 274, which forms a hydrophobic tunnel. Notably, the above residues involved in the inhibitor/substrate binding, as well as the catalysis, have been found to be conserved during the course of evolution among class I HDACs. Notably, the carbonyl oxygen of the acetyl-lysine substrate forms a hydrogen bond with the hydroxyl moiety of Y306. Aside from the catalytic Zn2+ ion, the enzyme activity of HDAC8 is dependent on the presence of the monovalent ion, K+ [60]. The crystal structure of HDAC8 shows the presence of two binding sites for K+ [58]. The first K+ binding site (K1) is located in the vicinity of the enzyme catalytic machinery, and it is hexacoordinated (octahedral geometry) with His 180 (carbonyl oxygen of the main chain), Asp 176 (oxygen atom of the main chain and side chain), Leu 200 (carbonyl oxygen of the main chain), and Ser 199 (O). Notably, His 180 and Asp 176 are the common residues coordinated with both the catalytic Zn2+ as well as the K+ ion. The second binding site for K+ ion (K2) is located 15Å away from the catalytic Zn2+ ion. It is hexacoordinated (octahedral geometry) with F189, T192, V195, Y225 as well as two water molecule.

The crystal structure of HDAC8-substrate complex (pdb 2V5W) elucidates the role of an aspartate residue (D101) in the substrate binding [59]. Asp 101 resides on the L2 loop and its carboxylate moiety makes two consecutive hydrogen bonds with the backbone of the p53-derived deacetylated peptide substrate as shown in Figure 1.3. Mutation of Asp 101 to Ala abolishes the HDAC8 catalytic activity, signifying its role in substrate binding. More importantly, the Asp residue has been found to be strictly conserved among different HDAC isozymes, further emphasizing its importance in the substrate binding.

Based on the crystallographic studies, a mechanism of the HDAC8 catalyzed reaction has been proposed. The zinc ion plays a pivotal role in the entire catalytic process.

HDAC8 has several intriguing features which distinguish it from other HDAC isozymes. It lacks the 50-111 AAs segment extending beyond the C-terminal of the catalytic domain which is utilized to recruit other co-repressor/transcription factors [57]. The crystallographic studies of HDAC8 with the structurally diverse hydroxamate inhibitors, namely, TSA, SAHA, M-334 and CRA-A, reveal an inherent malleability of its active site pocket, which is primarily due to the presence of the L1 loop (S30-K38) [57].