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Review
. 2020 Oct;34(10):13140-13155.
doi: 10.1096/fj.202001301RR. Epub 2020 Aug 30.

Critical review of non-histone human substrates of metal-dependent lysine deacetylases

Tasha B Toro  1 , Terry J Watt  1
Affiliations
Review

Critical review of non-histone human substrates of metal-dependent lysine deacetylases

Tasha B Toro et al. FASEB J. 2020 Oct.

Abstract

Lysine acetylation is a posttranslational modification that occurs on thousands of human proteins, most of which are cytoplasmic. Acetylated proteins are involved in numerous cellular processes and human diseases. Therefore, how the acetylation/deacetylation cycle is regulated is an important question. Eleven metal-dependent lysine deacetylases (KDACs) have been identified in human cells. These enzymes, along with the sirtuins, are collectively responsible for reversing lysine acetylation. Despite several large-scale studies which have characterized the acetylome, relatively few of the specific acetylated residues have been matched to a proposed KDAC for deacetylation. To understand the function of lysine acetylation, and its association with diseases, specific KDAC-substrate pairs must be identified. Identifying specific substrates of a KDAC is complicated both by the complexity of assaying relevant activity and by the non-catalytic interactions of KDACs with cellular proteins. Here, we discuss in vitro and cell-based experimental strategies used to identify KDAC-substrate pairs and evaluate each for the purpose of directly identifying non-histone substrates of metal-dependent KDACs. We propose criteria for a combination of reproducible experimental approaches that are necessary to establish a direct enzymatic relationship. This critical analysis of the literature identifies 108 proposed non-histone substrate-KDAC pairs for which direct experimental evidence has been reported. Of these, five pairs can be considered well-established, while another thirteen pairs have both cell-based and in vitro evidence but lack independent replication and/or sufficient cell-based evidence. We present a path forward for evaluating the remaining substrate leads and reliably identifying novel KDAC substrates.

Keywords: HDAC; KDAC; histone deacetylases; substrate specificity.

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Conflict of interest statement

Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1.
Figure 1.
The post-translational acetylation cycle.
Figure 2.
Figure 2.
Representative crystal structures of human KDAC catalytic domains. Crystal structures for all four class I members (KDACs 1, 2, 3, and 8; cyan), two class IIa members (KDACs 4 and 7; orange), and one class IIb member (KDAC6 second catalytic domain, magenta) have been solved. Structural alignment reveals the significant similarity in overall structure, especially within classes. All structures include the catalytic zinc atom (gray sphere). The KDAC8 structure also includes the active site water molecule (red sphere) and a synthetic substrate peptide bound to the active site (stick representation, colored by element); the peptide remains intact because this structure of KDAC8 has a Tyr306Phe mutation that renders the enzyme inactive without perturbing the structure. The Protein Databank ID of each structure is provided, and in all cases the first KDAC chain present in the structure was selected for this representation.
Figure 3.
Figure 3.
Minimal mechanism of metal-dependent KDACs. (A) The Zn2+ ion lowers the pKa of the water molecule, allowing for deprotonation by a histidine residue and nucleophilic attack on the acetyllysine residue. A tyrosine residue contributes a hydrogen bond to stabilize the tetrahedral intermediate, which collapses to from the free lysine and acetic acid. After a proton rearrangement, the products are released. Further discussion of possible additional transition states and contributing residues can be found in Chen et. al. (B) Alignment of the active site of reported KDAC structures shows the similarity in residue positions (cyan, orange, and magenta for classes I, IIa, and IIb, respectively). Three residues coordinating the zinc atom (gray sphere from KDAC8 structure, center of image), as well as two histidine residues and one tyrosine residue that contribute to catalytic activity. The class IIa KDACs have a histidine in place of the tyrosine (left side of image), which does not occupy the same position as the tyrosine and causes the changes in activity described in the main text. Catalytic water (red sphere) and a crystallized substrate (colored by element) are from the KDAC8 structure. All PDB codes are the same as Figure 2.
Figure 4.
Figure 4.
Relationships between experimental methods and the identification of high confidence substrates. Substrate leads can be found using either cell-based methods that determine acetylation status of a target protein with respect to some manipulation of a KDAC or by in vitro activity assays utilizing recombinant KDAC enzyme. Using any of these experimental methods generates substrate leads. A putative substrate has supporting evidence from at least one experiment of each type. Identification of a specific acetylated lysine residue that is the site of modification by the KDAC, additional cell-based verification by a second experimental method, and independent replication is necessary to identify the target protein as a substrate with high confidence. Additional methods (dashed ovals) can provide supporting evidence, but do not directly address the question of whether the target protein is a KDAC substrate. Other methods of probing KDAC activity and interactions (dotted ovals) are not relevant for determining whether a non-histone target protein is a KDAC substrate.
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