Purification of metal-dependent lysine deacetylases with consistently high activity

Toro, Tasha B.; Painter, Richard G.; Haynes, Rashad A.; Glotser, Elena Y.; Bratton, Melyssa R.; Bryant, Jenae R.; Nichols, Kyara A.; Matthew-Onabanjo, Asia N.; Matthew, Ashley N.; Bratcher, Derek; Perry, Chanel D.; Watt, Terry J.

doi:10.1016/j.pep.2017.08.009

Cited by 3 publications

(11 citation statements)

References 33 publications

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“…To create KDAC8 derivatives, D101A, D101E, D101N, and D101R mutations were introduced using polymerase chain reaction-based site-directed mutagenesis into either the pFastbac1 (D101E) or pJE (D101A, D101N, and D101R) plasmid containing human KDAC8. E. coli expression was performed as previously described to obtain KDAC8 and KDAC8 derivatives . For insect cell expression of KDAC6 and KDAC8 D101E, pFastbac1-based plasmids were transformed into DH10Bac cells (Life Technologies) to create bacmids that were isolated using a ZR Bac DNA miniprep kit (Zymo Research).…”

Section: Methodsmentioning

confidence: 99%

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Lysine Deacetylase Substrate Selectivity: A Dynamic Ionic Interaction Specific to KDAC8

et al. 2021

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Lysine acetylation and deacetylation are critical for regulation of many cellular proteins. Despite the importance of this cycle, it is unclear how lysine deacetylase (KDAC) family members discriminate between acetylated proteins to react with a discrete set of substrates. Potential short-range interactions between KDAC8 and a known biologically relevant peptide substrate were identified using molecular dynamics (MD) simulations. Activity assays with a panel of peptides derived from this substrate supported a putative ionic interaction between arginine at the −1 substrate position and KDAC8 D101. Additional assays and MD simulations confirmed this novel interaction, which promotes deacetylation of substrates. Verification that a negatively charged residue at the 101 position is necessary for the ionic interaction and observed reactivity with the substrates was performed using KDAC8 derivatives. Notably, this interaction is specific to KDAC8, as KDAC1 and KDAC6 do not form this interaction and each KDAC has a different specificity profile with the peptide substrates, even though all KDACs could potentially form ionic interactions. When reacted with a panel of putative human KDAC substrates, KDAC8 preferentially deacetylated substrates containing an arginine at the −1 position. KDAC8 D101-R(−1) is a specific enzyme−substrate interaction that begins to explain how KDACs discriminate between potential substrates and how different KDAC family members can react with different subsets of acetylated proteins in cells. This multi-pronged approach will be extended to identify other critical interactions for KDAC8 substrate binding and determine critical interactions for other KDACs.

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Section: Methodsmentioning

confidence: 99%

“…Purification of KDACs was performed as previously described . Briefly, cells were lysed and KDACs were purified on the basis of the presence of the C-terminal His 6 tag using TALON metal affinity resin (Takara).…”

Section: Methodsmentioning

confidence: 99%

Lysine Deacetylase Substrate Selectivity: A Dynamic Ionic Interaction Specific to KDAC8

et al. 2021

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“…The His 6 tag is then cleaved using tobacco etch virus protease during dialysis, followed by a second affinity column to obtain highly purified enzyme with identical activity from either expression system. 32 The resulting protein contains cobalt as the active site metal, which has previously been demonstrated to have comparable activity to the zinc enzyme. 8 The expected metal content of 1:1 cobalt:KDAC8 was verified by ICPMS, and saturation confirmed by activity assays in the presence of 100-fold excess Co 2+ as previously described.…”

Section: Methodsmentioning

confidence: 98%

“…KDAC8 was expressed in both E. coli and insect cells, and then purified as described previously. 22,32 Briefly, KDAC8-His 6 is affinity purified using Talon Co 2+ resin (Clontech). The His 6 tag is then cleaved using tobacco etch virus protease during dialysis, followed by a second affinity column to obtain highly purified enzyme with identical activity from either expression system.…”

Section: Methodsmentioning

confidence: 99%

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Chelatable trace zinc causes low, irreproducible KDAC8 activity

Toro

Edenfield

Hylton

et al. 2018

Analytical Biochemistry

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Acetylation is an important regulatory mechanism in cells, and emphasis is being placed on identifying substrates and small molecule modulators of this post-translational modification. However, the reported in vitro activity of the lysine deacetylase KDAC8 is inconsistent across experimental setups, even with the same substrate, complicating progress in the field. We detected trace levels of zinc, a known inhibitor of KDAC8 when present in excess, even in high-quality buffer reagents, at concentrations that are sufficient to significantly inhibit the enzyme under common reaction conditions. We hypothesized that trace zinc in solution could account for the observed variability in KDAC8 activity. We demonstrate that addition of chelators, including BSA, EDTA, and citrate, and/or the use of a phosphate-based buffer instead of the more common tris-based buffer, eliminates the inhibition from low levels of zinc as well as the dependence of specific activity on enzyme concentration. This results in high KDAC8 activity that is consistent across buffer systems, even using low concentrations of enzyme. We report conditions that are suitable for several assays to increase both enzyme activity and reproducibility. Our results have significant implications for approaches used to identify substrates and small molecule modulators of KDAC8 and interpretation of existing data.

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Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for Residues Following Acetyllysine

2023

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Lysine acetylation is a post-translational modification that is reversed by lysine deacetylases (KDACs). The goal of this work was to identify determinants of substrate specificity for KDACs, focusing on short-range interactions occurring with residues immediately following the acetyllysine. Using a fluorescence-based in vitro assay, we determined the activity for each enzyme with a limited panel of derivative substrate peptides, revealing a distinct reactivity profile for each enzyme. We mapped the interaction surface for KDAC6, KDAC8, and KDAC1 with the +1 and +2 substrate residues (with respect to acetyllysine) based on enzyme–substrate interaction pairs observed in molecular dynamics simulations. Characteristic residues in each KDAC interact preferentially with particular substrate residues and correlate with either enhanced or inhibited activity. Although nonpolar aromatic residues generally enhanced activity with all KDACs, the manner in which each enzyme interacted with these residues is distinct. Furthermore, each KDAC has distinctive interactions that correlate with lower activity, primarily ionic in nature. KDAC8 exhibited the most diverse and widest range of effects, while KDAC6 was sensitive only to the +1 position and KDAC1 selectivity was primarily driven by negative selection. The substrate preferences were validated for KDAC6 and KDAC8 using a set of peptides derived from known acetylated proteins. Overall, we determined how KDAC6, KDAC8, and KDAC1 achieve substrate specificity with residues following the acetyllysine. These new insights into KDAC specificity will be critical for identifying novel substrates of particular KDACs, designing KDAC-specific inhibitors, and demonstrate a general framework for understanding substrate specificity for other enzyme classes.

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Purification of metal-dependent lysine deacetylases with consistently high activity

Cited by 3 publications

References 33 publications

Lysine Deacetylase Substrate Selectivity: A Dynamic Ionic Interaction Specific to KDAC8

Lysine Deacetylase Substrate Selectivity: A Dynamic Ionic Interaction Specific to KDAC8

Chelatable trace zinc causes low, irreproducible KDAC8 activity

Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for Residues Following Acetyllysine

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