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

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.

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

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.

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

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.

Chelatable trace zinc causes low, irreproducible KDAC8 activity.

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.

Purification of metal-dependent lysine deacetylases with consistently high activity.

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.

Lysine Deacetylases Exhibit Distinct Changes in Activity Profiles Due to Fluorophore Conjugation of Substrates.

Lysine deacetylases (KDACs) are enzymes that reverse the post-translational modification of lysine acetylation. Thousands of potential substrates, acetylated protein sequences, have been identified in mammalian cells. Properly regulated acetylation and deacetylation have been linked to many biological processes, while aberrant KDAC activity has also been linked to numerous diseases. Commercially available peptide substrates that are conjugated to fluorescent dye molecules, such as 7-amino-4-methylcoumarin (AMC), are commonly used to monitor deacetylation in studies addressing both substrate specificity and small molecule modulators of activity. Here, we have compared the activity of several KDACs, representing all major classes of KDACs, with substrates in the presence and absence of AMC as well as peptides for which tryptophan has been substituted for AMC. Our results unequivocally demonstrate that AMC has a significant effect on activity for all KDACs tested. Furthermore, in neither the nature of the effect nor the magnitude is consistent across KDACs, making it impossible to predict the effect of AMC on a particular enzyme-substrate pair. AMC did not affect acetyllysine preference in a multiply acetylated substrate. In contrast, AMC significantly enhanced KDAC6 substrate affinity, greatly reduced Sirt1 activity, eliminated the substrate sequence specificity of KDAC4, and had no consistent effect with KDAC8 substrates. These results indicate that profiling of KDAC activity with labeled peptides is unlikely to produce biologically relevant data.

Endogenous expression of inactive lysine deacetylases reveals deacetylation-dependent cellular mechanisms.

Acetylation of lysine residues is an important and common post-translational regulatory mechanism occurring on thousands of non-histone proteins. Lysine deacetylases (KDACs or HDACs) are a family of enzymes responsible for removing acetylation. To identify the biological mechanisms regulated by individual KDACs, we created HT1080 cell lines containing chromosomal point mutations, which endogenously express either KDAC6 or KDAC8 having single inactivated catalytic domain. Engineered HT1080 cells expressing inactive KDA6 or KDAC8 domains remained viable and exhibited enhanced acetylation on known substrate proteins. RNA-seq analysis revealed that many changes in gene expression were observed when KDACs were inactivated, and that these gene sets differed significantly from knockdown and knockout cell lines. Using GO ontology, we identified several critical biological processes associated specifically with catalytic activity and others attributable to non-catalytic interactions. Treatment of wild-type cells with KDAC-specific inhibitors Tubastatin A and PCI-34051 resulted in gene expression changes distinct from those of the engineered cell lines, validating this approach as a tool for evaluating in-cell inhibitor specificity and identifying off-target effects of KDAC inhibitors. Probing the functions of specific KDAC domains using these cell lines is not equivalent to doing so using previously existing methods and provides novel insight into the catalytic functions of individual KDACs by investigating the molecular and cellular changes upon genetic inactivation.

KDAC8 with High Basal Velocity Is Not Activated by N-Acetylthioureas.

Lysine deacetylases (KDACs) are enzymes that reverse the post-translational modification of lysine acetylation. Recently, a series of N-acetylthioureas were synthesized and reported to enhance the activity of KDAC8 with a fluorogenic substrate. To determine if the activation was general, we synthesized three of the most potent N-acetylthioureas and measured their effect with peptide substrates and the fluorogenic substrate under multiple reaction conditions and utilizing two enzyme purification approaches. No activation was observed for any of the three N-acetylthioureas under any assayed conditions. Further characterization of KDAC8 kinetics with the fluorogenic substrate yielded a kcat/KM of 164 ± 17 in the absence of any N-acetylthioureas. This catalytic efficiency is comparable to or higher than that previously reported when KDAC8 was activated by the N-acetylthioureas, suggesting that the previously reported activation effect may be due to use of an enzyme preparation that contains a large fraction of inactive enzyme. Further characterization with a less active preparation and additional substrates leads us to conclude that N-acetylthioureas are not true activators of KDAC8 and only increase activity if the enzyme preparation is below the maximal basal activity.

ESPSearch: a program for finding exact sequences and patterns in DNA, RNA, or protein.

ESPSearch is a computer program for rapidly identifying nucleic acid or amino acid sequences of any length within any source sequence from promoters to entire genomes to protein libraries. ESPSearch utilizes a user-constructed database to identify many sequences simultaneously, including target sequences with wildcards and mismatches and user-specified patterns of those recognized sequences. Here we use ESPSearch to identify a variety of possible binding sites for dimeric artificial transcription factors within several p53 recognition sites and the promoter of the BAX gene. Heterodimeric and homodimeric proteins are designed using human zinc fingers by identifying groups of zinc finger binding sites meeting particular pattern constraints. ESPSearch is also used to estimate the specificity of each artificial transcription factor by searching the entire genome. Next, the specificity of several possible small interfering RNA (siRNA) sequences is determined by searching both the whole genome and the library of known human mRNAs. Finally, ESPSearch identifies proteins containing different forms of the LXXLL motif used in nuclear receptor-coactivator interactions from the human proteome, making use of user-defined groups of amino acids. ESPSearch could also be applied to other tasks involving sequence and pattern recognition on small and large scales. ESPSearch is freely available at http://web.chemistry.gatech.edu/-doyle/espsearch/.

Inquiry-based experiments for large-scale introduction to PCR and restriction enzyme digests.

Polymerase chain reaction and restriction endonuclease digest are important techniques that should be included in all Biochemistry and Molecular Biology laboratory curriculums. These techniques are frequently taught at an advanced level, requiring many hours of student and faculty time. Here we present two inquiry-based experiments that are designed for introductory laboratory courses and combine both techniques. In both approaches, students must determine the identity of an unknown DNA sequence, either a gene sequence or a primer sequence, based on a combination of PCR product size and restriction digest pattern. The experimental design is flexible, and can be adapted based on available instructor preparation time and resources, and both approaches can accommodate large numbers of students. We implemented these experiments in our courses with a combined total of 584 students and have an 85% success rate. Overall, students demonstrated an increase in their understanding of the experimental topics, ability to interpret the resulting data, and proficiency in general laboratory skills.

KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay.

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady-state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry-based experiments and cell-based experiments will allow the identification of specific KDAC-substrate pairs and lead to a better understanding of the biological consequences of these interactions.

An improved 96-well turbidity assay for T4 lysozyme activity.

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

Purification and characterization of enzymes from yeast: an extended undergraduate laboratory sequence for large classes.

Providing a project-based experience in an undergraduate biochemistry laboratory class can be complex with large class sizes and limited resources. We have designed a 6-week curriculum during which students purify and characterize the enzymes invertase and phosphatase from bakers yeast. Purification is performed in two stages via ethanol precipitation and anion exchange chromatography, and students perform both direct and coupled enzyme assays. By completion of the experimental series, students are able to identify which enzymes they have purified and have obtained kinetic parameters for one. This experimental series requires minimal instructor preparation time, is cost effective, and works with multiple sections of large groups of students. Students participating in this sequence showed increases in conceptual understanding of biochemical concepts as measured through in-class assessments and anonymous surveys.

Identification of novel peptide substrates for protein farnesyltransferase reveals two substrate classes with distinct sequence selectivities.

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a(2) position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca(1)a(2)X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a(2) and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.