Terry J. Watt

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.

The push and pull of land use policy: reconstructing 150 years of development and conservation land acquisition.

The growth of human populations and their resource needs have stressed the conservation of natural land resources. Many policies and programs have been implemented to address the pressures on land resources and notwithstanding this pressure, significant acquisition of land for conservation has occurred throughout history in the U.S., and internationally. Here we assess the on-the-ground result of the evolution of land use policies in California as a pioneer forerunner, in the form of acquisition of land for conservation (i.e. Open Space), and its impact on the rest of the U.S. and beyond. To this end we describe the timeline and spatial representation of the growth of California’s conservation network over the last 150 years, and link it to the history of land use policies. We then assess whether conservation land acquisition has consistently grown through time or occurred in specific decades. About ¼ of the state is now designated Open Space. Fewer and larger areas conserved and acquired at the beginning of the 20th century; the conservation network was complemented with a larger number of smaller sized properties. Despite acquisition of land in every decade, the process was uneven (E = 0.3 for California, E = 0.14±0.08 average for the state’s counties), mostly due to the large acquisitions and land set asides in the 1900s, followed by 1930s and 1940s. This process was a result of a comprehensive set of legislation that evolved through time, and resulted from the competing needs for development and conservation. Even with the impressive 174,000 km2 of public lands in California, the future of California’s natural infrastructure and natural heritage cannot rely solely on these public lands, nor public agencies and their resources. Critically a future course of land preservation relying on the purchase of new lands – in California and beyond – for conservation is tremendously expensive.

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.

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.

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.

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.

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.

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.

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.