An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

Shreenidhi Rajkumar; Danielle Dixon; Andrew M. Lipchik; Joshua J. Gruber

doi:10.3791/66054

JoVE Journal > Biochemistry

生化学

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

Published: January 26, 2024

doi:

10.3791/66054

Shreenidhi Rajkumar¹, Danielle Dixon¹, Andrew M. Lipchik², Joshua J. Gruber¹

¹Departments of Internal Medicine and the Green Center for Reproductive Biology Sciences,University of Texas Southwestern Medical Center, ²Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences,Wayne State University

概要

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

Abstract

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Introduction

Among the numerous eukaryotic acetyltransferases, HAT1 was the initial histone acetyltransferase to be isolated¹^,²^,³. Subsequent investigations have firmly established its pivotal role in chromatin replication, particularly in the synthesis of new nucleosomes during S-phase⁴. Our research endeavors led to the recognition that HAT1 is highly stimulated by epidermal growth factor (EGF) treatment in mammary cells⁵. Furthermore, it has come to light that HAT1 is required for rapid cell proliferation and tumor formation in vivo⁶^,⁷^,⁸^,⁹. Data indicate that HAT1 is critical in coordinating anabolic and epigenetic processes for cell division, driving tumor growth.

HAT1 di-acetylates the amino-terminal tail of histone H4 on lysines 5 and 12 in complex with the chaperone protein Rbap46, which binds the histone and presents the amino-terminus to HAT1. Histone tetramers or disomes¹⁰, together with HAT1/Rbap46 and other histone chaperones¹¹, are then imported to the nucleus. Histones are then released to be deposited at the replication fork, or other sites to support gene activation or repression. The function of the HAT1 di-acetylation mark on histone H4 is not fully understood. It is likely quickly removed within a span of 15-30 min by the action of histone deacetylases¹²^,¹³^,¹⁴^,¹⁵ after H4 is inserted into chromatin. Thus, the HAT1 di-acetylation mark is not propagated in chromatin and may not serve a true epigenetic role, although a role in the recruitment of chromatin-modifying enzymes to nascent chromatin has been postulated¹². Also, HAT1 does not directly acetylate chromatin; its activity is restricted to soluble histones.

The development of small-molecule histone acetyltransferase inhibitors has been hampered by nonspecific and low-throughput assays, often resulting in the generation of biologically reactive compounds¹⁶^,¹⁷. The gold-standard assay to measure acetyltransferase activities requires the use of ³H-acetyl-coA, which limits throughput and requires radiation. Nonetheless, recently, specific and highly potent small molecule acetyltransferase inhibitors targeting CBP/p300¹⁸ and KAT6A/B¹⁹^,²⁰ have been described and confirmed through the use of ³H-acetyl-CoA. Moving forward, improved assays to achieve better throughput and avoid laboratory hazards are being devised.

Recent advances in acetylation monitoring²¹ have used click chemistry to enable enzymatic reaction monitoring. There are a variety of click-enabled precursors that are accessible by simple synthetic routes or available for purchase that can be incorporated into enzyme reactions. These reactions are typically carried out in recombinant systems, although cell-based assays are also feasible²². The advantage of click-enabled co-factors and substrates is that screening can directly measure enzyme activity without the need for coupled read-out systems that are often perturbed by screening compounds and require additional handling steps. This allows for inhibitor treatments only during the enzymatic step, whereas all downstream functionalization and detection steps are carried out after extensive washing to remove compounds, thus limiting the potential for assay interference to occur. These advantages make the design of click-enabled assays preferable to coupled assays that commonly rely on the detection of free coenzyme A.

One important consideration is the acceptance of click-enabled co-factors into the enzyme active site. Existing click-enabled co-factors may not be fully compatible with the active site optimized for the native co-factor. Structural information and modeling can be used to design amino acid substitutions to enlarge the active site to incorporate altered substrates²³. This may enable screening with improved enzyme kinetics and lower substrate and enzyme levels. The drawback of this approach is that altered catalytic pockets may not identify inhibitors that interact strongly with the native enzyme. Ultimately, a combination of approaches is required to identify and validate potential enzyme inhibitors.

Here, we describe a method developed to purify and assay HAT1 enzyme activity using the click co-factor 4-pentynoyl-CoA²⁴. This assay (Figure 1) uses the native enzyme sequence in complex with its required partner protein Rbap46, which has been shown to boost enzyme activity. Purification of the enzyme from human cells allows for enzyme activation in cellulo, which may preserve stimulating post-translational modifications important for full enzyme activity. Design and optimization of recombinant enzyme assays for high-throughput chemical screens have successfully been used to identify and characterize HAT1 small molecule inhibitors.

Protocol

1. Method 1: Producing and purifying recombinant HAT1/Rbap46 complex

Thawing, recovering, and expanding HEK293f cells
1. Thaw 1-10 million HEK293f mammalian cells²⁶ into 30 mL freestyle 293 expression media in a 100 mL flask. Incubate in 8% CO₂at 37 °C while rotating at 60 rpm.
2. The next day, count and check cell viability, then adjust rotation speed to 120 rpm. Expand to 300 mL culture in a 1 L flask, maintaining seeding density at 500,000 cells/mL and splitting cells before density exceeds 3 x 10⁶ cells/mL.
HEK293f transfection
1. Seed 5 x 10⁵ cells/mL in 300 mL culture in a 1 L flask and culture for 24 h. The next day, count cells to ensure they are between 7.5 x 10⁵ to 1.2 x 10⁶ cells/mL.
2. Prepare a mixture of 300 µg of pHEK-FLAG-HAT1 and 300 µg of pHEK-Rbap46 plasmid DNAs in 30 mL of PBS. Add 1.2 mL of polyethylenimine (PEI) to the DNA/PBS and mix. Incubate for 20 min at RT. Add the DNA/PBS/PEI mix to the 300 mL culture and incubate at 37 °C for 48 h at 120 rpm.
Harvesting cells
1. Count cells to ensure they fall between 1.7 x 10⁶ to 2.5 x 10⁶ cells/mL. At this cell density, add forskolin to a final concentration of 12.5 µM to activate HAT1 and incubate cells for 30 min, 37 °C, 120 rpm.
2. Pellet the cells at 300 x g for 5 min, wash once with 30 mL of PBS, and snap-freeze in liquid N₂. Store at -80 °C until protein purification or proceed directly to purification.
HAT1/Rbap46 complex FLAG-bead purification
1. Lyse cells and prepare protein extract.
  1. Prepare lysis buffer by adding one tablet of protease inhibitor cocktail (PIC) to 50 mL of RSB-500 (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 25 mM MgCl₂).
  2. Thaw the cell pellet from 300 mL culture on ice and lyse with 40 mL of ice-cold lysis buffer with 0.1% Triton X-100. Sonicate on ice, then spin down at 10,000 x g for 10 min at 4°C. Collect all supernatant into one 50 mL tube on ice, which now is the protein extract.
2. Prepare FLAG beads.
  1. Split the FLAG beads into four 15 mL conical tubes by adding 400 µL of M2-FLAG agarose bead slurry into each tube containing 5 mL of 0.1 M Glycine pH 3.5, 0.01% Triton X-100. Shake each tube vigorously for 5 s by hand.
  2. Pulse centrifuge for 30 s at 1000 x g at 4 °C, aspirate supernatant, wash twice with 5 mL of RSB-500 + 0.1% Triton X-100 (Tx-100; no protease inhibitor), and incubate on ice.
3. Perform FLAG immunoprecipitation.
  1. Add 10 mL of protein extract to each 15 mL conical tube containing washed beads. Incubate the tubes at 4 °C with inverted rotation for at least 90 min or overnight.
  2. Wash the bound beads 5x in 10 mL of RSB-500 + 0.1% Tx-100. For each wash, invert the tube 2x-5x to resuspend beads, then pellet at 1000 x g for 1 min at 4 °C. Finally, wash 1x in RSB-100 (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 25 mM MgCl₂) + 0.1% Tx-100.
4. FLAG peptide elution: Elute the HAT1 complex in 1.5 mL of Elution Buffer (EB) containing 0.5 mg/mL FLAG peptide. Incubate at 4 °C for at least 1 h or overnight. Pulse centrifuge the eluted beads at 1000 x g for 30 s at 4 °C and collect the supernatant, which contains the purified HAT1/Rbap46 complex.
5. Concentrate protein and remove FLAG peptide.
  1. Add eluate to a 20 mL, 10,000 Dalton cutoff filter tube. Bring the volume up to 15 mL with EB. Spin at 2500 x g for 15 min at 4 °C, repeating twice with an additional 15 mL of EB each time.
  2. Recover the final eluate from the filter tube and check the protein concentration by A260. Expect a concentration of 1 mg total protein in 6 mL of EB (per 300 mL starting culture). Check protein purity by SDS-PAGE, Coomassie, and immunoblotting, and store aliquots at -80 °C.

2. Method 2: HAT1 acetyl-click standard curve

Preparing standard curve
1. Synthesize a positive control H4 N-terminal peptide with the sequence: SCRG[Pra]GGKGLG[Pra]GGAKRHRKVLRGG[Lys(Biotin)], where [Pra] denotes Propargylglycine.
2. Resuspend the Pra peptide to 0.1 mg/mL in DMSO. Mix the Pra peptide with biotinylated histone H4 peptide (1-23-GGK-biotin) to create a standard curve (Figure 2; volumes found in Table 1). Standard curves may be made fresh prior to plate binding or, if made in advance, mixed with 20 µL of 8 M urea and stored at -20 °C until step 3.3.

3. Method 3: HAT1 acetyl-click assay

Assembling acetylation reactions with test inhibitors
1. Assemble acetylation reactions in duplicate in 0.2 mL PCR tubes or 96-well PCR plates from the following components: biotinylated histone H4 peptide (1-23-GGK-biotin) resuspended in DMSO to 0.1 mg/mL (34.8 µM), HAT1 enzyme pre-diluted in EB, 20x buffer (1M Tris pH 8.5, 0.1% NP40), 2 mM DTT, 4-pentynoyl-CoA dissolved in water to 1 mg/mL (1 mM). A 20 µL reaction comprises 10 µL of enzyme, 1 µL of H4 peptide, 1 µL of 20x buffer, 1 µL of DTT pre-mixed and aliquoted to wells of a 96-well PCR plate on ice.
2. Add 1 µL of DMSO (negative control) or test compound dissolved at 1-10 µM, or H4K12CoA²⁵ (or suitable positive control inhibitor), per well. Mix by gentle pipetting and incubate for 10 min on ice to allow enzyme: inhibitor complexes to form.
Acetylation reaction continuation
1. Combine 2 µL of 4-pentynoyl-CoA with 4 µL of water, then add to the wells. Gently mix by pipetting, centrifuge at 300 x g for 30 s at RT to collect the contents, and incubate at 37 °C for 1 h in capped tubes or plates with resealable foil.
2. Process the contents directly for reaction products or quench with 20 µL of 8 M urea and store at -20 °C until processing.
Peptide binding to Neutravidin plate
1. Add reaction contents with or without urea to bovine serum albumin (BSA) pre-blocked black Neutravidin-coated 96-well plates containing 80 µL of PBST (PBS + 0.1% Tween-20) per well.
2. Add standard curve peptides in duplicate to their own wells containing 80 µL of PBST. Bind peptides with gentle orbital shaking for 1 h at room temperature (RT).
Wash: After binding is complete, aspirate the liquid from the wells and wash the wells with 200 µL of PBST 3x 15 strokes (180 µL stroke volume) using a plate washer.
Click chemistry reaction
1. Prepare the reagents for the click reaction as follows: 100 mM Tris(3-hydroxypropyltriazolymethyl)amine (THPTA) ligand in water and 20 mM CuSO₄ in water. THPTA prevents Cu(II) catalyzed hydrolysis and quenches radicals and peroxides generated from O₂/Cu/ascorbate.
2. Add the THPTA/Cu mixture to 300 mM sodium ascorbate in water and 2.5 mM biotin azide in DMSO. One-click reaction contains 140 µL of PBS, 10 µL of THPTA, 10 µL of CuSO₄, 10 µL of sodium ascorbate, 20 µL of biotin-azide. Always mix the click reagents fresh, then dispense 190 µL to each well, seal the plate, and incubate at 37°C for 1 h.
Wash: Aspirate the liquid from the wells and wash the plate 3x with PBST (180 µL stroke volume, 15 strokes for each wash).
Streptavidin-horseradish peroxidase binding: Dilute streptavidin-HRP (0.224 mg/mL) 1:10 in streptavidin (0.224 mg/mL), then further dilute 1:1000 in PBST. Add Steptavidin-HRP: streptavidin mix (100 µL per well) and incubate at RT for 1 h with gentle orbital shaking.
Wash 3x with PBST as in the previous steps.
Amplex red oxidation
1. Combine the Amplex red detection reagents as follows: 4.45 mL of NaHPO₄ buffer (1x = 50 mM final concentration), 50 µL amplex red (20 mM diluted in DMSO), 500 µL of diluted H₂O₂.
2. Dilute H₂O₂ from 30% stock to 3% in 1x NaHPO₄ buffer, then add 22.7 µL of 3% H₂O₂ into 977 µL of 1x NaHPO₄ buffer (this is the H₂O₂ used for the amplex reaction). Add 100 µL of the amplex red reaction mixture per well, incubate at RT for 30 min protected from light, then detect the fluorescence excitation/emission 571/585 nm using a standard fluorescence plate reader.
Calculation of enzyme inhibition: Calculate percent inhibition (Figure 3) according to the following formula: , where D is the fluorescence value of control reactions treated with DMSO only, X is the value of reactions treated with test compounds, and BG is the value of background wells (H4 peptide control, no enzyme added).
Dose curve: Select test compounds that are found to inhibit HAT1 enzyme activity for repeat assays, with the compound serially diluted. Determine the compound dilutions empirically. For example, start with 2 mM stock concentration, then serially dilute 1:3 for eight dilutions. Then dilute 1:20 into enzyme assays yielding 100 µM top dose.
1. Plotting the curve: Use least squares regression to fit dose-response inhibition curves (using percent inhibition values from step 3.10) in data analysis software (GraphPad Prism) and derive IC₅₀ values for each compound (Figure 4).
Acetylation reaction with acetyl-CoA:
1. Use this to confirm enzyme activity with the native co-factor acetyl-CoA instead of 4-pentynoyl-CoA. Carry out HAT1 acetylation assays as described in steps 3.1-3.2 with acetyl-CoA in place of 4-pentynoyl-CoA.
2. Spot the reaction products onto nitrocellulose membranes (1-2 µL), allow them to dry, then dot-blot with anti-H4-lysine-12-acetyl or anti-H4-lysine-5-acetyl antibodies. Quantify the immunoblot signal by densitometry.

Representative Results

Standard curves in duplicate (16 wells) should be included on every plate to ensure proper assay performance. Standard curve data should be set up in table form, with a range of 100% to 0% according to the ratio of Pra-containing peptide to native H4 peptide in solution (Table 1). Amplex red signal will be the highest in 100% pra/0% native H4 peptide wells, and lowest in 0% pra/100% native H4 peptide wells. After fluorescence has been detected and wells are averaged, the resulting standards graph should fit a straight line (Figure 2), although saturation of signal at concentrations above 50% Pra can be observed. Improper mixing of Pra to H4 control peptides may result in little to no amplex red signal or nonlinear changes in amplex red signal between 0-50% Pra. Proper signal and curve results indicate that the click chemistry reaction has occurred, and the data can be analyzed accordingly.

It is important to always include triplicate control reactions: DMSO-treated reactions serve as negative controls, and H4K12CoA-treated reactions serve as positive controls for enzyme inhibition, respectively. These should be included on every plate to ensure robust assay performance and allow for Percent Inhibition calculations. Positive control wells that contain 1 µM of H4K12CoA should have a low amplex red signal, which is similar to the 0% Pra/100% H4 peptide wells. Negative control wells containing DMSO should have a signal that is similar to the fluorescent output of wells with 50% Pra/50% H4 peptide. Test compound wells will contain varying amplex signals. Average duplicate, or triplicate, wells to get a mean value of the signal with standard error.

Calculate enzyme inhibition with the formula above where D is the DMSO fluorescent output, X is the experimental output, and BG is the output from the H4 peptide without enzyme or the 0% well on the standard curve. Fluorescence output from the test compound reactions will be less than DMSO if the test compound inhibits enzyme activity and will result in positive inhibition percent values. The opposite effect will be seen in reactions where no enzyme inhibition has occurred. Example data of test compounds screened at two concentrations (1 µM and 10 µM) are depicted in Figure 3. Serial dilutions of the compounds can also be tested using this assay, where the IC₅₀of the compound can be found with non-linear curve fitting of the data (Figure 4).

Figure 1: HAT1 acetylation/acylation click chemistry schematic. The HAT1 Acetylation click reaction occurs with purified HAT1 and Rbap46 protein in a solution with biotinylated histone H4 peptide, 4-pentynoyl-CoA, and test compounds. After reactions are completed, the solution is bound to a neutravidin-coated plate, and click chemistry with copper sulfate and biotin-azide is completed. The final addition of streptavidin-HRP and amplex red results in red fluorescent output. Comparison of amplex red fluorescence intensities determines HAT1 acetylation activity. This figure has been modified with permission from Gaddameedi et al.²⁴. Please click here to view a larger version of this figure.

Figure 2: Example standard curve. After averaging the standard curve values for each pair, graph the means ± SEM against the positive control Pra peptide percentages. The resulting curve should resemble the shown curve and fit a linear line. This assay has a limit of detection (LOD) of 1.8% ± 0.89% and a limit of quantitation (LOQ) of 5.4% ± 3.1%²⁴. The standard curve may saturate for Pra percentages >50%. Please click here to view a larger version of this figure.

Figure 3: Testing new compounds in triplicate. Acetyl-click assay was used to assess the inhibitory activity of newly synthesized compounds. Compounds were diluted to 10 µM and 1 µM for testing in triplicate, with median indicated. Please click here to view a larger version of this figure.

Figure 4: Serial dilution of test compounds in HAT1 acetyl click assay. Compounds were diluted from 100 µM to 0.01 µM and tested in duplicate. The resulting dose curves show that compounds A and C inhibit HAT1 activity by nearly 100% for several dilutions. Data were then used to calculate IC₅₀ of compounds to be used in further experiments. Data are mean ± SEM of duplicate experiments. R² values for nonlinear fits are 0.84, 0.78, and 0.94 for compounds A, B, and C, respectively. Please click here to view a larger version of this figure.

% Positive Control	Positive Control Peptide [2.5 rxns] “pra” diluted to 0.1 mg/mL in DMSO (alkyne-containing)	H4 Peptide [2.5 rxns] diluted to 0.1 mg/mL in DMSO
100	2.5 µL	-
75	1.875 µL	0.625 µL
50	1.25 µL	1.25 µL
25	0.625 µL	1.875 µL
10	1 µL	9 µL
5	1.25 µL (of 1:10)	1.25 µL
1	1 µL (of 1:10)	9 µL
0	-	2.5 µL
Total	8.5 µL	25.5 µL

Table 1: Dilution of Pra control peptide to H4 peptide. Ratios of positive control "Pra" peptide to H4 peptide that are used to create a standard curve. After Pra stock is initially resuspended at 4 mg/mL in DMSO, it must be diluted 1:40 in DMSO to a final concentration of 0.1 mg/mL. Biotinylated H4 peptide must also be diluted to 0.1 mg/mL (34.8 µM) in DMSO. Then, each peptide should be mixed together in the shown proportions. Each standard will be added in duplicate to the 96-well plate. Fluorescence output values should be averaged together to create the standard curve.

Discussion

In the past decade, click chemistry became prominent²⁰, enabling the precise design of interacting chemical structures. Within this context, various bioorthogonal covalent connections²¹ have emerged as promising options for forming complexes in their natural environment. Click chemistry employs pairs of functional groups that exhibit rapid and selective reactions, commonly known as "click reactions." These reactions occur efficiently in environmentally friendly, gentle aqueous conditions. Here, we present the development of a platform designed for identifying and characterizing HAT1 acetyltransferase activity²⁴. This platform utilizes a high-throughput, peptide-based enzymatic assay enabled by click chemistry.

One of the key advantages of this assay is that it relies on the human HAT1/Rbap46 enzyme complex obtained from human cell purification rather than a bacterial source. Moreover, this method directly measures enzymatic activity on the peptide substrate, eliminating the need for coupled reactions that might be susceptible to nonspecific inhibition. While antibody-based methods are available for assessing enzyme activity²¹, the acetyl-click assay offers distinct advantages by avoiding biological variability and antibody-related costs. Furthermore, we have successfully validated its high-throughput capabilities in 96-well plates, making it feasible to conduct extensive chemical screens. The click chemistry approach can likely be adapted for use with other acetyltransferases as well. For example, recombinant acetyltransferases could be incubated with other histone peptides that mimic their natural substrates in combination with 4-pentynoyl-CoA to induce peptide acylation. Then the subsequent assay steps for peptide binding, functionalization, and amplex red oxidation could be quickly adapted. Care should be taken to design appropriate positive and negative controls for standard curves to allow for assay quantitation.

Certain limitations of the assay should be kept in mind. First, although this protocol has been optimized for HAT1, not all acetyltransferases may accept pentynoyl-CoA as an acetyl-CoA mimetic. Thus, the development of additional acyl-donor molecules could be considered²⁶, or mutagenesis of the catalytic pocket may be required to allow acceptance of click-compatible co-factors²⁷^,²⁸. Second, as affinity for click-enabled co-factors may differ from the natural co-factor, it is recommended to use secondary follow-up assays to confirm results, for example, antibody-based detection (see step 3.12). Finally, additional confirmatory enzymatic, biophysical, and cellular assays should be performed to confirm results. Small molecule binding to protein complexes can be confirmed by instruments that measure surface plasmon resonance, for example²⁹.

There are a few critical steps to highlight to ensure the assay runs smoothly and the corresponding results are accurate. The exponential growth of 293f cells should be evident prior to and after DNA transfection to achieve good protein expression. Plasmids that yield high protein expression in transient transfection systems should be employed, for example, the pHEK-293 plasmid used here. HAT1 enzyme is activated by forskolin in this protocol, but similar results have been achieved with orthovanadate, suggesting post-translational modifications are necessary for HAT1 activity. We have observed that purified HAT1/Rbap46 enzyme preparations are relatively stable and can survive freeze/thaw cycles without loss of enzyme activity. However, we recommend freezing enzyme aliquots in small amounts. We do not typically use enzymes after more than one freeze/thaw cycle.

During the acetyl-click assay, it is important to always include triplicate control reactions: DMSO-treated reactions serve as negative controls, and H4K12CoA-treated reactions serve as positive controls for enzyme inhibition, respectively. DMSO-treated enzyme reaction fluorescence values should fall on the linear standard curve between 25 and 50 % positive Pra peptide to avoid signal saturation. This may require carefully titrating the amount of HAT1/Rbap46 enzyme in control reactions prior to drug testing. For example, the HAT1/Rbap46 enzyme can be diluted 1:1, 1:5, 1:10, 1:25, 1:50, 1:75, and 1:100 in EB, then used in acetyl-click reactions to determine the amount of enzyme required to generate signal between 25%-50% of Pra peptide. We have found that 100 nM enzyme is typically sufficient but should be determined empirically for each new preparation of enzyme. The purpose of mixing streptavidin-HRP with streptavidin is to decrease the amplex oxidation signal. The dilution of streptavidin can be adjusted to the output signal as needed. If a rapid signal is generated, it may be necessary to read the plate before 30 min to avoid signal saturation. During initial assay optimization, we suggest reading at 5 min, 15 min, and 30 min. Serial reads can be performed without influencing signal. If the assay is well calibrated the amplex red signals generated between 5 min and 30 min should scale linearly.

開示

The authors have nothing to disclose.

Acknowledgements

We thank George Zheng for providing H4K12CoA. We thank members of the Gruber Lab for helpful discussions and feedback. We thank support from the NIH/NCI (1K08CA245024), CPRIT (RR200090, RP210041), and the V Foundation (V2022-022).

Materials

4P CoA	Cayman Chemical	10547	Click chemistry co-factor
Amplex Red	Fisher Sci	A12222	Fluorescence substrate
Biotin-PEG-Azide	Alfa Aesar	J64996MC	Click chemistry
Copper Sulfate	Sigma-aldrich	7758-98-7	Click chemistry
DMSO	Fisher Scientific	67-68-5	diluent
DTT	Acros Organics	03-12-3483	reducting agent
Forskolin	VWR	102987-310	Protein expression
Freestyle 293 Expression Medium	Thermo Fisher	12338018	Media
Freestyle 293-F cells	Thermo Fisher	R790-07	Protein expression
H4-peptide/1-23-GGK-biotin	Anaspec	AS65097	peptide substrate
HEPES	Sigma-aldrich	7365-45-9	EB buffer
Hydrogen peroxide 30% solution	Sigma-aldrich	Z00183-99-0	initiator
M2 FLAG antibody slurry	Millipore-Sigma	A2220	Protein purification
Macrosep 10K Filter (Pall Lab)	VWR	89131-980	Protein purification
Neutravidin Plate	Thermo Sci	15127	BSA-pre-blocked
NP40 (IGEPAL)	MP Biomedical	198596	20x buffer
pHEK-293 plasmid	Takara Bio	3390	Protein expression
Phosphate Buffered Saline 10x	Alfa Aesar	Z00082-33-6	wash buffer
Pra peptide	Genscript	Custom synthesis	biotinylated
Sodium Ascorbate	Sigma-aldrich	134-03-2	Click chemistry
Sodium chloride	Sigma-aldrich	7647-14-5	EB buffer
Sodium phosphate	VWR International	7558-80-7	buffer
Streptavidin	EMD Millipore	189730	competitor
Streptavidin-HRP	Cell Signaling	3999S	enzyme
THPTA ligand	Fisher Sci	1010-500	Click chemistry
Tris base	Sigma-aldrich	77-86-1	20x buffer
Triton-X 100	VWR International	9002-93-1	EB buffer
Tween-20	Sigma-aldrich	9005-64-5	Wash buffer
Urea	Sigma-Aldrich	57-13-6	quencher

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Rajkumar, S., Dixon, D., Lipchik, A. M., Gruber, J. J. An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation. J. Vis. Exp. (203), e66054, doi:10.3791/66054 (2024).

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation