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).

1. Method 1: Producing and purifying recombinant HAT1/Rbap46 complex
<ol>
	<li>Thawing, recovering, and expanding HEK293f cells
	<ol>
		<li>Thaw 1-10 million HEK293f mammalian cells26 into 30 mL freestyle 293 expression media in a 100 mL flask. Incubate in 8% CO2 at 37 &#176;C while rotating at 60 rpm.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>HEK293f transfection
	<ol>
		<li>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.</li>
		<li>Prepare a mixture of 300 &#181;g of pHEK-FLAG-HAT1 and 300 &#181;g 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 &#176;C for 48 h at 120 rpm.</li>
	</ol>
	</li>
	<li>Harvesting cells
	<ol>
		<li>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 &#181;M to activate HAT1 and incubate cells for 30 min, 37 &#176;C, 120 rpm.</li>
		<li>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 &#176;C until protein purification or proceed directly to purification.</li>
	</ol>
	</li>
	<li>HAT1/Rbap46 complex FLAG-bead purification
	<ol>
		<li>Lyse cells and prepare protein extract.
		<ol>
			<li>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).</li>
			<li>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&#176;C. Collect all supernatant into one 50 mL tube on ice, which now is the protein extract.</li>
		</ol>
		</li>
		<li>Prepare FLAG beads.
		<ol>
			<li>Split the FLAG beads into four 15 mL conical tubes by adding 400 &#181;L 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.</li>
			<li>Pulse centrifuge for 30 s at 1000 x g at 4 &#176;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.</li>
		</ol>
		</li>
		<li>Perform FLAG immunoprecipitation.
		<ol>
			<li>Add 10 mL of protein extract to each 15 mL conical tube containing washed beads. Incubate the tubes at 4 &#176;C with inverted rotation for at least 90 min or overnight.</li>
			<li>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 &#176;C. Finally, wash 1x in RSB-100 (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 25 mM MgCl2) + 0.1% Tx-100.</li>
		</ol>
		</li>
		<li>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 &#176;C for at least 1 h or overnight. Pulse centrifuge the eluted beads at 1000 x g for 30 s at&#160;4 &#176;C and collect the supernatant, which contains the purified HAT1/Rbap46 complex.</li>
		<li>Concentrate protein and remove FLAG peptide.
		<ol>
			<li>Add eluate to a 20 mL, 10,000 Dalton&#160;cutoff filter tube. Bring the volume up to 15 mL with EB. Spin at 2500 x g for 15 min at 4 &#176;C, repeating twice with an additional 15 mL of EB each time.</li>
			<li>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 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Method 2: HAT1 acetyl-click standard curve
<ol>
	<li>Preparing standard curve
	<ol>
		<li>Synthesize a positive control H4 N-terminal peptide with the sequence: SCRG[Pra]GGKGLG[Pra]GGAKRHRKVLRGG[Lys(Biotin)], where [Pra] denotes Propargylglycine.</li>
		<li>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 &#181;L of 8 M urea and stored at -20 &#176;C until step 3.3.</li>
	</ol>
	</li>
</ol>
3. Method 3: HAT1 acetyl-click assay
<ol>
	<li>Assembling acetylation reactions with test inhibitors
	<ol>
		<li>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 &#181;M), 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 &#181;L reaction comprises 10 &#181;L of enzyme, 1 &#181;L of H4 peptide, 1 &#181;L of 20x buffer, 1 &#181;L of DTT pre-mixed and aliquoted to wells of a 96-well PCR plate on ice.</li>
		<li>Add 1 &#181;L of DMSO (negative control) or test compound dissolved at 1-10 &#181;M, 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.</li>
	</ol>
	</li>
	<li>Acetylation reaction continuation
	<ol>
		<li>Combine 2 &#181;L of 4-pentynoyl-CoA with 4 &#181;L 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 &#176;C for 1 h in capped tubes or plates with resealable foil.</li>
		<li>Process the contents directly for reaction products or quench with 20 &#181;L of 8 M urea and store at -20 &#176;C until processing.</li>
	</ol>
	</li>
	<li>Peptide binding to Neutravidin plate
	<ol>
		<li>Add reaction contents with or without urea to bovine serum albumin (BSA) pre-blocked black Neutravidin-coated 96-well plates containing 80 &#181;L of PBST (PBS + 0.1% Tween-20) per well.</li>
		<li>Add standard curve peptides in duplicate to their own wells containing 80 &#181;L of PBST. Bind peptides with gentle orbital shaking for 1 h at room temperature (RT).</li>
	</ol>
	</li>
	<li>Wash: After binding is complete, aspirate the liquid from the wells and wash the wells with 200 &#181;L of PBST 3x 15 strokes (180 &#181;L stroke volume) using a plate washer.</li>
	<li>Click chemistry reaction
	<ol>
		<li>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.</li>
		<li>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 &#181;L of PBS, 10 &#181;L of THPTA, 10 &#181;L of CuSO4, 10 &#181;L of sodium ascorbate, 20 &#181;L of biotin-azide. Always mix the click reagents fresh, then dispense 190 &#181;L to each well, seal the plate, and incubate at 37&#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Wash: Aspirate the liquid from the wells and wash the plate 3x with PBST (180 &#181;L stroke volume, 15 strokes for each wash).</li>
	<li>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 &#181;L per well) and incubate at RT for 1 h with gentle orbital shaking.</li>
	<li>Wash 3x with PBST as in the previous steps.</li>
	<li>Amplex red oxidation
	<ol>
		<li>Combine the Amplex red detection reagents as follows: 4.45 mL of NaHPO4 buffer (1x = 50 mM final concentration), 50 &#181;L amplex red (20 mM diluted in DMSO), 500 &#181;L of diluted H2O2.</li>
		<li>Dilute H2O2 from 30% stock to 3% in 1x NaHPO4 buffer, then add 22.7 &#181;L of 3% H2O2 into 977&#160;&#181;L of 1x NaHPO4 buffer (this is the H2O2 used for the amplex reaction). Add 100 &#181;L 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.</li>
	</ol>
	</li>
	<li>Calculation of enzyme inhibition: Calculate percent inhibition (Figure 3) according to the following formula: <img alt="Equation 1" src="/files/ftp_upload/66054/66054eq01.jpg" style="margin: auto;" />, 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).</li>
	<li>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 &#181;M top dose.
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Acetylation reaction with acetyl-CoA:
	<ol>
		<li>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.</li>
		<li>Spot the reaction products onto nitrocellulose membranes (1-2 &#181;L), 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.</li>
	</ol>
	</li>
</ol>

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

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We have filed patents describing the HAT1 acetyl-click assay (PCT/US20/29395). J.J.G. reports consulting agreements with Sharma Therapeutics, LLC and Guidepoint and research support (to his institution) from Hummingbird Biosciences.

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 &#34;click reactions.&#34; These reactions occur efficiently in environmentally friendly, gen...

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.&#160;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.

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

an acetyl-click chemistry assay to measure histone acetyltransferase 1 acetylation

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.

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 ...

Watch this Scientific Journal Video about Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening at JoVE.com

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

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.

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

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

an-acetyl-click-chemistry-assay-to-measure-histone-acetyltransferase

Research

JoVE Journal

Biochemistry

730 Views.  University of Texas Southwestern Medical Center. 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.

Video: Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

Chemistry

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Cancer Research

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

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

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Quantitative Immunofluorescence to Measure Global Localized Translation

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Assays for Validating Histone Acetyltransferase Inhibitors

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue