Name: deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates
Uploaded: 2016-02-27T14:00:00.000+00:00
Duration: 14 min 32 s
Description: Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

The project described here was supported by a grant from NIH/NCI (NCI-R01CA182506-01A1) as well as by the Robert H. Lurie Comprehensive Cancer Center - The Lefkofsky Family Foundation/Liz and Eric Lefkofsky Innovation Research Award to A.V. We would like to thank members of the laboratory (Carol O&#39;Callaghan and Elizabeth Anne Wayne) for critical reading and editing this manuscript.

1. In Vitro Deacetylation Assay
<ol>
	<li>Purification of SIRT2
	<ol>
		<li>Prepare a 10 cm culture dish of HEK 293T cells cultured in 8 ml DMEM with 10% FBS and antibiotics and grown in a 37 &#176;C, 5% CO2 tissue culture incubator to be about 70% confluent the next day.</li>
		<li>The next day, co-transfect 4 &#181;g pCDH-puro-GFP-SIRT2-Flag (lentivector made in our lab), 4 &#181;g pCMV- dR8.2 dvpr (packaging vector) and 0.5 &#181;g pCMV-VSV-G (envelope vector) 18 using polyethylenimine (PEI) at a ratio of 3 &#181;l PEI/&#181;g DNA.</li>
		<li>Change media after 12 hr.</li>
		<li>Collect supernatant after 36-48 hr.</li>
		<li>Remove debris by centrifugation at 1,000 x g for 5 min at 4 &#176;C.</li>
		<li>After filtering through 0.22 &#181;m filters, make 1 ml aliquots of SIRT2 lentivirus and keep at -80 &#176;C.</li>
		<li>Thaw SIRT2 lentivirus on ice (it is important to store the virus at -80 &#176;C when the virus is not used).</li>
		<li>Infect a 6-well plate of 80-90% confluent HEK 293T cells with 1 ml of lentivirus in the presence of 8 &#181;g/ml polybrene.</li>
		<li>Place virus-infected cells in 37 &#176;C incubator until the next day (24 hr).</li>
		<li>Replace medium after 24 hr.</li>
		<li>48 hr after infection, check infection efficiency by detecting GFP positive cells under a fluorescent microscope.</li>
		<li>If infection efficiency is good (at least 40-50% infected cells), replace medium with culture medium containing 2 &#181;g/ml puromycin to select for stable SIRT2 over expressing cells. 
		Note: This takes approximately 2 weeks, and the medium is changed every 3-4 days. Non-infected cells die over this selection period and only infected cells with the SIRT2 lentivirus grow.</li>
		<li>Analyze expression of Flag-tagged SIRT2 by western blotting using an anti-Flag antibody (1:1,000 dilution) after running 40 &#181;g of total protein on a 10% SDS-PAGE gel before starting the purification process (recommended) 19,20.</li>
		<li>To purify active SIRT2, split cells in 1:3 ratio by trypsinization in order to have enough dishes of HEK 293T cells stably overexpressing Flag-SIRT2 (Ten 10 cm dishes will allow purification of enough protein for subsequent experiments).
		<ol>
			<li>To trypsinize the cells, remove culture medium and eliminate residual serum by rinsing cells with sterile DPBS. Slowly add 2.5 ml of a 0.25% Trypsin-EDTA solution to cover the cell monolayer, incubate at room temperature for 30-45 sec. After removing the Trypsin-EDTA solution, incubate at 37 &#176;C until cells start to detach. Then add culture medium and transfer cells to new dishes.</li>
		</ol>
		</li>
		<li>Remove culture medium, wash cells twice in PBS and collect cells by trypsinization followed by centrifugation at 1,000 x g for 10 min at 4 &#176;C.</li>
		<li>Lyse cell pellet in 500 &#181;l lysis buffer A (20 mM HEPES pH 7.9, 18 mM KCl, 0.2 mM EGTA, 1.5 mM MgCl2, 20% Glycerol, 0.1% NP-40) including a protease inhibitors cocktail and 1 &#181;M TSA for 30 min at 4 &#176;C under constant rotation.</li>
		<li>Centrifuge at 14,000 x g for 15 min at 4 &#176;C and transfer supernatant to a new tube.</li>
		<li>Perform immunoprecipitation using an anti-Flag antibody conjugated to agarose beads as described below.
		<ol>
			<li>Add 200-300 &#181;l of anti-Flag antibody conjugated to agarose beads&#160;to the protein lysate (10-20 mg total protein).</li>
			<li>Incubate overnight at 4 &#176;C with constant agitation.</li>
			<li>Collect immunoprecipitates by centrifugation at 1,000 x g for 5 min at 4 &#176;C.</li>
			<li>Wash the pellet 5 times with 10 ml lysis buffer A at 1,000 x g for 5 min at 4 &#176;C. After each wash, pellet the beads and replace the supernatant with buffer A.</li>
		</ol>
		</li>
		<li>Prepare 1x Flag peptide solution (including a protease inhibitors cocktail and 1 &#181;M TSA in PBS).</li>
		<li>Add 500 &#181;l of 1x Flag peptide solution to the agarose beads and incubate for 30 min at 4 &#176;C under constant rotation.</li>
		<li>Collect supernatant by centrifugation at 6,000 x g for 2 min at 4 &#176;C.</li>
		<li>Repeat previous two steps.</li>
		<li>Remove beads from supernatant by using filter tubes and centrifugation at 15,000 x g for 1 min at 4 &#176;C.</li>
		<li>Concentrate eluted sample until the final protein concentration is 1 &#181;g/&#181;l using an ultrafiltration membrane.</li>
		<li>Check purification process by performing electrophoresis using 1 &#181;g of the eluted sample.</li>
		<li>Stain gel with a commercially available staining solution to confirm SIRT2 purification.</li>
		<li>Keep purified SIRT2 at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Purification of Acetylated Protein-substrate
	<ol>
		<li>Prepare 10 cm x 10 cm culture dishes with HEK 293T cells to be about 70% confluent the next day.</li>
		<li>The next day, co-transfect cells with 5 &#181;g of a Flag tagged plasmid vector expressing the protein-substrate as well as 5 &#181;g of the specific acetyl-transferase which can acetylate the protein using PEI at a ratio of 3 &#181;l PEI/&#181;g DNA. 
		Note: If the specific acetyl-transferase is not known, co-transfect with a mixture of known histone acetyl-transferases (HATs) such as p300, CBP, GCN5, Tip60 and PCAF.</li>
		<li>Change medium after 12 hr.</li>
		<li>The day before lysing the cells, treat the cells with 1 &#181;M TSA and 2 mM nicotinamide (NAM) overnight by replacing the culture medium with medium containing TSA/NAM to maximize acetylation levels of the protein-substrate by inhibiting deacetylases.</li>
		<li>48 hr after transfection, remove culture medium, wash cells twice in PBS and collect cells by trypsinization followed by centrifugation at 1,000 x g for 10 min at 4 &#176;C.</li>
		<li>Lyse cell pellet in 500 &#181;l lysis buffer A per dish (20 mM HEPES pH 7.9, 18 mM KCl, 0.2 mM EGTA, 1.5 mM MgCl2, 20% Glycerol, 0.1% NP-40) including a protease inhibitors cocktail, 1 &#181;M TSA and 2 mM NAM for 30 min at 4 &#176;C under constant rotation.</li>
		<li>Centrifuge at 14,000 x g for 15 min at 4 &#176;C and transfer supernatant to a new tube.</li>
		<li>Perform immunoprecipitation using an anti-Flag antibody conjugated to agarose beads as described below.
		<ol>
			<li>Add 200-300 &#181;l of anti-Flag antibody conjugated to agarose beads&#160;to the protein lysate (10-20 mg total protein).</li>
			<li>Incubate overnight at 4 &#176;C with constant agitation.</li>
			<li>Collect immunoprecipitates by centrifugation at 1,000 x g for 5 min at 4 &#176;C.</li>
			<li>Wash the pellet 2 times with 10 ml lysis buffer A at 1,000 x g for 5 min at 4 &#176;C. After each wash, pellet the beads and replace the supernatant with lysis buffer A.</li>
			<li>Wash the pellet 3 times with 10 ml lysis buffer A without NAM at 1,000 x g for 5 min at 4 &#176;C. After each wash, pellet the beads and replace the supernatant with lysis buffer A. It is important not to carry over any NAM to the deacetylation reaction.</li>
		</ol>
		</li>
		<li>Prepare 1x Flag peptide solution (including protease inhibitors and 1 &#181;M TSA in PBS).</li>
		<li>Add 500 &#181;l of 1x Flag peptide solution to the agarose beads and incubate for 30 min at 4 &#176;C under constant rotation.</li>
		<li>Collect supernatant by centrifugation at 6,000 x g for 2 min at 4 &#176;C.</li>
		<li>Repeat previous two steps.</li>
		<li>Remove beads from supernatant by using filter tubes and centrifugation at 15,000 x g for 1 min at 4 &#176;C.</li>
		<li>Using an ultrafiltration membrane, concentrate eluted sample until the protein concentration is 1 &#181;g/ul.</li>
		<li>Perform electrophoresis by running 1 &#181;l of the eluted sample on a 4-12% SDS-PAGE gel.</li>
		<li>Confirm acetylation of protein-substrate by western blotting using antibodies against Ac-K (1:1,000 dilution) and the protein-substrate (dilution depends on the specific antibody used).</li>
		<li>Keep purified acetylated protein-substrate at -80 &#176;C.</li>
	</ol>
	</li>
	<li>In Vitro Deacetylation Reaction
	<ol>
		<li>Prepare the deacetylation buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2 0.5 &#181;M TSA)</li>
		<li>Prepare 3 different reactions in different tubes in 20 &#181;l final volume (Table 1).</li>
		<li>Include additional reactions to confirm deacetylation of the protein-substrate by SIRT2 (optional). Add 2 &#181;g of purified deacetylation null mutant SIRT2 (this can be done using the protocol described in 1.1 after using a pCDH-puro-GFP-SIRT2H187Y-Flag lentivector) to the reaction instead of deacetylase active SIRT2 or add 10 mM nicotinamide (NAM) to the reaction to inhibit the deacetylation activity of SIRT2 (Table 1).</li>
		<li>Incubate reactions for 3 hr at 30 &#176;C under constant agitation.</li>
		<li>Stop the reaction by adding 20 &#181;l of 2x sample buffer and boil the samples for 10 min at 95 &#176;C.</li>
		<li>Perform electrophoresis by running the reaction mixture on a 4-12% SDS-PAGE gel and detect the acetylation status of the protein-substrate by western blotting using an anti Ac-K antibody (1:1,000 dilution) 19,20.</li>
	</ol>
	</li>
</ol>
2. In Vivo Deacetylation Assay
<ol>
	<li>SIRT2 Overexpression in Cultured Cells
	<ol>
		<li>Prepare 10 cm culture dishes with HEK 293T cells stably overexpressing pCDH-puro-GFP-empty vector, pCDH-puro-GFP-SIRT2-Flag and pCDH-puro-GFP-SIRT2H187Y-Flag (as described in 1.1) to be about 70% confluent.</li>
		<li>Alternatively, prepare 10 cm culture dishes with cells about 70% confluent and perform transient overexpression using 5 &#181;g of the vectors mentioned above using PEI at a ratio of 3 &#181;l PEI/&#181;g DNA.</li>
		<li>Perform western blotting of total cellular extracts using an anti-Flag antibody (1:1,000 dilution) to demonstrate overexpression of Flag-tagged SIRT219.</li>
	</ol>
	</li>
	<li>RNA Interference to Knockdown SIRT2 in Cultured Cells
	<ol>
		<li>Prepare a 10 cm culture dish of HEK 293T cells to be about 70% confluent the next day.</li>
		<li>The next day, co-transfect 4 &#181;g pLKO1-puro-sh SIRT2 (lentivector) 19, 4 &#181;g pCMV- dR8.2 dvpr (packaging vector) and 0.5 &#181;g pCMV-VSV-G (envelope vector) 18 using polyethylenimine (PEI) at a ratio of 3 &#181;l PEI/&#181;g DNA. 
		Note: For knocking down SIRT2, we use simple hairpin shRNAs in the pLKO.1 lentiviral vector designed by The RNAi Consortium (TRC).</li>
		<li>Follow the procedure as described in 1.1 to produce shSIRT2 lentivirus and infect target cells to knockdown SIRT2.</li>
		<li>Perform western blotting of total cellular extracts using an anti-SIRT2 antibody (1:1,000 dilution) to demonstrate efficient SIRT2 knockdown after running 40 &#181;g of total protein on a 10% SDS-PAGE gel.</li>
		<li>Alternatively, prepare 10 cm culture dishes with cells about 70% confluent and perform transient knockdown of SIRT2 using siRNAs following manufacturer&#39;s instructions.</li>
		<li>Perform western blotting of total cellular extracts using an anti-SIRT2 antibody (1:1,000 dilution) to demonstrate efficient SIRT2 knockdown after running 40 &#181;g of total protein on a 10% SDS-PAGE gel.</li>
	</ol>
	</li>
	<li>Immunoprecipitation and Immunoblotting to Detect Acetylated Levels in Cultured Cells
	<ol>
		<li>12 hr before collecting cell pellets from either cells overexpressing SIRT2 (see 2.1 above) or SIRT2 knocked down cells (see 2.2 above), add 1 &#181;m TSA to the culture medium to inhibit non Class III histone deacetylases.</li>
		<li>Remove culture medium, wash cells twice in PBS and collect cells by trypsinization followed by centrifugation at 1,000 x g for 10 min at 4 &#176;C.</li>
		<li>Add 10 ml lysis buffer A (20 mM HEPES pH 7.9, 18 mM KCl, 0.2 mM EGTA, 1.5 mM MgCl2, 20% Glycerol, 0.1% NP-40) including a protease inhibitors cocktail, 1 &#181;M TSA and 2 mM NAM.</li>
		<li>Incubate at 4 &#176;C for 30 min under constant rotation.</li>
		<li>Collect supernatants by centrifugation at 20,000 x g for 15 minutes at 4 &#176;C.</li>
		<li>Calculate protein concentration by using a Bradford protein assay.</li>
		<li>Transfer 1 mg of total protein to new tubes in a final volume of 1 ml using lysis buffer A. Use Table 2 as a reference to prepare protein samples from cells either overexpressing SIRT2 or after SIRT2 knockdown.</li>
		<li>Perform an immunoprecipitation using an antibody against the specific protein-substrate together with Protein A/G (40 &#181;l bead slurry is recommended). Alternatively, use an anti Ac-K antibody conjugated to agarose beads (20 &#181;l bead slurry is usually enough) to immunoprecipitate all acetylated proteins.</li>
		<li>Incubate samples at 4 &#176;C under constant rotation overnight.</li>
		<li>Collect immunoprecipitates by centrifugation at 1,000 x g for 2 min at 4 &#176;C.</li>
		<li>Wash beads 5 times with 1 ml buffer A at 1,000 x g for 2 min at 4 &#176;C.</li>
		<li>Resuspend beads in 40 &#181;l of 2x sample buffer.</li>
		<li>Boil samples for 10 min at 95 &#176;C.</li>
		<li>Perform electrophoresis by running the eluted samples (40 &#181;l from step 2.3.12) on a 4-12% SDS-PAGE gel without disturbing the beads while transferring the eluted samples.</li>
		<li>Detect acetylated levels of the protein-substrate by Western blotting using an anti-Ac-K antibody (1:1,000 dilution) if a protein-substrate specific antibody was used for immunoprecipitation, or a specific antibody against the protein-substrate (follow recommendations for the specific antibody regarding dilution) if anti Ac-K beads were used for the immunoprecipitation.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+age+of+crosstalk:+phosphorylation,+ubiquitination,+and+beyond.">The age of crosstalk: phosphorylation, ubiquitination, and beyond.</a> Molecular Cell. 28, 730-738 (2007).</li><li>Gao, M., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+regulators:+control+of+protein+ubiquitination+and+ubiquitin-like+modifications+by+extracellular+stimuli.">Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli.</a> Molecular Cell. 19, 581-593 (2005).</li><li>Philp, A., Rowland, T., Perez-Schindler, J., Schenk, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+acetylome:+translating+targeted+proteomics+into+meaningful+physiology.">Understanding the acetylome: translating targeted proteomics into meaningful physiology.</a> American Journal of Physiology. Cell Physiology. 307, C763-C773 (2014).</li><li>Allfrey, V. G., Faulkner, R., Mirsky, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+and+Methylation+of+Histones+and+Their+Possible+Role+in+the+Regulation+of+Rna+Synthesis.">Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 51, 786-794 (1964).</li><li>Shahbazian, M. D., Grunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+site-specific+histone+acetylation+and+deacetylation.">Functions of site-specific histone acetylation and deacetylation.</a> Annual Review of Biochemistry. 76, 75-100 (2007).</li><li>L'Hernault, S. W., Rosenbaum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+alpha-tubulin+is+posttranslationally+modified+in+the+flagella+during+flagellar+assembly.">Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly.</a> The Journal of Cell Biology. 97, 258-263 (1983).</li><li>Kim, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+and+functional+diversity+of+lysine+acetylation+revealed+by+a+proteomics+survey.">Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.</a> Molecular Cell. 23, 607-618 (2006).</li><li>Choudhary, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysine+acetylation+targets+protein+complexes+and+co-regulates+major+cellular+functions.">Lysine acetylation targets protein complexes and co-regulates major cellular functions.</a> Science. 325, 834-840 (2009).</li><li>Smith, K. T., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+the+acetylome.">Introducing the acetylome.</a> Nature Biotechnology. 27, 917-919 (2009).</li><li>Roth, S. Y., Denu, J. M., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetyltransferases.">Histone acetyltransferases.</a> Annual Review of Biochemistry. 70, 81-120 (2001).</li><li>Ozden, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+of+MnSOD+directs+enzymatic+activity+responding+to+cellular+nutrient+status+or+oxidative+stress.">Acetylation of MnSOD directs enzymatic activity responding to cellular nutrient status or oxidative stress.</a> Aging. 3, 102-107 (2011).</li><li>Anderson, K. A., Hirschey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+acetylation+regulates+metabolism.">Mitochondrial protein acetylation regulates metabolism.</a> Essays in Biochemistry. 52, 23-35 (2012).</li><li>Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+lysine+acetylation+controls+the+activity+of+the+mitochondrial+enzyme+acetyl-CoA+synthetase+2.">Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10224-10229 (2006).</li><li>Haberland, M., Montgomery, R. L., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+histone+deacetylases+in+development+and+physiology:+implications+for+disease+and+therapy.">The many roles of histone deacetylases in development and physiology: implications for disease and therapy.</a> Nature Reviews. Genetics. 10, 32-42 (2009).</li><li>Finkel, T., Deng, C. X., Mostoslavsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+biology+and+physiology+of+sirtuins.">Recent progress in the biology and physiology of sirtuins.</a> Nature. 460, 587-591 (2009).</li><li>Houtkooper, R. H., Pirinen, E., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+as+regulators+of+metabolism+and+healthspan.">Sirtuins as regulators of metabolism and healthspan.</a> Nature reviews. Molecular Cell Biology. 13, 225-238 (2012).</li><li>Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+landscape+of+lysine+acetylation+links+metabolism+and+cell+signalling.">The growing landscape of lysine acetylation links metabolism and cell signalling.</a> Nature Reviews. Molecular Cell Biology. 15, 536-550 (2014).</li><li>Stewart, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9, 493-501 (2003).</li><li>Kim, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT2+maintains+genome+integrity+and+suppresses+tumorigenesis+through+regulating+APC/C+activity.">SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.</a> Cancer Cell. 20, 487-499 (2011).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT2+directs+the+replication+stress+response+through+CDK9+deacetylation.">SIRT2 directs the replication stress response through CDK9 deacetylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 13546-13551 (2013).</li><li>Weinert, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+mapping+of+the+Drosophila+acetylome+demonstrates+a+high+degree+of+conservation+of+lysine+acetylation.">Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation.</a> Science Signaling. 4, ra48 (2011).</li><li>Still, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mitochondrial+acetylation+dynamics+highlights+prominent+sites+of+metabolic+regulation.">Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.</a> The Journal of Biological Chemistry. 288, 26209-26219 (2013).</li><li>Sadoul, K., Wang, J., Diagouraga, B., Khochbin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tale+of+protein+lysine+acetylation+in+the+cytoplasm.">The tale of protein lysine acetylation in the cytoplasm.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 970382 (2011).</li><li>Preble, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+and+purification+of+mitochondria+for+transplantation+by+tissue+dissociation+and+differential+filtration.">Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.</a> Journal of Visualized Experiments : JoVE. , e51682 (2014).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments : JoVE. , (2011).</li><li>He, W., Newman, J. C., Wang, M. Z., Ho, L., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+sirtuins:+regulators+of+protein+acylation+and+metabolism.">Mitochondrial sirtuins: regulators of protein acylation and metabolism.</a> Trends in Endocrinology and Metabolism: TEM. 23, 467-476 (2012).</li></ol>

The authors have nothing to disclose.

Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in ...

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table><tbody><tr><td>cell culture dishes</td><td>Denville Scientific Inc.</td><td>T1110 and T1115</td><td></td></tr><tr><td>pCDH-puro-GFP lentiviral vector</td><td>System Biosciences</td><td>CD513B-1</td><td></td></tr><tr><td>pCMV- dR8.2 dvpr (packaging vector)</td><td>Addgene</td><td>8455</td><td></td></tr><tr><td>pCMV-VSV-G (envelope vector)</td><td>Addgene</td><td>8454</td><td></td></tr><tr><td>polyethylenimine (PEI)&amp;nbsp;</td><td>Polysciences Inc.</td><td>24885</td><td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td></tr><tr><td>0.22 &amp;mu;m filters</td><td>Denville Scientific Inc.</td><td>F5512</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>H9268</td><td></td></tr><tr><td>fluorescent microscope</td><td>Carl Zeiss MicroImaging Inc.</td><td>Axiovert 200</td><td></td></tr><tr><td>puromycin</td><td>Invivogen</td><td>A11138-03</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>anti-Flag antibody</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>34596</td><td></td></tr><tr><td>protease inhibitors coctail 100x</td><td>Biotool</td><td>B14001</td><td></td></tr><tr><td>Trichostatin A (TSA)</td><td>Sigma</td><td>T8552</td><td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class III HDACs (sirtuins)</td></tr><tr><td>anti-Flag agarose beads</td><td>Sigma</td><td>A2220</td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>rotator</td><td>Thermo Scientific</td><td>415220Q</td><td></td></tr><tr><td>filter tubes</td><td>Millipore</td><td>UFC30HV00</td><td></td></tr><tr><td>Flag peptide&amp;nbsp;</td><td>Sigma</td><td>F3290</td><td></td></tr><tr><td>Vivaspin Centrifugal Concentrator</td><td>Sartorius Stedim Biotech S.A.</td><td>VS0102</td><td></td></tr><tr><td>SimplyBlue SafeStain solution&amp;nbsp;</td><td>Invitrogen</td><td>LC6060</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Techologies</td><td>NP0007</td><td></td></tr><tr><td>Tris-HCl pH 7.5</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>NAD&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma</td><td>N0632</td><td>required cofactor for sirtuins</td></tr><tr><td>nicotinamide (NAM)</td><td>Sigma</td><td>72340</td><td>selective inhibitor class III HDACs (sirtuins)</td></tr><tr><td>pLKO.1 lentiviral vector&amp;nbsp;</td><td>Addgene</td><td>8453</td><td></td></tr><tr><td>SIRT2 si RNA&amp;nbsp;</td><td>Qiagen</td><td>GS22933</td><td></td></tr><tr><td>anti-SIRT2 antibody</td><td>Proteintech</td><td>15345-1-AP</td><td></td></tr><tr><td>Bradford protein assay</td><td>BIO-RAD</td><td>500-0006</td><td></td></tr><tr><td>anti Ac-K agarose beads</td><td>Immunechem</td><td>ICP0388</td><td></td></tr></tbody></table>

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.

In order for a protein to be considered as a legitimate deacetylation target for any enzyme with deacetylation activity, both in vitro and in vivo deacetylation assays need to be performed to establish the interplay between the deacetylase and its substrate. For the in vitro deacetylase assay, the purification of both the deacetylase and the acetylated protein substrate is required before the assay can be done. Here we use the cytoplasmic sirtuin SIRT2 as the sp...

Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

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.

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 ...

deacetylation-assays-to-unravel-interplay-between-sirtuins-sirt2

Research

JoVE Journal

Chemistry

8.2K Views.  Feinberg School of Medicine, Northwestern University. 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.

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

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Biochemistry

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

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

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

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.

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

High-Throughput Thermal Shift Assays for Protein-Ligand Interactions

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Biology

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

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

Summary

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Chapters in this video

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

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

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

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

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization