The overall goal of this protocol is to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins. Despite the increasing number of proteins found to be acetylated, arguing for a widespread presence of acetylation, specific enzymes regulating these modification have not yet been identified for most of these proteins. The main advantage of these assays is that by establishing a protein as a legitimate substrate of a specific deacetylase, novel roles and functions of the deacetylase can be unraveled.
Though this method can provide insight into the identification of sirtuin 2-specific substrate, it can also be applied, with minor modifications, to on other members of the sirtuin family. To begin this procedure, prepare a 10 centimeter culture dish of HEK 293 T cells in eight milliliters of DMEM, with 10%FBS in antibiotics so that the cells will be about 70%confluent after growing overnight. On the following day, cotransfect the cells with the following:four micrograms of lentivector, expressing GFP and FLAG-tagged SIRT2, four micrograms of packaging vector and 0.5 micrograms of envelope vector.
Use polyethylenimine, or PEI, at a ratio of three microliters of PEI per microgram of DNA. 12 hour after transfection, change the medium. 36 to 48 hours later, collect the supernatant.
Remove debris by centrifugation at 1, 000 g for five minutes at four degrees Celsius. After filtering through a 0.22 micrometer filter, make one milliliter aliquots of SIRT2 lentivirus, and store at negative 80 degrees Celsius. When ready to infect the cells, thaw the SIRT2 lentivirus on ice.
Infect a six-well plate of 80 to 90%confluent HEK 293 T cells with one milliliter of lentivirus in the presence of eight micrograms per milliliter of Polybrene. Place the virus-infected cells in a 37-degree Celsius incubator for 24 hours. After 24 hours, replace the medium.
48 hours after infection, check the infection efficiency by detecting GFP-positive cells under a fluorescent microscope. If infection efficiency is at least 40 to 50%replace the medium with culture medium containing two micrograms per milliliter of Puromycin to select for stable SIRT2 overexpressing cells. Return the cells to the incubator.
The selection for stable SIRT2 overexpressing cells will take about two weeks, and the medium must be changed every three to four days. For SIRT2 purification, prepare at least 10 10-centimeter dishes of FLAG SIRT2-expressing HEK 293 T cells. After 24 hours, remove the culture medium, wash the cells twice in PBS and collect cells by trypsinization, followed by centrifugation at 1, 000 g for 10 minutes at four degrees Celsius.
Lyse the cell pellet in 500 microliters of lysis buffer A per dish that includes a protease inhibitor cocktail and one micromolar of TSA, a non-class three histone deacetylase inhibitor. Incubate for 30 minutes at four degrees Celsius under constant rotation. After 30 minutes, centrifuge at 14, 000 g for 15 minutes at four degrees Celsius.
Transfer the supernatant to a new tube. Perform immunoprecipitation using an anti-FLAG antibody conjugated to agarose beads. Add 200 to 300 microliters of the anti-FLAG antibody to the protein lysate and incubate at four degrees Celsius with constant agitation overnight.
On the following day, collect the immunoprecipitates by centrifugation at 1, 000 g and four degrees Celsius for five minutes. Wash the pellet five times with 10 milliliters of lysis buffer A.After the fifth wash, remove the supernatant and add 500 microliters of a one X FLAG peptide solution that includes a protease inhibitor cocktail and TSA to the agarose beads. Incubate at four degrees Celsius, under constant rotation, for 30 minutes.
Collect the supernatant by centrifugation at 6, 000 g for two minutes at four degrees Celsius. Remove the agarose beads from the supernatant by using filter tubes and centrifugation at 15, 000 g at four degrees Celsius for one minute. Lastly, use an ultrafiltration membrane to concentrate the eluted sample until the final protein concentration is one microgram per microliter.
Keep purified SIRT2 at negative 80 degrees Celsius. Begin this procedure by preparing 10 10-centimeter culture dishes of HEK 293 T cells, so they will be about 70%confluent the following day. On the following day, cotransfect the cells with five micrograms of a FLAG-tagged plasma infector expressing the protein-substrate and five micrograms of the specific acetyltransferase that can acetylate the protein.
If the specific acetyltransferase is not known, use a mixture of known histone acetyltransferases. Use PEI at a ratio of three microliters of PEI per microgram of DNA. Incubate the transfected cells for 12 hours.
Then, change the medium and incubate the cells for 24 hours. Treat the cells with one micromolar TSA and two millimolar nicotinamide for 12 hours by replacing the culture medium with medium containing TSA and nicotinamide. This will maximize acetylation levels of the protein-substrate by inhibiting deacetylases.
48 hours after transfection, remove the culture medium and wash the cells twice with PBS. Collect the cells by trypsinization, followed by centrifugation at 1, 000 g for 10 minutes at four degrees Celsius. To lyse the cell pellet, add 500 microliters of lysis buffer A per dish, that includes a protease inhibitor cocktail, one micromolar TSA and two millimolar nicotinamide, and incubate at four degrees Celsius, under constant rotation, for 30 minutes.
Centrifuge at 14, 000 g for 15 minutes at four degrees Celsius and transfer the supernatant to a new tube. Perform immunoprecipitation using an anti-FLAG antibody conjugated to agarose beads, as described in the text protocol. After concentrating the eluted sample to one microgram per milliliter, keep the purified acetylated protein-substrate at negative 80 degrees Celsius.
A critical step in this protocol is to confirm purification of the acetylated protein-substrate by protein, using a protein-specific antibody and anti-acetylation antibody prior to the in vitro deacetylation reaction. Prior to starting the in vitro deacetylation reaction, prepare deacetylation buffer B.Next, prepare three reactions in three different tubes, each in a total volume of 20 microliters, as shown in this table. All three tubes contain purified acetylated protein-substrate and deacetylation buffer B, but tube two also contains purified SIRT2, and tube three also contains purified SIRT2 and NAD plus.
To confirm deacetylation of the protein-substrate by SIRT2, an optional fourth reaction can be included, in which a purified deacetylase null mutant of SIRT2 is added instead of the deacetylase active SIRT2. Incubate the reactions at 30 degrees Celsius under constant agitation for three hours. Stop the reactions by adding 20 microliters of two X sample buffer to each tube, and boiling the samples at 95 degrees Celsius for 10 minutes.
Subsequently, the reaction mixtures are run on a four to 12%SDS page gel, and the acetylation status of the protein-substrate is detected by Western blotting, using an antibody against acetylated lysine. Using this protocol, wild-type SIRT2 and a deacetylation defective mutant, SIRT2 Hi87Y were purified at a final concentration of one microgram per microliter. Since both proteins are tagged with a FLAG peptide, the purification can be confirmed by immunoblotting, using an anti-FLAG antibody.
The protein-substrate and acetylated protein-substrate were also purified. Immunoblotting using an anti-acetyl lysine antibody confirmed increased acetylated levels of the purified protein in cells overexpressing the acetyltransferases. Total proteins were detected using an anti-FLAG antibody.
In an in vitro deacetylation assay, when the purified acetylated protein-substrate was incubated with wild-type SIRT2 or the deacetylation defective mutant in the presence of NAD plus, decreased acetylated levels were detected by immunoblotting, using an anti-acetyl lysine antibody in the presence of SIRT2, but not in the presence of the mutant. No deacetylation activity was observed when NAD plus was not included in the reaction mixture, confirming that SIRT2 requires NAD plus for its enzymatic function. Deacetylation was also inhibited when a sirtuin inhibitor, NAM, was added to the reaction mixture, which implies that the decrease in the acetylated levels of the protein is mediated by the deacetylase activity of SIRT2.
In order for a protein to be considered as a the deacetylation target for any enzyme, both in vitro and in vivo deacetylation assay need to be performed to establish the interplay between the deacetylase and each substrate. In vivo deacetylation assays in cells under specific experimental conditions can provide further insight regarding the interplay between the deacetylase and the potential substrate in physiological and pathophysiological scenarios, and establish the functional role of the deacetylation event, in a physiological context. Given the increasing interest regarding the role of sirtuins in regulating diverse cellular processes, the described deacetylation assays can be used with slight modifications to identify new substrates for other members of the sirtuin family, regardless of their subcellular localization.
Both in vitro and in vivo deacetylation assays carried out by specific deacetylases may be further combined with small-scale mass spectrometry analysis to reveal target lysines on the substrate to distinguish functional acetylation sites from non-regulatory acetylated lysines.
