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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. In Vitro Deacetylation Assay

1. Purification of SIRT2
   1. 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 °C, 5% CO₂ tissue culture incubator to be about 70% confluent the next day.
   2. The next day, co-transfect 4 µg pCDH-puro-GFP-SIRT2-Flag (lentivector made in our lab), 4 µg pCMV- dR8.2 dvpr (packaging vector) and 0.5 µg pCMV-VSV-G (envelope vector) ¹⁸ using polyethylenimine (PEI) at a ratio of 3 µl PEI/µg DNA.
   3. Change media after 12 hr.
   4. Collect supernatant after 36-48 hr.
   5. Remove debris by centrifugation at 1,000 x g for 5 min at 4 °C.
   6. After filtering through 0.22 µm filters, make 1 ml aliquots of SIRT2 lentivirus and keep at -80 °C.
   7. Thaw SIRT2 lentivirus on ice (it is important to store the virus at -80 °C when the virus is not used).
   8. Infect a 6-well plate of 80-90% confluent HEK 293T cells with 1 ml of lentivirus in the presence of 8 µg/ml polybrene.
   9. Place virus-infected cells in 37 °C incubator until the next day (24 hr).
   10. Replace medium after 24 hr.
   11. 48 hr after infection, check infection efficiency by detecting GFP positive cells under a fluorescent microscope.
   12. If infection efficiency is good (at least 40-50% infected cells), replace medium with culture medium containing 2 µg/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.
   13. Analyze expression of Flag-tagged SIRT2 by western blotting using an anti-Flag antibody (1:1,000 dilution) after running 40 µg of total protein on a 10% SDS-PAGE gel before starting the purification process (recommended) ^19,20.
   14. 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).
       1. 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 °C until cells start to detach. Then add culture medium and transfer cells to new dishes.
   15. 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 °C.
   16. Lyse cell pellet in 500 µl 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 µM TSA for 30 min at 4 °C under constant rotation.
   17. Centrifuge at 14,000 x g for 15 min at 4 °C and transfer supernatant to a new tube.
   18. Perform immunoprecipitation using an anti-Flag antibody conjugated to agarose beads as described below.
       1. Add 200-300 µl of anti-Flag antibody conjugated to agarose beads to the protein lysate (10-20 mg total protein).
       2. Incubate overnight at 4 °C with constant agitation.
       3. Collect immunoprecipitates by centrifugation at 1,000 x g for 5 min at 4 °C.
       4. Wash the pellet 5 times with 10 ml lysis buffer A at 1,000 x g for 5 min at 4 °C. After each wash, pellet the beads and replace the supernatant with buffer A.
   19. Prepare 1x Flag peptide solution (including a protease inhibitors cocktail and 1 µM TSA in PBS).
   20. Add 500 µl of 1x Flag peptide solution to the agarose beads and incubate for 30 min at 4 °C under constant rotation.
   21. Collect supernatant by centrifugation at 6,000 x g for 2 min at 4 °C.
   22. Repeat previous two steps.
   23. Remove beads from supernatant by using filter tubes and centrifugation at 15,000 x g for 1 min at 4 °C.
   24. Concentrate eluted sample until the final protein concentration is 1 µg/µl using an ultrafiltration membrane.
   25. Check purification process by performing electrophoresis using 1 µg of the eluted sample.
   26. Stain gel with a commercially available staining solution to confirm SIRT2 purification.
   27. Keep purified SIRT2 at -80 °C.
2. Purification of Acetylated Protein-substrate
   1. Prepare 10 cm x 10 cm culture dishes with HEK 293T cells to be about 70% confluent the next day.
   2. The next day, co-transfect cells with 5 µg of a Flag tagged plasmid vector expressing the protein-substrate as well as 5 µg of the specific acetyl-transferase which can acetylate the protein using PEI at a ratio of 3 µl PEI/µg 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.
   3. Change medium after 12 hr.
   4. The day before lysing the cells, treat the cells with 1 µM 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.
   5. 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 °C.
   6. Lyse cell pellet in 500 µl 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 µM TSA and 2 mM NAM for 30 min at 4 °C under constant rotation.
   7. Centrifuge at 14,000 x g for 15 min at 4 °C and transfer supernatant to a new tube.
   8. Perform immunoprecipitation using an anti-Flag antibody conjugated to agarose beads as described below.
      1. Add 200-300 µl of anti-Flag antibody conjugated to agarose beads to the protein lysate (10-20 mg total protein).
      2. Incubate overnight at 4 °C with constant agitation.
      3. Collect immunoprecipitates by centrifugation at 1,000 x g for 5 min at 4 °C.
      4. Wash the pellet 2 times with 10 ml lysis buffer A at 1,000 x g for 5 min at 4 °C. After each wash, pellet the beads and replace the supernatant with lysis buffer A.
      5. Wash the pellet 3 times with 10 ml lysis buffer A without NAM at 1,000 x g for 5 min at 4 °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.
   9. Prepare 1x Flag peptide solution (including protease inhibitors and 1 µM TSA in PBS).
   10. Add 500 µl of 1x Flag peptide solution to the agarose beads and incubate for 30 min at 4 °C under constant rotation.
   11. Collect supernatant by centrifugation at 6,000 x g for 2 min at 4 °C.
   12. Repeat previous two steps.
   13. Remove beads from supernatant by using filter tubes and centrifugation at 15,000 x g for 1 min at 4 °C.
   14. Using an ultrafiltration membrane, concentrate eluted sample until the protein concentration is 1 µg/ul.
   15. Perform electrophoresis by running 1 µl of the eluted sample on a 4-12% SDS-PAGE gel.
   16. 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).
   17. Keep purified acetylated protein-substrate at -80 °C.
3. In Vitro Deacetylation Reaction
   1. Prepare the deacetylation buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl₂ 0.5 µM TSA)
   2. Prepare 3 different reactions in different tubes in 20 µl final volume (Table 1).
   3. Include additional reactions to confirm deacetylation of the protein-substrate by SIRT2 (optional). Add 2 µg of purified deacetylation null mutant SIRT2 (this can be done using the protocol described in 1.1 after using a pCDH-puro-GFP-SIRT2^H187Y-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).
   4. Incubate reactions for 3 hr at 30 °C under constant agitation.
   5. Stop the reaction by adding 20 µl of 2x sample buffer and boil the samples for 10 min at 95 °C.
   6. 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.

2. In Vivo Deacetylation Assay

1. SIRT2 Overexpression in Cultured Cells
   1. 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-SIRT2^H187Y-Flag (as described in 1.1) to be about 70% confluent.
   2. Alternatively, prepare 10 cm culture dishes with cells about 70% confluent and perform transient overexpression using 5 µg of the vectors mentioned above using PEI at a ratio of 3 µl PEI/µg DNA.
   3. Perform western blotting of total cellular extracts using an anti-Flag antibody (1:1,000 dilution) to demonstrate overexpression of Flag-tagged SIRT2¹⁹.
2. RNA Interference to Knockdown SIRT2 in Cultured Cells
   1. Prepare a 10 cm culture dish of HEK 293T cells to be about 70% confluent the next day.
   2. The next day, co-transfect 4 µg pLKO1-puro-sh SIRT2 (lentivector) ¹⁹, 4 µg pCMV- dR8.2 dvpr (packaging vector) and 0.5 µg pCMV-VSV-G (envelope vector) ¹⁸ using polyethylenimine (PEI) at a ratio of 3 µl PEI/µg DNA.
      Note: For knocking down SIRT2, we use simple hairpin shRNAs in the pLKO.1 lentiviral vector designed by The RNAi Consortium (TRC).
   3. Follow the procedure as described in 1.1 to produce shSIRT2 lentivirus and infect target cells to knockdown SIRT2.
   4. Perform western blotting of total cellular extracts using an anti-SIRT2 antibody (1:1,000 dilution) to demonstrate efficient SIRT2 knockdown after running 40 µg of total protein on a 10% SDS-PAGE gel.
   5. Alternatively, prepare 10 cm culture dishes with cells about 70% confluent and perform transient knockdown of SIRT2 using siRNAs following manufacturer's instructions.
   6. Perform western blotting of total cellular extracts using an anti-SIRT2 antibody (1:1,000 dilution) to demonstrate efficient SIRT2 knockdown after running 40 µg of total protein on a 10% SDS-PAGE gel.
3. Immunoprecipitation and Immunoblotting to Detect Acetylated Levels in Cultured Cells
   1. 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 µm TSA to the culture medium to inhibit non Class III histone deacetylases.
   2. 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 °C.
   3. 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 µM TSA and 2 mM NAM.
   4. Incubate at 4 °C for 30 min under constant rotation.
   5. Collect supernatants by centrifugation at 20,000 x g for 15 minutes at 4 °C.
   6. Calculate protein concentration by using a Bradford protein assay.
   7. 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.
   8. Perform an immunoprecipitation using an antibody against the specific protein-substrate together with Protein A/G (40 µl bead slurry is recommended). Alternatively, use an anti Ac-K antibody conjugated to agarose beads (20 µl bead slurry is usually enough) to immunoprecipitate all acetylated proteins.
   9. Incubate samples at 4 °C under constant rotation overnight.
   10. Collect immunoprecipitates by centrifugation at 1,000 x g for 2 min at 4 °C.
   11. Wash beads 5 times with 1 ml buffer A at 1,000 x g for 2 min at 4 °C.
   12. Resuspend beads in 40 µl of 2x sample buffer.
   13. Boil samples for 10 min at 95 °C.
   14. Perform electrophoresis by running the eluted samples (40 µl from step 2.3.12) on a 4-12% SDS-PAGE gel without disturbing the beads while transferring the eluted samples.
   15. 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.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
cell culture dishes	Denville Scientific Inc.	T1110 and T1115
pCDH-puro-GFP lentiviral vector	System Biosciences	CD513B-1
pCMV- dR8.2 dvpr (packaging vector)	Addgene	8455
pCMV-VSV-G (envelope vector)	Addgene	8454
polyethylenimine (PEI)	Polysciences Inc.	24885	other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells
0.22 μm filters	Denville Scientific Inc.	F5512
polybrene	Sigma	H9268
fluorescent microscope	Carl Zeiss MicroImaging Inc.	Axiovert 200
puromycin	Invivogen	A11138-03
PBS	Corning	21-031-CM
anti-Flag antibody	Sigma	F3165
HEPES	Sigma	H3375
KCl	Sigma	P9541
Glycerol	Sigma	G5516
NP-40	Sigma	74385
MgCl2	Sigma	M9272
EGTA	Sigma	34596
protease inhibitors coctail 100x	Biotool	B14001
Trichostatin A (TSA)	Sigma	T8552	selective inhibitor of class I and II histone deacetylases (HDACs) but not class III HDACs (sirtuins)
anti-Flag agarose beads	Sigma	A2220
centrifuge	Eppendorf	5417R
rotator	Thermo Scientific	415220Q
filter tubes	Millipore	UFC30HV00
Flag peptide	Sigma	F3290
Vivaspin Centrifugal Concentrator	Sartorius Stedim Biotech S.A.	VS0102
SimplyBlue SafeStain solution	Invitrogen	LC6060
NuPAGE LDS sample buffer (4x)	Life Techologies	NP0007
Tris-HCl pH 7.5	Sigma	T5941
NAD+	Sigma	N0632	required cofactor for sirtuins
nicotinamide (NAM)	Sigma	72340	selective inhibitor class III HDACs (sirtuins)
pLKO.1 lentiviral vector	Addgene	8453
SIRT2 si RNA	Qiagen	GS22933
anti-SIRT2 antibody	Proteintech	15345-1-AP
Bradford protein assay	BIO-RAD	500-0006
anti Ac-K agarose beads	Immunechem	ICP0388