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 ep....

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 ....

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