Name: deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates
Uploaded: 2016-02-27T14:00:00
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...

<ol>
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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. 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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 border="0" cellpadding="0" cellspacing="0" width="1543">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">cell culture dishes</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">T1110 and T1115</td>
			<td style="width:728px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">pCDH-puro-GFP lentiviral vector</td>
			<td style="width:239px;">System Biosciences</td>
			<td style="width:119px;">CD513B-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV- dR8.2 dvpr (packaging vector)</td>
			<td style="width:239px;">Addgene</td>
			<td>8455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV-VSV-G (envelope vector)</td>
			<td style="width:239px;">Addgene</td>
			<td align="right">8454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylenimine (PEI)&#160;</td>
			<td>Polysciences Inc.</td>
			<td align="right" style="width:119px;">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 height="21">
			<td height="21" style="height:21px;">0.22&#956;m filters</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">F5512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polybrene</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H9268</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">fluorescent microscope</td>
			<td style="width:239px;">Carl Zeiss MicroImaging Inc.</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:239px;">Invivogen</td>
			<td style="width:119px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:239px;">Corning</td>
			<td style="width:119px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag antibody</td>
			<td style="width:239px;">Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">M9272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">EGTA</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">34596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitors coctail 100x</td>
			<td style="width:239px;">Biotool</td>
			<td style="width:119px;">B14001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichostatin A (TSA)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T8552</td>
			<td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag agarose beads</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rotator</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td style="width:119px;">415220Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">filter tubes</td>
			<td style="width:239px;">Millipore</td>
			<td>UFC30HV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flag peptide&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin Centrifugal Concentrator</td>
			<td>Sartorius Stedim Biotech S.A.</td>
			<td>VS0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain solution&#160;</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:119px;">LC6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS sample buffer (4x)</td>
			<td style="width:239px;">Life Techologies</td>
			<td style="width:119px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Ac-K antibody</td>
			<td style="width:239px;">Cell Signaling</td>
			<td style="width:119px;">9441</td>
			<td>several anti-Ac-K antibodies are available. In our hands, the Cell Signaling antibody exhibits the highest sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl pH 7.5</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T5941</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NAD+</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">N0632</td>
			<td>required cofactor for sirtuins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nicotinamide (NAM)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">72340</td>
			<td>selective inhibitor class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pLKO.1 lentiviral vector&#160;</td>
			<td style="width:239px;">Addgene</td>
			<td style="width:119px;">8453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SIRT2 si RNA&#160;</td>
			<td style="width:239px;">Qiagen</td>
			<td style="width:119px;">GS22933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-SIRT2 antibody</td>
			<td style="width:239px;">Proteintech</td>
			<td style="width:119px;">15345-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bradford protein assay</td>
			<td style="width:239px;">BIO-RAD</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti Ac-K agarose beads</td>
			<td style="width:239px;">Immunechem</td>
			<td>ICP0388</td>
			<td></td>
		</tr>
	</tbody>
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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...

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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 ...

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Chemistry

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