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

Il progetto qui descritto è stato sostenuto da una sovvenzione da NIH / NCI (NCI-R01CA182506-01A1), così come dal Robert H. Lurie Comprehensive Cancer Center - Il Lefkofsky Family Foundation / Liz e Research Award Eric Lefkofsky innovazione su AV Vorremmo per ringraziare i membri del laboratorio (Carol O&#39;Callaghan ed Elizabeth Anne Wayne) per la lettura critica e la modifica di questo manoscritto.

1. In Vitro deacetilazione Assay <ol><li> Purificazione di SIRT2 <ol><li> Preparare 10 cm coltura piatto di cellule HEK 293T coltivate in 8 ml DMEM con 10% FBS e antibiotici e coltivate a 37 ° C, 5% CO 2 incubatore coltura tissutale a circa il 70% confluenti il giorno successivo. </li><li> Il giorno successivo, il co-trasfezione 4 mg pCDH-puro-GFP-SIRT2-Flag (lentivector realizzato nel nostro laboratorio), 4 mg pCMV- dR8.2 dvpr (imballaggio del vettore) e 0,5 mg pCMV-VSV-G (vettore busta) 18 con polietilenimmina (PEI) in un rapporto di 3 ml PEI DNA / mg. </li><li> Cambiare media dopo 12 ore. </li><li> Raccogliere il surnatante dopo 36-48 ore. </li><li> Rimuovere i detriti per centrifugazione a 1000 xg per 5 minuti a 4 ° C. </li><li> Dopo che filtra attraverso 0,22 micron filtri, fare 1 ml aliquote di SIRT2 lentivirus e conservare a -80 ° C. </li><li> Scongelare lentivirus SIRT2 su ghiaccio (è importante conservare il virus a -80 ° C quando il virus non viene utilizzato). </li><li> Infect una piastra da 6 pozzetti di cellule 293T confluenti HEK 80-90% di 1 ml di lentivirus in presenza di 8 mg / ml polibrene. </li><li> Posizionare cellule infettate da virus a 37 ° C incubatore fino al giorno successivo (24 ore). </li><li> Sostituire media dopo 24 ore. </li><li> 48 ore dopo l&#39;infezione, controllare l&#39;efficienza infezione rilevando GFP cellule positive al microscopio a fluorescenza. </li><li> Se l&#39;efficienza di infezione è buono (almeno il 40-50% delle cellule infettate), sostituire media con terreno di coltura contenente 2 mg / ml puromicina per selezionare per SIRT2 stabile nel cellule che esprimono. Nota: Questo richiede circa 2 settimane, e il mezzo viene cambiato ogni 3-4 giorni. le cellule non infette muoiono in questo periodo di selezione e solo le cellule infettate con il lentivirus SIRT2 crescere. </li><li> Analizzare espressione di SIRT2 Flag-tag mediante western blotting utilizzando un anticorpo anti-Flag (1: 1.000 diluizione) dopo l&#39;esecuzione di 40 mg di proteine ​​totali su un gel SDS-PAGE 10% prima di avviare la purifprocesso icazione (consigliato) 19,20. </li><li> Per purificare SIRT2 attiva, celle divise in 1: 3 il rapporto con tripsinizzazione in modo da avere abbastanza piatti di cellule HEK 293T stabilmente sovraesprimono Flag-SIRT2 (Ten 10 cm piatti permetteranno di purificazione di proteine ​​sufficiente per esperimenti successivi). <ol><li> Per trypsinize le cellule, togliere terreno di coltura ed eliminare il siero residuo risciacquando cellule con DPBS sterile. Aggiungere lentamente 2,5 ml di una soluzione di tripsina-EDTA 0,25% a coprire il monostrato cellulare, incubare a temperatura ambiente per 30-45 sec. Dopo la rimozione della soluzione di tripsina-EDTA, incubare a 37 ° C finché le cellule iniziano a staccarsi. Quindi aggiungere terreno di coltura e trasferire le cellule a nuovi piatti. </li></ol></li><li> Rimuovere terreno di coltura, lavare le cellule due volte in PBS e raccogliere cellule mediante tripsinizzazione seguita da centrifugazione a 1.000 xg per 10 min a 4 ° C. </li><li> Pellet cellulare Lyse in 500 microlitri tampone di lisi A (20 mm HEPES pH 7.9, 18 mm KCl, 0,2 mM EGTA, 1,5 mm MgCl 2, 20% glicerolo, 0,1% NP-40) includente un cocktail inibitori della proteasi e 1 pM TSA per 30 min a 4 ° C sotto costante rotazione. </li><li> Centrifugare a 14.000 xg per 15 min a 4 ° C e trasferire il surnatante in una nuova provetta. </li><li> Eseguire immunoprecipitazione utilizzando un anticorpo anti-Flag coniugato agarosio perline come descritto di seguito. <ol><li> Aggiungere 200-300 ml di anticorpo anti-Flag coniugato con agarosio perline al lisato proteico (10-20 mg proteine ​​totali). </li><li> Incubare per una notte a 4 ° C con agitazione costante. </li><li> Raccogliere immunoprecipitati per centrifugazione a 1.000 xg per 5 minuti a 4 ° C. </li><li> Lavare il pellet 5 volte con 10 ml di tampone di lisi A a 1000 xg per 5 minuti a 4 ° C. Dopo ogni lavaggio, agglomerare le perline e sostituire il surnatante con tampone A. </li></ol></li><li> Preparare la soluzione Flag peptide 1x (tra cui un cocktail inibitori della proteasi e 1 micron TSA in PBS). </li><li> Aggiungere 500 ml di 1x bandiera peptidee soluzione ai agarosio e incubare per 30 min a 4 ° C sotto costante rotazione. </li><li> Raccogliere il surnatante per centrifugazione a 6.000 xg per 2 minuti a 4 ° C. </li><li> Ripetere due passaggi precedenti. </li><li> Rimuovere perline da surnatante utilizzando tubi filtranti e centrifugazione a 15.000 xg per 1 min a 4 ° C. </li><li> Concentrato campione eluito finché la concentrazione proteica finale è 1 mg / mL usando una membrana di ultrafiltrazione. </li><li> Controllare processo di purificazione da spettacolo elettroforesi utilizzando 1 mg del campione eluito. </li><li> Gel macchia con una soluzione colorante disponibile in commercio per confermare la purificazione SIRT2. </li><li> Mantenere SIRT2 purificata a -80 ° C. </li></ol></li><li> Purificazione di acetilato proteina-substrato <ol><li> Preparare 10 cm x 10 cm piatti della cultura con cellule HEK 293T essere di circa il 70% confluenti il ​​giorno successivo. </li><li> Le cellule giorno dopo, co-trasfezione con 5 mg di una bandiera con tag vettore plasmidico expressing proteina-substrato e 5 ug del specifica acetil-transferasi che può acetylate la proteina utilizzando PEI in un rapporto di 3 ml PEI DNA / mg. Nota: Se la specifica acetil-transferasi non è noto, co-trasfezione con una miscela di nota istone acetil-transferasi (copricapo), come p300, CBP, GCN5, Tip60 e PCAF. </li><li> Cambiare media dopo 12 ore. </li><li> Il giorno prima la lisi delle cellule, trattare le cellule con 1 micron TSA e 2 mm nicotinamide (NAM) durante la notte, sostituendo il terreno di coltura con terreno contenente TSA / NAM per massimizzare i livelli di acetilazione della proteina-substrato inibendo deacetilasi. </li><li> 48 ore dopo la trasfezione, rimuovere terreno di coltura, lavare le cellule due volte in PBS e raccogliere le cellule mediante tripsinizzazione seguita da centrifugazione a 1.000 xg per 10 min a 4 ° C. </li><li> Lyse pellet di cellule in 500 microlitri tampone di lisi A per piatto (20 mM HEPES pH 7.9, 18 mM KCl, 0,2 mM EGTA, 1,5 mM MgCl2, 20% glicerolo, 0,1% NP-40) includente una proteasi inhibitors cocktail, 1 pM TSA e 2 mM NAM per 30 minuti a 4 ° C sotto costante rotazione. </li><li> Centrifugare a 14.000 xg per 15 min a 4 ° C e trasferire il surnatante in una nuova provetta. </li><li> Eseguire immunoprecipitazione utilizzando un anticorpo anti-Flag coniugato agarosio perline come descritto di seguito. <ol><li> Aggiungere 200-300 ml di anticorpo anti-Flag coniugato con agarosio perline al lisato proteico (10-20 mg proteine ​​totali). </li><li> Incubare per una notte a 4 ° C con agitazione costante. </li><li> Raccogliere immunoprecipitati per centrifugazione a 1.000 xg per 5 minuti a 4 ° C. </li><li> Lavare il pellet 2 volte con 10 ml di tampone di lisi A a 1000 xg per 5 minuti a 4 ° C. Dopo ogni lavaggio, agglomerare le perline e sostituire il surnatante con tampone di lisi A. </li><li> Lavare il pellet 3 volte con 10 ml di tampone di lisi A senza NAM a 1000 xg per 5 minuti a 4 ° C. Dopo ogni lavaggio, agglomerare le perline e sostituire il surnatante con tampone di lisi A. E &#39;important non di riportare qualsiasi NAM alla reazione deacetilazione. </li></ol></li><li> Preparare la soluzione Flag peptide 1x (compresi gli inibitori della proteasi e 1 micron TSA in PBS). </li><li> Aggiungere 500 microlitri di soluzione di peptide 1x Flag alle perline di agarosio e incubare per 30 min a 4 ° C sotto costante rotazione. </li><li> Raccogliere il surnatante per centrifugazione a 6.000 xg per 2 minuti a 4 ° C. </li><li> Ripetere due passaggi precedenti. </li><li> Rimuovere perline da surnatante utilizzando tubi filtranti e centrifugazione a 15.000 xg per 1 min a 4 ° C. </li><li> Utilizzando una membrana di ultrafiltrazione, concentrare campione eluito finché la concentrazione proteica è 1 mg / ul. </li><li> Eseguire elettroforesi eseguendo 1 ml di campione eluito su un gel SDS-PAGE 4-12%. </li><li> Conferma acetilazione della proteina-substrato mediante western blotting utilizzando anticorpi contro Ac-K (1: 1.000 diluizione) e la proteina-substrato (diluizione dipende l&#39;anticorpo specifico utilizzato). </li><li> mantenere puricato acetilato proteina-substrato a -80 ° C. </li></ol></li><li> In Vitro deacetilazione Reazione <ol><li> Preparare il tampone di deacetilazione B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl 2 0,5 mM TSA) </li><li> Preparare 3 reazioni diverse nei diversi tubi in 20 ml di volume finale (Tabella 1). </li><li> Includi reazioni supplementari per confermare deacetylation della proteina-substrato da SIRT2 (opzionale). Aggiungere 2 microgrammi di purificato deacetilazione nulla SIRT2 mutante (questo può essere fatto utilizzando il protocollo descritto al punto 1.1 dopo aver usato un lentivector pCDH-puro-GFP-SIRT2 H187Y -Flag) per la reazione, invece di deacetilasi SIRT2 attivo o aggiungere 10 mM nicotinamide (NAM ) per la reazione di inibire l&#39;attività di deacetilazione SIRT2 (Tabella 1). </li><li> Incubare le reazioni per 3 ore a 30 ° C sotto costante agitazione. </li><li> Arrestare la reazione aggiungendo 20 l di tampone campione 2x e far bollire i campioni Fo 10 min a 95 ° C. </li><li> Eseguire elettroforesi eseguendo la miscela di reazione su un gel SDS-PAGE 4-12% e rilevare lo stato acetilazione della proteina-substrato mediante western blotting utilizzando un anticorpo anti Ac-K (1: 1.000 diluizione) 19,20. </li></ol></li></ol> 2. In Vivo deacetilazione Assay <ol><li> SIRT2 sovraespressione in cellule in coltura <ol><li> Preparare 10 piatti cm di coltura con cellule HEK 293T stabilmente sovraesprimono pCDH-puro-GFP-vettore vuoto, pCDH-puro-GFP-SIRT2-Flag e pCDH-puro-GFP-SIRT2 H187Y -Flag (come descritto in 1.1) da circa 70 % confluenti. </li><li> In alternativa, preparare 10 cm piastre di coltura con cellule circa il 70% confluenti ed eseguire sovraespressione transiente con 5 ug di vettori di cui sopra utilizzando PEI in un rapporto di 3 ml PEI DNA / mg. </li><li> Eseguire Western Blotting di estratti cellulari totali utilizzando un anticorpo anti-Flag (1: 1.000 diluizione) per dimostrare l&#39;iperespressione di Flag-tagged SIRT2 19. </li></ol></li><li> RNA interferenza di Knockdown SIRT2 in cellule in coltura <ol><li> Preparare un 10 centimetri cultura piatto di cellule 293T HEK essere di circa il 70% confluenti il ​​giorno successivo. </li><li> Il giorno successivo, il co-trasfezione 4 mg pLKO1-puro-sh SIRT2 (lentivector) 19, 4 mg pCMV- dR8.2 dvpr (imballaggio del vettore) e 0,5 mg pCMV-VSV-G (vettore busta) 18 utilizzando polietilenimmina (PEI) con un rapporto di 3 ml PEI DNA / mg. Nota: Per abbattere SIRT2, usiamo semplici shRNAs tornante nel vettore lentivirale pLKO.1 progettato dal consorzio RNAi (TRC). </li><li> Seguire la procedura descritta al punto 1.1 per la produzione di lentivirus shSIRT2 e infettare le cellule bersaglio per atterramento SIRT2. </li><li> Eseguire Western Blotting di estratti cellulari totali utilizzando un anticorpo anti-SIRT2 (1: 1.000 diluizione) per dimostrare efficiente atterramento SIRT2 dopo l&#39;esecuzione di 40 mg di proteine ​​totali su un gel SDS-PAGE 10%. </li><li> Alternativamente,preparare 10 cm piatti della cultura con cellule circa il 70% confluenti ed eseguire atterramento transitoria di SIRT2 utilizzando siRNA seguenti istruzioni del produttore. </li><li> Eseguire Western Blotting di estratti cellulari totali utilizzando un anticorpo anti-SIRT2 (1: 1.000 diluizione) per dimostrare efficiente atterramento SIRT2 dopo l&#39;esecuzione di 40 mg di proteine ​​totali su un gel SDS-PAGE 10%. </li></ol></li><li> Immunoprecipitazione e immunoblotting per rilevare i livelli ACETILATO in cellule in coltura <ol><li> 12 ore prima di raccogliere pellet cellulari sia da cellule overexpressing SIRT2 (vedi 2.1 sopra) o SIRT2 abbattuto cellule (vedi 2.2), aggiungere 1 micron TSA al mezzo di coltura per inibire la classe III deacetilasi degli istoni non. </li><li> Rimuovere terreno di coltura, lavare le cellule due volte in PBS e raccogliere cellule mediante tripsinizzazione seguita da centrifugazione a 1.000 xg per 10 min a 4 ° C. </li><li> Aggiungere 10 ml di tampone di lisi A (20 mm HEPES pH 7.9, 18 mm KCl, 0,2 mM EGTA, 1.5mM MgCl 2, 20% glicerolo, 0,1% NP-40) includente un cocktail inibitori della proteasi, 1 pM TSA e 2 mM NAM. </li><li> Incubare a 4 ° C per 30 min sotto rotazione costante. </li><li> Raccogliere surnatanti di centrifugazione a 20.000 xg per 15 minuti a 4 ° C. </li><li> Calcolare la concentrazione di proteine ​​utilizzando un Metodo di Bradford. </li><li> Trasferire 1 mg di proteine ​​totali di nuovi tubi in un volume finale di 1 ml con tampone di lisi A. Uso Tabella 2 come riferimento per preparare campioni di proteine ​​da cellule sia sovraespressione SIRT2 o dopo SIRT2 knockdown. </li><li> Eseguire un immunoprecipitazione utilizzando un anticorpo contro la proteina specifica-substrato insieme con proteina A / G (si consiglia 40 ml tallone liquami). In alternativa, utilizzare un anticorpo anti Ac-K coniugato con agarosio perline (20 ml tallone liquami di solito è sufficiente) per immunoprecipitare tutte le proteine ​​acetilati. </li><li> Incubare i campioni a 4 ° C sotto constant rotazione durante la notte. </li><li> Raccogliere immunoprecipitati per centrifugazione a 1.000 xg per 2 minuti a 4 ° C. </li><li> Lavare perline 5 volte con 1 ml di tampone A a 1.000 xg per 2 minuti a 4 ° C. </li><li> perline Risospendere in 40 ml di tampone 2x campione. </li><li> campioni bollire per 10 minuti a 95 ° C. </li><li> Eseguire l&#39;elettroforesi eseguendo i campioni eluiti (40 microlitri dal punto 2.3.12) su un gel SDS-PAGE 4-12% senza disturbare le perline durante il trasferimento dei campioni eluiti. </li><li> Rilevare livelli acetilati della proteina-substrato mediante Western blotting utilizzando un anticorpo anti-Ac-K (1: 1.000 diluizione) se un anticorpo specifico proteina-substrato è stato utilizzato per immunoprecipitazione, o un anticorpo specifico contro la proteina-substrato (seguire consigli l&#39;anticorpo specifico riguardante diluizione) se anti-perline Ac-K sono stati utilizzati per l&#39;immunoprecipitazione. </li></ol></li></ol>

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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>

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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><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.

Affinché una proteina per essere considerato come un bersaglio deacetilazione legittimo per qualsiasi enzima con attività di deacetilazione, sia in vitro e in vivo deacetilazione devono essere eseguiti per stabilire l&#39;interazione tra la deacetilasi e il suo substrato. Per il saggio deacetilasi in vitro, la purificazione sia della deacetilasi e il substrato proteico acetilato è richiesto prima del dosaggio può essere fatto. Qui usiamo il SIRT2 sirtuin ci...

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.

Deacetilazione saggi di svelare l&#39;interazione tra sirtuine (SIRT2) e specifici Protein-substrati

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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Research

JoVE Journal

Chemistry

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

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

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

Che utilizzano un sistema di arricchimento di immunoprecipitazione completa per identificare un profilo di modificazione post-traduzionale endogeno per proteine bersaglio

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

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Biochimica

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Misura simultanea di HDAC1 e HDAC6 attività in HeLa Cells utilizzando UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Chimica

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.

In Vitro Ubiquitination e Deubiquitination Saggi di Istoni Nucleosomici

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

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.

Arricchimento in un solo passaggio di un istone deacetilasi con tag TAP del fungo filamentoso Aspergillus nidulans per il saggio di attività enzimatica

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.

Analisi per la convalida degli inibitori dell'acetiltransferasi di Histone

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Ricerca sul cancro

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.

Rilevamento di Protein S-Acylation utilizzando 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 ...

Sviluppo di un test click-based per lo screening dell'attività dell'enzima HAT1

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.

Autore in primo piano: Sviluppo del test dell'acetil-clic per lo screening degli inibitori di HAT1

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.

Un test chimico acetil-click per misurare l'acetiltransferasi istonica 1

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

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.

Analisi di co-immunoprecipitazione per lo studio delle interazioni funzionali tra recettori e gli enzimi

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

Deacetilazione saggi di svelare l'interazione tra sirtuine (SIRT2) e specifici Protein-substrati

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Capitoli in questo video

Che utilizzano un sistema di arricchimento di immunoprecipitazione completa per identificare un profilo di modificazione post-traduzionale endogeno per proteine bersaglio

Misura simultanea di HDAC1 e HDAC6 attività in HeLa Cells utilizzando UHPLC-MS

In Vitro Ubiquitination e Deubiquitination Saggi di Istoni Nucleosomici

Arricchimento in un solo passaggio di un istone deacetilasi con tag TAP del fungo filamentoso Aspergillus nidulans per il saggio di attività enzimatica

Analisi per la convalida degli inibitori dell'acetiltransferasi di Histone

Rilevamento di Protein S-Acylation utilizzando Acyl-Resin Assisted Capture

Un test chimico acetil-click per misurare l'acetiltransferasi istonica 1

Un test chimico acetil-click per misurare l'acetiltransferasi istonica 1

Un test chimico acetil-click per misurare l'acetiltransferasi istonica 1

Analisi di co-immunoprecipitazione per lo studio delle interazioni funzionali tra recettori e gli enzimi