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Histone Ketoamide Adduction by 4-Oxo-2-nonenal Is A Reversible Posttranslational Modification Regulated by Sirt2 Yiwen Cui, Xin Li, Jianwei Lin, Quan Hao, and Xiang David Li ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00713 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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ACS Chemical Biology

Histone Ketoamide Adduction by 4-Oxo-2-nonenal Is A Reversible Posttranslational Modification Regulated by Sirt2 Yiwen Cui,†,‡,§ Xin Li,†,§ Jianwei Lin,† Quan Hao,*,‡ Xiang David Li*,† †

Department of Chemistry, ‡School of Biomedical Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China.

ABSTRACT: Lipid-derived electrophiles (LDEs) directly modify proteins to modulate cellular signaling pathways in response to oxidative stress. One such LDE, 4-oxo-2-nonenal (4-ONE), has recently been found to target histones and interfere with histone assembly into nucleosomes. Unlike other LDEs that preferentially modify cysteine via nucleophilic Michael addition, 4-ONE reacts with histone lysine residues to form a new histone modification, gamma-oxononanoylation (Kgon). However, it remains unclear whether Kgon can cause irreversible damage or be regulated by enzymes ‘erasing’ this non-enzymatic modification. Here, we report that human Sirt2 catalyzes the removal of histone Kgon. Among the tested human sirtuins, Sirt2 showed robust deacylase activity toward the Kgon-carrying histone peptides in vitro. We use alkynyl-4-ONE as a chemical reporter for Kgon to demonstrate that Sirt2 is responsible for removing histone Kgon in cells. Furthermore, we develop a ketone-reactive chemical probe to detect histones modified by endogenous 4-ONE in macrophages in response to inflammatory stimulation. Using this probe, we show Sirt2 as a deacylase able to control histone Kgon in stimulated macrophages. This study unravels a new mechanism for the regulation of LDE-derived protein posttranslational modifications, as well as a novel role played by Sirt2 as a histone Kgon deacylase in cytoprotective signaling responses.

Protein posttranslational modifications (PTMs) such as methylation, acetylation and phosphorylation play important roles in regulating protein structure and function.1 In cells, most protein PTMs are tightly controlled by enzymes that catalyze the addition and removal of these modifications. However, some PTMs are known to be installed into proteins in enzyme-independent manners. Under oxidative stress, the cellular polyunsaturated fatty acids (PUFAs) are readily targeted by reactive oxygen species (ROS) to generate a broad array of highly reactive electrophiles.2 These lipidderived electrophiles (LDEs), for example, 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE), will directly react with nucleophiles in DNA and proteins.3-5 Low LDE concentration normally leads to selective modifications on proteins, which are involved in the regulation of redox signaling pathways.2,6,7 An increased level of LDEs can cause toxic damages and is associated with pathogenesis of human diseases.5,8-10 The LDEs with ,-unsaturated alkenals or alkenones usually modify nucleophilic amino acid residues of proteins, including cysteine, histidine and lysine, via Michael addition.2 A recent study done by Marnett and co-workers found that lysine residues in histones can also be modified by 4-ONE, generating a stable ketoamide adduct as a novel histone PTM, which we hereafter refer to as lysine gamma-oxononanoylation (Kgon, Figure 1).11 It is well known that histone modifications can regulate chromatin structure and function in a wide range of DNA-associated processes such as gene transcription, DNA replication and damage repair.12-15 Indeed, Kgon was found to prevent assembly of

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nucleosomes,11 the basic repeating units of chromatin,16 suggesting that a high 4-ONE concentration in cells may damage histones and negatively impact the nucleosome dynamics. This result inspired us to investigate whether the histone Kgon is an irreversible oxidative damage, or could be removed by endogenous deacylases as a regulatory mechanism in response to oxidative stress. (Figure 1). To probe the reversibility of Kgon, we used alkynyl-4-ONE as a chemical reporter11 to monitor the dynamics of histone Kgon in cells. Alkynyl-4-ONE was used to label HeLa cells for three hours and then removed from the cell culture medium. After harvesting cells at different time points and extracting histones from the lysates, the alkynyl-4-ONE labeled histones were conjugated to a rhodamine-azide dye (Rho-N3) via Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC, or ‘click chemistry’). The histones were then resolved by SDS-PAGE and visualized by in-gel fluorescence scanning (Figure 2a). After removing the chemical reporter, the fluorescence intensity of the alkynyl-4-ONE-labeled histones increased in the first hour then faded gradually (Figure 2b). The initial increase in the fluorescence may be caused by the incomplete removal of intracellular alkynyl-4-ONE that leads to further modifications on histones. The subsequent fluorescence decrease suggests that the 4-ONE induced histone modifications, including Kgon, could be reversible, although the turnover of histones may also contribute to this process. Considering the amide linkage of histone Kgon, we hypothesized that potential deacylases might be responsible for the removal of this modification. To search for ‘erasers’ of histone Kgon, we focused on the sirtuins, a family of NAD-dependent lysine deacetylases.17-19 Recent studies demonstrated that sirtuins pose substrate promiscuity toward different lysine acyl modifications, including malonylation, succinylation, glutarylation, crotonylation and long chain fatty-acylation.20-27 It is therefore interesting to study whether Kgon could also be targeted by sirtuins. To this end, we incubated a histone H3 peptide carrying Kgon at Lys27 (H3K27gon), a known histone Kgon site,11 with human Sirt1, Sirt2, Sirt3, Sirt5 and Sirt6, respectively. The enzymatic reactions were then monitored by liquid chromatography-mass spectrometry (LC-MS). Among the five sirtuins tested, only Sirt2 showed robust activity to catalyze the removal of Kgon on H3K27. In addition, Sirt2 also exhibited deacylase activity toward another three histone peptides with Kgon at H3K23, H2BK116 and H4K79 (Figure 2c). In contrast, other sirtuins showed little or no detectable deacylase activity against these Kgon peptides (Figure 2c) with one exception in which Sirt3 catalyzed the hydrolysis of Kgon at H3K23 peptide (Figure S1). These results indicate that Sirt2 is a robust and promiscuous ‘eraser’ for histone Kgon in vitro. To test whether endogenous Sirt2 can regulate histone Kgon, we examined the effect of Sirt2 inhibition on the level of histone Kgon in cells. We first treated HeLa cells with a Sirt2-selective (AGK2) or a pan-sirtuin inhibitor (nicotinamide, NAM). The cells were then labeled by alkynyl-4-ONE and the labeled histones were visualized after ‘click’ to Rho-N3 as described above. The in-gel fluorescence analysis showed that both AGK2 and NAM treatments led to a significant increase in the fluorescence intensities of the alkynyl-4-ONE-labeled histones (Figure 3a), indicating that Sirt2 is involved in removing modifications induced by the electrophile. In addition to Kgon, alkynyl-4-ONE has also been shown to modify histidine residues on histones through Michael addition.11 However, the Michael adduct of histidine is not likely to be removed by Sirt2. Therefore, we reasoned that the increase in fluorescence of the labeled histones was caused by the accumulation of Kgon upon Sirt2 inhibition. Another line of evidence supporting Sirt2 as a Kgon ‘eraser’ came from the knockdown of Sirt2 by siRNA. In consistent with the result of Sirt2 inhibition, the labeling of histones with alkynyl-4-ONE was largely enhanced by treating the cells with Sirt2 siRNA (Figure 3b). In addition to Sirt2, we also examined the effects of the knockdown of Sirt1 or Sirt3 on the global histone Kgon level. This is because


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that while Sirt2 showed robust activity toward all the tested Kgon peptide in the in-vitro enzymatic assay, Kgon on H3K23 and H2BK116 peptides could also be removed by Sirt3 and Sirt1, respectively. As in Figure 2b and S2, Sirt1 knockdown caused little changes in the alkynyl-4-ONE-indeced labeling, whereas Sirt3 knockdown led to moderate enhancement in the labeling. Together, Sirt2 showed the highest activity to regulate histone Kgon level in living cells. While alkynyl-4-ONE allowed rapid detection of the modified histones, it was difficult to determine whether this chemical reporter was used at a concentration that reflects the level of endogenous 4ONE under physiological conditions. We therefore sought to detect the endogenous histone Kgon and its level change upon Sirt2 perturbation. We noticed that the ketone moiety renders Kgon a unique modification among all the currently known histone PTMs. As a well-characterized bioorthoganal functional group, ketone can be labeled via carbonyl condensation with amine nucleophiles.28,29 We therefore synthesized a hydrazide-functionalized biotin probe (biotin-NHNH2)30 to detect and enrich endogenous histone Kgon (Figure 4a). We first examined the reactivity of biotin-NHNH2 toward Kgon using a synthetic H3K27gon peptide. After the condensation reaction of H3K27gon with biotin-NHNH2, sodium cyanoborohydride (NaBH3CN) was added to reduce the generated imine for its propensity to be hydrolyzed back to ketone. The reaction mixture was analyzed using LC-MS. Notwithstanding a known slow reaction rate of ketone-hydrazide condensation,28,29 around 30% H3K27gon peptide was conjugated to biotin-NHNH2 within two hours (Figure S3). We then tested whether biotin-NHNH2 could detect the alkynyl-4-ONE modified histones. The histones were extracted from the HeLa cells treated with alkynyl-4-ONE. After the condensation with biotin-NHNH2 and reduction by NaBH3CN, the histones were then analyzed by western blotting using streptavidin-HRP. As expected, we detected a robust biotin signal with the alkynyl-4-ONE modified histones (Figure S4), suggesting that biotin-NHNH2 can potentially be applied to monitor endogenous histone Kgon. Finally, we used biotin-NHNH2 to examine histone Kgon by endogenous 4-ONE in response to inflammatory stimulation in macrophages. The fatty acid deficient RAW264.7 macrophages31 were first enriched by arachidonic acid (AA) that is known to generate 4-ONE and 4-HNE by ROS peroxidation. A chemically defined lipopolysaccharide, Kdo2-Lipid A (KLA), was then added to stimulate the macrophages for ROS generation. After harvesting the cells, histones were extracted and subjected to biotin-NHNH2 condensation, NaBH3CN reduction and streptavidin blotting (Figure 4b). The treatment of the cells with both AA and KLA resulted in a significantly increased biotin signal on the extracted histones, whereas AA or KLA alone caused only a slight increase in biotin signal, when compared with the untreated cells (Figure 4c). As biotin-NHNH2 labels only ketone functionality, we attributed this biotin signal increase to the histone adductions by 4-ONE, including both Kgon and the Michael addition products, in the stimulated macrophages. We next investigated the potential roles of Sirt2 in regulating histone Kgon during macrophage stimulation. Histones were extracted from the AAenriched and KLA-stimulated RAW264.7 cells with or without Sirt2 siRNA treatment, and subjected to biotin-NHNH2 condensation. Streptavidin blotting showed a much higher biotin signal on histones from the Sirt2 knockdown cells than those from the cells treated with control siRNA (Figure 4d). Despite that biotin-NHNH2 can label all types of histone 4-ONE adducts, the biotin signal increase induced by Sirt2 knockdown should be attributed to the increase in the histone Kgon level, as Sirt2 is incapable of reversing Michael addition reactions. This observation that endogenous histone Kgon accumulating upon Sirt2 knockdown again demonstrates that Sirt2 is a bona fide ‘eraser’ of histone Kgon in living cells. In summary, we have demonstrated that Sirt2 can catalyze the removal of histone Kgon, an oxidative



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stress induced histone modification, in vitro and in living cells. As a deacetylase, Sirt2 was previously reported to protect cells from oxidative stress by targeting metabolic enzymes and transcriptional factors to activate antioxidant proteins and promote their expressions.32 This study provides a new insight into cellular roles played by Sirt2 in response to oxidative stress, in which the enzyme could directly ‘repair’ the damaged proteins (i.e., histones) by removing the LDE-derived modification (Kgon). As Kgon was found to impair nucleosomes assembly,11 Sirt2, by ‘erasing’ Kgon, may help to maintain normal nucleosome dynamics. In addition, Kgon occurs on not only histones but also a variety of other proteins.3,4 To investigate whether Sirt2 or other deacylases are involved in the regulation of cellular Kgon will be the important next step of our study.

METHODS Detailed experimental procedures for cell culture, proteins expression and purification, western blotting, streptavidin blotting, synthesis of peptides, alkynyl-4-ONE and biotin-NHNH2 were described in SI. RNAi experiments. Human Sirt1 siRNA (15 nM, Thermo Fisher Scientific), Sirt2 siRNA (30 nM, Thermo Fisher Scientific), Sirt3 siRNA (30 nM, Thermo Fisher Scientific) and mouse Sirt2 siRNA 30 nM (Santa Cruz Biotechnology) were transfected into HeLa cell line and RAW264.7 cell line respectively with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Corresponding concentrations of control siRNA were used as negative controls. Following transfection, cells were then maintained in a humidified 37°C incubator with 5% CO2 for 48 h. Alkynyl-4-ONE labeling. HeLa cells were treated with 25 or 50 M of alkynyl-4-ONE for 3 h in a humidified 37 °C incubator with 5% CO2. For coincubation with Sirt2 selective inhibitors, HeLa cells were treated with AGK2 (50 M) or NAM (20 mM) overnight, followed by 50 M Alkynyl-4-ONE treatment for another 3 h. For the Sirt2 knockdown cells, after incubation with Sirt2 siRNA for 48 h, the HeLa cells were treated with 25 M of alkynyl-4-ONE for 3 h. After labeling, cells were harvested, washed once with ice-cold PBS and pelleted at 1000 rpm for 5 min. Cells were directly lysed or flash frozen in liquid nitrogen and stored at -80 °C. Histone extraction. An acid extraction method was used to isolate histones from HeLa and RAW264.7 cells31. Briefly, the harvested cell pellet was resuspended with lysis buffer (10 mM Tris–HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 2 mM PMSF, and Roche Complete EDTA free protease inhibitors) and incubated at 4 °C by rotating for 1 hr. The intact nuclei were pelleted by centrifuging at 10,000 g for 10 min at 4 °C. To extract histones, 0.4 N H2SO4 was added to resuspend the nuclei, followed by rotating at 4 °C overnight. After centrifuging to remove the nuclei debris, histones were precipitated by adding 100% trichloroacetic acid drop by drop (trichloroacetic acid final concentration 33%). The precipitated histones were pelleted at 16,000 g for 10 min at 4 °C and washed with ice-cold acetone twice. The air-dried protein pellet was dissolved with ddH2O and stored at −80 °C for later use. Cu(I)-Catalyzed Cycloaddition/Click Chemistry. To the prepared alkynyl-4-ONE labeled samples, 100 M rhodamine-azide was added, followed by tris(2-carboxyethyl)phosphine (TCEP, 1 mM), tris[(1benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA, 100 M) and finally the reactions were initiated by the addition of CuSO4 (1 mM). The reactions were incubated for 1 h at room temperature. In-gel fluorescence visualization. For the alkynyl-4-ONE labeled samples, the click chemistry reactions were quenched by adding 4 volume of ice-cold acetone to precipitate proteins. The mixture was placed at -20 °C overnight and centrifuged at 6,000 g for 5 min at 4 °C. The supernatant was


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discarded and the pellet was washed with ice-cold methanol twice and air-dried for 10 min. The proteins were resuspended in 1X LDS sample loading buffer (Invitrogen) and heated for 10 min at 85 °C. The samples were resolved by SDS-PAGE. The labeled proteins were visualized by scanning the gel on a Typhoon 9410 variable mode imager (excitation 532 nm, emission 580 nm). Enzymatic reactions. The enzymatic activities of human Sirtuins were measured by detecting the removal of Kgon modification from peptides. 1 M of Sirt1, 2, 3, 5 or 6 protein was incubated with 100 M of corresponding Kgon peptides and 1 mM of nicotinamide adenine dinucleotide (NAD) in a reaction buffer containing 20 mM Tris-HCl buffer (pH 7.5) and 1 mM DTT at 37 °C for 90 min. The reactions were stopped by adding 1/3 reaction volume of 20% TFA and frozen in liquid N2 immediately. Samples were then analyzed by LC-MS with a Vydac 218TP C18 column (150 mm, Grace Davison). Mobile phases used were 0.05% TFA in water (buffer A) and 0.05% TFA in 90 % ACN and 10 % water (buffer B). The flow rate for LC was 0.2 mL/min. The wavelength for UV detection was 220 nm. In vitro labeling of the Kgon-containing histone peptides and proteins using biotin-NHNH2. On peptide level, excessive biotin-NHNH2 (5 mM) was incubated with H3K27gon peptide (100 M) in 100 mM sodium phosphate buffer (pH 3.0) at 37 °C. 0.1 M NaBH3CN was added at 0 or 2 h to quenched the reaction. The reaction mixture was then analyzed by LC-MS. On histone level, HeLa cells were incubated with 25 M of alkynyl-4-ONE or DMSO for 3 h. Extracted histones were reacted with 5 mM biotin-NHNH2 at 37 °C. 0.1 M NaBH3CN was added at 2 h to quench the reaction. The reaction mixture was then analyzed by streptavidin blotting. Detection of endogenous histone Kgon in RAW264.7 cells. RAW264.7 cells were cultured in DMEM and enriched with 25 M arachidonic acid for 24 h. The media was removed and cells were washed once with PBS. Cells were then stimulated via the addition of 100 ng/L KLA for 24 h.11 Cells were harvested and extracted histones were incubated with biotin-NHNH2 (5 mM) in 100 mM sodium phosphate buffer (pH 3.0) at 37°C for 2 h. 0.1 M NaBH3CN was used to quench the reaction and the result was analyzed by streptavidin blotting.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website, including Figure S1-S11, and additional experimental methods.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] or [email protected] Author Contributions §These authors contributed equally to this work. Notes The authors declare no competing financial interest

ACKNOWLEDGMENT We acknowledge support from the Hong Kong Research Grants Council Collaborative Research Fund (CRF C7029-15G and C7037-14G), General Research Fund (GRF 17303114) and Early Career Scheme (ECS) (HKU 709813P). We acknowledge the University of Hong Kong for the Seed Funding Program (201511159093, 201411159101 and 201409160027). We acknowledge support from the National



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Natural Science Foundation of China (21572191).

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Figure 2

Figure 1

Figure 1. The proposed mechanism for the regulation of histone lysine gamma-oxononanoylation (Kgon)

Figure 2. Histone Kgon is a reversible modification. (a) Schematics for the detection of histone Kgon using chemical reporter alkynyl-4-ONE. (b) Analysis of histone Kgon dynamics. HeLa cells were labeled by alkynyl-4-ONE for 3 h and then the reporter was removed from cell culture medium. Cells were harvested at indicated time points and histones were extracted. The reporter-labeled histones were conjugated to Rho-N3 and visualized by in-gel fluorescence scanning. (c) LC-MS analyses of the enzymatic reactions of different sirtuins toward histone peptides carrying Kgon. The enzymes (1 M) were incubated with Kgon peptides (100 M) at 37 oC for 90 min, and then monitored by LC-MS. Black traces show total ion intensity for all ion species with m/z from 200 to 2000 (i.e., total ion counts, TIC); blue traces show ion intensity for the masses of Kgon peptides; magenta traces show ion intensity for the masses of deacylated peptides.

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Figure 3

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Figure 4

Figure 3. Sirt2 regulates histone Kgon in cells. (a) The inhibition of Sirt2 by NAM (20 mM) and selective Sirt2 inhibitor AGK2 (50 M) resulted in the increased levels of alkynyl-4-ONE-induced labeling on histones. (b) Level changes of alkynyl-4-ONEinduced labeling on histones upon Sirt1, Sirt2, and Sirt3 knockdown. HeLa cells were treated by chemical inhibitors or siRNAs, followed by alkynyl-4-ONE labeling. The histones were extracted and conjugated to Rho-N3 and visualized by in-gel fluorescence scanning. Immunoblotting of -tubulin was used as loading control. Rho: in-gel fluorescence, CB: Coomassie-blue staining.

Figure 4. Sit2 regulates histone Kgon in macrophages upon inflammatory stimulation. (a) Schematics for the detection of endogenous histone Kgon using biotin-NHNH2. (b) The procedure for the detection of endogenous histone Kgon level change

upon

inflammatory

stimulation

in

RAW264.7

macrophages. (c) Histones derived from RAW264.7 cells treated by both AA and KLA resulted in a significantly increased signal in streptavidin blotting. (d) Sirt2 knockdown resulted in an increased endogenous histone Kgon level. RAW264.7 cells were treated with Sirt2 or control siRNA for 24 h, followed by AA enrichment (24 h) and KLA stimulation (24 h). The histones were extracted and subjected to the reaction with biotin-NHNH2, followed by streptavidin blotting. H3 and -tubulin were used as loading controls. 2


Histone Ketoamide Adduction by 4-Oxo-2-nonenal ... - ACS Publications

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