Comparison of the Deacylase and Deacetylase Activity of Zinc

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Comparison of the deacylase and deacetylase activity of zinc-dependent HDACs Jesse J McClure, Elizabeth S. Inks, Cheng Zhang, Yuri K. Peterson, Jiaying Li, Kalyan Choudhary, Bradley Lee, Ashley Buchanan, Shiqin Miao, and C. James Chou ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 01 May 2017 Downloaded from http://pubs.acs.org on May 2, 2017

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ACS Chemical Biology

Comparison of the deacylase and deacetylase activity of zinc-dependent HDACs

Jesse J. McClure1†, Elizabeth S. Inks1†, Cheng Zhang2†, Yuri K. Peterson1, Jiaying Li1, Kalyan Chundru1, Bradley Lee1,3, Ashley Buchanan3, Shiqin Miao4, C. James Chou1*

1

Medical University of South Carolina, College of Pharmacy. Charleston, South Carolina.

2

China Agricultural University, Department of Applied Chemistry. Beijing, China.

3

College of Charleston. Charleston, South Carolina.

4

College of Pharmaceutical Sciences, Zhejiang University. Hangzhou, Zhejiang, China.

†

Authors contributed equally to the article

*

The corresponding author’s email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT The acetylation status of lysine residues on histone proteins has long been attributed to a balance struck between the catalytic activity of Histone Acetyl Transferases and Histone Deacetylases (HDAC). HDACs were identified as the sole removers of acetyl post-translational modifications (PTM) of histone lysine residues. Studies into the biological role of HDACs have also elucidated their role as removers of acetyl PTMs from lysine residues of non-histone proteins. These findings, coupled with high-resolution mass spectrometry studies that revealed the presence of acyl-group PTMs on lysine residues of non-histone proteins, brought forth the possibility of HDACs acting as removers of both acyl- and acetyl-based PTMs. We posited that HDACs fulfill this dual role, and sought to investigate their specificity. Utilizing a fluorescencebased assay and biologically relevant acyl-substrates, the selectivity of zinc-dependent HDACs toward these acyl-based PTMs were identified. These findings were further validated using cellular models and molecular biology techniques. As a proof of principal, an HDAC3 selective inhibitor was designed using HDAC3’s substrate preference. This resulting inhibitor demonstrates nanomolar activity and >30 fold selectivity toward HDAC3 compared to the other Class I HDACs. This inhibitor is capable of increasing p65 acetylation, attenuating NF-κB activation and thereby preventing downstream nitric oxide signaling. Additionally, this selective HDAC3 inhibition allows for control of HMGB-1 secretion from activated macrophages without altering the acetylation status of histones or tubulin.


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Increasing evidence suggests that lysine post-translational modifications (PTMs) play multiple and extensive roles in cell signaling, akin to the well-studied phosphorylation, methylation, or ubiquitinylation PTMs.1 Initial proteomic studies using high-resolution mass spectrometry have identified at least 3,600 lysine acetylation sites on over 1,750 proteins.2 In addition to lysine acetylation, a wider array of lysine acylations have gradually become recognized as important PTMs that control key cellular processes.3 These modifications include lysine-formylation, acetylation, propionylation, butyrylation, crotonylation, glutarylation, malonyl/succinylation, and myristoyl/palmitoylation.4-13 A common feature of these lysine acylations is most of them originate from coenzyme A (CoA) metabolites. The even numbered acyl groups such as acetyl and butyryl are likely derived from ß-oxidation pathways, and the more complex succinyl modification stems from succinyl-CoA, most commonly used in regulation of cellular energy homeostasis. This crosstalk between metabolism and PTM status suggest a role for these lysine modifications to regulate enzymes in metabolic pathways.14 Further, the identification of the diverse acyl-based PTMs has sparked studies focusing on the conditions under which they are attached and removed, leading to the demonstration that acylation of lysine residues is a non-specific process performed either through promiscuous Histone Acetyl Transferases or simply by non-catalytic chemical ligation.15 Unlike the promiscuous and even equilibrium-based ligation of acyl groups to lysine residues, the removal of these groups seems to be more carefully controlled.15 One enzyme family capable of removing glutaryl, malonyl/succinyl, and myristoyl/palmitoyl groups is the Sirtuins.9, 10, 13 As Class III members of the Histone deacetylase (HDAC) family of enzymes, Sirtuins are NAD+-dependent deacylases. Contrarily, Class I, II, and IV HDACs are metal-containing deacetylases. It has been suggested that Class I, II, and IV HDACs possess the ability to also deacylate lysines rather than an isolated ability to deacetylate them. With this knowledge, we asked if these relatively novel acyl groups were substrates for any zinc-dependent HDAC, and if so, questioned whether there was any level of specificity these isozymes displayed toward certain acyl substrates.



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RESULTS AND DISCUSSION

HDAC isozyme deacylase kinetic profiling. Our study began by developing 12 different aminomethylcoumarin-based fluorogenic substrates that would mimic biologically relevant acylgroup PTMs (Supplementary Figure 1a). These substrates were developed based on either known acids/acyl-CoA bound esters that have been found in the blood stream in high concentrations and are likely to be ligated to the ε-N terminus of lysine residues or known PTMs shown to exist via mass spectrometry ex vivo. The non-biologically relevant Trifluoroacetyl (TFA) substrate was utilized as a positive control for Class IIa HDACs and HDAC8 as it is the best-known substrate to be efficiently removed by these HDAC isozymes.16 Utilizing these substrates and recombinant human HDACs, all zinc-dependent HDAC isozymes were tested en bloc for their ability to deacylate each substrate, with particular interest for substrate cleaved over time with constant enzyme and substrate concentrations (Supplementary Figure 1b). As previously reported, HDACs 1, 2, 3, and 6 demonstrated the most robust deacetylase activity compared to all other HDAC isozymes.17 Also in line with external findings, Class IIa HDACs and HDAC8 only displayed the ability to deacylate the TFA-based substrate.18 No appreciable deacylase activity was seen for HDACs 10 and 11 which falls in line with a similarly performed study.17 In addition to this, we saw no appreciable activity of any isozyme toward our heptanoyl-, octanoyl-, glutaryl-, or adipoyl-based substrates (Supplementary Figure 1b). As such, the results of this experiment directed our focus toward more rigorous interrogation of the deacylase capacity of HDACs 1, 2, 3, and 6. HDACs 3 and 6 demonstrated appreciable deformylase activity with HDAC6 demonstrating higher catalytic activity as a deformylase than as a deacetylase with the concentrations of enzyme and substrate used; HDAC3 possessed by far the most diverse ability to deacylate a variety of substrates, including the TFA-based substrate, with a particular preference for deacylating the butyryl-, crotonyl- and valeryl-based substrates compared to


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HDACs 1 and 2;

and lastly, HDACs 1-3 were able to depropionylate with high catalytic

efficiency (Figure 1a and Supplementary Figure 1b). While there have been previous reports of HDAC3 possessing the ability to deacylate the TFA-based substrate17, we sought to determine if this finding was due to an impurity of one or more Class IIa HDACs in our HDAC3 solution. Briefly, HDAC3 was coincubated with TFA-substrate and vorinostat or diphenyl acetic hydroxamic acid (dPAHA). It has been previously shown that vorinostat possesses no appreciable inhibitory activity for Class IIa HDACs17 while dPAHA only possesses the ability to inhibit Class IIa HDACs.19 As expected, and in line with previous publications20, vorinostat, but not dPAHA, was capable of altering HDAC3’s ability to deacylate TFA substrate (Supplementary Figure 2). Therefore, we are confident in associating this deacylase ability with HDAC3. To further investigate the key findings from our initial screen, we performed Vmax kinetic analyses on HDACs 1, 2, 3, and 6 versus substrates that were deacylated by one or more of these isozymes. We determined values of Km, Vmax, kcat, and kcat/Km, the latter being the most well accepted measurement of catalytic efficiency (Table 1). Interestingly, the Km values of our formyl-based substrate vs HDACs 3 and 6 is nearly a log lower than the corresponding values for HDACs 1 and 2. The Vmax value for HDAC1, despite being higher than the value for HDAC6 is not likely to be achieved in vivo, as the concentration required to achieve this would be in the millimolar range. (Figure 1b and Table 1). HDAC6 is the most catalytically efficient deformylase, however, it is still a more efficient deacetylase. Surprisingly, HDACs 1-3 displayed remarkable catalytic efficiency as depropionylases. Despite this, there appears to be very little difference in selectivity or efficiency between the three isozymes. The last intriguing finding from this kinetic study was the deacylation ability of HDAC3 toward butyryl-, crotonyl-, and valerylbased substrates. It has been previously reported that HDAC3 was capable of deacylating crotonyl-substrates.21 Unlike the depropionylase ability of HDACs 1-3, the deacylase activity toward these substrates was very specific to HDAC3. This seems to stem from HDAC3’s ability



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to both bind these substrates more efficiently (lower Km values) as well as efficiently cleave these substrates (higher kcat/Km values) from the ε-N of lysine residues than HDACs 1 or 2 (Figure 1c and Table 1). Most interestingly, the crotonyl-substrate binds to HDAC3 with very high affinity, however, there is little substrate turnover (kcat) compared to the canonical acetylsubstrate (Table 1).

Interrogation of HDACs 3 and 6 as deformylases. Utilizing Hek293 cell lysates and various HDAC inhibitors, we sought to determine if this newly discovered deformylase activity both translated into a more robust cellular-based model and if it was affected by traditional small molecule inhibitors. Vorinostat, a Class I and HDAC6 inhibitor17; Tubastatin A (tubA), an HDAC6 specific inhibitor22; and PD-106, an HDACs 1-3 inhibitor23 were used to interrogate the individual and combined inhibitory effects of these small molecules against HDAC isozymes’ cellular deformylase activity (Figure 2a). The pan-inhibition of vorinostat at 1 µM demonstrated the ability to inhibit both deacetylation and deformylation, in line with our kinetics study. The selective HDAC6 inhibitor Tubastatin A at 0.5 µM, well above its IC50 of 95% pure via LC/MS, 1H and

13

C NMR. All HDAC isozymes were purchased from BPS

biosciences with >90% purity with the exceptions of HDACs 4 and 7. Chemical reagents were purchased from Bachem, Sigma-Aldrich, or Fisher Scientific. Cells were purchased through ATCC and cultured according to ATCC guidelines. Detailed methods can be found in Supporting Information.

ACKNOWLEDGEMENT The authors would like to thank C. Beeson and R. Schnellmann for their comments and discussions. We also thank of the Medical University of South Carolina Mass Spectrometry and NMR core facilities.

FUNDING SOURCES This work was supported by the National Institute of Health/National Cancer Institute (CA163452 to C.J.C.), the National Institute of General Medical Sciences (P20GM103542) from the National Institutes of Health, South Carolina Clinical and Translational Research Institute UL1TR001450, and in part by pilot research funding from an American Cancer Society Institutional Research Grant awarded to the Hollings Cancer Center, Medical University of South Carolina. The authors declare no competing financial interests.

SUPPORTING INFORMATION The following are made available free of charge via the internet at http://pubs.acs.org Supplementary Figures 1-7



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Supplementary Table 1 Supplementary Methods Supplementary Chemistry and Characterization

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45. Chen, X., Barozzi, I., Termanini, A., Prosperini, E., Recchiuti, A., Dalli, J., Mietton, F., Matteoli, G., Hiebert, S., and Natoli, G. (2012) Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages, Proc. Natl. Acad. U. S. A. 109, E2865-2874. 46. Kiernan, R., Bres, V., Ng, R. W., Coudart, M. P., El Messaoudi, S., Sardet, C., Jin, D. Y., Emiliani, S., and Benkirane, M. (2003) Post-activation turn-off of NF-kappa B-dependent transcription is regulated by acetylation of p65, J. Biol. Chem. 278, 2758-2766. 47. Hatano, E., Bennett, B. L., Manning, A. M., Qian, T., Lemasters, J. J., and Brenner, D. A. (2001) NFkappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis, Gastroenterology 120, 1251-1262. 48. Griscavage, J. M., Wilk, S., and Ignarro, L. J. (1996) Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NFkappa B, Proc. Natl. Acad. U. S. A. 93, 3308-3312. 49. Jones, E., Adcock, I. M., Ahmed, B. Y., and Punchard, N. A. (2007) Modulation of LPS stimulated NFkappaB mediated Nitric Oxide production by PKCepsilon and JAK2 in RAW macrophages, J. Inflamm. 4, 23. 50. Lu, B., Wang, H., Andersson, U., and Tracey, K. J. (2013) Regulation of HMGB1 release by inflammasomes, Protein Cell 4, 163-167. 51. Lu, B., Nakamura, T., Inouye, K., Li, J., Tang, Y., Lundback, P., Valdes-Ferrer, S. I., Olofsson, P. S., Kalb, T., Roth, J., Zou, Y., Erlandsson-Harris, H., Yang, H., Ting, J. P., Wang, H., Andersson, U., Antoine, D. J., Chavan, S. S., Hotamisligil, G. S., and Tracey, K. J. (2012) Novel role of PKR in inflammasome activation and HMGB1 release, Nature 488, 670-674. 52. Willingham, S. B., Allen, I. C., Bergstralh, D. T., Brickey, W. J., Huang, M. T., Taxman, D. J., Duncan, J. A., and Ting, J. P. (2009) NLRP3 (NALP3, Cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways, J. Immunol. 183, 2008-2015. 53. Stammler, D., Eigenbrod, T., Menz, S., Frick, J. S., Sweet, M. J., Shakespear, M. R., Jantsch, J., Siegert, I., Wolfle, S., Langer, J. D., Oehme, I., Schaefer, L., Fischer, A., Knievel, J., Heeg, K., Dalpke, A. H., and Bode, K. A. (2015) Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1beta via a Caspase-1-Independent Mechanism, J. Immunol. 195, 5421-5431.



a Formyl Acetyl Propionyl Butyryl Crotonyl Valeryl Hexanoyl TFA

Deacylase Activity

15

10

5

6 C

3 D H

H

D

A

A

C

2 C A D H

H

D

A

C

1

0

b 20000

Formyl

15000

Velocity

HDAC3

10000 HDAC6 5000

HDAC1 HDAC2

0 0

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200

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c 12000

Valeryl HDAC3

Velocity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 1 Acyl-substrate profiling. a) Results of en bloc acyl-substrate profiling screen against HDACs 1, 2, 3, and 6. Y-axis units in µmole substrate cleaved µmole enzyme-1 min-1. b) Vmax study of formyl-substrate vs HDACs 1, 2, 3, and 6. Y-axis units in pmole substrate cleaved s-1 mg enzyme-1. c) Vmax study of valeryl-substrate vs HDACs 1, 2, and 3. Y-axis units in pmole substrate cleaved s-1 mg enzyme-1. n = 3; error bars are S.E.M.



b vorinostat

tubastatin A

PD-106

entinostat

120

% Deacylase Activity

a

Formyl Acetyl

100 80

**

**

**

60

**

40

**

20

-1 06 +P D **

20

6

3

C A si H

D

si H

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si H

3+ 6

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A D H

% Deformylase Activity

C

6 C

600 400

*

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1 C H

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C 3/ N C A

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oR

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0

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H

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H

HH3

**

1000

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HDAC1

f

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e Hsp90

oR

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-1 06 **

40

sc ra m

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bl e

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C

AcTub

80

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HDAC6

100

D

HDAC3

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si H

si H

HDAC1

% Deformylation Activity

A D

D si H

Hsp90

PD

C

C A

A D

d

tu bA

3+

6

3 C

1 C A si H

D si H

c

sc ra m

bl e

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C tr l vo rin os ta t

0

pc D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 2 Role of HDACs 3+6 in cellular deformylation. a) Chemical structures of small molecule inhibitors of HDACs 1, 2, 3, and 6. b) Comparison of effect on deformylase and deacetylase activities of Hek293 lysates with various inhibitors for 24 hours; data normalized to control deacylase activity; vorinostat and PD-106 used at 1 µM, tubA used at 0.5 µM. c) Western blot analysis of Hek293 cells with various siRNA transfections demonstrating selective and specific knockdown of targeted HDACs. d) Deformylase activity of siRNA treated Hek293 lysates; data normalized to scramble deacetylase activity. e) Western blot analysis of Hek293 cells transfected with various vectors to overexpress targeted HDACs. f) Deformylase activity of transfected Hek293 cells; data normalized to pcDNA3.1+ vector transfected cells. b, d, and f are n = 3; error bars are S.E.M. c and e are representative of n ≥ 2 experiments. * = p-value < 0.01. ** = p-value < 0.0001. All statistical analyses were Dunnett’s multiple comparisons of means to the means of their respective controls.



a % Deacylase Activity

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Acetyl Valeryl

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+P D tu bA

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pc D

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Figure 3 Role of HDAC3 in cellular devalerylation. a) Comparison of effect on devalerylase and deacetylase activities of Hek293 lysates with various inhibitors; data normalized to control deacylase activity; vorinostat and PD-106 used at 1 µM, tubA used at 0.5 µM. b) Devalerylase activity of siRNA treated Hek293 lysates; data normalized to scramble deacetylase activity. c) Devalerylase activity of transfected Hek293 cells; data normalized to pcDNA3.1+ vector transfected cells. Data collected after 24 hours of treatment. n = 3; error bars are S.E.M. * = pvalue < 0.01. ** = p-value < 0.0001. All statistical analyses were Dunnett’s multiple comparisons of means to the means of their respective controls.


l

HH3

50k

18k

HH3 1.5

2.0

* *

* *

d

* * sc ra s i mb H le D s i AC H 3 D A si C H 6 D A C 3+ 6

1.0

0.5

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0.5

*


*

2

0

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50k

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HH3

-1 06 +

tu bA

Tubulin Acetylation

*

vo r i Ct en nos rl tin ta o t PD sta -1 t PD -1 tu 06 06 b +t A ub A

PD

vo rin os ta en tin t os ta PD t -1 06 tu bA

c

6

2.0

1.0

Formylation

tr

b **

18k

HH3 am si bl e H D A si C H 3 s i DA H D C6 A C 3+ 6

AcHH4

sc r

AcHH3 **

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1.0

Valerylation

4

Histone Acetylation vo rin Ct en os rl tin ta o t PD sta -1 t PD 0 -1 tu 6 06 b +t A ub A

AcTub 10

0

am si bl e H D A si C H 3 s i DA H D C6 A C 3+ 6

18k 2.0

e

sc ra m si b H le D s i AC H 3 D A si H C6 D A C 3+ 6

Hsp90

HH3 8

sc r

100k Formylation

50k

vo r C en ino trl tin st o at PD sta PD -1 t -1 t 06 06 ub +t A ub A

100k

Valerylation

C

a

vo r C en ino trl tin sta o t PD sta PD -1 t -1 t 06 06 ub +t A ub A

C tr vo l enrino t s PD ino tat s tu -10 tat bA 6 PD -1 06 +t ub A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 C tr vo l enrino t s PD ino tat s tu -10 tat bA 6 PD -1 06 +t ub A

ACS Chemical Biology Page 28 of 37

25

20

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** **

*

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10 5

0

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*

*

*

*

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Figure 4 Roles of HDACs 3 and 6 on global cellular formylation and valerylation. a) left: Western blot analysis of Hek293 cells treated with small molecule HDAC inhibitors. right: Quantification of histone and tubulin acetylation normalized to HH3 levels. b) left: Global formylation levels of Hek293 cells treated with HDAC inhibitors. right: Quantification of global formylation normalized to HH3 levels. c) left: Global formylation levels of Hek293 cells treated with siRNA. right: Quantification of global formylation normalized to HH3 levels. d) left: Global valerylation levels of Hek293 cells treated with HDAC inhibitors. right: Quantification of global valerylation normalized to HH3 levels. e) left: Global valerylation levels of Hek293 cells treated with siRNA. right: Quantification of global valerylation normalized to HH3 levels. All inhibitors used at 1 µM, except tubA at 0.5 µM. Data recorded after 24 hours of treatment, representative western blots of n ≥ 3 experiments; error bars are S.E.M. * = p-value < 0.01. ** = p-value < 0.0001. All statistical analyses were Dunnett’s multiple comparisons of means to the means of their respective controls.



d

% Deacetylase Activity

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a

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Log [2c] (M)

c % Deacetylase Activity

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-5

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Figure 5 Substrate-specificity driven development of HDAC3 selective inhibitor. a) IC50 of HDACs 1-3. The crotonyl-subtrate serves as a fairly potent and selective HDAC3 inhibitor. IC50 values for HDACs 1-3 respectively: 46.5 µM, 18.1 µM, 6.42 µM. b) IC50 of HDACs 1-3 vs 2. c) IC50 of HDACs 1-3 vs 2a. d) IC50 of HDACs 1-3 vs 2b. e) IC50 of HDACs 1-3 vs 2c. All data normalized to vehicle (DMSO) control. Graphs a-c fit via GraphPad Prism log(inhibitor) vs. normalized response – variable slope parameters. n ≥ 3; error bars are S.E.M.


C

tr l

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p65

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Nucleus

a

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vo r in o tu sta bA t

l

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vo rin os ta PD t -1 06 tu bA


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Figure 6 Effects of HDAC inhibition on NF-κB p65 acetylation and inflammatory responses. a) Western blot analysis of RAW264.7 cells treated with various HDAC inhibitors; vorinostat and PD-106 used at 1 µM, tubA at 0.5 µM. b) NO concentration secreted from RAW264.7 treated cells; normalized to control treated viable cell concentration. 1 µM used for all inhibitors. n ≥ 3. c) Western blot analysis of RAW264.7 cells. HMGB-1 secretion monitoring with ponceau stain as loading control. Cells were treated for six hours. 1 µM vorinostat and 0.5 µM tubA used. d-f) Western blot analysis of LPS-treated RAW264.7 cells. Nuclear and cytosolic fractions split. 2a and vorinostat used to assess regulation of HDACs on Ac-p65 subcellular localization. Representative westerns of n ≥ 2 experiments; error bars are S.E.M. ** = p-value < 0.0001.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

HDAC1 Formyl Acetyl Propionyl Butyryl Crotonyl Valeryl HDAC2 Formyl Acetyl Propionyl Butyryl Crotonyl Valeryl HDAC3 Formyl Acetyl Propionyl Butyryl Crotonyl Valeryl HDAC6 Formyl Acetyl Propionyl Butyryl Crotonyl Valeryl

Km (µM) 342 36.6 13.1 49.1 2.40 102 Km (µM) 313 36.6 8.53 21.9 3.20 122 Km (µM) 39.1 17.3 8.06 1.03 0.114 9.99 Km (µM) 37.2 1.93 161 N.D. N.D. N.D.

Vmax (pmole s-1 kcat (min-1) mg protein-1) 12300 8.24 24800 16.6 9850 6.62 1910 1.28 802 0.380 5330 3.56 Vmax (pmole s-1 kcat (min-1) mg protein-1) 5980 4.01 3 4.00x10 2.69 4.90x103 3.29 804 0.540 1010 0.485 2880 1.93 Vmax (pmole s-1 kcat (min-1) mg protein-1) 15600 9.30 26900 16.0 13900 8.31 2560 1.53 758 0.362 11100 6.63 Vmax (pmole s-1 kcat (min-1) mg protein-1) 1.00x104 19.1 2130 4.04 2 9.00x10 1.72 N.D. N.D. N.D. N.D. N.D. N.D.

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kcat/Km (M-1 s-1) 402 7580 8410 435 2670 582 kcat/Km (M-1 s-1) 214 1230 6440 412 2520 264 kcat/Km (M-1 s-1) 3970 15400 17200 24700 53700 11100 kcat/Km (M-1 s-1)


8580 34800 178 N.D. N.D. N.D.

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Table 1 Kinetic profile of HDACs 1, 2, 3, and 6. Results are mean values of n = 3 experiments. S.E.M. < 10% of mean in all cases.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Keywords: Histone Deacetylase, HDAC, deacylation, formylation, valerylation, crotonylation, HDAC3, selective inhibitor.


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HDACs 3 and 6 show deacylase ability toward formyl groups. HDAC3 shows deacylase ability toward propionyl, butyryl, crotonyl, and valeryl groups.


Comparison of the Deacylase and Deacetylase Activity of Zinc

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