HDAC11 Regulates Type I Interferon Signaling Through Defatty-Acylation of SHMT2
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HDAC11 regulates type I interferon signaling through defatty-acylation of SHMT2 Ji Caoa,b, Lei Sunc, Pornpun Aramsangtienchaia,1, Nicole A. Spiegelmana, Xiaoyu Zhanga, Weishan Huangd,2, Edward Setoc, and Hening Lina,e,3 aDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; bZhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, 310058 Hangzhou, China; cGeorge Washington University Cancer Center, Washington, DC 20037; dDepartment of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and eHoward Hughes Medical Institute, Cornell University, Ithaca, NY 14853 Edited by Vern L. Schramm, Albert Einstein College of Medicine, Bronx, NY, and approved January 31, 2019 (received for review September 5, 2018) The smallest histone deacetylase (HDAC) and the only class IV interaction. These findings provide insights into the immuno- HDAC member, HDAC11, is reported to regulate immune activa- modulatory function of HDAC11. tion and tumorigenesis, yet its biochemical function is largely unknown. Here we identify HDAC11 as an efficient lysine defatty- Results acylase that is >10,000-fold more efficient than its deacetylase HDAC11 Is an Efficient Lysine Defatty-Acylase. To study the enzy- activity. Through proteomics studies, we hypothesized and later matic activity of HDAC11, we first tested recombinant HDAC11 biochemically validated SHMT2 as a defatty-acylation substrate of purified from HEK293T cells (SI Appendix, Fig. S1A) on H3K9 HDAC11. HDAC11-catalyzed defatty-acylation did not affect the peptides bearing different acyl groups (Fig. 1A). Using an enzymatic activity of SHMT2. Instead, it affects the ability of HPLC-based assay, we found that HDAC11 could efficiently SHMT2 to regulate type I IFN receptor ubiquitination and cell sur- remove long-chain fatty-acyl groups (myristoyl, palmitoyl, and 3- face level. Correspondingly, HDAC11 depletion increased type I IFN hydroxydodecanoyl) from H3K9 peptides (Fig. 1 A and B). signaling in both cell culture and mice. This study not only dem- Surprisingly, HDAC11 failed to remove smaller acyl groups, onstrates that HDAC11 has an activity that is much more efficient including octanoyl and decanoyl, from H3K9 peptides (Fig. 1 A than the corresponding deacetylase activity, but also expands the and B). Many other acyl groups tested, including lipoyl, succinyl, BIOCHEMISTRY physiological functions of HDAC11 and protein lysine fatty acyla- and glutaryl, on H3K9 peptides were not HDAC11 substrates tion, which opens up opportunities to develop HDAC11-specific (Fig. 1A). We also used a few different peptide sequences. We inhibitors as therapeutics to modulate immune responses. detected demyristoylation activity of HDAC11 on H2BK12 and TNFα peptides, but did not detect any deacetylation activity on HDAC11 | lysine fatty acylation | SHMT2 | IFNAR1 | interferon H2BK12 and α-tubulin peptides (SI Appendix, Fig. S1B). Mouse HDAC11 also exhibited efficient demyristoylation activity (SI istone deacetylases (HDACs) regulate many biological Appendix, Fig. S1C). Since N-terminal glycine myristoylation is a Hfunctions by removing acetyl groups from lysine residues on well-known posttranslational modification, we also tested whether proteins (1, 2). There are 18 HDACs in mammals, classified into HDAC11 could catalyze glycine demyristoylation. No glycine + four classes. Class III HDACs (known as sirtuins) are NAD - dependent, while class I, II, and IV HDACs are all zinc-dependent. Significance Class I HDACs, consisting of HDAC1, 2, 3, and 8, are homologous to yeast Rpd3. Class II HDACs are homologous to yeast Hda1 and HDAC11 is the only class IV member of the histone deacetylase are further divided into two subclasses: IIa (HDAC4, 5, 7, 9) and (HDAC) family, and very little is known about its biological IIb (HDAC6, 10) (1, 3). Class IV HDAC is comprised solely of function. The work here reveals its efficient and physiologically HDAC11 (4, 5). relevant activity. The regulation of SHMT2 and interferon sig- HDAC11 is normally found in brain, testis, and immune cells naling expands the known biological function of protein lysine such as antigen-presenting cells (APCs) (5, 6). Up-regulation of fatty acylation, which has only recently started to be appreci- HDAC11 has been found in many cancer cells (7, 8) and ated. Furthermore, a compelling molecular mechanism is pro- interleukin-13 (IL-13) treated B cells (9). It has been reported that posed to connect HDAC11 to immune response. The finding HDAC11 negatively regulates IL-10 production in APCs by opens exciting opportunities to develop HDAC11-specific inhibi- binding to the IL-10 promoter and promoting histone deacetyla- tors to treat human diseases that would benefit from increased tion, thus influencing immune activation (10). However, the type I interferon signaling, such as viral infection, multiple sclerosis, deacetylase activity of HDAC11 reported is rather weak, and its and cancer. ability to directly deacetylate histones has not been demonstrated. The lack of efficient deacetylase activity has prevented the iden- Author contributions: J.C., L.S., P.A., E.S., and H.L. designed research; J.C., L.S., P.A., X.Z., tification of more substrate proteins that could help to understand W.H., and E.S. performed research; N.A.S. contributed new reagents/analytic tools; J.C. the biological function of HDAC11. and H.L. analyzed data; and J.C. and H.L. wrote the paper. Recently, several HDACs have been shown to hydrolyze acyl The authors declare no conflict of interest. lysine modifications that are distinct from acetyl lysine (11–15). This article is a PNAS Direct Submission. In particular, SIRT5 can efficiently remove succinyl and malonyl This open access article is distributed under Creative Commons Attribution-NonCommercial- groups (16, 17), while SIRT6 can efficiently remove long-chain NoDerivatives License 4.0 (CC BY-NC-ND). fatty acyl groups in vitro and in vivo (18). Herein we identify a 1Present address: Department of Biochemistry, Faculty of Science, Burapha University, defatty-acylase activity of HDAC11 that is >10,000 times more 20131 Chonburi, Thailand. efficient than its deacetylase activity. Two other laboratories at a 2Present address: Department of Pathobiological Sciences, School of Veterinary Medicine, similar time found the in vitro defatty-acylation activity (19, 20), Louisiana State University, Baton Rouge, LA 70803. and we coordinated the deposition of the results on BioRxiv. 3To whom correspondence should be addressed. Email: [email protected]. This potent defatty-acylase activity of HDAC11 led us to iden- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tify its substrate SHMT2, a lysine-acylated protein, as well as 1073/pnas.1815365116/-/DCSupplemental. an immunoregulatory pathway mediated by HADC11/SHMT2 Published online February 28, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1815365116 PNAS | March 19, 2019 | vol. 116 | no. 12 | 5487–5492 Downloaded by guest on September 24, 2021 Fig. 1. HDAC11 is an efficient lysine defatty-acylase. (A) HDAC11 activity on H3K9 peptides bearing different acyl groups. (B) Representative HPLC assay data of HDAC11 activity on myristoyl and acetyl H3K9 peptides. (C) Representative HPLC assay data of HDAC11 WT and Y304H mutant activity on myristoyl H3K9 peptide. (D) Kinetic parameters of HDAC11 on myristoyl and acetyl H3K9 peptides. demyristoylation activity was detected on p21-activated kinase 2 (SILAC) to identify proteins with increased Alk14 labeling in (PAK2) and Gα peptides (SI Appendix, Fig. S1D). HDAC11 knockout (KO) HAP1 cells (Fig. 2B and SI Appendix, To rule out that the defatty-acylation activity was from a Fig. S2C). The SILAC experiments were carried out in two in- contaminating protein in the HDAC11 preparation, we further dependent repeats. The HDAC11 KO cells were cultured with constructed and purified four catalytic mutants of HDAC11 that heavy amino acids, while the corresponding control cells were lack the general acid catalytic residue (Y304H) or zinc-binding cultured with light amino acids. The cells were labeled with residues (D181A, H183A, and D261A). Wild-type (WT) HDAC11, Alk14, and the alkyne-labeled proteins were conjugated to a but not the catalytic mutants, could remove myristoyl group from the C SI Appendix E biotin tag (biotin-azide) via click chemistry. The modified pro- H3K9 peptide (Fig. 1 and ,Fig.S1 ). Furthermore, we teins were pulled down with streptavidin, treated with hydrox- also detected the demyristoylation activity of recombinant HDAC11 Saccharomyces cerevisiae SI Appendix F ylamine, and analyzed by mass spectrometry. A protein with purified from ( ,Fig.S1 ). heavy/light (H/L) ratio higher than 1 in SILAC could be a po- We have previously identified SIRT2 and SIRT6 as functional tential substrate of HDAC11. To remove false positives and lysine defatty-acylases (18, 21). Compared with SIRT2 and SIRT6, HDAC11 exhibited the most efficient and specific simplify downstream validation studies, we further filtered our SI Appendix G data using the following criteria: H/L ratio ≥1.2, H/L variabil- demyristoylation activity ( , Fig. S1 ). Kinetics ≤ studies (Fig. 1D) showed that the Km value for the myristoyl ity 30%, and with at least two unique peptides identified. Based H3K9 peptide was 17.3 μM, and catalytic efficiency (kcat/Km on these criteria, we identified 7 and 71 proteins in the two re- 4 −1 −1 SI