HDAC6 regulates antibody-dependent intracellular neutralization of viruses via deacetylation of TRIM21

Abstract

Tripartite motif–containing protein 21 (TRIM21) is a cytosolic antibody receptor that targets the internalized virus–antibody complex to the proteasome for degradation. However, the precise mechanism regulating TRIM21 activity is unknown. Here we show that TRIM21 is a substrate of histone deacetylase 6 (HDAC6) and that its function is regulated by acetylation. HDAC6 interacts with TRIM21 through its PRYSPRY motif and deacetylates TRIM21 at lysine 385 and lysine 387, thus promoting its homodimerization. Inhibiting HDAC6 activity increases TRIM21 acetylation, and hyperacetylation blocks TRIM21 dimerization and ubiquitination, preventing its binding to the virus–antibody complex and its degradation via the ubiquitin–proteasome pathway. HDAC6 depletion or inhibition increases virus accumulation in cells, indicative of an impaired capacity for antibody-dependent intracellular neutralization of viruses, whereas TRIM21 acetylation-deficient K385/387R mutant rescues HDAC6 depletion–caused ADIN impairment. These findings provide evidence for HDAC6 as a novel regulator of TRIM21-mediated intracellular innate immunity. Tripartite motif–containing protein 21 (TRIM21) is a cytosolic antibody receptor that targets the internalized virus–antibody complex to the proteasome for degradation. However, the precise mechanism regulating TRIM21 activity is unknown. Here we show that TRIM21 is a substrate of histone deacetylase 6 (HDAC6) and that its function is regulated by acetylation. HDAC6 interacts with TRIM21 through its PRYSPRY motif and deacetylates TRIM21 at lysine 385 and lysine 387, thus promoting its homodimerization. Inhibiting HDAC6 activity increases TRIM21 acetylation, and hyperacetylation blocks TRIM21 dimerization and ubiquitination, preventing its binding to the virus–antibody complex and its degradation via the ubiquitin–proteasome pathway. HDAC6 depletion or inhibition increases virus accumulation in cells, indicative of an impaired capacity for antibody-dependent intracellular neutralization of viruses, whereas TRIM21 acetylation-deficient K385/387R mutant rescues HDAC6 depletion–caused ADIN impairment. These findings provide evidence for HDAC6 as a novel regulator of TRIM21-mediated intracellular innate immunity. Tripartite motif-containing protein 21 (TRIM21) belongs to the TRIM family of proteins that share a common multidomain architecture comprising an N-terminal RING domain with E3 ubiquitin ligase activity, a B-box domain, and a helical coiled-coil domain. The C-terminal PRYSPRY motif of TRIM21 determines its binding specificity to the Fc region of IgG antibody (1James L.C. Keeble A.H. Khan Z. Rhodes D.A. Trowsdale J. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function.Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17400754): 6200-620510.1073/pnas.0609174104Crossref PubMed Scopus (255) Google Scholar). TRIM21 was first reported to interact with the Fc domain of human IgG1 in a yeast two-hybrid screen (2Yang Y. Eversole T. Lee D.J. Sontheimer R.D. Capra J.D. Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain.Scand. J. Immunol. 1999; 49 (10354373): 620-62810.1046/j.1365-3083.1999.00547.xCrossref PubMed Scopus (30) Google Scholar). The significance of this interaction was revealed by a model for antibody-dependent intracellular neutralization (ADIN) involving the nonenveloped DNA virus adenovirus (AdV) type 5 (3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar). During ADIN, TRIM21 functions as both a sensor and an effector (3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar, 4McEwan W.A. Tam J.C. Watkinson R.E. Bidgood S.R. Mallery D.L. James L.C. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21.Nat. Immunol. 2013; 14 (23455675): 327-33610.1038/ni.2548Crossref PubMed Scopus (255) Google Scholar). When antibody-bound AdV enters the target cell, TRIM21 homodimerizes through its coiled-coil domain and binds with high affinity to the Fc domain of the antibody via its PRYSPRY motif (1James L.C. Keeble A.H. Khan Z. Rhodes D.A. Trowsdale J. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function.Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17400754): 6200-620510.1073/pnas.0609174104Crossref PubMed Scopus (255) Google Scholar, 3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar, 4McEwan W.A. Tam J.C. Watkinson R.E. Bidgood S.R. Mallery D.L. James L.C. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21.Nat. Immunol. 2013; 14 (23455675): 327-33610.1038/ni.2548Crossref PubMed Scopus (255) Google Scholar, 5Keeble A.H. Khan Z. Forster A. James L.C. TRIM21 is an IgG receptor that is structurally, thermodynamically, and kinetically conserved.Proc. Natl. Acad. Sci. U. S. A. 2008; 105 (18420815): 6045-605010.1073/pnas.0800159105Crossref PubMed Scopus (141) Google Scholar). This interaction activates the E3 ligase activity of TRIM21 and triggers its monoubiquitination and lysine-63 polyubiquitination, which targets the AdV–antibody complex to the proteasome for degradation (6Hauler F. Mallery D.L. McEwan W.A. Bidgood S.R. James L.C. AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23091005): 19733-1973810.1073/pnas.1210659109Crossref PubMed Scopus (77) Google Scholar, 7Fletcher A.J. Mallery D.L. Watkinson R.E. Dickson C.F. James L.C. Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (26150489): 10014-1001910.1073/pnas.1507534112Crossref PubMed Scopus (79) Google Scholar). TRIM32 and TRIM25, two important members of TRIM family, play a critical role in antiviral infections (8Fu B. Wang L. Ding H. Schwamborn J.C. Li S. Dorf M.E. TRIM32 senses and restricts influenza A virus by ubiquitination of PB1 polymerase.PLoS Pathog. 2015; 11 (26057645): e100496010.1371/journal.ppat.1004960Crossref PubMed Scopus (99) Google Scholar, 9Sanchez J.G. Sparrer K.M.J. Chiang C. Reis R.A. Chiang J.J. Zurenski M.A. Wan Y. Gack M.U. Pornillos O. TRIM25 binds RNA to modulate cellular anti-viral defense.J. Mol. Biol. 2018; 430 (30342007): 5280-529310.1016/j.jmb.2018.10.003Crossref PubMed Scopus (46) Google Scholar). The catalytic activity of the RING domains of these two proteins is governed by their oligomerization through coiled-coil domains (10Koliopoulos M.G. Esposito D. Christodoulou E. Taylor I.A. Rittinger K. Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity.EMBO J. 2016; 35 (27154206): 1204-121810.15252/embj.201593741Crossref PubMed Scopus (109) Google Scholar, 11Streich Jr., F.C. Ronchi V.P. Connick J.P. Haas A.L. Tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism.J. Biol. Chem. 2013; 288 (23408431): 8209-822110.1074/jbc.M113.451567Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). TRIM21 also dimerizes to bind IgG (12Bottermann M. Foss S. van Tienen L.M. Vaysburd M. Cruickshank J. O'Connell K. Clark J. Mayes K. Higginson K. Hirst J.C. McAdam M.B. Slodkowicz G. Hutchinson E. Kozik P. Andersen J.T. et al.TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (30209217): 10440-1044510.1073/pnas.1806314115Crossref PubMed Scopus (36) Google Scholar, 13Dickson C. Fletcher A.J. Vaysburd M. Yang J.C. Mallery D.L. Zeng J. Johnson C.M. McLaughlin S.H. Skehel M. Maslen S. Cruickshank J. Huguenin-Dezot N. Chin J.W. Neuhaus D. James L.C. Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21.Elife. 2018; 7 (29667579): e3266010.7554/eLife.32660Crossref PubMed Scopus (41) Google Scholar). However, the precise mechanism regulating TRIM21 activity is unknown. Posttranslational modifications regulate protein localization, activity, and interaction with other cellular molecules; acetylation and ubiquitination modify lysine residues of a protein, and their interaction controls the stability and function of the target protein. Given that TRIM21 ubiquitination is a critical step in ADIN (3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar, 6Hauler F. Mallery D.L. McEwan W.A. Bidgood S.R. James L.C. AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23091005): 19733-1973810.1073/pnas.1210659109Crossref PubMed Scopus (77) Google Scholar, 7Fletcher A.J. Mallery D.L. Watkinson R.E. Dickson C.F. James L.C. Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (26150489): 10014-1001910.1073/pnas.1507534112Crossref PubMed Scopus (79) Google Scholar, 14Xue B. Li H. Guo M. Wang J. Xu Y. Zou X. Deng R. Li G. Zhu H. TRIM21 promotes innate immune response to RNA viral infection through Lys27-linked polyubiquitination of MAVS.J. Virol. 2018; 92 (29743353): e00318-e0032110.1128/JVI.00321-18Crossref PubMed Scopus (82) Google Scholar), we speculated that TRIM21 is acetylated and that this influences the process of ADIN. In our previous work, we identified TRIM21 as a putative substrate of histone deacetylase 6 (HDAC6) and determined by MS that TRIM21 acetylation in the liver was 5.77-fold higher in HDAC6 KO mice compared with their WT counterparts (15Zhang L. Liu S. Liu N. Zhang Y. Liu M. Li D. Seto E. Yao T.P. Shui W. Zhou J. Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.Protein Cell. 2015; 6 (25311840): 42-5410.1007/s13238-014-0102-8Crossref PubMed Scopus (47) Google Scholar), suggesting that TRIM21 is deacetylated by HDAC6. Based on the subcellular localization and observed acetylation of TRIM21, in this study we investigated whether HDAC6 regulates TRIM21-mediated ADIN. We found that HDAC6 deacetylated TRIM21; inhibition of HDAC6 activity resulted in TRIM21 hyperacetylation, which prevented its dimerization and polyubiquitination. Moreover, HDAC6 inhibition or depletion impaired virus removal through ADIN. These results demonstrate that the regulation of TRIM21 by HDAC6 plays an essential role in the cellular response to viral infection. In our previous work, we identified the cytosolic protein TRIM21 as a putative substrate of HDAC6 (15Zhang L. Liu S. Liu N. Zhang Y. Liu M. Li D. Seto E. Yao T.P. Shui W. Zhou J. Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.Protein Cell. 2015; 6 (25311840): 42-5410.1007/s13238-014-0102-8Crossref PubMed Scopus (47) Google Scholar). To confirm this possibility, we first examined the subcellular distribution of TRIM21 and HDAC6 by immunofluorescence microscopy. Endogenous TRIM21 colocalized with HDAC6 (Fig. 1a), as did exogenous TRIM21 that was diffusely distributed in the cytoplasm (Fig. 1b, upper panel). However, exogenous TRIM21 also appeared to form rodlike shapes that was only weakly colocalized with HDAC6 (Fig. 1b, lower panel). To determine whether HDAC6 interacts with TRIM21 in cells, we immunoprecipitated endogenous HDAC6 from HEK293T cell lysates. Endogenous TRIM21 was precipitated with an HDAC6 antibody, but not with the control IgG (Fig. 1c). Conversely, endogenous HDAC6 was precipitated with an anti-HA antibody, indicating an interaction between HDAC6 and HA-tagged TRIM21 (Fig. 1d). Similarly, exogenous HA-tagged TRIM21 was precipitated with exogenously expressed GFP-tagged HDAC6 (Fig. 1e), and vice versa (Fig. 1f). In vitro pulldown experiments using recombinant His-tagged TRIM21 and Myc/DDK-tagged HDAC6 showed that HDAC6 directly interacted with TRIM21 in the absence of any other cellular proteins (Fig. 1g). We next constructed GFP-tagged truncated HDAC6 (Fig. 2a) and FLAG-tagged truncated TRIM21 (Fig. 2b) to analyze the interaction between HDAC6 and TRIM21. The Δ(460–840) deletion mutant of HDAC6 lacking the DD2 deacetylase domain lost its ability to bind TRIM21 (Fig. 2a), suggesting that these two proteins interact through deacetylation. Conversely, the TRIM21 fragment lacking the PRYSPRY motif failed to precipitate with GFP-HDAC6 (Fig. 2b), indicating that HDAC6 binds to TRIM21 through this motif. The association between HDAC6 and TRIM21 suggests that TRIM21 is a target of HDAC6. To test this possibility, we treated HEK293T cells with the HDAC6 inhibitor tubacin (16Haggarty S.J. Koeller K.M. Wong J.C. Grozinger C.M. Schreiber S.L. Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation.Proc. Natl. Acad. Sci. U. S. A. 2003; 100 (12677000): 4389-439410.1073/pnas.0430973100Crossref PubMed Scopus (897) Google Scholar) and performed immunoprecipitation with an antibody against acetyl-lysine (AcK) or the TRIM21 epitope tag. Endogenous TRIM21 showed increased acetylation upon tubacin treatment (Fig. 2c). HDAC6 inhibition also led to increased acetylation of overexpressed TRIM21 (Fig. 2, d and e). To confirm the regulatory relationship between the two proteins, we coexpressed GFP-tagged HDAC6 with FLAG-tagged TRIM21 or HA-tagged TRIM21; anti-AcK or anti-HA immunoprecipitate was analyzed by Western blotting. HDAC6 overexpression markedly reduced the levels of acetylated TRIM21 (Fig. 2, f and g). To identify HDAC6-specific deacetylation sites in TRIM21, we constructed acetylation-deficient mutants of TRIM21, in which the lysines within the PRYSPRY motif were replaced with arginines (K314R, K351R, K366R, K374R, K385/387R, K455R). We found that HDAC6-mediated deacetylation of TRIM21 was remarkably impaired by K385/387R mutant and mildly impaired by K341R or K351R mutants (Fig. 2h), suggesting Lys-385 and Lys-387 as the major sites of TRIM21 that are deacetylated by HDAC6. These data suggest that HDAC6 deacetylates TRIM21. TRIM21 forms homodimers through its coiled-coil domain (17Wang D. Buyon J.P. Yang Z. Di Donato F. Miranda-Carus M.E. Chan domain of promotes protein formation and in vitro PubMed Scopus Google Scholar), which is for its binding with AdV–antibody (3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar). We investigated whether HDAC6-mediated deacetylation of TRIM21 its dimerization by Inhibiting HDAC6 activity by tubacin treatment impaired the ability of TRIM21 to form (Fig. a and Conversely, overexpression of WT but not the TRIM21 dimerization (Fig. Given that dimerization is critical for the formation of rodlike shapes by TRIM21 (17Wang D. Buyon J.P. Yang Z. Di Donato F. Miranda-Carus M.E. Chan domain of promotes protein formation and in vitro PubMed Scopus Google Scholar, A. G. A. G. S. L. D. E. S. S. A. S. A. The tripartite motif family cell J. PubMed Scopus Google Scholar), we examined the of HDAC6 inhibition on the subcellular localization of TRIM21. treatment increased the of cells rodlike TRIM21 (Fig. d and its dimerization (Fig. a and TRIM21 is to ubiquitination (3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar, 7Fletcher A.J. Mallery D.L. Watkinson R.E. Dickson C.F. James L.C. Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (26150489): 10014-1001910.1073/pnas.1507534112Crossref PubMed Scopus (79) Google Scholar, T. T. of Ro52 PubMed Scopus Google Scholar), which influences its activity as a regulator of intracellular viral We examined the of HDAC6 on TRIM21 with tubacin reduced TRIM21 ubiquitination (Fig. whereas overexpression of but not the mutant led to an in TRIM21 ubiquitination (Fig. We to the association of acetylation with ubiquitination by using constructed acetylation-deficient mutants of TRIM21. of TRIM21 ubiquitination revealed that K351R and mutants resulted in a in ubiquitination (Fig. In our immunoprecipitation revealed that mutant but not K351R mutant increased the dimerization of TRIM21 (Fig. indicating Lys-385 and Lys-387 acetylation are critical for the regulation of TRIM21 in with previous findings K. K. T. protein Ro52 by its PubMed Scopus Google Scholar), the rodlike TRIM21 did not with ubiquitin (Fig. indicating that this form of TRIM21 is not the ubiquitination of TRIM21 in response to tubacin treatment is with its rodlike shapes (Fig. d and e). These results that hyperacetylation of TRIM21 by HDAC6 inhibition blocks its dimerization and TRIM21 functions as a of ADIN. We investigated whether HDAC6 is an regulator of this process by of the viral neutralization a gene were with an antibody against the protein of to form which were to for the of cells was analyzed by (Fig. We first performed the experiments using WT and HDAC6 KO and observed that the of cells was higher in the in the (Fig. and suggesting that cells lacking HDAC6 were to intracellular of We HDAC6 to the of HDAC6 in this HDAC6 levels were reduced in cells with and compared with with the control (Fig. The of cells was higher in cells of HDAC6 (Fig. and with the results using To determine whether the of HDAC6 on its deacetylase activity, we treated cells with tubacin for infection. This increased the infection (Fig. and indicating that HDAC6 activity cells against viral infection. To the of HDAC6-mediated deacetylation in ADIN, we overexpressed FLAG-tagged or mutants in cells (Fig. with the critical role of Lys-385 and Lys-387 acetylation in TRIM21 dimerization regulation (Fig. K385/387R mutant HDAC6 ADIN (Fig. and These findings demonstrate that HDAC6 deacetylates TRIM21 to ADIN. our identified HDAC6 as a regulator in a of cellular cell D. S. Y. L. J. D. Liu M. Zhou J. deacetylase HDAC6 promotes by regulating cell in an Cell. PubMed Scopus Google Scholar, D. X. Zhang L. B. S. Liu R. Liu M. Zhou J. deacetylase 6 and protein function to regulate the of Cell. 5 PubMed Scopus Google Scholar, L. Liu N. S. X. Zhou J. Liu M. Li D. HDAC6 regulates cell and play a role in the Biol. PubMed Scopus Google Scholar, C. A. R. Y. A. A. M. Wang Yao T.P. HDAC6 is a PubMed Scopus Google Scholar), J. Yang Y. Li D. Liu M. Zhou J. of and is for HDAC6 to 2015; 5 PubMed Scopus Google Scholar, Y. J. Liu M. Li D. Li Y. X. D. J. G. R. 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Here we a mechanism by which HDAC6 cells against Based on our we a model in which that cells are by TRIM21, which HDAC6. interaction in the cytoplasm to the deacetylation of TRIM21 and its through the coiled-coil domain. The homodimers bind via the Fc domain of the antibody and target the to the proteasome for degradation through and polyubiquitination. In the absence of TRIM21 which the formation of TRIM21 AdV–antibody are not via TRIM21-mediated ADIN, in viral of the viral and of viral proteins (Fig. The antiviral of HDAC6 in reported S. Zhang Y. M. of viral and of the response to infection by are regulated by the acetylation and phosphorylation of its 2013; PubMed Scopus (46) Google Scholar, S.J. Lee Lee D.J. S. J. Yao T.P. Lee et regulates cellular viral RNA by deacetylation of J. 2016; 35 PubMed Scopus (79) Google Scholar, D. L. Yang M. Wang L. X. Wang J. Li Z. X. Liu N. Li Z. H. Li N. Zhou J. et of to virus infection in cells and in Cell. 2015; 6 PubMed Scopus Google Scholar). 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Lee et regulates cellular viral RNA by deacetylation of J. 2016; 35 PubMed Scopus (79) Google Scholar). cells, are by and that to cells are by the cytosolic Fc receptor TRIM21 and by TRIM21-mediated ADIN (3Mallery D.L. McEwan W.A. Bidgood S.R. Towers G.J. Johnson C.M. James L.C. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21045130): 19985-1999010.1073/pnas.1014074107Crossref PubMed Scopus (331) Google Scholar, N. S. H. James L.C. P. and intracellular of Google Scholar). In this we that HDAC6 promotes the neutralization of intracellular antibody-bound through deacetylation of TRIM21, evidence for the antiviral of HDAC6. data revealed that Lys-385 and Lys-387 were critical for the regulation of TRIM21 acetylation, and ubiquitination at and Lys-387 suggesting a between acetylation and ubiquitination at Lys-385 and Lys-387 K385/387R mutant was to HDAC6 ADIN impairment. Given that both the TRIM21 dimerization and ubiquitination are for ADIN, we that HDAC6-mediated Lys-385 and Lys-387 deacetylation promotes TRIM21 which ubiquitination at thus in ADIN. The of antibody-bound by TRIM21 not on but on the of intracellular which function as is that TRIM21-mediated ADIN as an intracellular mechanism to virus that is by the antibody that TRIM21-mediated ADIN on HDAC6 activity as a basis for the of for viral infection. in this study the TRIM21, HDAC6 and A anti-HA and were from the or or for immunofluorescence and or for and tubacin were from the gene were from for and mutant were by of the the as J. Liu M. J. Li H. H. T. Y. Zhou P. Y. Yang Y. Yang Y. F. Guo H. Zhang L. S. et phosphorylation blocks HDAC6 ubiquitination and degradation to the of Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). deletion mutants were constructed by and the were as GFP-HDAC6 GFP-HDAC6 and for and were by of the the and The deletion mutants of were directly and by and the were by and with as a from cells was from and HDAC6 from HEK293T cells was from HDAC6 mice were a from Yao and to WT and HDAC6 experiments were in with the regulatory and were by the and of were from WT and HDAC6 as J. Lee Lee Yao T.P. deacetylase 6 regulates and Biol. 2007; PubMed Scopus Google Scholar). the mice with were by The were from the and and were The were and with at for were in in a and HEK293T cells were in with and at in a with were cells with HDAC6 and or the control from were cells with gene was by Western blotting. cells on were with for with in for and with in for at was performed using and in were at in to the The cells were with and the were with were with an or a were and and proteins were by and to a that was with in for with the and were using to the the proteins were at with which were and to Western blotting. The experiments were at and the of was determined by with the of The protein levels were to the immunoprecipitated or proteins and the was determined by with the control of the control was to were at 105 cells in and to a gene a of infection of for cells and for were with antibody at for at the were to the cells were and cells were examined by between were with the was the data are in the or from the upon antibody-dependent intracellular neutralization adenovirus acetyl-lysine

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