YopJ-mediated Dual Acetylation At The Serine And Lysine Residues And Its Function

INTRODUCTION Human Yersinia pestis infection causes the deadly disease called bubonic plague commonly spreading through rodents and fleas. To establish infection, Y. pestis expresses multiple outer membrane proteins that are secreted and adhesive to host epithelial cells capable of suppressing or resisting host immune responses by various mechanisms. Generally, Yersinia outer proteins （Yops） are effectors counteracting host innate immune responses including the pro-inflammatory signaling pathways of mitogen-activated protein （MAP） kinase （MAPK） and nuclear factor Kappa B（NF-κB）. Among them, YopJ is a 32 kD protein with 288 amino acids known to block cytokine production and inducing apoptosis of the infected cells. YopJ is also a serine acetyltransferase that is known to counteract the aforementioned inflammatory responses by acetylation of the serine residues in I kappa B kinase （IKK） and MAPK kinases （MKKs）. YopJ-mediated serine acetylation is shown to prevent serine phosphorylation, and subsequently block MAPK signaling and NF-κB activation, leading to significantly reduced production of both pro-inflammatory and anti-apoptotic host cytokines. YopJ is also reported to inhibit NF-κB activation and pro-inflammatory cytokine production by reducing the activation of eukaryotic initiation factor 2 （elF2） in yeast and mammalian cells. Thus, YopJ-mediated serine acetylation has a great impact on the innate immune responses of the infected hosts.Lys-acetylation is a well-known post translational modification （PTM） in higher eukaryotes and has a great impact on a wide range of biological processes such as metabolism, RNA modification, nuclear morphology, gene expression and mitochondrial function. Lysine acetyltransferases and their counterparts for the reverse reaction, best-known as histone acetyltransferases （HATs） and histone deacetylases （HDACs）, act cooperatively to tightly regulate the acetylation of histone and non-histone proteins for gene expression, cell development and cancer development. However, the interaction between lysine and serine acetyltransferases has not been examined.In this study, we aim to characterize the molecular effects of bacterial acetyltransferase YopJ on serine and Lys-acetylation by mimicking Y. pestis infection in human cells. Using shotgun proteomics and ’label-free quantification, acetylation at the serine and lysine residues with and without YopJ are identified and compared. Specifically, Ser- and Lys-acetylation at different positions of membrane-associated E3 ubiquitin ligase MARCH8 was characterized. Specific antibodies directed against MARCH8 acetylated serine residues were produced. Immunoassays were used to confirm YopJ-induced Ser- and Lys-acetylation in MARCH8. YopJ catalytic mutant study indicated that YopJ-induced Ser- and Lys-acetylation in MARCH8 was dependent on its catalytic activity. Our data support the conclusion that bacterial acetyltransferase YopJ targets the protein substrates for acetylation at both the lysine and serine residues in the host cells.Objective: To corroborate YopJ-mediated dual acetylation at the serine and lysine residues and its functionMethods:Recombinant plasmid construction and expression in HeLa cells The YopJ cDNA （Genbank accession # KP 641301） was chemically synthesized,MARCH8 cDNA （Genbank accession # NM₀01002266, 1-291 AA） was amplified from the total RNA of K562 cells by RT-PCR. A truncated MARCH8（Genbank accession # NM₀01002266, 1-156 aa） was amplified by RT-PCR.The cDNAs were sub-cloned into the pcDNA3.0-flag expression vector and their sequences verified by DNA sequencing. YopJ catalytic mutant C172A was generated by site-specific mutagenesis and confirmed by DNA sequencing. The recombinant plasmids were purified using Omega Maxi-Kits and then transfected into HeLa cells by Lipofectamine according to the manufacturer’s protocol. HeLa cells were harvested 24-48 hrs post transfection.Protein extraction The transfected cells were rinsed by PBS and lysed in RIPA lysis buffer with protease and phosphotase inhibitor cocktails, HDAC inhibitors （sodium butyrate and nicotinamide）, leupeptin, pepstatin and aprotinin, either sonicated for 15 seconds, or kept on ice for 40min with vigorous vortex for 20s per 10 minutes. The cell lysates were centrifuged for 10min at 14000rpm, and the supernatants were collected, protein concentration were determined by BCA kit and stored at -80℃ until further use.in-solution tryptic digestion Cell lysates from different transfected cells or the proteins from immunoprecipitation were digested with trypsin as previously described. Briefly, protein samples were dissolved in 200μl of 0.1 M Tris/HCI（PH 8.5） containing 8M Urea and loaded on a filter unit. After centrifugation,the proteins on the filter were washed twice with 100μl of 50mM ammonium bicarbonate, re-suspended in 40μl of 50mM ammonium bicarbonate with trypsin, incubated at 37 ℃ for 24 h in a wet chamber. The digested peptides were collected by centrifugation and desalted with a C18 spin column following manufacturer’s protocol. The purified peptides were dried in SpeedVac and stored at -80 ℃ until further use.Immunoprecipitation and in-gel tryptic digestion HeLa cells were co-transfected with MARCH8 and YopJ; cell lysates were prepared. The flag-tagged MARCH8 was immunoprecipitated with the anti-flag antibody following manufacture’s standard protocol. The precipitated proteins were separated on SDS-PAGE and stained by Coomassie Bright Blue. The protein bands corresponding to MARCH8 were excised from the gel, cut into 1 mm slices, and digested overnight with trypsin following the modified in-gel digestion protocol. The digested peptides were desalted by C18 column ZipTip for LC-MS/MS analysis.LC-MS/MS analysis -2 μg of the trypsin-digested peptides were injected into a C18 column （200x φ0.075 mm） and eluted with a 120 min linear gradient（5-35% acetonitrile with 0.1% formic acid） followed by the Orbitrap Elite massspectrometer. Data were acquired in a data-dependent mode, in which MS/MS fragmentation was performed for the 20 most intense peaks of every full MS scan. MS/MS spectra were searched against the human protein dabase（UniProt: human-20151224, 149,731 sequences, 47,136,486 residues） using Mascot and Sequest, parts of the Proteome Discoverer 1.4 data analysis package. MS/MS spectra were searched with a maximum mass tolerance of 10 ppm for the precursors, 0.8 Da for fragments, dynamic modifications of lysine and serine acetylation and methionine oxidation, and missed cleavage of 2. The thresholds for Mascot lonScore and Sequest XCorr as accepting individual MS/MS spectra were 38 and 1.22 x charges, respectively. All modification site assignments were confirmed by manual spectrum interpretation.Label-free quantification The abundance of the acetylated peptides was quantified by label-free method using Progenesis LC-MS software （version 4.1; Nonlinear Dynamics, UK）. Briefly, the acquired raw data were transformed to the mzxml format, searched by Proteome Discoverer 1.4 with mascot, high confident peptides were imported into Progenesis LC-MS software. The ion intensity maps from multiple analysis were examined for defects. The best dataset was selected as the reference for data alignment. Peptide ions with charge state of +1 or > 4 were excluded. Peptides were also searched against a decoy database to determine False Discovery Rate （FDR）. For quantification,the unique peptide validated by MS （high confident peptide, label-free score>30） was chosen and calculated by summing the abundance of all peptides allocated to a specific site. Statistics analysis was based on the ratio of the sum of all acetylated peptides at a specific site to that of the non-acetylated peptides to that of a set of reference peptides（HQGVMVGMGQK） from a- actin.MARCH8 Sac71 polyclonal antibody production Polyclonal antibody specific to MARCH8 Sac71 was produced following a protocol modified from pan-acetyl antibody production. A peptide encompassing acetylated Sac71 of MARCH8, Ac-T（Sac）ITPSSQDICRICHCEGDC-NH2, was synthesized and verified by LC-MS/MS that more than 90% of the peptides were the acetylated peptide. The corresponding non-acetylated peptide,Ac-TSITPSSQDICRICHCEGDC-NH2, was also synthesized and verified by LC-MS/MS. The acetylated peptide was conjugated to BSA or KLH by a conjugation kit and verified by Coomassie Blue staining that the peptide was successfully conjugated. The New Zealand rabbits were firstly immunized by injection （s.c.） of 400 μg conjugated-proteins with CFA （Freund’s Adjuvant,Complete, Sigma, F5881）. Four weeks later, another 400μg conjugated-protein with IFA was immunized. The last injection （i.v.） was performed three weeks later with 200μg conjugated-proteins in PBS. 10 days after the last injection, blood samples were collected from rabbit ear and the antibody titer was determined by ELISA. All blood samples were collected in 15 days after the last injection. The anti-sera were passed twice through the affinity chromatography column coupled with the unmodified peptide to deplete the antibodies for the same peptide lacking the acetylated Sac71. The depleted anti-sera were affinity purified with the acetylated Sac71 peptide.Finally, the specificity of the Sac71 antibody was confirmed by the dot-blot peptide competition assay and western-blot competition assay. All animal work was permitted by Ethic Committee on Animal Experiments of Medical School of Shandong University and conducted according to relevant national and international guidelines.Immunoprecipitation-Western blotting （IP-WB） The flag-tagged MARCH8 was first immunoprecipitated with the anti-flag antibody following the standard protocol from manufacture. The immunoprecipitated proteins were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked by 5% BSA. For detection, the membrane was incubated with the primary antibodies against the flag-tag （mouse）, pan-acetyl lysine, or MARCH8 Sac71. After washing, the targeted proteins were stained by fluorescence-conjugated secondary antibody （IRDye 800CW or IRDye 680 conjugated IgG, LI-COR）. The membranes were scanned with Odyssey infrared imaging system （Li-COR）.In-vitro acetylation assay with recombinant YopJ Cell lysates HeLa cell lysate with MARCH8 transfection was aliquoted equally into three fractions;the recombinant MARCH8 in each fraction was immobilized on the resin by incubation with the anti-flag antibody; the immobilized MARCH8 was washed four times by PBS and prepared for in vitro acetylation reaction. As controls,the immobilized MARCH8 was incubated with 200μl cell lysate transfected with pcDNA3.0 vector （V） in the presence of 50μM acetyl-CoA, protease inhibitor cocktail, 1mM PMSF, 10mM Nicotinamide and 10mM Sodium Butyrate. For in vitro acetylation reaction, the immobilized MARCH8 was incubated with the cell lysate transfected with YopJ （Y） or YopJ C172A mutant （C172A）,respectively. After incubation at 37℃ for 1h, the immobilized MARCH8 was washed by PBS four times, recovered by boiling in SDS-loading buffer and analyzed by Western-blotting using antibodies against the flag, MARCH8 Sac71, or anti-Pan-K. Signal intensities of the Ser-acetylated MARCH8, and the Lys-acetylated MARCH8 were quantified by Li-COR software and normalized against the control levels of MARCH8.In-vitro acetylation assay with affinity-purified YopJ HeLa cell lysate with flag-tagged YopJ or YopJ C172A mutant transfection was incubated with the anti-flag antibody and affinity-purified by the protein G resin. The bound YopJ was washed with PBS, eluted with 0.2M Glycine （PH=3.0）,neutralized with 1M Tris-Cl （PH=8.5） and dialyzed against autoclaved TBS for in vitro acetylation reaction. HeLa cell lysate with flag-tagged MARCH8 transfection was aliquoted equally into three fractions; the recombinant MARCH8 in each fraction was immobilized on the protein G resin after incubation with the anti-flag antibody; the immobilized MARCH8 was washed four times by PBS and prepared for in vitro acetylation reaction. For in vitro acetylation reaction,the immobilized MARCH8 was incubated with the affinity-purified YopJ or YopJ C172A mutant, respectively, in the presence of 50μM acetyl-CoA, protease inhibitor cocktail, 1mM PMSF, 10mM Nicotinamide and 10mM Sodium Butyrate. As controls, the immobilized MARCH8 was incubated in the same buffer without YopJ. After incubation at 37℃ for 1h, the immobilized MARCH8 was washed by PBS four times, recovered by boiling in SDS-loading buffer and analyzed by Western-blotting using antibodies against the flag, MARCH8 Sac71, or anti-Pan-K. Signal intensities of the Ser-acetylated MARCH8, and the Lys-acetylated MARCH8 were quantified by Li-COR software and normalized against the control levels of MARCH8.In vitro ubiquitination assays The flag-tagged E3 ubiquitin ligase MARCH8 was purified from the cell lysates by immunoprecipitation using anti-flag antibody, and used directly for in vitro ubiquitination. The E2 ubiquitination conjugating enzyme （UbcH5a） and ubiquitin activating enzyme （UBE1） were purchased from Boston Biochem. Briefly, the in vitro reaction mixture （40μl）included 50 nM rabbit UBE1, 2 μM UbcH5a （E2）, 28 μM ubiquitin 5 mM ATP,50 mM Tris-HCI （pH 7.5）, 5 mM MgCl2 and 2 mM DTT, 25 pM MG-132 proteasome inhibitor. The mixture was incubated at 37 ℃ for 6 hrs and stopped by boiling in SDS-loading buffer. Samples were separated by 12%SDS-PAGE and immunoblotted with anti-ubiquitin antibody P4D1。Statistics analysis SPSS 19.0 software （SPSS Inc., USA） was used for statistical analysis. P-value<0.05 or FDR<0.01 was considered statistically significant. Statistics between two groups was performed using a two-tailed student’s t- test.Results:1.Bacterial serine acetyltransferase YopJ mediates protein acetylation at the serine and lysine residues in HeLa cells: Using shotgun proteomics followed by label-free quantification, we demonstrate an increase of dual acetylation in YopJ transfected human cells, including 10 Ser- （YopJ/non-YopJ 1.3-fold, p=0.02） and 8 Lys- （YopJ/non-YopJ 3.5-fold, p=0.00003） acetylation sites.2.Acetylation of ubiquitin E3 ligase MARCH8 at the serine and lysine residues by bacterial serine acetyltransferase YopJ: Using IP-LC-MS/MS,we proved that YopJ expression augments acetylation of membrane-associated E3 ubiquitin ligase MARCH8 at the serine residue Sac44, Sac71 and Sac253, and the lysine residue Kac247 and Kac252.Acetylation at Sac253 and Sac44 increased by 28±11 （p=0.002） and 31 ±8（p=0.002） folds in YopJ transfected HeLa cells, respectively. In consistent, the acetylated lysines at Kac252 and Kac247 were augmented for 36±18（p=0.0003） and 18±8 （p=0.01） times, respectively.3.YopJ-mediated dual acetylation of MARCH8 at serine and lysine is confirmed by immunoassays in vivo and in vitro: To further confirm YopJ-mediated acetylation of MARCH8 at the serine and lysine residues, the antibodies were produced against Sac71 of MARCH8 by immunization of rabbits with a synthetic peptide Ac-T（Sac）ITPSSQDICRICH CEGDC-NH2,dot-blot and competition assay proved the specificity of the antibody. Using IP-WB, we proved that when cotransfected with YopJ, the level for serine-acetylation in MARCH8 were significantly increased by YopJ expression（1.8±0.5, p=0.027, n=3）. Similarly, the level for Lys-acetylation in MARCH8 was also significantly up-regulated （1.9±0.4, p=0.012, n=3）.Consistently, our data from independent immunoassays also confirmed YopJ-mediated acetylation of MARCH8 at both the Ser and Lys residues in HeLa cells. In support, the C172A mutant, which was known to reduce the acetyltransferase activity of YopJ, was also demonstrated to reduce the dual acetylation at Ser and Lys, indicating that the augmentation of Ser- and Lys- acetylation was dependent on the catalytic activity. Next we examined the dual acetylation activity of YopJ in vitro using the purified MARCH8 protein and recombinant YopJ cell lysate. The flag-tagged MARCH8 protein was immobilized on the resin by immune-affinity purification and incubated with recombinant YopJ and YopJ mutant C172A cell lysates in the presence of acetyl-coenzyme A and HDAC inhibitors. The levels of Ser- and Lys-acetylation were examined by Western-blotting using MARCH8 Sac71 and Pan-acetyl lysine antibodies.Consistently, acetylation at the Ser- as well as Lys- residues was enhanced in comparison with the control; however, both activities were reduced when YopJ was replaced by the C172A mutant. Thus, YopJ was consistently responsible for the acetylation at the Ser and Lys residues. To further prove the direct acetylation of YopJ at the Ser- and Lys- residues, we repeated the in vitro acetylation assay using the purified MARCH8 protein and affinity-purified YopJ,the result is consistent with that using recombinant YopJ cell lysate, thus proved the direct dual acetylation of YopJ.4. YopJ-mediated dual acetylation at serine and lysine elevates auto-ubiquitination of MARCH8 and the elevation is catalytic activity-dependent. To address whether YopJ-mediated dual acetylation was involved in any cellular or biological functions, auto-ubiquitination of MARCH8 was tested in vitro using ubiquitin activating enzyme （UBE1）, the E2 ubiquitination conjugating enzyme （UbcH5a） and the transiently expressed E3 ubiquitin ligase MARCH8. Results indicated that cotransfection of YopJ significantly enhanced the auto-ubiquitination level of MARCH8. In addition,the C172A mutant of YopJ reduced auto-ubiquitination of MARCH8 comparison with the MARCH8 control, implying this process was also dependent on the catalytic activity. Thus, these data demonstrated that bacterial YopJ-mediated dual acetylation at serine and lysine simultaneously enhanced auto-ubiquitination of MARCH8.Conclusion: In conclusion, our study provides evidence for YopJ-mediated dual acetylation at serine and lysine, suggesting a connection between Ser-and Lys-acetylation networks. Specifically, YopJ-mediated dual acetylation is involved in auto-ubiquitination of ubiquitin ligase MARCH8, suggesting a general role in regulation of MARCH8-mediated ubiquitination in human cells.Histone acetylation adds an acetyl group on the lysine residue commonly within the N-terminal tail protruding from the histone core of the nucleosome and is important for chromosome structure and function in gene transcription and chromatin remodeling. Acetylation may also occur on other residues additional to lysine but have not been thoroughly investigated at the proteomics level. Here we report a wide tolerance acetylation study mimicking the addition of 42±0.5 Da delta mass modification on undefined amino acid residues of histones by shotgun proteomics usinng liquid chromatography-tandem mass spectrometry. A multi-blind spectral alignment algorithm with a wide peptide tolerance revealed frequent occurrence of 42±0.5 Da modifications at lysine （K）, serine （S） and threonine （T） residues in human histones from kidney tissues. Precision delta mass analysis identified acetylation （42.011 ± 0.004 Da） and trimethylation （42.047 ± 0.002 Da）modifications within the delta mass range. Specific antibody was produced to validate the acetylated T22 of histone H3 （H3T22ac） by immune assays. Thus,we demonstrated that the wide tolerance acetylation approach identified histone acetylation as well as modification variants commonly associated with acetylation at undefined residues additional to lysine.