A Proteomics Strategy for the Identification of FAT10-Modified Sites

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A Proteomics Strategy for the Identification of FAT10-Modified Sites by Mass Spectrometry Ling Leng, Changming Xu, Chao Wei, Jiyang Zhang, Boya Liu, Jie Ma, Ning Li, Weijie Qin, Wanjun Zhang, Chengpu Zhang, Xiaohua Xing, Linhui Zhai, Fan Yang, Mansheng Li, Chaozhi Jin, Yanzhi Yuan, Ping Xu, Jun Qin, Hongwei Xie, Fuchu He, and Jian Wang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr400395k • Publication Date (Web): 18 Jul 2013 Downloaded from http://pubs.acs.org on July 20, 2013

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Journal of Proteome Research

Xie, Hongwei; College of Mechatronic Engineering and Automatic Control, He, Fuchu; State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, National Center for Protein Sciences Beijing Wang, Jian; Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Beijing Proteome Research Center

ACS Paragon Plus Environment


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A Proteomics Strategy for the Identification of FAT10-Modified Sites by Mass Spectrometry

Ling Leng‡ §, Changming Xu¶§, Chao Wei&, Jiyang Zhang¶, Boya Liu‡ , Jie Ma‡ , Ning Li‡ , Weijie Qin‡ , Wanjun Zhang‡ , Chengpu Zhang‡ , Xiaohua Xing‡ , Linhui Zhai‡ , Fan Yang‡ , Mansheng Li‡ , Chaozhi Jin‡ , Yanzhi Yuan‡ , Ping Xu‡ , Jun Qin‡

‡

‡‡

, Hongwei Xie¶, Fuchu He‡ *, Jian Wang‡

*

State Key Laboratory of Proteomics, Beijing Proteome Research Center,

National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206, China ¶

Department of Automatic Control, College of Mechatronics and Automation,

National University of Defense Technology, Changsha, 410073, China National Engineering Research Center for Protein Drugs, Beijing 102206, China &

Institute of Basic Medical Science, Chinese Academy of Medical Science and

School of Basic Medicine, Peking Union Medical College, Beijing, 10005, China ‡‡

Baylor College of Medicine, Houston, TX 77030, USA

KEYWORDS: FAT10, post translational modification, mass spectrometry 1


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ABSTRACT: The ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) is uniquely expressed in mammals. The fat10 gene is encoded in the MHC class I locus in the human genome and is related to some specific processes, such as apoptosis, immune response and cancer. However, biological knowledge of FAT10 is limited owing to the lack of identification of its conjugates. FAT10 covalently modifies proteins in eukaryotes, but only a few substrates of FAT10 have been reported until now, and no FATylated sites have been identified. Here, we report the proteome-scale identification of FATylated proteins by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 175 proteins with high confidence as FATylated candidates. A total of 13 modified sites were identified for the first time by a modified search of the raw MS data. The modified sites were highly enriched with hydrophilic amino acids. Furthermore, the FATylation of hnRNP C2, PCNA and PDIA3 were verified by a co-immunoprecipitation assay. We confirmed that most of the substrates were covalently attached to a FAT10 monomer. The functional distribution of the FAT10 targets suggests that FAT10 participates in various biological processes, such as translation, protein folding, RNA processing and macromolecular complex assembly. These results should be very useful for investigating the biological functions of FAT10.

2



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INTRODUCTION Ubiquitin (Ub), a small protein with 76 amino acid residues, covalently modifies other proteins by forming a covalent isopeptide linkage between its C terminus and a lysine of the target protein. A few ubiquitin-like proteins have been identified that share 14% to 52% sequence similarity with Ub1. These proteins can be classified into two groups: (a) the ubiquitin-domain proteins, which cannot covalently modify other proteins, and (b) the ubiquitin-like modifiers (Ubls), which covalently modify target proteins2. The Ubls include SUMO, NEDD8, ISG15 and FAT101. The conjugation of the Ubls to their substrates plays critical roles in various processes, such as protein degradation, cell cycle transcription, apoptosis and heterochromatin remodeling3, 4. The large-scale separation and identification of Ubl-modified proteins provide useful clues to understand their functions and natural activities 5, 6. As one of the Ubls, FAT10 has two ubiquitin-like domains with 29% and 36% identity to Ub at its N- and C-termini, respectively7. The fat10 gene is encoded in the MHC class I locus in the human genome and is thought to be related to specific processes, such as apoptosis, immune response and cancer7-9. Although FAT10 was identified more than ten years ago, its protein conjugation system is unclear. Endogenous FAT10 is induced upon stimulation of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) and forms conjugates with its substrates, suggesting that FAT10 is related to the immune response. FAT10 is rapidly degraded by the 26S proteasome in mammalian cells10, 11. 3


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FAT10 is constitutively expressed in mature dendritic cells and B cells8, 12 and the proinflammatory cytokines IFN-γ and TNF-α induce FAT10 expression in various other cells13, 14. The overexpression of FAT10 leads to cell apoptosis through the caspase-dependent cascade with cytokine induction7. Mice with a knockout of the fat10 gene were more susceptible to spontaneous apoptotic death than wild-type mice15. FAT10 regulates chromosomal stability by a reduction in the kinetochore localization of MAD2, a spindle-assembly check-point protein, during the prometaphase stage of the cell-cycle13,

16

.

FAT10 has been reported to mediate the activation of the NF-κB pathway17. FAT10 is upregulated in hepatocellular carcinoma and various cancers 9, 18. but the mechanisms of this upregulation are unclear. The enzymatic conjugation cascade of FAT10 to its substrates resembles the ubiquitination system, which includes a FAT10-activating enzyme (E1) and a FAT10-conjuating enzyme (E2). FAT10 and ubiquitin share a common E1, UBA6 (also named E1-L2), to catalyze the ATP-dependent adenylation of the C-terminal carboxylate and transfer the activated FAT10 to a conserved cysteine on E119. Then, the FAT10 thioester intermediate is transferred to an E2 enzyme, USE120. Whether USE1 directly transfers FAT10 to an ε-amino group of a lysine within the substrate or needs the assistance of a FAT10-protein ligase (E3) remains to be determined. The FATylated substrates are rapidly degraded by the 26S proteasome10. Recently, the degradation of FAT10 was shown to require ubiquitination21. To date, several substrates of 4



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FAT10 have been identified, including ubiquitin-specific E1-activating enzyme UBA1, E2 conjugating enzyme USE1, tumor suppressor p53, autophagosomal receptor p62/SQSTM1 and huntingtin11, 20, 22-24. The fast degradation and low abundance of FAT10-modified proteins makes it difficult to identify FATylated substrates. Additionally, a large residual peptide (a 13-a.a tag) is left on the substrates after trypsin digestion, which produces complicated MS/MS fragmentation patterns. No FATylated sites have been identified by mass spectrometry until now. The identification of these FATylated proteins should facilitate an understanding of the biochemical mechanisms and biological functions of FAT10. In this study, we used a proteomics method to identify 175 potential FATylated proteins and 13 FATylated sites. EXPERIMENTAL SECTION DNA Constructs The gene sequence corresponding to FAT10 was amplified by PCR using a human brain cDNA library as a template. The DNA sequence of protein G was synthesized and sub-cloned with the fat10 sequence into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). As a control, the FAT10 mutant FAT10∆GG coding sequence was amplified by PCR and then subcloned into pcDNA3.1 with an N-terminal protein G tag. Myc-FAT10 and Myc-FAT10∆GG were generated by PCR. The resulting DNA fragments were cloned into the pCMV-Myc vector (Clontech, Palo Alto, CA, USA). Flag-HNRNP C2, Flag-PCNA and Flag-PDIA3 were generated by PCR using a human liver 5


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cDNA library as a template. The resulting DNA fragments were cloned into the pFLAG-CMV vector (Sigma, St. Louis, MO, USA). Cell Lines, Immunoprecipitation and Immunoblotting Stable HeLa cell lines overexpressing pcDNA3.1-protein G-FAT10 and pcDNA3.1-protein G-FAT10∆GG were obtained after selection with 500 ng/ml G418 (Sigma). The HeLa cells and HEK293 cells were cultured in DMEM supplemented with 10% fetal calf serum. The transfection of HeLa cells and HEK293 cells was performed using Lipofectamine 2000 (Invitrogen) and a transfection reagent (Origene, Rockville, MD, USA). The Myc-FAT10, Myc-FAT10∆GG,

Flag-PCNA,

Flag-HNRNP-C2

and

Flag-PDIA3

were

expressed in HEK293 cells after induction with TNF-α (40 ng/ml) and IFN-γ (40 ng/ml) for 24 h. To immunoprecipitate the FAT10 conjugates, the HEK293 cells were washed three times with PBS and lysed in lysis buffer (20 mM HEPES, 50 mM NaCl, 0.5% Triton, 1 mM NaF, 1 mM DTT, 1 mM Na3VO4 and 0.5% sodium protease inhibitors). The lysates were centrifuged (13,000 rpm for 10 min at 4°C). After a 2-h incubation at 4°C with α-Flag antibodies, the lysates were incubated for another 4 h at 4°C with protein A/G agarose. After collection and extensive washing of the bound proteins, the samples were boiled for 10 min and separated on SDS-PAGE, followed by western blot analysis. Antibodies The following antibodies were used: anti-Flag (A8592-1 MG, Sigma), anti-Myc 6



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(sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FAT10 (sc-67203, Santa Cruz Biotechnology), and anti-PCNA (#2586, Cell Signaling Technology, Beverly, MA, USA). Affinity Purification Assays Stable HeLa cell cultures (2.64×108 cells) were cultured for 24 h at 37°C, induced with TNF-α (40 ng/ml) and IFN-γ (40 ng/ml) for 24 h, harvested by centrifugation at 900 rpm for 5 min at 4°C and then washed 3 times using the same method. The cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.5], 125 mM NaCl, 5% glycerol, 0.2% NP-40, 1.5 mM MgCl2, 25 mM NaF, 1 mM Na3VO4 and protease inhibitors). After sonication, the cell debris was removed by centrifugation at 13200 rpm for 10 min at 4°C. We incubated the lysate with rabbit-IgG agarose (Sigma) at 4°C for 3 h. The bound proteins were washed with lysis buffer three times, and the agarose was then boiled in SDS sample buffer. The control for this experiment was prepared in the same manner. Mass Spectrometry and Data Analysis The mass spectrometric analysis of the purified samples was performed by LC-MS/MS using a Linear Ion Trap (LTQ) mass spectrometer (Thermo-Fisher Science, Bremen, Germany) equipped with a nanospray source and Eksigent high-performance liquid chromatography. The samples were analyzed by one-dimensional gel electrophoresis. The gel lanes were cut, sliced and subjected to in-gel digestion with trypsin. The LC separation was achieved by using a mobile phase from 1.95% ACN, 97.95% H2O, 0.1% FA (phase A) to 7


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79.95% ACN, 19.95% H2O, 0.1% FA (phase B), and the linear gradient was from 5% to 50% buffer B for 80 min at a flow rate of 300 nL/min. The LTQ mass spectrometer was operated in the data-dependent mode. A full scan survey MS experiment was performed (m/z range from 400 to 2000, automatic gain control target 5e5 ions, resolution 100,000 at 400 m/z), and the 5 most abundant ions detected in the full scan were analyzed by MS2 scan events (automatic gain control target 1e4 ions, maximum ion accumulation time 200 ms). The normalized collision energy was 35%. Additionally, the other samples were analyzed using a LTQ-Orbitrap-velos mass spectrometer (Thermo-Fisher Science) coupled with Nano Acquity ultra performance liquid chromatography. The samples were analyzed by one-dimensional SDS-PAGE. The gel was stained with Coomassie blue. The gel lanes were cut, sliced and subjected to in-gel digestion with trypsin. The LC separation was achieved using a mobile phase from 2% ACN, 98% H2O, 0.1% FA (phase A) to 100% ACN, 0.1% FA (phase B), and the linear gradient was from 5% to 50% buffer B for 70 min at a flow rate of 350 nL/min. The LTQ mass spectrometer was operated in the data-dependent mode. A survey MS scan was performed on Orbitrap with m/z range from 300 to 1600, automatic gain control target of 1e6 ions, and resolution of 30,000 at 400 m/z. The 20 most abundant ions detected in the full scan were analyzed by MS2 scan events (automatic gain control target 5e4 ions, maximum ion accumulation time 150 ms). The normalized collision energy was 35%. 8



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All MS/MS spectra were first searched against a combined Swiss-Prot database [IPI human v3.82 (target+decoy) with 184208 entries] using X!Tandem25 (the latest CYCLONE release) with the following parameters: mass tolerance of 0.4 Da for fragment ion matches, [RK]|P as cleavage sites, full cleavage with up to 2 missed cleavages, methionine oxidation (M+15.99491)

as

a

variable

modification

and

cysteine

carboxyamidomethylation (C+57.022) as a fixed modification. For the LTQ and Orbitrap Velos data, a mass tolerance of [-2,4] Da and 15 ppm for the parent ion matches, respectively, were used. The Trans Proteomic Pipeline software26 (revision 4.5) (Institute of Systems Biology, Seattle, WA, USA) was then utilized to filter the search results by running PeptideProphet27, iProphet28 and ProteinProphet29. The sequences of the proteins with a ProteinProphet probability of 0.9 or higher were extracted to a FASTA file, and another open-source software ChopNSpice30 was used to create a new FASTA file with the following parameters: spice species, H. sapiens; spice sequence, FAT10; spice site, KX; spice mode, once per fragment; inclusion of unmodified fragments in the output; enzyme, trypsin (Lys/Arg, do not cleave at Pro); up to 2 protein miscleavages allowed; up to 1 miscleavage in the “spice sequence” allowed; output formatting, FASTA (single protein sequence); all cleaved sites (“J”) were marked; and comments retained in the FASTA format without line breaks in the FASTA output. To identify the conjugated peptides and sites by FAT10, UblSearch was developed using the new FASTA file generated by 9


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ChopNSpice. For a given MS/MS spectrum, UblSearch would find all candidate peptides within a given mass tolerance, including the linear peptides (without the FAT10 fragment) and conjugated peptides (with the FAT10 fragment). For the conjugated peptides, the lysines in the sequence would be sequentially considered the conjugated sites of the FAT10 fragment, and the corresponding theoretical fragment ions would be generated from not only the modified peptides but also the FAT10 fragments (Supplementary Fig. S1). For the linear peptides, the theoretical fragment ions were the same as normal database searches. Then, the X!Tandem scoring scheme was used to find the peptide matching best with the given MS/MS spectrum and calculate the expectation value of the peptide identification. All MS/MS spectra were then searched against the new FASTA file using UblSearch with the following parameters: consider [X]|[J] to be the cleavage site, full cleavage with zero missed cleavages, and the other parameters were same as the above settings. The MS/MS spectra of the FATylated peptides with a X!Tandem expectation value less than 0.1 were inspected manually for identification. To reduce the false positive results, only the FATylated peptides with scores higher than the score of the linear peptides previously identified by X!Tandem for the same MS/MS spectra were considered candidates. The FATylated peptides with scores higher than the corresponding linear peptides by X!Tandem for the same MS/MS were also manually inspected, although their expectation values were not less than 0.1. 10



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Moreover, two batches of MS data that produced from LTQ-Orbitrap were analyzed by MaxQuant31 (v1.3.0.5). The logarithm (log10) of the LFQ (label-free quantification) intensities were used to determine the specific enriched FATylated proteins. We defined the quantitative value of the two test data as QE1 (LFQ intensity E1) and QE2 (LFQ intensity E2), and the control data

as

QC1

(LFQ

intensity

QC1=0&&(QE1>0||QE2>0);

C1).

(2)

For

a

given

protein,

if:

QC1>0&&((QE1=0&&QE2-QC1

(1) ≥

2)||(QE2=0&&QE1-QC1≥2)); or (3) QC1>0&&(QE1-QC1≥1&&QE2-QC1≥1), it was considered as FATylated target protein. All the other cases (QC1> 0) were identified as non-specific proteins. RESULTS Protein G-tagged FAT10 is Covalently Conjugated to Target Proteins Due to the lack of an appropriate antibody to enrich endogenous FAT10 conjugates and because FAT10 is immediately degraded by the proteasome10, we constructed a N-terminal protein G-tagged FAT10 that could improve the affinity rate effectively32. In addition, a FAT10∆GG mutant was constructed, with the carboxyl-terminal diglycine motif replaced by the amino acids AACV7. Then, we investigated whether protein G-FAT10 covalently conjugates to target proteins normally. Wild-type protein G-FAT10 was transiently expressed in HeLa cells (Fig. 1A). After stimulation with TNF-α and IFN-γ, FAT10 was immunoprecipitated and detected by western blotting (Fig. 1B, first lane). The results showed that wild-type FAT10 with the protein G tag apparently formed 11


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protein conjugates in the HeLa cells. In contrast, the FAT10∆GG mutant lost the ability to modify the substrates (Fig. 1B, second lane). The light smeared band in the FAT10∆GG lane might be ubiquitinated FAT10. To verify that possibility, we included the FAT10K0 (with all lysines mutated to alanines) mutant and FAT10∆GGK0 double mutant as controls (Fig. 1C). The results confirmed that FAT10∆GGK0 does not form protein conjugates in HeLa cells. The Optimal Conditions for FATylation in Stable HeLa Cells To facilitate the enrichment of the FAT10 conjugates, a stable HeLa cell line with protein G-tagged FAT10 was constructed. We cultured HeLa cells transfected with a vector that encodes protein G-tagged FAT10, followed by selection with G418. Finally, we obtained a HeLa cell line that stably expressed protein G-FAT10. At the same time, a HeLa cell line with protein G-FAT10∆GG was

constructed.

The

expression

of

protein

G-FAT10

and

protein

G-FAT10∆GG was determined by western blotting (Supplementary Fig. S2). Next, we verified that stably expressed protein G-FAT10 covalently conjugates to target proteins in HeLa cells. First, we confirmed that the endogenous FAT10 collects conjugates after induction with TNF-α and IFN-γ (Fig. 2A). Second, the lysates of the HeLa cells stably expressing protein G-FAT10 and the protein G-FAT10∆GG mutant were immunoprecipitated with rabbit-IgG agarose, and the immunoprecipitates were analyzed by western blotting with the anti-FAT10 antibody. The results showed that wild-type protein G-FAT10 is covalently conjugated to its target proteins whereas the protein G-FAT10∆GG 12



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mutant barely collects any conjugates (Fig. 2B). To determine the optimal conditions for FATylation, increased equal amounts of TNF-α and IFN-γ were added to induce the FAT10 activity. The concentration of 40 ng/ml for each TNF-α and IFN-γ was shown to be appropriate for FATylation (Fig. 2C). Then, we stimulated the HeLa cells with TNF-α and IFN-γ (40 ng/ml) for different times. The results showed that stimulation with cytokines for 24 hours was suitable for FATylation (Fig. 2D). Affinity Purification of FATylated Proteins The FAT10 conjugates were purified by immunoprecipitation with rabbit IgG using the aforementioned conditions in HeLa cells. To avoid the degradation of the FAT10 conjugates by the 26S proteasome, a proteasome inhibitor, MG132, was added. Approximately 1×108 HeLaproteinG-FAT10 cells were used for the immunoprecipitation assay. The FATylated proteins were detected by western blotting with the anti-FAT10 antibody using the cell lysate, purified section and flow-through section. The results showed that FATylated proteins are highly enriched after affinity purification (Fig. 3A, lane 2). The purified sample was run on SDS-PAGE (Fig. 3B) and stained with Coomassie blue. Then, the top part of the gel that contained the FAT10 conjugates was cut into slices, digested with sequencing-grade trypsin and analyzed by mass spectrometry. The affinity purification assay was repeated four times. Identification of FATylated Proteins and FATylated Sites by LC-MS/MS To obtain more comprehensive coverage of FATylated proteins, the purified 13


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samples were analyzed by LTQ-MS and LTQ Orbitrap Velos. The large number of background proteins (non-specific binders) that are co-captured during the purification of FATylated candidates is a well-documented phenomenon. Thus, we identified potential FATylated candidates by comparing the test with the control purification. First, the theoretical molecular weights of the identified proteins were compared with the experimental molecular weights on SDS-PAGE. Only those proteins that had a higher experimental molecular weight were considered potential FATylated substrates. In total, 190 proteins (with a ProteinProphet probability of 0.9 or higher, corresponding to a false positive rate of 1%) were identified in more than two of the four purification assays using the X!Tandem search engine. Third, two batches of the MS data that produced from LTQ-Orbitrap were analyzed by MaxQuant using a label-free quantification method. Finally, 15 non-target proteins were removed, and the remaining 175 target proteins were identified as potential FATylated proteins (Supplementary Table S1 and S2). Among the potential FATylated proteins, twenty proteins only from LTQ-MS were not analyzed by MaxQuant. An isopeptide bond is formed between the C-terminal carboxyl group of FAT10 and the ε-amino group of a lysine residue within the substrate protein 7. A FAT10 fragment with 13 amino acids will remain on the target peptide after trypsin digestion. The large residual FAT10 peptide makes it difficult to find the modified site with common search engines. To unbiasedly identify the FAT10-modified sites, a ChopNSpice30 strategy was used in combination with 14



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UblSearch,

which

was

developed

based

on

Page 16 of 39

X!Tandem

to

identify

ubiquitin-like-conjugated sites. The FAT10 peptide fragment was considered as a variable modification of lysines. A total of 13 FATylated sites were identified (Table 1), including 11 FAT10 conjugated sites that were identified unambiguously (Fig. 4 and Supplementary Fig. S3). All 13 identified proteins only have one FATylated site, and six of these proteins were identified in only one of the four repeated assays. Interestingly, unlike the consensus sequence (ψKxE/D, with ψ being a hydrophobic amino acid and x being any amino acid) of SUMO, the examination of a six-amino-acid window adjacent to the modified lysines revealed that the modified lysines had a slight tendency to be localized in regions enriched in hydrophilic residues, such as Glu, Lys and Asp (Supplementary Fig. S4). Consistent with the SUMO consensus sequence33, 34, the second amino acid adjacent to the modified lysine was often Glu or Asp. Surprisingly, the third and fourth amino acids after the modified site also had a high likelihood of being Glu or Asp. Whether these conserved amino acids represent a common rule remains to be resolved by finding more FATylated sites. A sequence logo was provided to clearly show the potential preservation at the FATylation site (Supplementary Fig. S4). This potential preservation remains to be confirmed by finding more FATylated sites. Validation of FATylated Proteins by Immunoprecipitation Assay To validate the FATylated results of the proteomics method, we randomly selected a subset of three proteins (hnRNP C2, PCNA and PDIA3) identified 15


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by MS to analyze their FATylation by co-immunoprecipitation assays. The Flag-tagged proteins were transfected into HEK293 cells with Myc-tagged FAT10. The cell lysates were immunoprecipitated with a Flag antibody and detected by western blotting using an anti-Myc antibody. First, a protein with a FATylated site, hnRNP C2 (heterogeneous nuclear ribonucleoprotein proteins), was validated. We showed that FAT10 covalently modified hnRNP C2 (Fig. 5A). A band at 65 kDa appeared with the co-transfection of Flag-hnRNP C2 and Myc-FAT10 but not in the control lane, which indicates that a single FAT10 forms conjugates with hnRNP C2. To confirm that FAT10 directly modifies hnRNP C2, rather than the modification being mediated by ubiquitin or other Ubls, a FAT10∆GG mutant was included as a negative control. As expected, FAT10∆GG did not form conjugates with hnRNP C2 (Fig. 5B). Then, we chose two proteins (PCNA, proliferating cell nuclear antigen; PDIA3, protein disulfide isomerase A3) from the potential FATylated candidates. Both proteins were shown to be covalently modified by FAT10 after stimulation with TNF-α and IFN-γ (Fig. 5C and 5E). Moreover, we confirmed that endogenous PCNA is FATylated in HeLa cells (Fig. 5D). In contrast, FAT10∆GG lost the ability to form conjugates with PDIA3 (Fig. 5E). A known substrate of FAT10, p62, was included

as

a

positive

control,

and

similar

results

were

obtained

(Supplementary Fig. S5). Interestingly, only conjugates with a monomer of FAT10 were detected for all three proteins. These validation results also confirmed that only a small fraction of the total protein was covalently modified 16



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by FAT10. Functional Distribution of FATylated Candidates The FATylated candidates participate in various biological processes, with the largest number involving translation, protein folding, RNA processing and macromolecular complex assembly. For example, CCT (chaperonin containing TCP-1) is a large complex that assists in the folding of proteins. We identified six subunits of the CCT complex as FATylated candidates: CCT1, CCT4, CCT5, CCT6A, CCT7 and CCT8. The coatomer is a cytosolic protein complex that reversibly associates with Golgi non-clathrin-coated vesicles, which mediate biosynthetic protein transport from the ER via the Golgi up to the trans-Golgi network35. Three subunits (COPA, COPB1 and COPG) of the coatomer were identified as FATylated candidates. Consistent with previous reports of the function of FAT107,

36

, many apoptosis-related proteins were identified as

candidates, such as PDCD6IP (programmed cell death 6 interacting protein) and annexin A1. Further studies on the relationships among these proteins will improve the understanding of the role of FAT10 during the cell death process. The identification of the components of the ubiquitination enzyme cascades and 26S proteasome as candidate FATylated proteins further supports the idea that the FATylation of proteins is associated with proteasome-mediated protein degradation. UBA1 (ubiquitin-like modifier-activating enzyme 1, also named as UBE1), which has been reported previously as a substrate of FAT1022, was identified as a candidate. The ubiquitin-protein ligase, TRIM21, mediates the 17


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intracellular antibody-mediated proteolysis pathway37. Whether the FATylation of TRIM21 affects this process deserves further study. Interestingly, a SUMO-protein ligase, RanBP2, was found to be FATylated. Whether it is degraded by FATylation or functions as an E3 ligase of FAT10 remains to be determined. Ubiquitin was also repeatedly identified, which may due to the covalent

modification

of

FAT10

by

ubiquitin.

Moreover,

the

proteasome-associated protein ECM29 and 26S proteasome non-ATPase regulatory subunit 2 (PSMD2) were found to be FATylated and might be related to FATylation. DISCUSSION The ubiquitin-like protein FAT10 was discovered in 1996 by chromosomal sequencing of the human HLA-F locus8. This ubiquitin-like-protein has a number of interesting features, such as expression in mature B cells and dendritic cells, synergistic induction by the pro-inflammatory cytokines TNF-α and IFN-γ, modification of substrates and the ability to induce apoptosis. However, we still know little about the functional roles of FAT10. An in-depth understanding of the roles of FAT10 requires the identification of its substrates and their FATylated sites. To date, only a few substrates of FAT10 have been reported, such as UBA1, USE1, p53, p62 and huntingtin. However, no unambiguous FATylated sites have been identified for those proteins. In this study, we constructed stable HeLa cell lines that express protein G-tagged FAT10, which facilitates the process of purifying FAT10-modified 18



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proteins. Although the exogenous stable expression of protein G-FAT10 partially induces the apoptosis of the HeLa cells, the FATylated proteins are significantly enriched by affinity purification. The FAT10 conjugates were subsequently identified by mass spectrometry. The MS-based methods have been successfully used to identify the substrates and sites of many types of post-translational modifications, such as phosphorylation, ubiquitination and SUMOylation. However, few proteins modified by FAT10 have been identified, which may be due to the lack of arginine or lysine in the proximity of the carboxyl terminus of FAT10, which leads to a large residual peptide left on the substrates after trypsin digestion and complicated fragmentation spectra. To identify the sites modified by FAT10, UblSearch was developed based on the X!Tandem software using the new FASTA file created by the improved ChopNSpice30. Finally, 13 reliable FATylation sites were identified through manual verification of the mass spectra. To analyze whether a common rule exists around the FATylated sites, six amino acids were analyzed up- or downstream of the FATylated lysine. We found that hydrophilic amino acids, glutamic acid or lysine, have a high frequency upstream of the modified lysine. Arginine or lysine often occur downstream of the modified lysine. The second amino acid after the modified lysine is often glutamic acid or asparagine, which is similar to the SUMO consensus sequence. These results indicate that a conserved FAT10 consensus sequence may be confirmed by identifying more FATylated 19


Page 20 of 39

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peptides. Our work identified 175 potential FATylated proteins with various biological functions.

The

reliability

of

this

dataset

was

validated

using

immunoprecipitation assays of FATylated proteins. We confirmed that three proteins were covalently modified by FAT10 after stimulation with TNF-α and INF-γ (Fig. 5), which indicates that the dataset of FATylated proteins has a high confidence level. The FATylated candidates are involved in a variety of functions,

including

translation,

protein

folding,

RNA

processing,

macromolecular complex assembly and apoptosis. The functional roles of the FATylation of these proteins need further study. For example, we identified thirty proteins involved in RNA processing, in which hnRNPs bind pre-mRNA and nucleate the assembly of 40S hnRNP particles. hnRNP C2 was confirmed as a substrate of FAT10 (Fig. 5A). Therefore, the FATylation of these proteins might play a role in the early steps of spliceosome assembly and pre-mRNA splicing. We also observed that many of the substrates were not specific to FATylation. For example, Myosin-9 is also modified by ISG1538. Both ISGylation and FATylation need IFN induction, which suggests that different types of post-translational modifications might cooperate with each other. The SUMOylation of hnRNPs has been reported to decrease their binding to nucleic acids39. PCNA has multiple functions in DNA replication, repair and chromatin

remodeling40,

which

are

regulated

by

20


ubiquitination

and


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Page 22 of 39

SUMOylation41. Interestingly, few histone proteins, which are reported to be modified

by

methylation,

acetylation,

phosphorylation,

ubiquitination,

SUMOylation, ISGylation, citrullination and ADP-ribosylation42,

43

, were

identified as FAT10 candidates. Whether the FATylation of histones has a function in the epigenetic regulation of gene expression remains to be answered. Furthermore, the identification of the components of the ubiquitination or Ubls

enzyme

cascades

supports

FATylation

being

related

to

proteasome-mediated protein degradation. We identified a reported substrate of FAT10, UBA1 (E1), but the exact site was not found. Two E3 ligases of ubiquitination or SUMOylation, TRIM21 and RanBP2, may participate in the FATylation process. These results are consistent with the known functions of FAT10. However, it should be cautioned that these molecules were identified in FAT10-overexpression cells. Further studies are needed to confirm their native modifications and biological functions. In summary, we performed a proteome-scale purification of FAT10-modified targets that should provide useful insights into the molecular mechanisms and cellular functions of FATylation. ASSOCIATED CONTENT Supporting Information Table S1 shows the list of potential FATylated proteins. Table S2 shows the list of proteins with quantitative values by MaxQuant. Figure S1-S5 show the 21


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theoretical fragment pattern of the FAT10- conjugated peptide, the expression of the proteins G-FAT10 and protein G-FAT10-∆GG in stable HeLa cell lines, the MS/MS spectra of FAT10-conjugated peptides and sites, the six amino acid window adjacent to the FATylated sites and a co-immunoprecipitationed assay of FAT10 modifying p62. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *J.

W.:

e-mail,

[email protected];

phone,

+86-10-80705118;

fax,

+86-10-80705155. F.H.: e-mail, [email protected]; phone, +86-10-68171208; fax, +86-10-80705155. Author Contributions §These authors contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We thank Dr. Marcus Groettrup for the FAT10K0 expression construct. This work was supported in part by the Special Funds for Major State Basic Research of China (2011CB910600), the National High-Tech Research and Development

Program

(2012AA020201),

the

National

International

Cooperation Project (2011DFB30370), Beijing Municipal Natural Science Foundation (5122014) and Beijing NOVA program (2011014). 22



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Page 24 of 39

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29


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Figure Legends Figure 1. Protein G-FAT10 covalently modifies its substrates. (A) HeLa cells were transfected with protein G-tagged wild-type FAT10 or FAT10 mutants. The cell lysates were detected by western blotting with the anti-FAT10 antibody. (B-C) HeLa cells were transfected with protein G-tagged wild-type FAT10 or FAT10 mutants as indicated and stimulated with TNF-α and IFN-γ. MG132, a proteasome inhibitor, was

added to avoid the degradation of

the

FAT10-modified proteins. The cell lysates were immunoprecipitated with rabbit-IgG agarose and detected by western blotting with the anti-FAT10 antibody. -, the control with wild-type FAT10 transfection but without stimulation with TNF-α and IFN-γ; WT, wild-type FAT10; ∆GG, FAT10∆GG; K0, FAT10K0; ∆GGK0, FAT10∆GGK0. The total cell lysate was loaded on 12% SDS-PAGE and analyzed by western blot. The immunoblots were repeated at least three times. Figure 2. The optimal conditions for FAT10 modification. (A) Endogenous FAT10 and its conjugates in the lysates of the cells were immunoprecipitated with the FAT10 antibody and detected by western blotting with the FAT10 antibody. (B) ProteinG-FAT10 conjugates were immunoprecipitated with rabbit-IgG agarose and detected by western blotting with the anti-FAT10 antibody. (C) Stable HeLa cells expressing the protein G-FAT10 were stimulated with equal amounts of TNF-α and IFN-γ (0, 20, 40, 80 ng/ml) for 24 h to induce the FAT10 conjugates. The FAT10 conjugates were pulled down by rabbit IgG and detected with the FAT10 antibody. (D) Stable HeLa cells 30



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expressing the protein G-FAT10 were stimulated with equal amounts (40 ng/ml for each of the cytokines) of TNF-α and IFN-γ for 0, 12, 24, and 36 hours to induce the FAT10 conjugates. The FAT10 conjugates were pulled down by rabbit IgG and detected by the FAT10 antibody. The immunoblots were repeated three times. Figure 3. Affinity purification of the FAT10-modified proteins. (A) The samples were resolved by SDS-PAGE and probed with the anti-FAT10 antibody. WCL, whole cell lysate; Purification, the affinity purified FAT10 conjugates; Flow-through, the protein sample from the flow-through. (B) The SDS-PAGE gel of the FAT10-modified proteins. The gel was visualized by Coomassie blue staining. The affinity purification assay was repeated four times. Figure 4. MS/MS spectra of FAT10-conjugated peptides and sites. The peptides were identified by searching the database of modified sequences (generated by ChopNSpice) with UblSearch. In addition, the b/y ions on both sides of lysine were evaluated to confirm the conjugated sites. In (A), (B), (C), both the peptide sequence and the conjugated sites were confirmed. In (D), only the peptide sequence was confirmed as the conjugated sites could not be confirmed unambiguously. Figure 5. Validation of FAT10 candidates by a co-immunoprecipitation assay. (A, B, C and E), the HEK293 cells were transfected with the indicated plasmids and treated with TNF-α, IFN-γ and MG132. After 24 h, the cells were collected. The Flag antibody was used to immunoprecipitated hnRNP C2, PCNA, and 31


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PDIA3. The immunoprecipitates were then probed with the anti-Myc-HRP antibody. D, the HeLa cells were induced by TNF-α and IFN-γ. A proteasome inhibitor, MG132, was added to the culture. After 24 h, the cells were collected and immunoprecipitated with the anti-FAT10 antibody and IgG control. The immunoblots were repeated three times.

32



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Page 34 of 39

Table 1. Fat10-conjugated Peptides and Sites Iidentified by MS/MS IPI accession Protein Name

Peptide sequence

Residue

Mass accuracy

modified

(Da/ppm)

Score

Expect

Charge

number

value

IPI00009841.6

RNA-binding protein EWS isoform 1

(R)K&KPPMNSMRGGLPPR(E)

3

K452

1.866

31.3

1.20E-02

IPI00420014.2

Isoform 1 of U5 small nuclear

(R)DILCGAADEVLAVLKNEK&LR(D)

3

K147

2.309

28.4

2.30E-02

(K)LK&GDDLQAIKK(E)

3

K176

3.204

26.8

5.60E-01

ribonucleoprotein 200 kDa helicase

IPI00477313.3

Isoform C2 of Heterogeneous

nuclear ribonucleoproteins

IPI00221091.9

40S ribosomal protein S15a

(K)SINNAEK&R(G)

3

K19

7*

24.3

1.30E-01

IPI00221325.3

E3 SUMO-protein ligase RanBP2

(K)SNNSETSSVAQSGSESKVEPKK(C)

3

—

3.455

29.6

1.70E-02

IPI00295851.4

Coatomer subunit beta

(R)SLGEIPIVESEIK&K(E)

3

K494

-12*

32.5

4.50E-03

IPI00604620.3

Nucleolin

(K)EALNSCNK&REIEGR(A)

3

K545

-6*

32.5

4.90E-02

IPI00743335.3

Isoform 1 of Myosin-Ic

(R)ELCIKNMVWK&YCR(S)

3

K813

0.146

40.8

5.10E-04

IPI00019502.3

Isoform 1 of Myosin-9

(R)RNAEQYK&DQADK(A)

3

K1862

-1.573

28.6

2.60E-02

IPI00220573.4

Myosin regulatory light chain 12A

(R)FTDEEVDELYREAPIDKK(G)

3

—

0.744

23.2

5.00E-02

IPI00001734.3

Phosphoserine aminotransferase

(R)IGNAKGDDALEK&R(F)

3

K363

-1.143

35.8

1.80E-02

IPI00027442.4

Alanyl-tRNA synthetase,

(K)K&AEEIANEMIEAAKAVYTQDCPLAAAK(A)

3

K651

1.851

32

4.60E-02

IPI00844215.1

Isoform 1 of Spectrin alpha chain

(K)K&FDDFQK(D)

3

K1132

-1.377

26.5

3.90E-02

&, Fat10-conjugated sites; —, the conjugated site is not unambiguously identified; *, ppm, the results were from LTQ-Orbitrap,

and the other results were from LTQ-MS.

33


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Graphical Abstract

34



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Figure 1 24x16mm (600 x 600 DPI)


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Figure 2 177x201mm (300 x 300 DPI)



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Figure 5 105x65mm (300 x 300 DPI)


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A Proteomics Strategy for the Identification of FAT10-Modified Sites

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