Characterization of Protein Serotonylation via Bioorthogonal Labeling

Jul 23, 2014 - ABSTRACT: Protein serotonylation is a transglutaminase-mediated ... the complete profiling of serotonylation targets in a proteome rema...
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Characterization of Protein Serotonylation via Bioorthogonal Labeling and Enrichment Jason Ching-Yao Lin,†,§,∥,# Chi-Chi Chou,†,‡,# Zhijay Tu,† Lun-Fu Yeh,† Shang-Chuen Wu,†,⊥ Kay-Hooi Khoo,†,‡,§,⊥ and Chun-Hung Lin*,†,§,⊥ †

Institute of Biological Chemistry, ‡Core Facilities for Protein Structural Analysis, and §Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, 128 Academia Road Section 2, Taipei 11529, Taiwan ∥ Department of Chemistry, National Tsing Hua University, 101 Kuang-Fu Road Section 2, Hsinchu 30013, Taiwan ⊥ Institute of Biochemical Sciences, National Taiwan University, 1 Roosevelt Road Section 4, Taipei 10617, Taiwan S Supporting Information *

ABSTRACT: Protein serotonylation is a transglutaminase-mediated phenomenon whose biological mechanism of protein serotonylation is not yet fully understood, as the complete profiling of serotonylation targets in a proteome remains a critical challenge to date. Utilizing an alkyne-functionalized serotonin derivative bioorthogonally coupled to a cleavable linker, we developed a method to selectively enrich serotonylated proteins in a complex sample. With online nanoflow liquid chromatography and LTQ-Orbitrap Velos hybrid mass spectrometer detection, we identified 46 proteins with 50 serotonylation sites at their glutamine residues. Mass spectrometric analysis also generated direct residue-level evidence of various biological processes such as transglutaminase-chaperon interactions as well as actin assembly. An enrichment workflow utilizing click chemistry and on-bead digestion allowed us to achieve sitespecific identification of protein serotonylation by mass spectrometry, and results obtained hereby also provided a great foundation in the elucidation of the true roles of protein serotonylation in biological systems. KEYWORDS: Click chemistry, serotonylation, protein modifications, proteomics



INTRODUCTION Protein serotonylation, the transamidation of 5-HT onto glutamine residues, is a phenomenon in a large class of posttranslational modifications in which transglutaminase conjugates various biogenic monoamines to initiate signaling and activations. Since the discovery that small GTPases such as Rho and Rab were potentially serotonylated,1 other candidates such as actin and fibronectin as well as filamin A and myosin have also been investigated.2,3 However, the exact biological role remains only partially clarified despite the belief that a significant number of biological pathways are potentially linked to protein serotonylation.4 Aside from one reported case of directly using biotinylated 5-HT to identify serotonylated proteins,2 most studies relied on antibodies or radiolabeled 5HT for protein-level characterization. The complete profiling of serotonylation targets in a proteome remains a critical challenge to-date. The ubiquitous and wide availability of biogenic monoamines in nature often lead to the generation of ineffective antibodies on which most rely to enrich transamidated proteins. We recently propargylated the 5-OH moiety of serotonin to generate a serotonin derivative, 5-PT, which could be functionalized with various reporter groups via click chemistry to investigate protein serotonylation.5 We sought to enrich modified proteins to attempt to elucidate the mechanistic nature of protein serotonylation. The presence of © 2014 American Chemical Society

biotin, however, may lead to ionization difficulties during mass spectrometry, and thus we considered the incorporation of an intramolecular disulfide bond to be cleaved with reducing agents such as 2-ME. Bioorthogonal conjugations to isolate and characterize proteins have been widely used, most notably with activity-based probes for the purpose of enzymatic profiling or proteomic analyses.6−9 Such a construct allowed the enrichment of serotonylated peptides or proteins from cell lysates, providing for the first time a proteome-wide characterization of targets of TGase 2-mediated protein serotonylation.



EXPERIMENTAL SECTION

Peptide Level Enrichment

Hras 57−68 (DTAGQEEYSAMR) was transamidated in vitro by 2 mU liver TGase 2 (Sigma) in saline-buffered activity solution of 25 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol, 10 mM CaCl2, pH 7.4 in the presence of 1 mM 5-PT overnight at 37 °C. Samples were then vacuum-dried and desalted with C18 ZipTip (Millipore). Serotonylated peptides were clicked with 1 mM TBTA, 0.5 mM BiotinCTA-N3 (Figure 1a) in DMSO, 0.3 mM CuSO4, 3 mM sodium Received: April 3, 2014 Published: July 23, 2014 3523

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Figure 1. Design of a cleavable bioorthogonal system to probe protein serotonylation. (a) Simplified synthetic scheme of Biotin-CTA-N3, the disulfide linker. (b) TGase 2 cross-links 5-PT onto glutamine residues and creates an adduct giving off ammonia as a side product. Glutamine resides that are serotonylated by 5-PT can be conjugated with azide-functionalized biotinyl linkers via Huisgen cycloaddition, producing a stable triazole ring. When a cleavable moiety is integrated into the linker, for example, a disulfide structure, the biotin moiety can be removed to elute the desired proteins or peptides. (c) Upon alkylation of free cysteines in SW480 lysates by iodoacetamide, the orthogonally conjugated biotin moiety (0.1 mM) can be specifically attached to proteins serotonylated by 5-PT (1 mM) and cleanly reduced by 2-ME (2.5%).

DPBS, and 10% ethanol in DPBS, three additional times in DPBS, followed by once in 25 mM ammonium bicarbonate, pH 8.5. On-bead digestion proceeded with 80 μg of sequencinggrade modified trypsin (Promega) overnight in 25 mM ammonium bicarbonate, pH 8.5. To analyze efficiency, we collected and reduced the flowthrough with 10% 2-ME for 45 °C for 100 min to analyze efficiency. The precipitate was washed three times with DPBS and 30% acetonitrile in DPBS and three additional times in water before elution with 10% 2ME at 45 °C for 100 min and collection of the supernatant after centrifugation for mass spectrometric analysis.

ascorbate in DMSO for 1 h prior to enrichment by streptavidinconjugated magnetic beads (GE Healthcare) overnight at 4 °C. After washing with water, serotonylated peptides were eluted by incubation in 10% 2-ME for 0.25 h for mass spectrometric analysis. Enrichment of Serotonylated Proteins

SW480 cultures were prepared and lysed as described in Supporting Information. Lysates (1.4 mg of total proteins) were serotonylated by the addition of TGase 2 at the ratio of 20:1 (lysate:TGase), 2 mM 5-PT in transglutaminase activity buffer at 25 °C for 4 h. Without reduction, proteins were alkylated by 200 mM iodoacetamide for 1 h. To test whether the use of detergents would improve enrichment efficiency, we incubated samples with 400 mM urea for 1 h for denaturation. After desalting with Zeba spin columns (Thermo), samples were then clicked with 1 mM sodium ascorbate, 0.3 mM CuSO4, and 0.25 mM Biotin-CTA-N3 for 3 h and again desalted prior to enrichment overnight with 1:1 high-performance streptavidin Sepharose (GE Healthcare) at 4 °C. After centrifugation, the beads were washed with a succession of 1% SDS in Dulbecco’s PBS (DPBS, Cellgro), 4 M urea in DPBS,

Mass Spectrometric Analysis

Samples were reconstituted in 5% acetonitrile and 0.1% formic acid and loaded onto a C18 column of 75-μm × 250 mm (nanoACQUITY UPLC BEH130, Waters). The peptides mixtures were separated by online nanoflow liquid chromatography using nanoAcquity system (Waters) with a linear gradient of acetonitrile from 5−50% and 0.1% formic acid in 95 min, followed by 85% acetonitrile in 1 min and held for another 15 min at a constant flow rate of 300 nL min−1. Peptides were detected in an LTQ-Orbitrap Velos hybrid mass 3524

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Figure 2. Proteomic workflow utilizing bioorthogonal conjugation to characterize protein serotonylation. Serotonylated proteins from SW480 lysate can be selectively enriched and identified by MS/MS after iodoacetamide alkylation, desalting, and cycloaddition reactions with the disulfide linker and precipitated with streptavidin. The hits were then digested on-bead, leaving peptides conjugated with biotin immobilized on the resin after a series of washes. An elution step in the presence of 2-mercaptoethanol then cleaves serotonylated peptides from the solid support, availing such samples for spectrometric analysis.

use of mild reducing agents such as 2-ME. When coupled to a propargylated serotonin derivative, elution gave two possible products (Figure 1b) with unique precursor signatures. To verify the stability of intramolecular disulfides with Biotin-CTAN3, we performed serotonylation reactions using SW480 in vitro and used immunoblotting to confirm the cleavability of intramolecular disulfides with reducing SDS-PAGE and immunoblotting (Figure 1c). One can observe that upon the addition of 2-ME, bands indicative of biotinylation diminished to near background levels. The presence of unique mass signatures from bioorthogonal conjugation combined with a cleavable biotin linker alleviated issues of nonspecific binding and endogenous biotinylated proteins appearing as falsepositives in mass spectrometric profiling. Starting then from a reference peptide, Hras 57−68, we sought to determine the possible ion fragmentation under MALDI- and ESI-MS/MS after linker conjugation. To do so, Hras 57−68 after serotonylation was desalted, conjugated with Biotin-CTA-N3, affinity-purified by streptavidin, and reduced via 2-ME. Results indicate a net addition of 376.1 u onto Gln 61 under MALDI-MS/MS (Figure S1 in the Supporting Information), while the dominant precursor ion in the elutant under MALDI-MS was the 2-ME-eluted standard peptide at m/ z 1658.38. The relative incorporation of efficiency of 5-PT onto the reference peptide was ∼70%, while the conjugation of Biotin-CTA-N3 postelution by 2-ME was essentially near completion, as observed by MALDI-MS (Figure S2a in the Supporting Information). We also observed a recovery rate of 52% by comparing relative intensities of eluted peptide postelution (Figure S2b in the Supporting Information). We then utilized Biotin-CTA-N3 to profile serotonylated proteins in SW480 (a human colorectal adenocarcinoma cell line) lysate to understand the potential biological connections between serotonin linked to signaling and their effectors. Because detergents might adversely affect the efficiency of click chemistry10 and methanol/acetone precipitation techniques resulted in generally lower protein recoveries, we devised an enrichment strategy that relied on size-exclusion exchange resins for desalting and opted for click reactions under nondenaturing conditions (Figure 2). The sample after serotonylation was first carbamidomethylated by iodoacetamide and desalted before linker conjugation. A second desalting step

spectrometer (Thermo Scientific) using a data-dependent CID Top20 method in positive ionization mode. For each cycle, fullscan MS spectra (m/z 300−2000) were acquired in the Orbitrap at 60 000 resolution (at m/z 400) after accumulation to a target intensity value of 5 × 106 ions in the linear ion trap. The 20 most intense ions with charge states ≥2 were sequentially isolated to a target value of 10 000 ions within a maximum injection time of 100 ms and fragmented in the highpressure linear ion trap by low-energy CID with normalized collision energy of 35%. The resulting fragment ions were scanned out in the low-pressure ion trap at the normal scan rate and recorded with the secondary electron multipliers. Ion selection threshold was 500 counts for MS/MS, and the selected ions were excluded from further analysis for 20 s. An activation q = 0.25 and activation time of 10 ms were used. Standard mass spectrometric conditions for all experiments were: spray voltage, 1.8 kV; no sheath and auxiliary gas flow; heated capillary temperature, 200 °C; predictive automatic gain control (AGC) enabled; and an S-lens RF level of 69%. MS and MS/MS raw data were processed with Proteome Discoverer version 1.4 (Thermo Scientific), and the peptides were identified from the MS/MS data searched against the UniProtKB/Swiss-Prot database using the Mascot search engine 2.4.1 (Matrix Science). Search criteria used were as follows: trypsin digestion; considered variable modifications of glutamine serotonylation (+300.10448 Da), 2-ME reduced form of glutamine serotonylation (+376.10277 Da), methionine oxidation (+15.9949 Da), and cysteine carboxyamidomethylation (+57.0214 Da); up to three missed cleavages were allowed; and mass accuracy of 10 ppm for the parent ion and 0.6 Da for the fragment ions. The significant peptide hits defined as peptide score must be higher than Mascot significance threshold (p < 0.05) and therefore considered highly reliable, and manual interpretation confirmed agreement between spectra and peptide sequence.



RESULTS AND DISCUSSION We synthesized a short linker Biotin-CTA-N3 (Figure 1a) by functionalizing the terminal amines of cystamine with azide and biotin. Disulfide exchanges can be effectively minimized with proper alkylation of free thiols prior to conjugation and relatively stable until the elution stage, which required only the 3525

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Table 1. Subset of Identified Proteins and Sites of Serotonylation As Well As Selected References of Protein−TGase Interactions and Associationsa protein name

peptide sequence

sites

14−3−3 protein zeta/delta

LAEQAER SVTEQGAELSNEER NPFDTFFGQR KTETQEK ETIEQEK VELQELNDR QTVSWAVTPK DVNQQEFVR DSYVGDEAQSK NEGSESAPEGQAQQR

Q15 Q32 Q118 Q24 Q37 Q108 Q854 Q12 Q59 Q183

heat shock protein 40 thymosin beta-4 vimentin alpha-2-macroglobulin 40S ribosomal protein S19 actin Y-box transcription factor a

references 21 22, 23 24 25 26 27 2, 28 29

Modified glutamine residues are bolded. A full table of all proteins can be found in Table S1 in the Supporting Information.

folding and assembly of proteins in the endoplasmic reticulum.14 Another process previously implicated was the assembly of actin, which utilized Radixin in the process. Interestingly, Radixin also interacted with GNA13, which in itself had been shown to interact with Rho guanine nucleotide exchange factors 1;15,16 coupled to the evidence that RanBP-1 was also serotonylated, one might conclude with confidence that members of the small GTPase family and their interacting partners were indeed modulated differentially by TGase 2, as previously hypothesized. Interestingly, 14−3−3 Zeta (YWHAZ), which regulates Raf-1 and Akt signaling, was also found to be serotonylated. In addition, we also identified several proteins very closely related to those previously reported, such as myosin light chain and prolamin. Transglutaminase and its possible interactions with heat shock proteins such as Hsp70, Hsp20, and Hsp27 had been and demonstrated on a cellular level,17−19 and our results here further supported previous findings with direct residue-level evidence that upon signaling events triggered by the influx of serotonin, various heat-shock proteins were targeted for sustained activation to alleviate stresses exerted during potential apoptotic risks. To visualize the possible pathways affected by protein serotonylation, we performed a STRING-search20 to generate a network map linking our protein hits together. Herein we noticed a central node, Ran, interconnecting several clusters of interacting proteins at nodes YWHAZ, PCNA, and RANBP1 across key functions such as transcription and translation, transport, cytoskeletal functions, and responses to stress as well as apoptosis. There is also overlapping association of cellular functions among those closely associated with apoptosis and stress responses from vimentin through plectin (PLEC), ribosomal protein S19 (RPS19), and translation initiating factor 4H (EIF4H) downstream. The inclusion of stressinduced phosphorprotein 1 (STIP1), an important element in mediating interactions with several heat shock proteins, and Hsp40 in the same cluster reaffirms the role of heat shock protein serotonylation by TGase 2 as an action in facilitating cellular responses to stress. Transglutaminases had been found to have a wide range of substrate specificity, and any irregularity in amine donors, for example, rapid influx of an unintended signal, could potentially cause a drastic and possibly unintentional shift in equilibrium. Current findings suggested that upon the influx of calcium and various biogenic monoamines through ion channels and monoamine transporters, tissue transglutaminase would activate to initiate signaling cascades throughout the cell,4 a phenomenon collectively known as

removed unreacted linkers to maximize recovery yields during enrichment. Washes with mild denaturants as suggested11 removed nonspecific binding and left only biotinylated proteins on-bead. Digestion and additional washes further removed unbound and nonserotonylated peptides, availing uniquely modified peptides for elution with 2-ME. Utilizing on-bead digestion, we were able to identify 62 unique peptides with 58 peptides assigned to existing proteins as well as 50 modified sites on 46 proteins (Table 1 and S1). The use of 2-ME as the elution agent for the Biotin-CTA-N3-linked peptides generated two possible mass increments on precursor ions of serotonylated glutamines, m/z 300.1 and m/z 376.1, for the thiolate and 2-ME-linked glutamine, respectively. We observed an approximately even distribution of both variants in our experiments, with some peptides exhibiting both products. Other reducing agents such as dithiothreitol, which produced a single thiolated product ion in our experimentation (data not shown), or tris(2carboxyethyl)phosphine could also be used in place of 2-ME, although further optimizations and experimental steps such as a subsequent alkylation stage may be required. Additionally, harsher eluting conditions such as a makeup of 0.1% trifluoroacetic acid, 10% 2-ME, and 30% acetonitrile did not drastically improve the number of matches. For this application, CID was preferred over HCD for its scan rate and the more sensitive identification of modified peptides than HCD (Tables S2 and S3 and Figure S3 in the Supporting Information). We attempted a similar experiment to characterize protein serotonylation in vivo by first stimulating SW480 cultures with 5-PT and calcium chloride. Enrichment results included peptides with identical modification sites as some of those observed in vitro, but the number of peptides was significantly fewer. An example of this is DNA polymerase delta catalytic subunit, which was found to be serotonylated at Q1062 both in vitro and in vivo (data not shown). These mass shifts provided unique identification signatures for site-specific identification, a positive feature difficult to achieve with antibody-based enrichment methods. Among the possible candidates, actin, for instance, was previously identified as a target of protein serotonylation. TGase 2 appears to have a preference for nonpolar and aliphatic regions for glutamine serotonylation (Figure S4 in the Supporting Information). In addition, among the hits, we observed several proteins belonging or associating with the heat-shock protein family. PSMD9, for instance, acts as a chaperon during the assembly of the 26S proteasome. DNAJB1, or Hsp40, has also been shown to interact with STUB112 and HSPA4;13 GRP78 (alternatively, HspA5) is involved in the 3526

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Figure 3. General implication of protein serotonylation. (a) MS/MS spectra of tryptic peptides ANYDVLESQK from the protein 26S proteasome non-ATPase regulatory subunit 9 (PSMD9, above) and DSYVGDEAQSK from actin (below), which afforded [M + H]+ precursor ions at m/z 1542.7 and 1498.6, respectively. Underlined serotonylated residue is underlined. Filled circles above the labeled ions indicate serotonylated product ions with a mass increment of m/z 376.1 in the presence of 2-ME or m/z 300.1 without 2-ME. Open circles (○) and asterisks (*) denote product ions carrying additional neutral loss of H2O and NH3, respectively. (b) Network view of interactions among hits found to be serotonylated, with line between nodes, indicating the existence of association. Nodes labeled in orange were serotonylated proteins. Associations suggest that such a mechanism may be directly responsible for prolonging critical activations of these processes. (c) Schematic illustration of the “hormone effect” theory. Serotonin is transported into cells via two possible routes, coupled receptors or transport channels. Transports through channels effectively enrich the amount of intracellular serotonin. Activated TGase 2 then cross-links protein targets with the signaling molecule covalently, effectively prolonging the signaling process. The activated processes then proceed normally under a sustained fashion, resulting in longer signaling effects. Findings here suggest a similar phenomenon may occur with heat shock proteins.

“the hormone effect” (Figure 3c). Such facilitated transports were thought to promote longer lasting effects of signaling, and our analyses revealed intimate links among groups of proteins, whose actions could be well-enhanced by this effect, for example, the preservation of protein folding through chaperons. We, however, observed relatively low levels of serotonylation compared with quantities previously predicted, with a highest observed efficiency of 63% (40 serotonylated peptides among a total of 64). While this finding may be improved by further optimizations of enrichment methods, it is more likely that because of the extremely tight regulation of transglutaminase activity at the cellular level only a small subset of proteins is actually modified with specific biogenic amines at a time. The flowthrough, for instance, only contained one modified peptide, suggesting that most of the modified peptides were retained onbeads prior to elution (data not shown). The relatively low levels of protein serotonylation may result in the underrepresentation of modified peptides when porous materials such as sepharose, which can adsorb peptides nonspecifically, are used for enrichment. Our workflow enabled site-specific profiling of serotonylation from a complex sample, which was

previously unachieved. Peptide-level enrichment would hypothetically offer high enrichment yields than the current proteinlevel approach, although in reality the difference may be offset due to difficulties in removing the unreacted linkers. The unremoved biotinyl linkers may saturate available binding sites, requiring a greater deal of optimization with potentially diminishing improvements. As with the field of proteomics, there exists an inherent “chicken-and-egg” problem, in which it may be difficult to elucidate the biological function of a particular feature without a list of potential candidates, and it is equally difficult to generate a confident list of hits without understanding at least a small subset of the biological functions. TGase 2, while ubiquitous in nature, may only have selectivity for a small set of targets when serotonin is used as the amine donor. This suggests both that this process is highly specific in nature and that it is a relatively uncommon event triggered only by extreme stimuli. However, because most targets of serotonylation, such as PSMD9 and various small GTPases, were also heavily posttranslationally modified, it might actually be the inter-regulation of all posttranslational modifications in the presence of serotonyla3527

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Laboratory, Institute of Biological Chemistry, Academia for the synthesis of Hras 57-68.

tion that was responsible for regulating protein activation. It becomes exceedingly important then first to assess the role and mechanism of tissue transglutaminase in more global events such as inflammation, redox homeostasis, or necrosis to gain a better understanding of protein serotonylation. Because 5-PT has similar biophysical properties as serotonin and could be used in vivo to observe endogenous protein serotonylation, for instance, Hsp40 (Figure S4 in the Supporting Information), its use could be extended to provide useful insights into protein serotonylation as well as a variety of different protein monoaminylations by similarly functionalizing other biogenic amines as substrates of transglutaminase.



ABBREVIATIONS TGase 2, tissue transglutaminase or transglutaminase type 2; 2ME, 2-mercaptoethanol; 5-PT, 5-propargyltryptamide. 5-HT, 5hydroxytryptamine; TBTA, tris[(1-benzy-1H-1,2,3,-triazol-4yl)methyl]amine





CONCLUSIONS While it was commonly believed that transglutaminase freely utilized biogenic mono- or polyamines indiscriminately to modify protein targets primarily to evade proteolysis, our results here suggested that protein modifications involving transglutaminase appeared to be selective and specific. While transglutaminase exhibited a wide range of amine donor specificity, differences in their relative chemical relativity suggested that transglutaminase did not indiscriminately modify proteins with free and random amines. Out of all possible signaling pathways, each biogenic amine serves a distinct purpose. Our results isolated 50 sites on 46 proteins in the SW480 proteome prone to serotonylation, and results here could provide a better understanding of global events with which tissue transglutaminase was connected. A method to enrich monoaminylated proteins for profiling, such as the one illustrated here, provides a first peek at some of the possible targets of transglutaminase involvement beyond small GTPases and assembly elements. Undoubtedly, with further optimizations and detailed biochemical investigations, the mechanism and significance of protein serotonylation would be fully elucidated in time.



ASSOCIATED CONTENT

S Supporting Information *

General materials, cell cultures, lysis and blotting, synthesis of Biotin-CTA-N3, and the mass spectra of identified peptides. An expanded table detailing the list of proteins and serotonylated sites is also available. This material is available free of charge via the Internet at http://pubs.acs.org.



REFERENCES

(1) Walther, D. J.; Peter, J. U.; Winter, S.; Holtje, M.; Paulmann, N.; Grohmann, M.; Vowinckel, J.; Alamo-Bethencourt, V.; Wilhelm, C. S.; Ahnert-Hilger, G.; Bader, M. Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release. Cell 2003, 115 (7), 851−862. (2) Watts, S. W.; Priestley, J. R.; Thompson, J. M. Serotonylation of vascular proteins important to contraction. PLoS One 2009, 4 (5), e5682. (3) Penumatsa, K. C.; Fanburg, B. L. Transglutaminase 2-mediated serotonylation in pulmonary hypertension. Am. J. Physiol.: Lung Cell. Mol. Physiol. 2014, 306 (4), L309−L315. (4) Walther, D. J.; Stahlberg, S.; Vowinckel, J. Novel roles for biogenic monoamines: from monoamines in transglutaminasemediated post-translational protein modification to monoaminylation deregulation diseases. FEBS J. 2011, 278 (24), 4740−4755. (5) Lin, J. C.; Chou, C. C.; Gao, S.; Wu, S. C.; Khoo, K. H.; Lin, C. H. An in vivo tagging method reveals that Ras undergoes sustained activation upon transglutaminase-mediated protein serotonylation. ChemBioChem 2013, 14 (7), 813−817. (6) Slack, J. L.; Causey, C. P.; Luo, Y.; Thompson, P. R. Development and use of clickable activity based protein profiling agents for protein arginine deiminase 4. ACS Chem. Biol. 2011, 6 (5), 466−476. (7) Gartner, C. A.; Elias, J. E.; Bakalarski, C. E.; Gygi, S. P. Catchand-release reagents for broadscale quantitative proteomics analyses. J. Proteome Res. 2007, 6 (4), 1482−1491. (8) Salisbury, C. M.; Cravatt, B. F. Click chemistry-led advances in high content functional proteomics. QSAR Comb. Sci. 2007, 26 (11− 12), 1229−1238. (9) Tsai, C. S.; Liu, P. Y.; Yen, H. Y.; Hsu, T. L.; Wong, C. H. Development of trifunctional probes for glycoproteomic analysis. Chem. Commun. 2010, 46 (30), 5575−5577. (10) Yang, Y.; Yang, X.; Verhelst, S. H. Comparative analysis of click chemistry mediated activity-based protein profiling in cell lysates. Molecules 2013, 18 (10), 12599−12608. (11) Yang, Y.; Hahne, H.; Kuster, B.; Verhelst, S. H. A simple and effective cleavable linker for chemical proteomics applications. Mol. Cell. Proteomics 2013, 12 (1), 237−244. (12) Ballinger, C. A.; Connell, P.; Wu, Y.; Hu, Z.; Thompson, L. J.; Yin, L. Y.; Patterson, C. Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol. Cell. Biol. 1999, 19 (6), 4535−4545. (13) Oh, W. K.; Song, J. Cooperative interaction of Hsp40 and TPR1 with Hsp70 reverses Hsp70-HspBp1 complex formation. Mol. Cell 2003, 16 (1), 84−91. (14) Hendershot, L. M.; Valentine, V. A.; Lee, A. S.; Morris, S. W.; Shapiro, D. N. Localization of the gene encoding human BiP/GRP78, the endoplasmic reticulum cognate of the HSP70 family, to chromosome 9q34. Genomics 1994, 20 (2), 281−284. (15) Johnson, E. N.; Seasholtz, T. M.; Waheed, A. A.; Kreutz, B.; Suzuki, N.; Kozasa, T.; Jones, T. L.; Brown, J. H.; Druey, K. M. RGS16 inhibits signalling through the G alpha 13-Rho axis. Nat. Cell Biol. 2003, 5 (12), 1095−1103. (16) Hart, M. J.; Jiang, X.; Kozasa, T.; Roscoe, W.; Singer, W. D.; Gilman, A. G.; Sternweis, P. C.; Bollag, G. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science 1998, 280 (5372), 2112−2114.

AUTHOR INFORMATION

Corresponding Author

*Phone: +886-2-2789-0110. Fax: +886-2-2651-4705. E-mail: [email protected]. Author Contributions #

J.C.-Y.L. and C.-C.C. contributed equally.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Financial support was provided by Academia Sinica and the Ministry of Science and Technology (100-2113M-001-021MY3 to C.H.L. and NSC 102-2319-B-001-003 to the Core Facilities for Protein Structural Analysis) of Taiwan. We thank Drs. Yu-Yin Shih and Yu-Yao Tseng for their kind assistance with bioinformatics analysis and the Peptide Synthesis 3528

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(17) Boroughs, L. K.; Antonyak, M. A.; Johnson, J. L.; Cerione, R. A. A unique role for heat shock protein 70 and its binding partner tissue transglutaminase in cancer cell migration. J. Biol. Chem. 2011, 286 (43), 37094−37107. (18) Caccamo, D.; Condello, S.; Ferlazzo, N.; Curro, M.; Griffin, M.; Ientile, R. Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress. Amino Acids 2013, 44 (1), 151−159. (19) Boros, S.; Kamps, B.; Wunderink, L.; de Bruijn, W.; de Jong, W. W.; Boelens, W. C. Transglutaminase catalyzes differential crosslinking of small heat shock proteins and amyloid-beta. FEBS Lett. 2004, 576 (1−2), 57−62. (20) Jensen, L. J.; Kuhn, M.; Stark, M.; Chaffron, S.; Creevey, C.; Muller, J.; Doerks, T.; Julien, P.; Roth, A.; Simonovic, M.; Bork, P.; von Mering, C. STRING 8–a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 2009, 37, D412−D416. (21) Kristensen, A. R.; Gsponer, J.; Foster, L. J. A high-throughput approach for measuring temporal changes in the interactome. Nat. Methods 2012, 9 (9), 907−909. (22) Karpuj, M. V.; Becher, M. W.; Springer, J. E.; Chabas, D.; Youssef, S.; Pedotti, R.; Mitchell, D.; Steinman, L. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat. Med. 2002, 8 (2), 143−149. (23) Borrell-Pages, M.; Canals, J. M.; Cordelieres, F. P.; Parker, J. A.; Pineda, J. R.; Grange, G.; Bryson, E. A.; Guillermier, M.; Hirsch, E.; Hantraye, P.; Cheetham, M. E.; Neri, C.; Alberch, J.; Brouillet, E.; Saudou, F.; Humbert, S. Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase. J. Clin. Invest 2006, 116 (5), 1410−1424. (24) App, C.; Knop, J.; Huff, T.; Sticht, H.; Hannappel, E. Thymosin beta4 and tissue transglutaminase. Molecular characterization of cyclic thymosin beta4. Protein J. 2013, 32 (6), 484−492. (25) Gupta, M.; Greenberg, C. S.; Eckman, D. M.; Sane, D. C. Arterial vimentin is a transglutaminase substrate: a link between vasomotor activity and remodeling? J. Vasc. Res. 2007, 44 (5), 339− 344. (26) Borth, W. Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 1992, 6 (15), 3345− 3353. (27) Chen, J.; Zhao, R.; Semba, U.; Oda, M.; Suzuki, T.; Toba, K.; Hattori, S.; Okada, S.; Yamamoto, T. Involvement of cross-linked ribosomal protein S19 oligomers and C5a receptor in definitive erythropoiesis. Exp. Mol. Pathol 2013, 95 (3), 364−375. (28) Nemes, Z., Jr.; Adany, R.; Balazs, M.; Boross, P.; Fesus, L. Identification of cytoplasmic actin as an abundant glutaminyl substrate for tissue transglutaminase in HL-60 and U937 cells undergoing apoptosis. J. Biol. Chem. 1997, 272 (33), 20577−20583. (29) Willis, W. L.; Hariharan, S.; David, J. J.; Strauch, A. R. Transglutaminase-2 mediates calcium-regulated crosslinking of the Ybox 1 (YB-1) translation-regulatory protein in TGFbeta1-activated myofibroblasts. J. Cell. Biochem. 2013, 114 (12), 2753−2769.

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dx.doi.org/10.1021/pr5003438 | J. Proteome Res. 2014, 13, 3523−3529