Production of Free Methylarginines Via the Proteasome and Autophagy Pathways in Cultured Cells
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MOLECULAR MEDICINE REPORTS 4: 615-620, 2011 Production of free methylarginines via the proteasome and autophagy pathways in cultured cells TAKUMA SHIRAKAWA1, KOICHIRO KAKO1, TAKASHI SHIMADA1, YUSUKE NAGASHIMA1, AYUMI NAKAMURA1, JUNJI ISHIDA1,2 and AKIYOSHI FUKAMIZU1,2 1Graduate School of Life and Environmental Sciences; 2Life Science Center, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan Received December 3, 2010; Accepted April 8, 2011 DOI: 10.3892/mmr.2011.488 Abstract. ω-NG-monomethylarginine (MMA) and asymmetric regulation of transcriptional control, RNA processing, DNA ω-NG, ω-NG-dimethylarginine (ADMA), are endogenous repair and signal transduction. This modification is catalyzed competitive inhibitors for three isoforms of nitric oxide by protein arginine methyltransferases (PRMTs) via the meth- synthase (NOS). Although free methylarginines are thought to ylation of nitrogen atoms on the guanidinium side chains of be liberated through the intracellular proteolysis of proteins arginine residues within proteins with S-adenosylmethionine methylated by protein arginine methyltransferases (PRMTs), as a methyl-donor (1,2). PRMTs are classified into two the degradation pathways of the arginine-methylated proteins main categories: type I enzymes, which catalyze the forma- involved in the biosynthesis of free methylarginines have yet tion of ω-NG-monomethylarginine (MMA) and asymmetric to be determined. In this study, the biosynthesis of free methy- ω-NG, ω-NG-dimethylarginine (ADMA) (3-9), and type II larginines with cultured cells was analyzed as follows: first, we enzymes, which generate MMA and symmetric ω-NG, ω-N'G- established a method for quantifying trace amounts of free dimethylarginine (SDMA) (10-12). intracellular methylarginines by means of ultra high-perfor- These methylarginine derivatives are believed to be mance liquid chromatography (UPLC). Second, we determined released into the cytoplasm following proteolysis, since no the type of methylation produced in the cultured cell lines using direct methylation reaction of arginine as a solitary amino matrix-assisted laser desorption/ionization quadrupole ion trap acid has been observed (13). Among the derivatives, MMA time-of-flight tandem mass spectrometry (MALDI-QIT-TOF/ and ADMA, but not SDMA, are endogenous inhibitors of MS). Finally, we investigated whether methylarginines are nitric oxide (NO) synthase (NOS) through competition with generated via the proteasome and autophagy pathways, the free L-arginine residue (L-Arg), a specific substrate for primary intracellular protein degradation systems. By using NOS (14,15). In the vasculature, NO is produced by consti- specific inhibitors for each pathway, we found that the blockade tutively expressed endothelial-specific NOS (eNOS). This of proteasome activity reduced the amount of free ADMA and endothelial-derived NO diffuses into the smooth muscle cell symmetric ω-NG, ω-N'G-dimethylarginine (SDMA), while the layer and activates soluble guanylate cyclase followed by inhibition of autophagy significantly reduced cellular ADMA smooth muscle relaxation (13). While it is clear that increased only. These results suggest that both the proteasome and plasma concentrations of ADMA are correlated with various autophagy pathways play an essential role in the production of cardiovascular pathologies (15), and that the administration free methylarginines. of ADMA has significant cardiovascular effects (16), less is known regarding the mechanisms behind the biosynthesis of Introduction methylarginines. The aim of the present study was to examine whether the degradation of endogenous protein via the Protein arginine methylation has recently been established as proteasome and autophagy pathways affects the production of an important post-translational modification involved in the methylarginines. Materials and methods Correspondence to: Professor Akiyoshi Fukamizu, Life Science Chemicals and reagents. L-Arg, MMA, ADMA, SDMA, Center, TARA, University of Tsukuba, Tsukuba, Ibaraki 305-8577, N-propyl-L-arginine (N-PLA) and chloroquine were Japan purchased from Sigma-Aldrich (St. Louis, MO, USA). E-mail: [email protected] Cbz-L-Leu-L-Leu-L-Leu-al (MG132) and epoxomicin were from the Peptide Institute (Osaka, Japan). High-performance Key words: protein arginine methyltransferase (PRMT), liquid chromatography (HPLC)-grade methanol and aceto- methylarginine, proteasome, autophagy, matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem nitrile were from Wako (Tokyo, Japan). A Bond Elut SCX mass spectrometry, ultra high-performance liquid chromatography cartridge was obtained from Varian (Harbor City, CA, USA). The AccQ-Tag Ultra-Fluor™ derivatization kit (borate buffer 616 SHIRAKAWA et al: PROTEOLYTIC PRODUCTIONS OF METHYLARGININES and reagent) was obtained from Waters (Milford, MA, USA). 40˚C with an acetonitrile gradient in ammonium acetate buffer Angiotensin II (Ang II) and bradykinin 1-7, calibration (pH 6.0) at a flow rate of 0.25 ml/min. Mobile phase solutions standards for MALDI-QIT-TOF/MS, were purchased from A and B were made up of 4 and 30% acetonitrile, respectively, Sigma-Aldrich. 2,5-Dihydroxybenzoic acid (DHBA) matrix in 0.1 M sodium acetate pH 6.0. The gradient conditions were of MALDI-MS grade was obtained from Shimadzu GLC A:B = 100:0 for 25 min, then 93:7 for >45 min, hold at 93:7 for (Tokyo, Japan). 75 min, flushing with 0:100 for 5 min, then a return to initial conditions of 100:0 for >1 min. The column was allowed to Cell culture. The human embryonic kidney 293T (HEK293T) re-equilibrate for 10 min. The injection volume was 7.5 µl. and lung epithelial A549 cell lines were maintained in For the assessment of purity, the fractionated peaks Dulbecco's Modified Eagle's Medium (DMEM; Nissui, were re-chromatographed by HPLC (Alliance 2695 sepa- Tokyo, Japan) supplemented with 10% heat-inactivated fetal ration module and 414 fluorescence detector, Waters). bovine serum (Life Technologies, Carlsbad, CA, USA) at Chromatographic separation was achieved on a Cosmosil 37˚C in a humidified atmosphere containing 5% CO2. πNAP column (5 µm, 4.6x150 mm) obtained from Nacalai Tesque (Kyoto, Japan). The column was operated at 30˚C. Sample preparations. HEK293T and A549 cells were After sample injection (100 µl), separation was performed separately plated at a density of 1x107 cells. After 48 h, the under isocratic conditions with 25% methanol as a solvent, cells were treated with proteasome or autophagy inhibitors and maintained for 45 min. In order to elute strongly bound and harvested in PBS. The supernatants were removed by compounds, the column was flushed with 100% methanol centrifugation at 1,000 x g for 2 min, and the precipitated cells for 5 min and re-equilibrated under isocratic conditions for were re-suspended in ice-cold Milli-Q™ water. After boiling 10 min prior to the next injection. In both the UPLC and for 10 min, the samples were incubated for 5 min at room HPLC systems, fluorescence from the analytes was detected temperature and then maintained on ice for 10 min. Cell debris at λex 250 nm and λem 395 nm. Data were collected using and insoluble matter were removed by centrifugation at 16,000 Empower™ 2 HPLC software for Windows (Waters). x g for 10 min. Protein concentrations in the lysates were deter mined using Bio-Rad protein assay reagent (Bio-Rad, MALDI-QIT-TOF/MS. DHBA matrix (10 mg) was dissolved Hercules, CA, USA), and the cell lysates were divided into in 1 ml of 40% acetonitrile and 0.1% trifluoroacetic acid. aliquots of 1.8 ml. Each aliquot was acidified with 200 µl of Matrix solution (0.5 µl) and 1% sample (0.5 µl) dissolved in 10% 5-sulfosalicylic acid and dissolved in Milli-Q water to a methanol and water were deposited on a MALDI plate and total volume of 2.0 ml. Samples were vortexed, maintained on left to dry at room temperature for the preparation of sample ice for 10 min for complete protein precipitation, and centri- spots. External calibrations were achieved using the standard fuged at 16,000 x g for 10 min. The supernatant was reserved reagents Ang II [monoisotopic mass (M+H)+ = 1046.5], and and maintained on ice until solid-phase extraction (SPE). bradykinin 1-7 [(M+H)+ = 757.4]. Tandem mass experiments with MALDI-QIT-TOF/MSn (n=1, 2, or 3) were performed SPE and derivatization. Deproteinized samples were subjected using argon gas as the collision gas on an AXIMA-QIT mass to crude fractionation on a Bond Elut SCX cation exchange spectrometer (Shimadzu, Kyoto, Japan) equipped with a cartridge as previously reported (17). After final elutions nitrogen laser (337 nm) with the collision-induced dissocia- of SPE, eluted basic molecules were dried with Savant tion control value set at 160 (19). SpeedVac™ concentrator (Thermo Fisher Scientific, Waltham, MA, USA). Amino acid derivatization with AccQ-Tag reagents Standard curves. Calibration standards for the methylarginine was conducted as previously described (17). Briefly, 25 µl of derivatives (0, 6.25, 12.5 and 25.0 ng) were prepared from either a standard mix solution or cell extract was combined stock solutions (0.4 mg/ml in 0.1 M HCl) diluted in Milli-Q with 25 µl of AccQ-Tag Ultra borate buffer, then 10 µl of water and stored at -20˚C. For the preparation of the calibra- AccQ-Tag reagent previously dissolved in 1.0 ml of AccQ-Tag tion curves, the cell lysates were spiked with standards for Ultra reagent diluent was added. The reaction was allowed to arginine derivatives and an internal standard (200 ng N-PLA) proceed for 10 min at room temperature. and subjected to SPE, derivatization and chromatography as described above. Calibration curves were calculated by plotting Antibodies and Western blotting. Monoclonal antibodies against the ratio of the peak area of analytes to the area of the internal ubiquitin (P4D1) and β-actin were purchased from Santa Cruz standard (N-PLA) vs. analyte quantity. For the determination of Biotechnology (Santa Cruz, CA, USA) and Sigma-Aldrich, the recovery rate, standard solutions (25 ng of MMA, ADMA respectively.