title: Production of free methylarginines via the proteasome and autophagy pathways in cultured cells.
abstract: ω-NG-monomethylarginine (MMA) and asymmetric ω-NG, ω-NG-dimethylarginine (ADMA), are endogenous competitive inhibitors for three isoforms of nitric oxide synthase (NOS). Although free methylarginines are thought to be liberated through the intracellular proteolysis of proteins methylated by protein arginine methyltransferases (PRMTs), the degradation pathways of the arginine-methylated proteins involved in the biosynthesis of free methylarginines have yet to be determined. In this study, the biosynthesis of free methylarginines with cultured cells was analyzed as follows: first, we established a method for quantifying trace amounts of free intracellular methylarginines by means of ultra high‑performance liquid chromatography (UPLC). Second, we determined the type of methylation produced in the cultured cell lines using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry (MALDI-QIT-TOF/MS). Finally, we investigated whether methylarginines are generated via the proteasome and autophagy pathways, the primary intracellular protein degradation systems. By using specific inhibitors for each pathway, we found that the blockade of proteasome activity reduced the amount of free ADMA and symmetric ω-NG, ω-N'G-dimethylarginine (SDMA), while the inhibition of autophagy significantly reduced cellular ADMA only. These results suggest that both the proteasome and autophagy pathways play an essential role in the production of free methylarginines.
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Takuma
authors.initials: T
authors.lastname: Shirakawa
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kako
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimada
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nagashima
authors.forename: Ayumi
authors.initials: A
authors.lastname: Nakamura
authors.forename: Junji
authors.initials: J
authors.lastname: Ishida
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Fukamizu
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008745:Methylation; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019323:omega-N-Methylarginine / Q000032:analysis
chemical_list: D007976:Leupeptins; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D019323:omega-N-Methylarginine; C024917:symmetric dimethylarginine; C018524:N,N-dimethylarginine; D002738:Chloroquine; D001120:Arginine; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin