Production of free methylarginines via the proteasome and autophagy pathways in cultured cells
| Autor: | Ayumi Nakamura, Takashi Shimada, Akiyoshi Fukamizu, Junji Ishida, Yusuke Nagashima, Koichiro Kako, Takuma Shirakawa |
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| Rok vydání: | 2011 |
| Předmět: |
Proteasome Endopeptidase Complex
Cancer Research Methyltransferase Arginine Leupeptins Proteolysis Methylation Biochemistry Cell Line chemistry.chemical_compound Biosynthesis Autophagy Genetics medicine Humans Molecular Biology Chromatography High Pressure Liquid omega-N-Methylarginine medicine.diagnostic_test biology Chloroquine Cell biology Nitric oxide synthase Oncology Proteasome chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Molecular Medicine Oligopeptides Proteasome Inhibitors Intracellular |
| Zdroj: | Molecular Medicine Reports. |
| ISSN: | 1791-3004 1791-2997 |
| DOI: | 10.3892/mmr.2011.488 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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