한빛사논문
- 조회471
Mi-Ock Baek1, Hye-Jeong Cho2, Do Sik Min3, Cheol Soo Choi4,5,6* & Mee-Sup Yoon1,2,6*
1Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, Republic of Korea; 2Lee Gil Ya Cancer and Diabetes Institute, Incheon, Republic of Korea; 3College of Pharmacy, Yonsei University, Incheon, Republic of Korea; 4Korea Mouse Metabolic Phenotyping Center, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Republic of Korea; 5Department of Internal Medicine, Gil Medical Center, Gachon University, Incheon, Republic of Korea; 6Department of Molecular Medicine, Gachon University College of Medicine, Incheon, Republic of Korea
*Correspondence to: Mee-Sup Yoon and Cheol Soo Choi
Abstract
Background
Muscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C-terminal unique attached sequence motif (UNE) domain of leucyl-tRNA synthetase (LRS-UNE-L) as an mTORC1 (mTOR complex1)-activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle-enhancing peptide.
Methods
In vitro Vps34 lipid kinase assay, phosphatidylinositol 3-phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS-UNE-L on the Vps34-PLD1-mTOR pathway. Adeno-associated virus (AAV)-LRS-UNE-L was transduced in C2C12 cells in vitro, in BaCl2-injured tibialis anterior (TA) muscles, and in 18-month-old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle-specific cell-permeable peptide M12 was fused with LRS-UNE-L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12-LRS-UNE-L was introduced into BaCl2-injured TA muscles of 15-week-old Pld1+/+ or Pld1−/− mice, and its effect was analysed by measurement of cross-sectional area of regenerating muscle fibres.
Results
The LRS-UNE-L expression restored amino acid-induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases-independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS-UNE-L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid-treated control cells, but it did not affect autophagic flux. AAV-delivered LRS-UNE-L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin-like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV-LRS-UNE-L increased the size of regenerating muscle fibres in BaCl2-injured TA muscles (124%, P = 0.0279) (n = 9–10), but it did not change the muscle fibre size of TA muscles in old mice. M12-LRS-UNE-L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2-injured TA muscles in a PLD1-dependent manner (116%, P = 0.0022) (n = 6).
Conclusions
Our results provide compelling evidence that M12-LRS-UNE-L could be a muscle-enhancing protein targeting mTOR.
논문정보
- Research article
- 2022년 02월 (BRIC 등록일 2022-03-11)
- 국내 연구진
- 의약학 > 분자세포의학
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