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We identified the C\u2010terminal unique attached sequence motif (UNE) domain of leucyl\u2010tRNA synthetase (LRS\u2010UNE\u2010L) as an mTORC1 (mTOR complex1)\u2010activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle\u2010enhancing peptide.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p><jats:italic>In vitro<\/jats:italic> Vps34 lipid kinase assay, phosphatidylinositol 3\u2010phosphate (PI(3)P) measurement, <jats:italic>in vivo<\/jats:italic> PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS\u2010UNE\u2010L on the Vps34\u2010PLD1\u2010mTOR pathway. Adeno\u2010associated virus (AAV)\u2010LRS\u2010UNE\u2010L was transduced in C2C12 cells <jats:italic>in vitro<\/jats:italic>, in BaCl<jats:sub>2<\/jats:sub>\u2010injured tibialis anterior (TA) muscles, and in 18\u2010month\u2010old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle\u2010specific cell\u2010permeable peptide M12 was fused with LRS\u2010UNE\u2010L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12\u2010LRS\u2010UNE\u2010L was introduced into BaCl<jats:sub>2<\/jats:sub>\u2010injured TA muscles of 15\u2010week\u2010old <jats:italic>Pld1<\/jats:italic><jats:sup><jats:italic>+\/+<\/jats:italic><\/jats:sup> or <jats:italic>Pld1<\/jats:italic><jats:sup><jats:italic>\u2212\/\u2212<\/jats:italic><\/jats:sup> mice, and its effect was analysed by measurement of cross\u2010sectional area of regenerating muscle fibres.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The LRS\u2010UNE\u2010L expression restored amino acid\u2010induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases\u2010independent manner (421%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.007 vs. LRS knockdown control cells). The LRS\u2010UNE\u2010L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0352), PI(3)P levels (146%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0039), and PLD1 activity (228%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0294) compared with amino acid\u2010treated control cells, but it did not affect autophagic flux. AAV\u2010delivered LRS\u2010UNE\u2010L domain augmented S6K1 phosphorylation (174%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0013), mRNA levels of myosin heavy chain (MHC) (122%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0282) and insulin\u2010like growth factor 2 (IGF2) (146%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.008), and myogenic fusion (133%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0479) in C2C12 myotubes. AAV\u2010LRS\u2010UNE\u2010L increased the size of regenerating muscle fibres in BaCl<jats:sub>2<\/jats:sub>\u2010injured TA muscles (124%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0279) (<jats:italic>n<\/jats:italic>\u00a0=\u00a09\u201310), but it did not change the muscle fibre size of TA muscles in old mice. M12\u2010LRS\u2010UNE\u2010L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl<jats:sub>2<\/jats:sub>\u2010injured TA muscles in a PLD1\u2010dependent manner (116%, <jats:italic>P<\/jats:italic>\u00a0=\u00a00.0022) (<jats:italic>n<\/jats:italic>\u00a0=\u00a06).<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Our results provide compelling evidence that M12\u2010LRS\u2010UNE\u2010L could be a muscle\u2010enhancing protein targeting mTOR.<\/jats:p><\/jats:sec>","DOI":"10.1002\/jcsm.12947","type":"journal-article","created":{"date-parts":[[2022,2,18]],"date-time":"2022-02-18T03:23:20Z","timestamp":1645154600000},"page":"1277-1288","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Self\u2010transducible LRS\u2010UNE\u2010L peptide enhances muscle regeneration"],"prefix":"10.1002","volume":"13","author":[{"given":"Mi\u2010Ock","family":"Baek","sequence":"first","affiliation":[{"name":"Department of Health Sciences and Technology GAIHST, Gachon University  Incheon Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hye\u2010Jeong","family":"Cho","sequence":"additional","affiliation":[{"name":"Lee Gil Ya Cancer and Diabetes Institute  Incheon Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Do Sik","family":"Min","sequence":"additional","affiliation":[{"name":"College of Pharmacy Yonsei University  Incheon Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheol Soo","family":"Choi","sequence":"additional","affiliation":[{"name":"Korea Mouse Metabolic Phenotyping Center Lee Gil Ya Cancer and Diabetes Institute, Gachon University  Incheon Republic of Korea"},{"name":"Department of Internal Medicine, Gil Medical Center Gachon University  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