Non-canonical functions of leucyl-tRNA synthetase: Mechanism of cell growth and skeletal myogenesis

Son, Kook

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Description

Title

Non-canonical functions of leucyl-tRNA synthetase: Mechanism of cell growth and skeletal myogenesis

Author(s)

Son, Kook

Issue Date

2019-04-17

Director of Research (if dissertation) or Advisor (if thesis)

Chen, Jie

Doctoral Committee Chair(s)

Prasanth, Supriya

Committee Member(s)

• Chen, Jie
• Belmont, Andrew
• Chen, Lin-Feng

Department of Study

Cell & Developmental Biology

Discipline

Cell and Developmental Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Leucyl-tRNA synthetase (LRS)
• Aminoacyl-tRNA synthetase (AARS)
• Mammalian target of rapamycin (mTOR)
• Vacuolar protein sorting 34 (Vps34)
• Cell growth
• Skeletal myogenesis

Abstract

The mammalian target of rapamycin (mTOR) complex 1, mTORC1, is a master regulator of cell growth, proliferation, differentiation and metabolism, and it has emerged as an attractive target of intervention in several human diseases. mTORC1 assembles a signaling network that senses and transduces a variety of environmental and cellular cues, including amino acid sufficiency, which is a prerequisite for mTORC1 activation under all conditions. The mechanism of how cellular amino acids are sensed has been a long-standing question. Recent discoveries of several direct amino acid sensors have begun to illuminate an intricate sensing network that consists of redundancy and parallel pathways. In my dissertation work I have investigated the role of leucyl-tRNA synthetase (LRS) as a leucine sensor regulating mTORC1 activity in cell growth, myogenic differentiation, and skeletal muscle regeneration. Amino acid availability activates signaling by mTORC1. The class III PI-3-kinase Vps34 mediates amino acid signaling to mTORC1 by regulating lysosomal translocation and activation of the phospholipase PLD1. In collaboration with Dr. Mee-Sup Yoon, I identified leucyl-tRNA synthetase as a leucine sensor for the activation of Vps34-PLD1 upstream of mTORC1. LRS is necessary for amino acid-induced Vps34 activation, cellular PI(3)P level increase, PLD1 activation, and PLD1 lysosomal translocation. Leucine binding, but not tRNA charging activity of LRS, is required for this regulation. Moreover, we found that LRS physically interacts with Vps34 in amino acid-stimulatable non-autophagic complexes. Finally, purified LRS protein activates Vps34 kinase in vitro in a leucine-dependent manner. Collectively, our findings detailed in Chapter 2 provide compelling evidence for a direct role of LRS in amino acid activation of Vps34 via a non-canonical mechanism and fill a gap in the amino acid-sensing mTORC1 signaling network. mTOR also regulates skeletal myogenesis, but the signaling mechanism is distinct from that in cell growth regulation. A role of LRS in myogenesis has not been reported. In collaboration with Dr. Jae-Sung You, I found that LRS negatively regulates myoblast differentiation in vitro. This function of LRS is independent of its regulation of protein synthesis, and it requires leucine-binding but not tRNA charging activity of LRS. Local knockdown of LRS accelerates muscle regeneration in a mouse injury model, as well as the knockdown of Rag or Raptor. Further in vitro studies establish a Rag-mTORC1 pathway, which inhibits the IRS1-PI3K-Akt pathway, as a mediator of the non-translational function of LRS in myogenesis. BCLI-0186, an inhibitor previously reported to disrupt LRS-Rag interaction, promotes robust muscle regeneration with enhanced functional recovery in mice, but this effect is abolished by co-treatment with an Akt inhibitor. Taken together, our findings detailed in Chapter 3 reveal a novel function for LRS in controlling the homeostasis of myogenesis and suggest a potential therapeutic strategy to target a non-canonical function of a house-keeping protein. In Appendix A, I document the effort of an RNAi screening to identify novel regulators of myogenesis among the aminoacyl-tRNA synthetases (AARSs). A few of AARSs were discovered to be negative regulators based on the knockdown phenotypes, but the majority appeared to be positive regulators of myoblast differentiation. It is intriguing, aside from LRS, that only a small number of AARSs may have negative functions in myogenesis. This is not surprising considering the canonical role of AARSs in protein translation, which is necessary for the myogenic process. Further investigation of these candidates could potentially reveal new therapeutic approaches for the treatment of muscular diseases. Lastly, in Appendix B, I document my preliminary examination on the in vivo role of Vps34 in skeletal muscle regeneration. I have observed a mild decrease in size of regenerating myofibers from the human skeletal actin (HSA)-Cre driven Vps34 knockout skeletal muscles, but there was no statistically significant difference. I propose an alternative strategy using tamoxifen-inducible Myf5-CreER driven Vps34 knockout in muscle cells of the offspring to investigate the role of Vps34 in the early stage of muscle regeneration. Additionally, I suggest an experiment to probe the in vivo involvement of the Vps34-PLD1-mTOR-IGF2 pathway in skeletal myogenesis.

Graduation Semester

2019-05

Type of Resource

text

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Copyright and License Information

Copyright 2019 Kook Son

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