Elucidating The Regulatory Mechanisms Of The Mammalian Target Of Rapamycin Complex 1 (Mtorc1) In Protein Homeostasis
The proper balance between protein synthesis, maturation, and degradation is crucial for an organism to survive and prosper. Each of these processes is coupled to the cellular environment through a multitude of signaling events to maintain protein homeostasis (proteostasis). The costly process of protein synthesis is partially regulated through the mammalian target of rapamycin in complex 1 (mTORC1). mTORC1 acts as a metabolic hub for the cell monitoring energy levels, nutrients, and amino acid availability in order to increase anabolic processes including initiation and elongation during mRNA translation. Deregulation of mTORC1 signaling can be catastrophic for an organism leading to cancer, obesity, and age-related illness. This work focuses on the molecular events that promote these disease states when mTORC1 is hyperactivated, and further investigates a potential mechanism for the cell to cope with this dys-homeostasis. Here, I investigated the impact of hyperactive mTORC1 on the proteostasis network by inducing cytosolic and proteotoxic stress within cell cultures. Utilizing fibroblast knockouts, lentiviral knockdowns, plasmid transfections, and mTORC1 inhibitors, I was able to manipulate mTORC1 activity to elucidate its role in mRNA translation and overall proteostasis. I discovered that mTORC1 signaling is necessary for global cap-dependent protein synthesis, but attenuated cap-independent and IRES mRNA translation. Moreover, I demonstrated that the heat shock protein 70 (Hsp70) utilized a cap-independent mechanism of translation through its 5'UTR. Hyperactive mTORC1 signaling prevented the stress-induced preferential translation of HSP70, which inhibited cell recovery leading to cell death. These results uncovered an intimate connection between mTORC1 signaling and the stress response, highlighting how an increase in protein synthesis can imbalance a translational switch necessary to maintain proteostasis. Correspondingly, a decrease in mTORC1 signaling and protein synthesis can be beneficial to an organism leading to an increase in stress resistance and lifespan. To elucidate this mechanism I further investigated the effects of mTORC1 activity on protein synthesis and discovered an increase in protein quantity altered the quality of newly synthesized polypeptides. I demonstrated that hyperactive mTORC1 signaling decreased translation fidelity through its downstream target S6K, with no apparent influence on the chaperone network or ubiquitin proteasome system. An increase in S6K signaling promoted faster elongation rates, potentially leading to the observed mistranslation. Furthermore, partially inhibiting mTORC1 activity with rapamycin treatment restored protein quality by slowing down mRNA translation. My results reveal a mechanistic connection between protein quality and mTORC1 activity, which strengthen the role of nutrient signaling in proper cell growth and healthy aging. Collectively, my ex vivo results used to manipulate mTORC1 activity uncover molecular events highlighting mTORC1 as a key component of the proteostasis network. mTORC1 signaling favors the development of age-related pathologies and investigating the effects of its activity provides an array of downstream potential drug targets.
Protein Synthesis; mTOR; Protein Homeostasis
Lee, Siu Sylvia; Lis, John T
Ph.D. of Genetics
Doctor of Philosophy
dissertation or thesis