Investigations into the Mechanism of CB3A mediated mTORC1 Inhibition
Creators: name: Henkin, Anna  Julia
Publisher: name: Brandeis University
Year: 2023
Subjects: CB3A
Inhibitor
mTOR
mTORC1
Photoaffinity
TSC2
Molecular Biology

description: Mammalian Target of Rapamycin (mTOR) is a Ser/Thr kinase that acts as a master regulator of cellular metabolism and growth via its incorporation into two complexes, mTORC1 and mTORC2, that collectively control the cellular transition between anabolism and catabolism, respectively. Regulation of mTORC1 integrates nutrient, growth, and stress signals to enact precise control of anabolic processes like protein translation, fatty acid synthesis, and nucleic acid synthesis. These are all vital processes in the progression of metabolic and proliferative diseases, and mTORC1 is an attractive but complex target for drug development. The small molecule CB3A is a specific inhibitor of mTORC1-driven protein translation that acts through an unknown mechanism that is distinct from other mTOR inhibitors. Previous work in the lab has identified three proteins of interest in the search for a mechanistic binding partner: TSC2, a GTPase activating protein that inhibits mTORC1, UBQLN2, a proteasome shuttle factor that has a role in lysosomal acidification, and RNF152, an E3 ligase that inhibits re-activation of mTORC1 in the absence of growth factors or nutrients. The ultimate goal of this study is to investigate the novel mechanism of CB3A-mediated mTORC1 inhibition and to clarify the roles of RNF152, UBQLN2, and TSC2 in it.Chapter 1 of this thesis aims to: 1) review the background of mTOR and mTORC1 regulation, downstream effects, and role in disease, and 2) review the history of CB3A’s serendipitous discovery as a novel mTORC1 inhibitor, summarize the previous work carried out to uncover its mechanism of action, and provide additional background on protein pathways that are relevant to the mechanism of CB3A-mediated mTORC1 inhibition.
description: In the second chapter of this thesis, I will discuss work done to investigate a potential role for an E3 ligase, RNF152, in the mechanism of action of CB3A-mediated mTORC1 inhibition. RNF152-mediated ubiquitination plays a vital role in nutrient signal persistence within the mTORC1 regulatory network, and prior work in the lab led us to hypothesize that CB3A may act through modulation of RNF152 activity. Through the generation and experimental study of a CRISPR-Cas9 RNF152 KO cell line, we found that RNF152 is not required for CB3A-mediated mTORC1 inhibition.
description: The pro-anabolic activity of mTORC1 is regulated by a vast array of nutrient and stress sensing pathways, that work to transition the overall cellular metabolic apparatus from anabolism to catabolism and back over and over throughout a cell’s lifetime. The complex and often circular nature of these pathways have made photoaffinity labeling (PAL) an attractive option for probing the mechanistic binding partner(s) of CB3A, which have, so far, proved elusive. Photoaffinity labeling is a method that utilizes a photoactivatable analog of a ligand of interest to carry out specific, covalent labeling of biological molecules under native binding conditions. In Chapter 3, I detail the design, synthesis, and validation of three photoaffinity labeling compounds for use in further direct binding assays.
description: Chapter 4 details the development and optimization of a photoaffinity labeling and affinity purification workflow for the specific labeling and identification of candidates for CB3A’s mechanistic partner(s) in mTORC1 inhibition. This methodology identified two proteins that regulate opposing facets of the mTORC1 pathway, MAP2K1 and ATP6V1B2, as potential mechanistic binding partners that integrate many of our previous observations about CB3A into a compelling mechanistic model. The possibility of MAP2K1 as a mechanistic binding partner directly ties the mechanism of CB3A-mediated mTORC1 inhibition to TSC2 regulation. The involvement of the v-ATPase provides a connection to our prior data implicating UBQLN2 in the mechanism of CB3A-mediated mTORC1 inhibition.