Determine The Amino Acid Sensing Specificity Of Folliculin Case Study

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Experimental Design

Aim 1: Determine the amino acid sensing specificity of folliculin. Since folliculin’s novel amino acid sensing function discovery, I plan to further understand whether FLCN specifically senses a certain amino acid and if this level of detection is enough to elicit an mTORC1 response.

1.1. Determine if folliculin’s novel molecular function in amino acid sensing is triggered by glutamine. mTORC1 activation by amino acids requires FLCN where its lysosomal residence during starvation interacts with inactive Rag complex and exerts GAP activity to activate RagC/D during amino acid re-stimulation. Active RagC/D initiates the binding between raptor and the Rag complex to promote mTORC1 lysosomal translocation and its activation.
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Determine if FLCN’s binding glutamine capacity elicits a mTORC1 response. It’s imperative to evaluate whether FLCN glutamine sensitivity is relevant; does FLCN’s glutamine sensitivity trigger mTORC1 activation? Initial FLCN glutamine sensitivity investigation took place with high glutamine concentrations; but first, we need to evaluate if different glutamine concentration has varying mTORC1 activity effects and find the optimal glutamine concentration to use by evaluating P-S6 levels from cell lysates that have been glutamine starved and re-feed at various concentrations. Previous experiments show silencing FLCN potentiates amino acid-induced mTORC1 activity. Therefore, a rescue experiment will be conducted to test FLCN glutamine-induced mTORC1 activation; whereby, HEK293Tcells with reduced FLCN expression by shRNA will be subsequently supplemented with purified FLCN and stimulated with glutamine to test if purified FLCN addition with glutamine supplementation is enough to initiate mTORC1 activity. Purified FLCN will be obtained through immunoprecipitation of HEK293T cells expressing FLAG-FLCN. Lastly, if FLCN glutamine sensitivity initiates mTORC1 activation, does glutamine stimulation occur in a dose-dependent manner. In starved cells, FLCN interacts with RagB and dissociate when stimulated with amino acids. To analyze if dissociation occurs in a dose-dependent manner in respect to glutamine stimulation, I will analyze the dissociation upon increasing concentration of glutamine compared to Rap2A, a GTPase known to have no interactions with FLCN. This dose-dependent assay will begin by purifying RagB and Rap2A through immunoprecipitation with anti-FLAG from cell lysates expressing FLAG-RagB and FLAG-Rap2A that have been starved for 50mins. Once obtain purified proteins from starved cells, then increasing glutamine concentrations will be added to the purified protein mixture; subsequently, immunoblotted for FLCN of analyze FLCN-RagB protein interactions. If

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