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. …show more content…
This will be biochemically analyzed through co-immunoprecipitation of HEK293T cells stably expressing FLAG-RagB who are starved from 50mins and then re-stimulated with glutamine for 10mins and immunoblot for endogenous FLCN levels. If FLCN is glutamine sensitive, you should see a transition from protein interaction between FLCN and RagB seen under starved conditions to its dissociation upon glutamine stimulation. Finally, I will observe FLCN-FNIP RagC/D GAP activity, whereby FLCN complex stimulates GTP hydrolysis on RagC/D when supplied with amino acids. FLCN-FNIP GAP activity will be tracked by thin-layer chromatography (TLC) GTP hydrolysis in protein mixtures of purified FLAG-RagC/D and FLCN-FNIP2 from cell lysates that were either starved or starved then re-stimulated with glutamine, which are then mixed with radio labeled GTP, [-32P]GTP. I focused initial GAP analysis towards FLCN-FNIP2 because this complex had the strongest preference to hydrolyze GTP on RagC and RagD (Tsun). Purified FLCN-FNIP2 protein complex will be obtained by tandem affinity methods captured through co-immunoprecipitation from cell lysates expressing tandem tagged FLCN-FNIP2;
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. …show more content…
This will be biochemically analyzed through co-immunoprecipitation of HEK293T cells stably expressing FLAG-RagB who are starved from 50mins and then re-stimulated with glutamine for 10mins and immunoblot for endogenous FLCN levels. If FLCN is glutamine sensitive, you should see a transition from protein interaction between FLCN and RagB seen under starved conditions to its dissociation upon glutamine stimulation. Finally, I will observe FLCN-FNIP RagC/D GAP activity, whereby FLCN complex stimulates GTP hydrolysis on RagC/D when supplied with amino acids. FLCN-FNIP GAP activity will be tracked by thin-layer chromatography (TLC) GTP hydrolysis in protein mixtures of purified FLAG-RagC/D and FLCN-FNIP2 from cell lysates that were either starved or starved then re-stimulated with glutamine, which are then mixed with radio labeled GTP, [-32P]GTP. I focused initial GAP analysis towards FLCN-FNIP2 because this complex had the strongest preference to hydrolyze GTP on RagC and RagD (Tsun). Purified FLCN-FNIP2 protein complex will be obtained by tandem affinity methods captured through co-immunoprecipitation from cell lysates expressing tandem tagged FLCN-FNIP2;