Amino Acid Sensor Lab Report

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Aim 2: How does folliculin detect amino acids to regulate mTORC1 activity? Here, the goals are to elucidate the mechanism of action that enables folliculin to function as an amino acid sensor.

2.1. Determine the amino acid reservoir folliculin senses. Evidence points FLCN as a novel amino acid sensor. To resolve the biochemical mechanism FLCN employs, we need identify the specific amino acid reservoir FLCN senses. Lysosomes’ are the main amino acid reservoir and the ‘inside-out’ mechanism is a known lysosome sensing pathway that transduces luminal amino acid signals to the surface via the v-ATPase then relay signals to the Rag complex and activate mTORC1 (zoncou). Given FLCN-FNIP2 complex localizes to the lysosome during starvation and silencing
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How does FLCN interact with amino acids? Since FLCN may function of FLCN as an amino acid sensor gives rise to the understanding how FLCN would interact with amino acids. Not all amino acid sensors act the same, some directly bind to amino acids, while others relay the signals indirectly through protein interactions. Identifying FLCN amino acid reservoir allows us to determine how FLCN interacts with amino acids by providing insight whether the interaction is direct or …show more content…
Does FLCN require FNIP to sense amino acids? Folliculin exists in a complex by binding to either of its novel interacting proteins, FNIP1 or FNIP2. Truncation mutations commonly seen in FLCN gene of BHD patients promote the loss of binding with FNIPs (). Moreover, FLCN must exists as a complex to interact with the Rag GTPases because individual purified protein components abolish FLCN-FNIP interactions with RagA/C (Tsun). Further biochemical evidence discovered FLCN-FNIP GAP activity can only occur as a complex because when expressed individually potentiates its GTP hydrolysis function (Tsun). Interestingly, FLCN-FNIP1 complex expresses less GAP activity than FLCN-FNIP2 and, even, elicits the majority of its GAP activity upon RagD. However, FLCN-FNIP2 holds no preference and strongly initiates GTP hydrolysis on RagC and RagD, greater than FLCN-FNIP1; although, its greatest GAP activity upon RagC (Tsun). Since FLCN requires its binding partner to interact with the Rag complex and to perform its GAP activity, does FLCN requires its binding partners, FNIP1 or 2, to recognize amino acids to initiate mTORC1 activation? To begin, I will observe: FLCN lysosomal dissociation and FLCN GAP activity in HEK293 cells with FNIP1 or FNIP2 knockdown executed by shRNA against FNIP. To track FLCN amino acid-induced lysosomal dissociation, these cells are also designed by crispr methods to express endogenous fluorescently tagged FLCN and LAMP1. HEK293T cells will be seeded directly on

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