Structure And Function Of TBC1D7-TSC1 Complex And Castor1 Protein In MTOR Signaling Pathway

The mammalian target of rapamycin(mTOR)signaling pathway integrate diverse intracellular and extracellular signals to regulate organismal growth and metabolism.mTOR signaling pathway regulates many major biological activities and is implicated in many pathological conditions,including cancer,obesity,etc.In this paper,we chose the TSC(Tuberous Sclerosis Complex)protein complex and CASTOR1(Cytosolic arginine sensor for mTORC1 subunit 1)protein as the research object,which play important regulatory functions in the human mTOR signaling pathway,resolved their crystal structure and carried out a series of experiments to study the biological function of them.TSC complex acts as an important negative regulator in the human mTOR signaling pathway.TSC complex contains three subunits,TSC1,TSC2 and TBC1D7.We took the TSC1 and TBC1D7 protein as research objects,and resolved the crystal structure of TSC1(939-992)-TBC1D7(21-293)complex.The crystal structure reveals that TSC1 and TBC1D7 form a heterotetrameric structure in the form of 2:2,and the molecular interaction between TBC1D7 and TSC1 is mainly hydrophobic interaction.The MALSSEC experiment and analytical ultracentrifugation analysis demonstrated that of the TBC1D7(21-293)and TSC1(939-992)interact with each other in a progressive style.In vitro experiment and cellular experiments confirmed that TBC1D7 can stabilize the C terminal Coiled-coil structure of the TSC1 protein.CASTOR1 has been identified as the cytosolic arginine sensor for the mTORC1 pathway in mammalian cells,it can interact with GATOR2 and regulate mTOR pathway through singaling cascade.We determined the crystal structure of human CASTOR1 in complex with arginine.We found that CASTOR1 protein existed in the homodimerizc form,and two monomers interact with each other mainly through hydrophobic interaction and hydrogen bond.The crystal structure shows that the arginine molecule carved between the NTD and the CTD domains of CASTOR1.The GATOR2 binding site of CASTOR1 was found by structural alignment with Escherichia coli aspartate kinase,in vitro pull down assay using purified protein revealed that mutation of this surface patch disrupted CASTOR1's recognition and inhibition of GATOR2.DSC results shows that the stability of CASTOR1 protein was increased after it binding to arginine.Normal mode analysis shows that there is one kind of "open-close" movement between NTD and CTD of CASTOR1.These results suggest that arginine regulate the interaction with GATOR2 by affecting the relative motion between the NTD and CTD of CASTOR1 protein.The innovative points are as follows: First we sloved the crystal structures of TBC1D7(21-293)-TSC1(939-992)complex and CASTOR1 protein,some new protein-protein and protein-substrate binding sites were found,these structural findings explain how TBC1D7-TSC1 and CASTOR1 exert their biological function.Furthermore,this thesis propose some new interaction models and singaling transduction pathways,which can provide theroretical bases and new ideas for mTOR signaling related study.