OTU Domain–containing Ubiquitin Aldehyde-binding Protein 1 Suppresses MTOR Complex 1 Activity By Deubiquitinating Its Inhibitor DEPTOR

During the growth and development of organism,the response to surrounding microenvironment is crucial to cells.According to the different niche,cells have various physiological responses to ensure the normal physiological functions of the organism.Numerous studies have shown that mTORC1(Mammalian target of rapamycin complex 1)plays an extremely important role in this process.Amino acids,glucose,and various growth factors are able to activate mTORC1,which subsequently regulates a range of downstream responses,such as cell growth,proliferation,protein synthesis,and lipid metabolism.Since the abnormality of the mTORC1 signaling pathway can lead to series of diseases,the research on the regulation of the mTORC1 signaling pathway by the organism is also critical.At present,the regulation of the activation of mTORC1 signaling pathway has been studied concentratively and thoroughly,but there are less studies on its inactivation regulation mechanism.Our previous data showed that the mTORC1 signaling pathway can not keep in an activated state for prolonged stimulation of amino acids,but shows a tendency to increase first and then decrease.In order to find out the mechanism,we detected the expression levels of mTORC1 signaling pathway-related proteins and found that only the DEPTOR(DEP domaincontaining mTOR-interacting protein)expression level is controlled by amino acid signals,and significantly the change has negative correlation with mTORC1 activity.However,the study of the relationship between amino acid signal and the stability of DEPTOR protein have not been reported yet.Based on this,we have focused on the inactivation mechanism of the mTORC1 signaling pathway under long-term amino acid stimulation conditions.We found the DEPTOR,a novol and major negative regulator protein in this process,and then we identified the DEPTOR-specific deubiquitinating enzyme OTUB1(OTU Deubiquitinase,Ubiquitin Aldehyde Binding 1).We found that OTUB1 can specifically remove the ubiquitination of DEPTOR,and this deubiquitination function does not depend on the enzyme activity but through a non-canonical pathway.In this process,OTUB1's 88 th aspartate binds to E2(such as UBC13)and inhibits its function,blocking the formation of ubiquitylation chains and inhibiting ubiquitination of substrate proteins.Through structural analysis,we found that OTUB1 directly interacts with DEPTOR through its unique N-terminal domain,and the interaction is regulated by amino acid signals.The stimulation of amino acid signals can enhance the binding of them.OTUB1 can increase the protein stability of DEPTOR in both exogenous and endogenous conditions,prolonging the half-life of DEPTOR.In addition,we also examined the effect of OTUB1 on the mTORC1 signaling pathway.We found that OTUB1 inhibits the activity of the amino acid signal-mediated mTORC1 signaling pathway by deubiquitination of DEPTOR and stabilization of DEPTOR.At the same time,we had some functional studies on OTUB1.The experimental results proved that OTUB1 can influence autophagy,cell proliferation,and cell size through DEPTOR.In summary,this paper has discovered the molecular mechanism of the transition of the m TORC1 signaling pathway from hyperactivation to inactivation under longterm stimulation of amino acid.The key protein DEPTOR was found,and the stability mechanism of DEPTOR under amino acid conditions was also revealed.The DEPTOR deubiquitinating enzyme OTUB1 was identified,further elucidating the important role of ubiquitination modification in the regulation of mTOR signaling pathway.This paper provides a theoretical basis for the study of various m TOR-related diseases,and also prepares a new direction for identifying new diagnostic and therapeutic targets to diseases.