Studies On The Mechanism Of Transcription Factor EB(TFEB) Degradation By Ubiquitin Ligase Stub1

Objective:TFEB is one of the important transcription components of MiTF/TFE family,it is a factor of PI3k-Akt-mTOR signaling pathways downstream,its functional activity regulated by the mTOR receptor proteins.It is also involved in many pathological and physiological process,and is closely related to the occurrence of a lot diseases'development.In lysosome function,cell autophagy and lipid metabolism and energy metabolism it plays an important role,and also involved in cellular stress,immune response,clear,mitochondria,biological synthesis processes in cell.Under normal circumstances,TFEB in cytoplasm,function is inhibited,this is because in the condition of normal function,lysosome function is normal,dissolving enzyme on the surface of the body surface small molecules such as Rag proteins,GTP enzyme can recruit mTORC1 protein to the surface of a lysosome and activate mTORC1,at the same time,these small molecules can also recruit TFEB protein to the surface of a lysosome,here,the activity of mTORC1 can make TFEB phosphorylation,phosphorylation TFEB after no longer has the activity,function is inhibited,combined with inert protein 14-3-3,stayed in the cytoplasm.However,in hunger,stress,pressure,lysosome dysfunction,abnormal mitochondria damage and bacterial infection,TFEB function will be activated,at this time of mTORC1 release from the lysosome membrane and become less active,inactived mTORC1 no longer phosphorylation TFEB,in the related of the action of calcium ions TFEB occur to phosphorylation and have activity,free access to the nucleus,accordingly gene transcription and its corresponding function into full play.With the further understanding and research of TFEB,we found that TFEB is a substrate of ubiquitin STIP1?Stub1,STIP1 homomeric and protein 1 containing u-box region,which is also known as CHIP since its C end contains a protein that can interact with HSC70?.E3 ubiquitin ligase Stub1,plays an important role in substrate recognition and catalyzing ubiquitin transfer as well as determining the diversity and selectivity of ubiquitin substrates.Literature has shown that Stub1 can regulate the activity of TFEB,Stub1 priority combination of phosphorylation TFEB and through ubiquitin proteasome pathway make its degradation,making phosphorylation of TFEB and the non-phosphorylation of TFEB formed the different source of dimer decreased,and thus increase the number of active TFEB and nuclear translocation increases,thus regulate autophagy transcription and lysosome gene level.In other words,E3 ubiquitin ligase Stub1 can regulate autophagy,mitochondrial,lysosomal biosynthesis and other functions by regulating TFEB activity.However,as the effect of Stub1 on TFEB and degradation mechanism are still unclear,we have conducted in-depth research and exploration on this,which is the main research purpose and content of this topic.At the same time,we also found that VCP?Valosin-containing protein,including valerian casein peptide protein?is a kind of important type?adenosine triphosphate?ATP?enzyme,a kind of distribution of a wide range of atpase,N and C terminal mainly interact with proteins or supplementary protein,can combine many ubiquitin chains,responsible for identifying the substrate.Conservative,abundant in cells and structure in the endoplasmic reticulum associated protein degradation and autophagy plays an important role,it can be protein molecules from protein components,endoplasmic reticulum,and chromatin structure of large cell organelles membrane separate,promote its degradation by proteolytic enzyme,is essential for protein ubiquitin chemical degradation enzymes,exist in all eukaryotes and bacteria.In process of ubiquitin ligase Stub1 degradation substrate of TFEB,ligase Stub1 with combination of ubiquitin E2?enzyme?and connect the ubiquitin to target protein TFEB,secondly Stub1 needs to combine with VCP proteins,and then identify the substrate TFEB and transferred to the26s proteasome,26s proteasome can specifically identify the substrate protein with ubiquitin tag,and make it degradation.VCP proteins have been shown to be involved in a variety of cellular activities,including mitosis,homogenous membrane fusion,nuclear transcriptional regulation of endoplasmic reticulum related degradation mechanisms,and ubiquitin-dependent protein degradation.It has been reported that VCP is associated with apoptosis,invasion and metastasis of cancer.SVIP?small VCP-interacting protein?,is a kind of mutual combination of VCP protein and role of a small molecule protein,is associated with the secretion of very low density lipoprotein and transshipment.Studies have found that SVIP have the structure which contain of domain VIM that cancombined with VCP,Stub1 have the structure containing domain VBM that also can be combined with VCP protein,but the domain structural of VIM combined and interacted with VCP protein stronger than the domain structure of VBM,namely structure domain VIM can give priority to with VCP protein and form competitive inhibition on the VBM structure domain,therefore,SVIP can competitive inhibition Stub1 combined with VCP protein,inhibit the effect of Stub1toTFEB protein,but the result need to be studied and verified.Therefore,if we can clarify the effect and degradation mechanism of TFEB by Stub1 and whether SVIP can inhibit this effect,we can regulate the activity and function,synthesis and degradation of TFEB in many ways,so as to achieve the corresponding purpose.So we studied the other substrates of Stub1 and the mechanism of degradation,found that it is effect with the features of the domain of the substrates,then analysis the domain structure of could be combined with TFEB function and role,and build the missed function domain of TFEB mutants,observe Stub1 effect of wild TFEB and mutants.Through study the effect of degradation mechanism by Stub1 to TFEB,provide new theoretical basis for TFEB regulation,for the treatment of diseases and provide new ideas and methods of scientific research.Methods:?1?Using sequence alignment and molecular biology software,identified amino acid sequences on TFEB that might be associated with Stub1.?2?The Mutation1and Mutation2 mutants of TFEB were constructed by site-directed mutation technology,and the TFEB^??218-250?-Mutation1 and TFEB^??176-246?-Mutation2 mutants were detected and identified by RT-PCR.?3?Westernblot and immunofluorescence microconfocal technique were used to observe the expression of TFEB wild type and two mutant types in cells.?4?Westernblot and immunoprecipitation were used to observe the effect and binding of TFEB wild type and two mutant types to Stub1.?5?Using actinomycetin assay and ubiquitination detection to observe the degradation and ubiquitination of Stub1 to TFEB wild type and two mutant types.Results:?1?The TFEB amino acid sequences that may be associated with Stub1were found by sequence alignment and molecular biology software.?2?The TFEB^??218-250?-Mutation1 and TFEB^??176-246?-Mutation2 mutants of TFEB were successfully constructed by using site-directed mutation and RT-PCR technology.?3?Western blot and immunofluorescence results showed that both TFEB mutants and TFEB wild bodies can be expressed stably in cells,and they can be expressed in cells together with Stub1.Most of them are co-located in the nucleus,and some of them are co-located in the cytoplasm,which provides a basis for subsequent experimental observation.?4?Westernblot results and immunoprepitation observation showed that Stub1 had an effect on TFEB,and had different binding conditions for the wild type TFEB and the two mutants.Stub1 had an obvious binding effect on the wild type TFEB,while it had a weak binding effect or no effect on TFEB^??218-250?-Mutation1 and TFEB^??176-246?-Mutation2mutants.?5?Stub1 have the different degradation and ubiquitination effects on the wild TFEB and two mutants were also observed by actinomycin assay and ubiquitination assay.Conclusion:TFEB is a substrate of Stub1,Stub1can combination,ubiquitin and degradation of TFEB.For wild type TFEB,Stub1 has the obvious combination and ubiquitin degradation of it.For the two mutants of TFEB^??218-250?-Mutation1 and TFEB^??176-246?-Mutation2,Stub1 have weaker or no effect of them,such suggesting that Stub1 by acting on the specific field of TFEB to combination of it and then have the corresponding function.It is the important foundation of TFEB's degradation by Stub1,and this is also the important mechanism for SVIP competitive Stub1 inhibits the degradation of TFEB.This provides a new molecular basis for TFEB regulation,as well as a new therapeutic targets of some diseases and research progress of science.