The Function And Mechanism Of E3 Ligase TRIM21 Upregulate TGF? Signal Pathway

Cancer is a great threat to human life,and is often fatal,we are still only prevent cancer.The research for oncogene is on process early,however few breakthrough treatment results,so the cancer research is still a urgent work.According to previous research,TGF-? signaling pathways plays an important role in cancer development.In the early tumorigenesis,TGF-? can inhibit the occurrence of tumor,inhibit the growth of cancer cells,and promote cell apoptosis.In the development of malignant tumor,TGF-? can induce tumor cell growth inhibition and escape to immune surveillance,plays a role in promoting the formation of tumor,ectomesenchymal transformation can promote epithelial cells invasion,and angiogenesis tumor metastasis.TGF-? signaling pathways play the role of a double-edged sword,so the research on the mechanism in this signaling pathway is critical,is an opportunity to find new targets for cancer treatment.Moreover,protein of ubiquitination modification has been widely studied,found plays an important role in the degradation of protein,activity modefication,location and metabolism,and participate in a variety of physiological activity,such as signal transduction,cell growth,proliferation,apoptosis,migration and immune response.Therefore,protein ubiquitin modification research has the vital significance,study its role in the TGF-?signaling pathways,can reveal the occurrence of cancer development mechanism.In this research,we analyzed the cancer patient data,then we found that E3 ubiquitin ligase TRIM21 is abnormal amplification,deletion or mutation in different cancers,that means TRIM21 plays different roles in different cancers.Next,we found that the downstream factor and reportgene of TGF-? signaling pathway are significantly reduced by down-regulating gene TRIM21.Over-expression of wild-type TRIM21 are able to enhance TGF-? signaling pathway,while TRIM21 inactivation isoform which was found no such fuction,TRIM21-WT is also capable of enhancing apoptosis and growth inhibition of TGF-? cell.Then,we continue to work on TRIM21 is how to increase TGF-P signaling pathway,we simultaneously overexpressed TGF-? signaling pathway in the core factor and TRIM21,Co-Immunoprecipitation experiments,we found TRIM21 can interact with Smad4,this phenomenon is also tested in vitro pull down assay and endogenous levels.Next,we used the method by denaturing ubiquitination and found overexpression of wild-type levels of TRIM21 to promote the ubiquitination level of Smad4,but inactive isform is not.In TGF-? signaling transduct process,Smad4 and Smad2/3 form a complex shuttle into the nucleus together,regulate downstream genes,we found that TRIM21 can facilitate this process,reduced the expression of TRIM21 make weaken Smad4 affinity with Smad2/3,and Smad4 reduce accumulation in the nucleus.In addition,we found that deubiquitination enzyme USP15 can influence ubiquitination level of TRIM21 by mass spectrometry.We found that USP15 and TRIM21 can interact with each other,endogenous co-immunoprecipitation experiment also test the interaction,USP 15 can also combined with TRIM21 in vitro.Then,we make a lot of deletion constructs,identified domain responsible for the interaction.Finally,the evidence thet internal and external interaction determined to USP 15 downregulate ubiquitination of TRIM21 protein rely on its ubiquitination enzymes activity.In summary,in the study,we found that TRIM21 can enhance TGF-? signaling pathway activity,and their interact with Smad4 then increase its ubiquitination level,affect the formation of Smad4/2/3 complex and the location in the nucleus.In addition,identified USP 15 work as deubiquitination enzymes of TRIM21,able to interact with it and reduced its ubiquitination levels of TRIM21.We are still working on USP 15 can affect which function of TRIM21.