Mechanism Of DTX3L Degradation Of Toll-like Receptor 3 Through The Ubiquitin Proteasome Pathway

Toll-like receptor 3 is a number of Toll-like family,which mainly participate nonspecific immunity and specific immunity and triggers immune response to microbial infection.The agonist of TLR3 directly change tumor cells characteristics such as promotes apoptosis or inhibit the growth of tumor cell and so on.This research clarifies that DTX3 L induces the degradation of TLR3 via the ubiquitin-proteasome pathway and the degradation disorder of TLR3 indirectly inhibits migration and invasion capabilities.Adopting the CRISPR Cas9 to knock out TLR3 gene,we constructed H1299 cells with knockout of TLR3.The results of cloning assay,cell migration and invasion assays showed that the absence of TLR3 gene improve the ability of growth and inhibits the ability migration and invasion.The protein synthesis inhibitors were used to treat H1299 cells under different conditions,followed by Western blotting.The results showed that inhibition of proteasome degrading enzyme activity could enhance the stability of TLR3 protein.To further investigate the molecular mechanism of TLR3 degradation,we performed immunoprecipitation,overexpression of Ubiquitin(Ub)and western bloting.The results showed that TLR3 molecules physically interact with Ub ubiquitin and Ub can induce degradation of TLR3.Thus,it preliminarily revealed that TLR3 is degraded by the ubiquitin-proteasome pathway in H1299 cells.Ubiquitin-protein ligase specifically binds to the targeted protein and regulates its stability.The results of immunoprecipitation experiments and bio-mass spectrometry showed that TLR3 interacted with Deltex-3-like(DTX3L);Then overexpressing DTX3 L of different quality in H1299 cells,discovered that the protein level of TLR3 is in dose.The truncated mutants of TLR3 is constructed and it were transfected.The H1299 cells are treated with MG132 and the possible ubiquitination site is analyzed as K97,K102;And two point mutants were transfected into cells,through the half-life experiments TLR3-K97 is the most stable;these results indicate that the ubiquitination site of TLR3 is K97.Overexpression of DTX3 L,the protein level of TLR3 evidently reduced.Through cell migration and invasion experiments,it was found that the reduction of TLR3 can evidently reduce the migration and invasion of H1299 cells;In conclusion,DTX3 L induces TLR3 degradation in H1299 cells via the ubiquitinproteasome pathway.The TLR3 ubiquitination site is located at the K97 residue which decreases TLR3 levels in cells can lead to the inhibition of cell proliferation,migration and invasion.