Identification Of Latexin Complex And Its Mechanism In Anti-inflammatory Response

Latexin?LXN?,which containing 222 amino acids in length with molecular weight of 29kD,was first discovered and identified in the lateral neocortex of rats and acts as a marker of neurons in the lateral neocortex of developing rat brain.Functionally,LXN is a potent inhibitor of carboxypeptidase A?CPA?,and shares structural similarity with a cysteine protease inhibitor cystatin C,indicated LXN probably regulating tissue-specific protein degradation and turnover.More recently,the concept that LXN is a tumor-suppressor gene has been proved,as well as,LXN is also implicated in inflammation because it is expressed in macrophage and mast cells and can be induced by lipopolysaccharide,however,the knowledge on functions of LXN is extremely limited.My work mainly includes the following:In 2^nd chapter,the distribution and expression patterns of Latexin in mammals wereanalyzedbyWesternBlot,immunohistochemistryandcellular immunofluorescence techniques.In 3^rd chapter,the protein complexes were identified by proteomic strategies,and theinteractingproteinswereverifiedbyimmunoprecipitationand immunofluorescence.In 4^th chapter,the mechanism?s?how Latexin modulates inflammatory response in cancer cells were investigated.Firstly,we found that overexpression of Latexin inhibits NF-kB-Luc activity and inhibits TNF-?-induced inflammatory cytokine expression,such as ICAM1,VCAM1,and IL-6,suggesting that Latexin may inhibit inflammatory responses.Secondly,Latexin inhibits TNF-?-induced nuclear translocation and DNA binding activity of NF-kB,however,do not affect TNF-?-induced phosphorylation of IKK,indicating Latexin is downstream of the inhibitory inflammatory pathway.Since the translocation of NF-kB into the nucleus and activation are regulated by IkB?,Latexin plasmid or siRNA was employed to transfect in SKOV3 cells,we found that overexpression of Latexin inhibits TNF-?-induced degradation of IkB?,and thus prolongs the half-life of IkB?.It is well known that the degradation of IkB?was mainly performed by ubiquitylation in NF-kB signaling pathway.We found that overexpression of Latexin inhibited the ubiquitylation of IkB?,however,knockdown of Latexin promoted the ubiquitylation of IkB?.Interestingly,we found that after induction of TNF-?,the interaction between IkB?and RPS3 was enhanced.However,the interaction between IkB?and RPS3 was attenuated in Latexin overexpressed cell,suggesting that RPS3 mediates the ubiquitylation of IkB?,as well as the changes of IkB?ubiquitylation.To further explore the role of Latexin in affecting the ubiquitination of IkB?,we screened the protein HECTD1,which interacts with Latexin,by co-immunoprecipitation combined with mass spectrometry.HECTD1 is an E3 ubiquitin ligase.Using Western Blot technology,we verified the interaction between HECTD1 and Latexin,as well as RPS3,to form a complex.By knockdown HECTD1 and RPS3 in cells,the ubiquitylation of IkB?was found to be inhibited,indicating that HECTD1 and RPS3are involved in the ubiquitylation of IkB?.By analyzing the gene expression regulatory region of Latexin,it was found that it was regulated by the anti-inflammatory drug Retinoic Acid.Our experiment also proved that Retinoic acid induced the increase of Latexin expression,while HECTD1 expression was inhibited,which is the same as overexpression of Latexin.By knocking down Latexin in cells and treating with retinoic acid,the inhibitory effect of ubiquitination of IkB?was diminished,and the inhibition of inflammatory factors by retinoic acid indicates that Latexin mediates the anti-inflammatory process of retinoic acid.Our study found and demonstrated new mechanisms by which Latexin inhibits TNF-?-induced inflammatory responses:1)Latexin inhibits ubiquitination of IkB?;2)Latexin,HECTD1,and RPS3 form protein complexes,and Latexin down-regulates HECTD1expression;3)HECTD1 and RPS3 are involved in the ubiquitylation of IkB?;4)Latexin mediates the anti-inflammatory effects of retinoic acid.In conclusion,here,we reported that the ectopic expression of LXN could interacts with and down-regulates HECTD1 viaIkB?/RPS3pathway,thus contributing to increase the accumulation of IkB?in SKOV3 cells and finally lead to the inhibitory production of inflammatory cytokines.This study provides a novel mechanism by which LXN modulates inflammatory responses in cancer cells.