Heat Shock Protein 70 Combined With Traf6 Regulate Lps-activated Nf-¦Êb Signaling Pathway

HSP70,the member of HSP70 family,is consisted of N-terminal ATPase domain and C-terminal domain,which includes polypeptide binding domain and variable region.It has the function of molecular champerone,is the 'tumor antigen' or 'vector of tumor antigen'in tumor immunity and also regulates the signal pathway of apoptosis. TRAF6 was found by yeast two-hybrid assay using CD40 peptide as the probe in 1996. It is consisted of Ring finger domain,Zinc finger domain in N-terminal and Coiled-coil domain,TRAF-C domain in C-terminal. TRAF6 recognizes and binds to CD40 or the peptide domain of cytoplasmic portion of TRANCE-R(PXEXX Ar/Ac,where Ar is an aromatic and Ac an acidic residue).Upon stimulation ,interaction of TRAF6 with CD40 or TRANCE-R activates NF-κB transcription factors and mitogen-activated protein (MAP) kinases. A unique feature of TRAF6 is that, unlike other TRAFs, it also participates in IL-1R/TLR family signaling. LPS (Lipopolysaccharide,also named endotoxin) is the principal component of the outer membrane of Gram-negative bacteria. Monocytes orchestrate the innate immunity response to LPS by expressing a variety of inflammatory cytokines. LPS initiates cells through TLR4,which binds to MyD88, then IRAK binds with the complex and activates TRAF6.The ubiquitination of TRAF6 is needed during the process,then TAKl,a member of MAP3K,is activated by ubiquitinated TRAF6 and phospherylates IKK β .At last NF-κB is activated by phospherylated IKK β . NF-κB was first reported by Sen and Baltimore in 1986. It is one of the key point in the regulation of gene expression of pro-immunity cytokines,which induces the expression of pro-immunity cytokines,chemokines,cell adhesion molecules,and etc.In these years,the signal transduction system of NF-κB has been understanded relatively.Furthermore the initiated process,the regulation of gene expression and the relation with human diseases also have been recognized gradually.But there are still many aspects of the signal network need to be studied more deeply.The study aims at invatigating the regulation of HSP70 on the NF-κB pathway initiated by LPS.The deleted mutant genes of HSP70 or TRAF6 were inserted into eukaryotic expression plasmids. The recombinant plasmids were transformed into HEK 293 cell and then we study the binding of HSP70 and TRAF6.As a result we find that the ATPase domainand peptide binding domain of HSP70 bind with the TRAF-C domain of TRAF6,and HSP70 regulates NF-kB signal pathway initiated by LPS through binding with TRAF6.Furthermore,for solving hard transformation,we obtain the Raw264.7 cells which express HSP70 stably by transforming HSP70/pcDNA3 into the cells through lipofectmine and then adding G418.Whereafter we use the cells to study the effect of HSP70 on NF-kB signal pathway initiated by LPS,in the end we realize HSP70 inhibits the activation of NF-kB spathway.