The Function And Mechanism Of The Interaction Between YopE And P65in The Pathogenic Process Of Yersinia Pestis

YopE which interacte with membrane lotus associated protein RhoGTPase by the144“arginine finger”, is one of six secretion protein in the pathogenic process ofYersinia pestis. YopE binds to the GTPase and promotes efficient GTP hydrolysis,make the enzyme from active state into the non-active state, resulting in degradationof the actin cytoskeleton, YopE causes rounding and detachment of infected cella inculture.Exchange of arginine144with alanine located in the arginine finger motif,results in an inactive form of YopE that can no longer stimulate GTP hydrolysis.One transcription factor that serves as a key responder to changes in theenvironment is Nuclear transcription factor NF-κB (the Nuclear factor-kappa B), anevolutionarily highly conserved signaling module that plays a critical role in manybiological processes, widely present in mammalian cells. NF-κB play a crucial role inmany biological processes, such as inflammatory response, immune response, celldifferentiation and apoptosis, and other stress reactions.Our Lab screening YopE and p65interaction by the yeast two-hybrid assay,suggesting that they interaction may play an important role in the infection process ofthe Yersinia pestis. By exogenous and semi-endogenous co-immunoprecipitation,GST-Pull Down experiment to verify that YopE interact with p65does exist. In theearly transfected, the reporter gene experiments revealed that YopE significantactivation of NF-κB pathway. GST-Pull Down experiment to determine that theinteraction domain of YopE and p65were:YopE (1-49aa,88-99aa) and p65(201-312aa), YopE (88-99aa) peptide plays an important role in interaction withp65.YopE/YopE (R144A) significantly promote the phosphorylation of IκBα andpromote its degradation, and significantly promote the dissociation of p65and IκBα,promoting p65into the nucleus, increase NF-κB downstream target genes, notably theanti-witherdeath and inflammatory response genes. YopE promote cell survival ofHepG2and Raw264.7cells, and YopE (R144A) significantly supressed apoptosisinduce by BAY. We constructed YopE/YopE (R144A) induction expression system,using the system we verify the above experimental results.In addition, We also study the effect of YopE/YopE (R144A) on HeLa cellbiological characteristics.The experimental results show that YopE/YopE (R144A)significantly inhibited ROS production caused by Rosup, arrest cell cycle andpromote the expression of p21.In summary, in this study we found that YopE promote NF-κB activation throughinteraction with p65, and improve the viability of the cells. YopE may have play animportant role in the early infection process of Yersinia pestis.