TmTNF-? Mediated Tumor Cytotoxicity Induce TRAF2 Ubiquitination Degradation Via TNFR2

Tumor necrosis factor(TNF)is a proinflammatory cytokine,which has many biological effects.TNF-a is expressed on the cell membrane by 26 kDa transmembrane structure called tmTNF-?(transmembrane TNF-a),which can be cut to 17 kDa sTNF-a(soluble TNF-a)by metal matrix shear enzymes.Two Types of TNF-a through their specific receptor TNFR1 and TNFR2 mediate biological effect on tumor cells.Our previous study show that cell biological effects of tmTNF-? and sTNF-a are not the same.TmTNF-? can kill the tumor cell HL-60 with TNFR2 overexpression,which is tolerance to sTNF-a.The mechanisms are as follows: In TNFR2 signals,tmTNF-? not only can enhance the death signal molecules FADD,RIP1 and caspase-8,which can induce apoptosis,but induce TRAF2 and cIAP-1 ubiquitination degradation,downregulating anti-apoptotic molecule cFLIP,which inhibits the activation of NF-kB,reducing the survival signal and mediating the killing effect of tmTNF-? together.Ubiquitination is protein modification process after transcription,which is one of the important ways to regulate cellular protein function as well.The covalent bonding of ubiquitin is achieved through the sequential action of three key enzyme families: ubiquitin-activating enzymes E1,ubiquitin-carrier proteins E2,ubiquitin-protein ligases E3,who can specifically recognize target protein.Ubiquitin degradation refers to ubiquitin modified target protein in ubiquitin proteasome pathway(UPS)with the presence of ATP.It is reported that SIAH2 is 48 E3 ubiquitin ligase to TRAF2.Alignment of amino acid sequences of TNFR2 from various species show up to five highly conserved sequences within the intracellular region from one to five.These conserved sequences include module III,which include the amino acids responsible for inducing TRAF2 degradation and also contain two TRAF2 binding sites module ? and module V.We speculate that TNFR2 domain may recruit E3 ligase SIAH2 for TRAF2 ubiquitin degradation.But whether the killing effect is universal in tmTNF-? treatment via TNFR2,48 E3 ubiquitin ligase SIAH2 plays a role in TRAF2 ubiquitination degradation and the region recruits the 48 E3 ubiquitin ligase SIAH2 are still unknow.Thus,basing on the previous research,we sentence the scientific hypothesis: tmTNF-? can recruit the 48 E3 ubiquitin ligase SIAH2 in uncertain TNFR2 domain resulting in TRAF2 ubiquitination degradation for killing the tumor cells.This study aims at illuminating the universal of cell killing in tumor cells and further clarifying the molecular mechanisms in TRAF2 ubiquitination degradation through tmTNF-? stimulating mediated via TNFR2,which provides a new target and strategy for anti-tumor therapy.The main results are as follows: 1.TmTNF-? induced tumor cell HepG2 apoptosis via TNFR2(1)The choice of effect and target cellsFCM analyzed the expression of tmTNF-? was 43.7%,which can be served as effect cells.FCM tested hepatoma HepG2 cell line showed that high TNFR2 expression was up to 83.23±8.54% and low expression of TNFR1 was under 19.9±11.1%,which can be served as target cells.(2)TmTNF-? could kill HepG2 cell in mainly TNFR2 expression,while sTNF-a mediated HepG2 proliferationMTT assay showed that tmTNF-? effectively killed HepG2 cells with the cytotoxicity in 51.5%,which were in TNFR2 overexpression,while sTNF-a mediated HepG2 proliferation.(3)Transfection of TNFR1 siRNA had no effect on cytotoxicity in tmTNF-? treatmentMTT assay indicated that tmTNF-? could killed HepG2 cells with the cytotoxicity in 65.3% before transfecting TNFR1 siRNA,which had significant difference compared with the control group(P<0.001).After transfecting TNFR1 siRNA,the cytotoxicity in tmTNF-? treatment had no significant changes.Furthermore,the cytotoxicity in sTNF-a treatment had neither significant changes in lower killing rate 20.37±3.80%,compared with untransfect TNFR1 siRNA.(4)Transfection of TNFR2 siRNA could significant decrease the killing rateMTT assay indicated that tmTNF-? could killed HepG2 cells with the cytotoxicity in 67.2% before transfecting TNFR2 siRNA,which had significant difference compared with the control group(P<0.0001).After transfecting TNFR2 siRNA,the cytotoxicity in tmTNF-? treatment decrease to 22.4%(P<0.001),compared with untransfect TNFR2 siRNA.Furthermore,the cytotoxicity in sTNF-a treatment had neither significant changes in proliferation rate 20.03±5.78%,compared with no TNFR2 siRNA transfection.2.TmTNF-? can kill the tumor cells relying on TRAF2 ubiquitination degradation via TNFR2(1)TmTNF-?-TNFR2 could recruit E3 ubiquitin ligase SIAH2Co-IP indicated that E3 ubiquitin ligase SIAH2 could be recruited in tmTNF-?-TNFR2 signal complex,compared with the control group,while sTNF-a could significantly inhibit the recruitment of SIAH2 protein.(2)SIAH2 could promote TRAF2 ubiquitination degradation through the ubiquitin proteasome pathwaySIAH2 siRNA were transfected into target cells before.Co-IP showed that SIAH2 silence could not induce TRAF2 to form the polyubiquitin chain in tmTNF-?-TNFR2 signal complex,which pointed out that TRAF2 ubiquitination degradation relied on SIAH2 protein in tmTNF-? treatment.While under the effect of proteasome inhibitor MG132 in 4 hour,TRAF2 cannot form the polyubiquitin chain,which further confirmed that SIAH2 could promote TRAF2 ubiquitination degradation through the ubiquitin proteasome pathway.(3)TRAF2 ubiquitination degradation mediated by SIAH2 depended on protein synthesisWestern Blot analysis indicated that CHX could gradually reduce the E3 ubiquitin ligase SIAH2 protein in 0h,6h,12 h and 24 h,which downregulated TRAF2 ubiquitination degradation and inhibited the kill effect.(4)The III region(343-378)domain of TNFR2 could recruit SIAH2 for TRAF2 ubiquitination degradationFirstly,we successfully constructed TNFR2 mutants: ?424-439-TNFR2,?379-439-TNFR2 and ?343-439-TNFR2,which identified with Enzyme digestion and sequencing analysis.Then TNFR2 mutants were transfected in 293 T with 48 h.Co-IP indicated that SIAH2 could be recruited in ?424-439-TNFR2(deletion ? domain)and ?379-439-TNFR2(deletion ? and ? domain).Only in ?343-439-TNFR2(deletion ?,? and ? domain)could not SIAH2 be recruited.These above prompted that The III region(343-378)domain of TNFR2 could recruit SIAH2 for TRAF2 ubiquitination degradation.In summarize,our study show that tmTNF-? could kill HepG2 cell while sTNF-a mediated HepG2 proliferation,which rely on TNFR2 but not TNFR1 signal.Then SIAH2 could promote TRAF2 ubiquitination degradation through the ubiquitin proteasome pathway,which depend on protein synthesis.Last and most important,the III region(343-378)domain of TNFR2 could recruit SIAH2 for TRAF2 ubiquitination degradation.Our study clarify the molecular mechanisms in TRAF2 ubiquitination degradation through tmTNF-? stimulating mediated via TNFR2,which provides a new intervention targets and strategies.