1.Effect Of EST3 On TmTNF-α-induced Cytotoxicity And Its Underlying Mechanism 2.Correlation Of TmTNF-α Trimerization And Its Conservative Sequences

Part 1:Effect of EST3 tmTNF-a-induced cytotoxicity and its underlying mechanism.Tumor necrosis factor-a (TNF-a) as a multifunctional cytokine with a wide range of biological effects, can be involved in cell proliferation, differentiation, killing tumor cells and plays a role in immune regulation, etc.; And it is also an inflammatory mediator involved in fever and chronic infection. TNF-a exists in two biologically active forms, including 17kDa secreted TNF-a (sTNF-a) and the 26kDa transmembrane TNF-a (tmTNF-a). tmTNF-a is a precursor of sTNF-a, expressing mainly in activated monocytes and immune cells. tmTNF-a releases sTNF-a under the action of the TNF-converting enzyme (TACE/ADAM17).Preliminary work of our group confirmed that the two types of TNF-a induced significantly different type of cytotoxicity. Two types of TNF-a can bind TNFR, but induce cell death in the different way. tmTNF-a primarily induces apoptosis while sTNF-a induces necrosis. Further using of suppression subtractive hybridization, we obtained two types of TNF-a activated fragments which were differentially expressed genes and they were specifically related to tmTNF-a and we called it "EST3". Compared by the Blast, we confirmed that EST3 was belonged to S2 ribosomal protein family. But, the biological function and the mechanism of the molecule on tmTNF-a is still unclear. Construction of the positive and negative EST3 and the EST3 mutants in this study is also to discuss the important functional domains of EST3, which may have some effect on the cytotoxicity.The main results are as follows:一. Construction and identification of EST3 and EST3 mutants:We successfully constructed EST3 recombinant plasmid and EST3 mutants by using overlap PCR technology--EST3-pIRES2-GFP (wt EST3),Δ59-91-EST3-pIRES2-GFP (Lack of ribosomal protein S5 marker locus),Δ42-105-EST3-pIRES2-GFP (deletion Double-stranded mRNA binding domain of S5) andΔ16-22-EST3-pIRES2-GFP (Lack of tyrosine kinase phosphorylation site). It was confirmed that there were absence of any other point mutations, frame-shift and deletion mutations but expected mutations by restriction enzyme digestion, PCR and sequencing identification.二. EST3 in the positioning of eukaryotic cells:HepG2 cells were transfected with pTriEx-EST3-EGFP fusion expression plasmid and stained with PI. The results showed that: EST3 distributed in the cytoplasm and the nucleus.三. A model for Chemotherapy drugs induced cytotoxicity on HepG2 and K562: Using MTT assay to detect the cytotoxicity on HepG2 and K562 cells under different concentrations of cisplatin (CDDP),we decided the IC50 of the two types of cell lines were 15μg/ml and 13μg/ml. Because the two cell lines are moderately sensitive to CDDP, we chose cisplatin as the experimental drug in this model.四. Enhancement of the expression of EST3 can increase cytotoxicity on HepG2 of tmTNF-αand CDDP:1. The expression of EST3 recombinant plasmid while transfected into HepG2:Our early work suggests that there is little EST3 in HepG2 cells and HepG2 is insensitive to tmTNF-α. Therefore, in this study we transfected pTriEx-EST3(+) into HepG2 cells in order to observe the increased sensitivity of target cells on tmTNF-α. A specific band about 32kD was detected in wide type EST3 group by Western blot,which was significantly darker compared with the untransfected group, suggesting EST3 expressed successfully.2. Enhancement of the expression of EST3 can promote tmTNF-αand CDDP-mediated cytotoxicity:The results of MTT assay showed that, compared with the control group, the cytotoxicity of transfected HepG2 with pTriEx-EST3(+) was significantly increased (approximately by 20%) with the treatment of tmTNF-αand CDDμP. It implies that EST3 can enhance the sensitivity of tmTNF-αand CDDP on HepG2 target cells.3. Enhancement of the expression of EST3 can promote tmTNF-αand CDDP-mediated apoptosis:The results of Hoechst staining assay showed that:compared with the control group, high expression of EST3 can increase tmTNF-αand CDDP-induced apoptosis in HepG2 cells. The nuclei turned out to be chromatin condensation, deformation and fracture of the fluorescent fragments. The results suggest that high expression of EST3 can improve the sensitivity of HepG2 cells to apoptosis.4. Enhancement of the expression of EST3 can promote tmTNF-a and CDDP-mediated Gl cell apoptosis:Flow cytometry analysis showed that, with tmTNF-a and CDDP treatment, compared with the control group, HepG2 cells with high expression of EST3 showed significantly higher sub-G1 peaks. Meanwhile, G1 phase cells also significantly reduced (about 30%). It hints that EST3 can promote tmTNF-a and CDDP-mediated apoptosis of HepG2 cell and the apoptosis almost occurs in the G1 phase.五. Inhibition of EST3 expression can reduce tmTNF-a and CDDP induced cytotoxicity on the K562:1. Negative EST3 expression in K562 cells:Our early work suggests that there is many EST3 in K562 cells and K562 is sensitive to tmTNF-a. Therefore, in this study we transfected pTriEx-EST3(-) into K562 cells in order to observe the decreased sensitivity of target cells on tmTNF-a. A specific band about 32kD was detected in wide type EST3 group by Western blot,which was significantly lighter compared with the untransfected group, suggesting EST3 expressed successfully.2. Inhibition of EST3 expression can reduce tmTNF-a and CDDP induced cytotoxicity of K562 cells:The results of MTT assay showed that, compared with the control group, the cytotoxicity of transfected K562 with pTriEx-EST3(-) was significantly decreased (approximately by 29%and 31%respectively) with the treatment of tmTNF-a and CDDP. It implies that inhibition of EST3 expression can decrease the sensitivity of tmTNF-a and CDDP on K562 target cells.六. The relationship between EST3 domain and its function for promoting sensitivity of target cells to tmTNF-a and CDDP:1. Effect on the killing of HepG2 by tmTNF-a and CDDP after transfected by EST3 and EST3 mutants:Respectively, we transfected HepG2 cells with wtEST3,Δ16-22-EST3,Δ59-91-EST3 andΔ42-105-EST3 mutants to observe the impact of killing of tmTNF-a and CDDP. The results showed that the killing efficiency of tmTNF-a and CDDP on HepG2 cells transfected with wtEST3 orΔ16-22-EST3 mutant increased (13.8%and 21.3% respectively), but the killing rate between the two groups had no difference,compared with the control group. But the killing efficiency of tmTNF-αand CDDP on HepG2 cells transfected withΔ59-91-EST3 andΔ42-105-EST3 HepG2 was almost the same as the control group. These two mutants above of EST3 completely lost the function of promoting cytotoxicity. It implied the the functiion of EST3 on promoting the killing of target cells by tmTNF-αand cisplatin may be associated with ribosome function, and its tyrosine kinase phosphorylation site has nothing to do with its function.2. Effect on the apoptosis of HepG2 by tmTNF-αand CDDP after transfected by EST3 and EST3 mutants:Under the effect of tmTNF-αand CDDP, compared with the control group the transfected HepG2 cells with wtEST3 andΔ16-22-EST3 mutant went to apoptosis, and the nuclei showed chromatin condensation, deformation and fracture of the fluorescent fragments. Because of the emergence of similar cells, there was sub-G1 peak during the apoptotic course. But, after transfected with A59-91-EST3 and A42-105-EST3 mutants, the extent of apoptosis in HepG2cells significantly decreased, and the sub-G1 peak of the mutant-transfected cell group had no significant difference from the control group. this phenomenon Fully demonstrated that EST3 possibly through its protein translation rather than through the influence of kinase function, improved tmTNF-αand CDDP induced apoptosis of HepG2 cells.七. EST3 can promote proapoptotic protein synthesis, such as caspase-3, to increase tmTNF-αand CDDP induced cytotoxicity of HepG2 cells:1.Inhibition of protein translation by using cycloheximide (CHX):The experiment suggested that the role of ribosomal protein EST3 on promoting apoptosis had something with protein synthesis. In order to further confirm this relationship, we used the protein synthesis inhibitor CHX to function with EST3 to get a further confirmation on EST3 for promoting tmTNF-αand CDDP induced cytotoxicity.2. The impact of EST3 on caspase-3 expression in HepG2 cells:The results of western blot showed that,compared with the control group,if transfected with EST3,HepG2 cells could promote the expression of caspase-3; On the other hand, if inhibited EST3 expression, caspase-3 expression in the transfected cells was significantly reduced.It hint that EST3 may function to promote the pro-apoptotic molecules such as caspase-3 expression, in the target cells,thereby enhancing the target cells of anti-tmTNF-a and CDDP sensitivity.CONCLUSION:①high expression of EST3 can promote tmTNF-a and cisplatin (CDDP) induced cytotoxicity and apoptosis of HepG2;②inhibition of EST3 expression in K562 cells can reduce the cytotoxicity of tmTNF-a and cisplatin;③EST3 promoting tmTNF-aand CDDP induced cytotoxicity is associated with ribosome functional domain, and has nothing to do with its tyrosine kinase phosphorylation site;④EST3 can promote synthesis and expression of caspase-3. These results suggest that EST3 can promote certain pro-apoptotic proteins such as caspase-3 expression, thereby enhancing the target cells of anti-tmTNF-a and cisplatin sensitivity.Part 2:Correlation of tmTNF-a trimerization and its conservative sequencesTumor necrosis factor superfamily, (TNF-SF), including TNF-0, LT-β,CD27L, CD30L, CD40L, CD95L (FasL),41BB, OX40L, TRILL 20aTNF-and etc., Their common characteristic is the formation of trimer, although the amino acid sequences, spatial configuration, receptor and biological functions are not the same with each other. Yet a common feature of these membrane proteins is the tendency on the trimer, and trimer is the biologically active form of these molecules. They share the highly conserved extracellular amino acid sequence in their primary structures. Therefore, we hypothesized that members of the TNF superfamily and highly conserved sequence are likely to be common features for their trimer formation.Tumor necrosis factor (TNF-a) is a member of the TNF-SF, involved in inflammation, immune response, anti-tumor and endotoxin shock. Demonstrated by Molecular Bioinformatics, the highly conserved sequences are right in the connecting parts of tmTNF-a monomers, which includes 9-16,48-62,119-132,151-157 amino acid fragment. To elucidate the conserved sequences of TNF superfamily and TNF superfamily relationship between the structure of the trimer, the subject of these conserved sequences through mutation of the key amino acid sites may be involved in trimer formation to observe the formation of trimer and biological activity effect to confirm the formation of the trimer in the key amino acids. The results of this study are as follows:一. Construction and identification of tmTNF-αand tmTNF-a mutant recombinant plasmids:Using overlapping PCR, synthesizing fragments for the missing tmTNF-a mutants (Δ54-59,Δ119-126,Δ119-124,Δ119-122,Δ119-120,Δ115-118) and point mutants (H15P, G54F, G54N, I58A, I58Y, G121F, F124A, F124S, L126T, F152S and G153F), After colony PCR, restriction enzyme digestion and DNA sequencing, we confirmed mutations as expected without other point mutation, frameshift and deletion mutations, suggesting that mutants were successfully constructed.2. Transfection and expression analysis of tmTNF-a and its mutants:After transfection 293T cells with tmTNF-a and its mutants, we can detect GFP expression as tmTNF-a and its mutant transfection efficiency, also we can use tmTNF-a antibody to detect the expression of tmTNF-a and its mutants. The results showed that the transfection efficiency of each mutant, compared with the expression of wild-type tmTNF-a, had no significant difference, except I58Y, F124A, F121S,and G153F, suggesting that these mutants almost had no effect on protein synthesis and expression on the membrane.3. The biological function of conservative amino acid mutated tmTNF-a:On the using cytotoxic experiments confirmed that the cytotoxic effect of tmTNF-a mutants missing fragments fromΔ54-59 andΔ119-126's, almost entirely disappeared. About theΔ119-126 missing fragment, from the lack of reduction of 8 to the lack of two amino acids, there was still no cytotoxicity of tmTNF-a; Conserved sites of point mutations in the H15P, G54F, G54N,158Y and L126T mutation of tmTNF-a coused a completely loss of cytotoxicity. While about the other sites mutation on tmTNF-a, the killing effect reduced different degrees (40%-80%inhibition). Implying tmTNF-a conserved sequence mutation can affect the biological function of tmTNF-a. 4. Effect on their trimer formation of conservative amino acid mutations in tmTNF-a: Chemical cross-linking tmTNF-a and its mutants to detect the effect on their trimer formation. The results showed that wild-type tmTNF-a, G54N-tmTNF-a and I58A-tmTNF-a can form clearly dimer and trimer after cross linking, and H15P, G54F and 158Y of point mutants and all the deletion mutants can form only in the single position of specific bands, suggesting that trimer formation of tmTNF-a is due to not only the conserved sequence mutation sites, but also the nature of amino acids. It implied that the mutant tmTNF-a killing function may be related to the formation of the trimer.五. tmTNF-a conservative site mutation on the release of sTNF-a and its biological function:1. The supernatant of transfected cell with tmTNF-a and and its mutants has weakened cytotoxicity:Collecting the supernatant of tmTNF-a and its mutants after transfected for 48 hours and the cytotoxicity of the supernatant was observed by MTT. The results showed that in the culture supernatant of wild-type tmTNF-a transfected cell group, the cytotoxicity significantly high, while in the mutants transfected cells groups, the culture supernatant of lost the killing effect(H15P, G54F, I58Y and F124S);These results suggest that, tmTNF-a conservative mutations significantly affect the cytotoxic effect of the supernatant and sTNF-a monomer lost their function, or tmTNF-a monomer can not be digested by TACE into the secretory molecules.2.. Effect on the release of sTNF-a on tmTNF-a conservative amino acid mutations:In order to confirm the speculation, we used ELISA to assay the sTNF-a.content in cell culture supernatant of tmTNF-a and its mutants transfected cells.The results showed that the supernatant of tmTNF-a transfected cell group had a large quantity of sTNF-a (up to 593.29pg/ml), while the supernatants sTNF-a of the deletion mutants group was not detected;and in the point mutation group, H15P, I58Y and F124S not detected, and G54F, G54N, and I58A had a detectable secretion of sTNF-a and had no statistical difference., compared with the wild type tmTNF-a,.It hint that tmTNF-a may be hydrolyzed to sTNF-a due to lack of conserved sequence, or some mutation, which may be related to the loss trimer formation. 六. the biological effect of FasL and FasL mutants corresponding to the conservative sites:1. Construction and identification of FasL and FasL mutant recombinant plasmids: These results above confirmed that His 15 and Gly54 are the key conservative sites in tmTNF-αtrimer formation, so we have the corresponding sites in the FasL, and we constructed for the same sites mutants (H49P and G88F), by colony PCR, restriction enzyme digestion and DNA sequencing, we confirmed the expected mutation in addition to outside without other point mutation, frameshift and deletion mutations, suggesting that mutant was successfully constructed.2. Annexin V-PI assay for detecting the mutant FasL cytotoxic effect:Using 5mM PHA stimulated Jurkat T cells for 12 hours, the Fas expression rate was about 30% and AICD rate was 35%, and thus using this state of Jurkat T cells as target cells and FasL and its mutant transfected 293T as effector cells, mix them to observe the induction of apoptosis. The results showed that:only the wild-type FasL transfected cells had killing effect, and its mutants lost cytotoxicity, suggesting that the conservative sequences of TNF superfamily family members play as important role.in maintaining their biological effects.Conclusions:(1) The conserved sequence 54-59 and 119-126 fragments of tmTNF-αplays an important role in biological activity and trimer formation, the latter fragment shortened to two amino acid deletion can still has this effect. (2) tmTNF-αconserved sequence His 15, Gly54 and Ile58 amino acid sites is the key to the formation of the trimer. (3) Members in the TNF superfamily dilapidating TNF-αtrimer formation, not only may lose their killing activity, and may inhibit the production of soluble molecules. (4) tmTNF-αconservative amino acids also affect other members of TNF family, such as the cytotoxic effect of FasL, which is associated with the formation of the trimer.The preliminary study to clarify tmTNF-αconservative sequence to form trimer is essential for its biological functions, and it is the same for the other members of TNF superfamily, such as FasL killing effect; And this provides an important experimental basis for the study the relationship structure and function between tmTNF-αand TNF superfamily.