Relationship Between Interaction Ability Of Thioredoxin With Apoptosis-regulating Kinase1 And Amino Acid Sites Of Thioredoxin

Backgroud and Aims:Apoptosis signal-regulating kinase 1(ASK1) was identified as a member of the family of mitogen-activated protein kinase(MAPK), which could be activated by various oxidation materials or tumor necrosis factor-α. So it plays a key pathogenic role in many cardiovascular diseases, which can induce the apoptosis by activating the C-Jun N-terminal kinase (JNK) and p38 MAP kinase passways.Thioredoxin (Trx) is a 12KDa small protein, which expressed all living cells. It could inhibit the apoptosis of ASK1 by combining with it, and plays an important role in antioxidation. It is characterized by the reduction/oxidation (redox) active site sequence Trp-Cys-Gly-Pro-Cys-Lys. The two cysteine residues within the redox center provide the sulfhydryl involved in the antioxidation and antiapoptosis of Trx. We kwon Trx is composed by 105 amino acid sites. And many studies find that Trx could combine with ASK1,and inhibit it's apoptosis also associated with the two cystenie redidues. So,we want to kwon whether there are another amino acid sites could relevant to the combination of Trx and ASK1, and play an important role in the antioxidant and antiapoptosis of Trx. So, this article through mutate the Trx Cys32,Cys35 and Tyr49 amino acids sites in order to study the relationship between the interaction ability of Trx with ASK1 and amino acid sites of Trx.Methods:Part one : The construction and transfection of plasmids.Construct the wide-type Trx with expression plasmids of His epitope-tagged Trx(WT-trx His tag,TrxA),the mutation of C32S(hTrx-C32S His tag,TrxB) and C35S(hTrx-C35S His tag,TrxC),which is mutated cystenie to serine ,and construct the Y49F,which is mutated the tyrosine to phenylalanie(hTrx-Y49F His tag,TrxD) plasmids. The HEK293A cells were cultured at 80% confluence in 24-well plates for 24 hours and transfacted . Diluted the HD transfection reagent and plasmids Trx and its mutants and ASK1(hemagglutinin epitope-tagged mutant forms of ASK1,ASK1 HA tag) respectively with Opti. The ratio of Trx and ASK1 is 4:1. Room temperature for 15min, and then transfected into HEK293A cells,5% CO₂ ,37℃incubator incubation. Cells were harvested at 24 hours after transfection, and cells lysate A were used for protein assays. For immunoprecipitation to analyze protein interaction in vivo, different concentrations(0,0.5,1,3mmol/L ) of H₂O₂ were incubated for 20min before the extraction of protein.Part two: the interaction of Trx and its mutant with ASK1.24 hours after co-transfenction the Trx and its mutant with ASK1 (TrxA+ASK1,TrxB+ASK1,TrxC+ASK1 and TrxD+ASK1), detection the cell apoptosis by TUNEL and Capase-3 . After incubate cells with H₂O₂ for 20min, detect the dissociation degree of the protein-protein complex by immunoprecipitation in order to explore the mechanism of how TrxA and its mutant to inhibited the ASK1-induced apoptosis.Results:1. The plasmids of TrxA and its mutants were successfully constructed.2. The plasmids of ASK1, TrxA and its mutants were successfully transformed, amplificated and extracted.3. Just transfected the ASK1 group, the cell apoptosis evidenced by TUNEL and Caspase-3 showed significant increased compared to CON group; When transfected the TrxA, the cell apoptosis was reduce compared to the ASK1 group, but more reduced in the TrxB, TrxC and TrxD group. Moreover, there was no obvious difference between the three groups.4. Under the hydrogen peroxide intervention, the combination of TrxB and ASK1 was more closer as well as the TrxC and TrxD groups compared to the group of TrxA.Conclusions:1. The mechanism of Trx-depentent antiapoptosis was that Trx could associated with ASK1, and inhibited the ASK1-induced cell apoptosis.2. The combination ability of Trx with ASK1 was closed related to the Trx's amino acid sites, for example the Cys32 and Cys35 could enhance the ability, but it's Tyr49 also involved in the combination.3. ASK1 could be released from Trx–ASK1 complex by the appearance of hydrogen peroxide. But different mutant of Trx played different role in the combination of Trx and ASK1. So, Trx showed different antiapoptosis.