人硫氧还蛋白同ASK1的结合与硫氧还蛋白氨基酸位点的关系

Relationship between interaction ability of thioredoxin with apoptosis signal-regulating kinase-1 and amino acid sites of thioredoxin

目的:通过对硫氧还蛋白(Trx)突变体与凋亡信号调节激酶-1(ASK1)结合能力的研究,揭示Trx的氨基酸位点与其抗氧化抗凋亡性的关系,进一步阐述Trx在细胞凋亡中的作用。方法:构建人类野生型Trx(pcDNA-hTrx-H is tag,TrxA)和人类Trx 49位突变体(pcDNA-hTrx-Y49F-H is tag,TrxB)及ASK1质粒(pCDNA-ASK1-HA tag)。用TrxA、TrxB质粒分别与ASK1质粒共转染入HEK-293A细胞。实验分为对照组(CON)、ASK1质粒转染组、TrxA+ASK1质粒共转染组及TrxB+ASK1质粒共转染组。于转染24 h后,裂解细胞,裂解细胞前20 m in加入不同浓度的过氧化氢。将裂解产物进行W estern b lot检测转染的结果,运用免疫共沉淀(co-immunoprec ip itation)法检测ASK1与Trx在过氧化物作用下解离的程度,采用TUNEL法及检测Caspase-3的活性法检测HEK-293A细胞的凋亡。结果:与ASK1转染组细胞的凋亡指数(73.8±6.93)%及Caspase-3活性[(69.3±3.57)nmol/(h.mg蛋白)]相比,TrxA+ASK1共转染组细胞的凋亡率(49.8±4.62)%明显减少,Caspase-3的活性降低[(51.4±3.61)nmol/(h.mg蛋白)],(P<0.05)。TrxB+ASK1共转染组的凋亡率(22.4±5.07)%更少,Caspase-3的活性降低更明显[(30.1±2.48)nmol/(h.mg蛋白)](P<0.01)。与TrxA+ASK1共转染组相比,TrxB+ASK1共转染组的细胞凋亡数及Caspase-3的活性下降更明显(P<0.05)。此外,在过氧化氢干预下,TrxB与ASK1的结合同TrxA与ASK1的结合相比较,前者更紧密。结论:Trx中第49位的酪氨酸突变为苯丙氨酸后,Trx与ASK1的结合能力增强,Trx抗凋亡的作用明显增加。说明Trx与ASK1的结合能力与Trx的氨基酸位点明显相关,进而可影响Trx抗细胞凋亡的能力。

AIM ： To investigate the relationship between the interaction ability of thioredoxin （Trx） with apoptosis signal-regulating kinase-1 （ ASK1 ） and the amino acid sites of Trx with which ASK1 interacts. METHODS ： WT-Trx-His tag （ TrxA, wild-type Trx） plasmids, hTrx-Yd9F-His tag （ TrxB, mutating the Trx tyrosine 49 to phenylalanine） mutant plasmids and ASK1-HA tag （ASK1） plasmids were constructed, and plasmids of TrxA or TrxB with ASK1 plasmids were co-transfected into HEK 293A cells using HD transfection reagent. Twenty hours after transfection, cells were harvested for evaluation of apoptosis and caspase-3 activity or further treated with hydrogen peroxide at different concentrations （0, 0. 5, 1,2 mmol/L） for an additional 20 min for evaluation of the interaction between Trx and ASK1. Transfection efficiency was evaluated by Western blot. Interaction of Trx with ASK1 was tested by co-immunoprecipitation. Apoptosis was tested by TUNEL, and caspase-3 activity was detected by ELISA. RESULTS： ASK1, TrxA and TrxB were effectively transfected into the cells. ASK1 transfection increased the apoptosis and caspase-3 activity of the cells. TrxA and ASK1 co-transfection attenuated the apoptosis and caspase-3 activity promoted by ASK1 transfection. Compared with TrxA and ASK1 co-transfection, TrxB and ASK1 co-transfection increased the interaction of Trx with ASK1 and further decreased the apoptosis and caspase-3 activity induced by ASK1 transfection. In addition, after pretreatment with H202, ASK1 is more susceptible to interact with TrxB than TrxA. CONCLUSION： Our study demonstrates that mutating the 49 tyrosine of Trx to phenylalanine promotes the interaction ability between Trx and ASK1 and, therefore, enhances the anti-apoptosis ability of Trx. Our present study further indicates that the interaction ability of Trx with ASK1 is involved in the amino acid sites of Trx with which ASK1 interacts.