Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.
Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.
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DOI：
10.1016/j.redox.2017.01.018
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发表时间：
2017-04
期刊：
影响因子：
11.4
通讯作者：
Jiménez A
中科院分区：
文献类型：
--
作者：
Calderón A;Ortiz-Espín A;Iglesias-Fernández R;Carbonero P;Pallardó FV;Sevilla F;Jiménez A
Thioredoxins （Trxs）是细胞氧化还原调控的关键成分，通过控制许多靶蛋白的氧化还原状态起作用，并已被证明在细胞存活和生长中发挥重要作用。Trx系统在植物细胞核中的存在很少受到关注，植物Trx的核靶点尚未得到最终确定。因此，我们对Trxs在这个细胞区室中的功能知之甚少。之前，我们研究了PsTrxo1的细胞内定位，并证实了它在线粒体中存在，有趣的是，在标准生长条件下，它也存在于细胞核中。为了研究PsTrxo1的细胞核功能，我们利用豌豆叶片纯化细胞核，通过亲和层析技术鉴定出增殖细胞核抗原（PCNA）作为PsTrxo1的靶点。斑点印迹和双分子荧光互补（BiFC）实验证实了这种蛋白-蛋白相互作用，表明两种蛋白在细胞核中相互作用。此外，PsTrxo1在先前氧化的重组PCNA蛋白上显示出二硫还原酶活性。同时，我们研究了PsTrxo1过表达对烟草亮黄-2 （TBY-2）细胞培养的影响。显微镜和流式细胞术分析显示，PsTrxo1过表达增加了转化细胞系的细胞增殖率，在细胞培养开始时（第1天和第3天）和培养较长时间后（第9天）细胞周期的S期百分比较高，与PCNA蛋白上调相一致。此外，在PsTrxo1过表达的细胞中，细胞总谷胱甘肽含量减少，但细胞核中谷胱甘肽的积累保持不变，特别是在培养结束时，与非过表达细胞不同，有丝分裂指数更高。这些结果表明，Trxo1可能通过其与细胞PCNA和谷胱甘肽的联系参与了TBY-2培养的细胞周期进程。PCNA是PsTrxo1在植物细胞核中的靶标。PsTrxo1能够在体外还原氧化的PCNA。过表达PsTrxo1 TBY-2的细胞增殖和有丝分裂指数较高。过表达（Oex）细胞的谷胱甘肽含量较低，PCNA蛋白含量较高。在Oex细胞中，细胞核GSH在培养结束时保持不变。
Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein–protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione. PCNA is a target of PsTrxo1 in cell plant nucleus. PsTrxo1 is able to reduce oxidized PCNA in vitro. Higher proliferation and mitotic index occur in overexpressing PsTrxo1 TBY-2 cells. Overexpressing (Oex) cells have less glutathione content and more PCNA protein. Nuclear GSH is maintained at the end of the culture in Oex cells.