Ancín María, Fernandez-Irigoyen Joaquin, Santamaria Enrique, Larraya Luis, Fernández-San Millán Alicia, Veramendi Jon, Farran Inmaculada
Institute for Multidisciplinary Research in Applied Biology (IMAB), Universidad Pública de Navarra (UPNA), Campus Arrosadia, 31006 Pamplona, Spain.
Instituto de Agrobiotecnologia (IDAB), Consejo Superior de Investigaciones Científicas (CSIC)-Gobierno de Navarra, Avenida Pamplona 123, 31192 Mutilva, Spain.
Antioxidants (Basel). 2022 Oct 4;11(10):1979. doi: 10.3390/antiox11101979.
Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.
翻译后氧化还原修饰为主要细胞过程的调控提供了一种重要机制。硫氧还蛋白（Trxs）是这种调控机制中的关键参与者，是催化硫醇-二硫键交换反应的普遍存在的蛋白质。在叶绿体中，Trx f、Trx m和NADPH依赖性硫氧还蛋白还原酶C（NTRC）已被确定为通过将电子传递给下游靶酶来传递氧化还原信号的物质。在过去几年中，已鉴定的Trx靶标的数量大幅增加，但其中大多数是使用缺乏同工型特异性的体外方法确定的。在此背景下，我们开发了一种基于将His标签的Trx f、Trx m或NTRC单半胱氨酸突变体过表达到植物中的新方法。过表达的突变Trxs能够在植物中与靶蛋白形成稳定的混合二硫键，将其固定在填充有Ni-NTA琼脂糖的亲和柱上，并用二硫苏糖醇洗脱共价连接的靶标，并通过基于质谱的蛋白质组学进行鉴定。该方法分别鉴定出Trx f、Trx m和NTRC的6个、9个和42个新的潜在靶标，并实现了NTRC与Trxs之间明显的特异性。功能分析表明，这些靶标参与了多个细胞过程。
