UMR 1347 Agroécologie AgroSup Dijon/INRA/Université de Bourgogne, Pôle Mécanisme et Gestion des Interactions Plantes-microorganismes - ERL CNRS 6300, Dijon, France.
Biochem J. 2012 Oct 15;447(2):249-60. doi: 10.1042/BJ20120257.
NO has important physiological functions in plants, including the adaptative response to pathogen attack. We previously demonstrated that cryptogein, an elicitor of defence reaction produced by the oomycete Phytophthora cryptogea, triggers NO synthesis in tobacco. To decipher the role of NO in tobacco cells elicited by cryptogein, in the present study we performed a proteomic approach in order to identify proteins undergoing S-nitrosylation. We provided evidence that cryptogein induced the S-nitrosylation of several proteins and identified 11 candidates, including CDC48 (cell division cycle 48), a member of the AAA+ ATPase (ATPase associated with various cellular activities) family. In vitro, NtCDC48 (Nicotiana tabacum CDC48) was shown to be poly-S-nitrosylated by NO donors and we could identify Cys(110), Cys(526) and Cys(664) as a targets for S-nitrosylation. Cys(526) is located in the Walker A motif of the D2 domain, that is involved in ATP binding and was previously reported to be regulated by oxidative modification in Drosophila. We investigated the consequence of NtCDC48 S-nitrosylation and found that NO abolished NtCDC48 ATPase activity and induced slight conformation changes in the vicinity of Cys(526). Similarly, substitution of Cys(526) by an alanine residue had an impact on NtCDC48 activity. More generally, the present study identified CDC48 as a new candidate for S-nitrosylation in plants facing biotic stress and further supports the importance of Cys(526) in the regulation of CDC48 by oxidative/nitrosative agents.
NO 在植物中具有重要的生理功能，包括对病原体攻击的适应反应。我们之前证明， cryptogein，一种由卵菌 Phytophthora cryptogea 产生的防御反应诱导剂，触发烟草中的 NO 合成。为了解析 cryptogein 引发烟草细胞中 NO 的作用，在本研究中，我们采用蛋白质组学方法来鉴定发生 S-亚硝基化的蛋白质。我们提供的证据表明， cryptogein 诱导了几种蛋白质的 S-亚硝基化，并鉴定出 11 个候选蛋白，包括 CDC48（细胞分裂周期 48），一种 AAA+ATP 酶（与各种细胞活动相关的 ATP 酶）家族成员。在体外，NO 供体显示 NtCDC48（Nicotiana tabacum CDC48）被多 S-亚硝基化，并且我们可以鉴定 Cys(110)、Cys(526)和 Cys(664)作为 S-亚硝基化的靶标。Cys(526)位于 D2 结构域的 Walker A 基序中，该基序参与 ATP 结合，并且先前在果蝇中报道其受到氧化修饰的调节。我们研究了 NtCDC48 S-亚硝基化的后果，发现 NO 消除了 NtCDC48 的 ATP 酶活性，并在 Cys(526)附近诱导轻微的构象变化。同样，Cys(526)被丙氨酸残基取代对 NtCDC48 活性有影响。更一般地说，本研究鉴定出 CDC48 作为植物在面临生物胁迫时 S-亚硝基化的新候选蛋白，并进一步支持 Cys(526)在氧化/亚硝化试剂调节 CDC48 中的重要性。
