Regulation of plant metabolic function mediated by protein S-nitrosylation

半胱氨酸残基的S亚硝化是一种关键的氧化还原修饰，调节着广泛的蛋白质功能，这个过程涉及到活性氮物种，如S亚硝硫醇(RSNO)，它们可以修饰蛋白质的硫醇部分。本研究试图探讨：(1)拟南芥中RSNO代谢在调节蛋白质S-亚硝化中的重要性，尤其是S-亚硝基谷胱甘肽还原酶(GSNOR)作为决定细胞RSNO水平和S-亚硝化程度的关键酶的调节作用；(2)这种翻译后修饰可能参与某些植物代谢功能的调节，如氮的同化。(1)当受到NO2、NO供体和硝酸盐等RNS的影响时，拟南芥植物的RSNO水平增加，其中相当大一部分被回收到高分子组分中。这表明，对这些Inorg…的反应，累积的RsNO具有蛋白质性质更多的惰性氮氧化物。用生物素转换法监测了野生型植物中S亚硝化蛋白水平的变化，发现NO供体处理促进了野生型植物中蛋白质的S亚硝化。然而，GSNOR的过表达减轻了这种翻译后修饰的程度，增加的水平远低于野生型植物，支持了该酶在调节蛋白质S-亚硝化方面的重要性。(2)从氮同化的调节方面研究了GSNOR过表达的生理后果。与野生型植株相比，过量表达GSNOR的转基因植株比野生型植株吸收和同化了更多的硝酸盐和NO2来源的氮素。由于GSNOR在催化作用上与无机氮的同化过程无关，因此这些结果表明GSNOR可能参与了蛋白质的S亚硝化，参与了氮同化的调节。较少

S-Nitrosylation of cysteine residues is a critical redox modification that regulates a broad spectrum of protein functions, and this process involves reactive nitrogen species (RNS) such as S-nitrosothiols (RSNO) that are known to modify the thiol moiety of proteins. This research attempted to examine: (1) the importance of RSNO metabolism in modulating protein S-nitrosylation in higher plants, with special focus on the regulatory role of S-nitrosoglutathione reductase (GSNOR) as a key enzyme determining cellular RSNO levels and the degree of S-nitrosylation; and (2) the possibility that this posttranslational modification may participate in the regulation of certain plant metabolic functions such as nitrogen assimilation.(1) When exposed to RNS such as NO_2, NO donors and nitrate, Arabidopsis plants increased the level of RSNO, a substantial proportion of which was recovered in high-molecular fractions. This suggests the proteinous nature of accumulated RSNO in response to these inorg … More anic nitrogen oxides. Using biotin-switch method, the changes in S-nitrosylated protein levels were monitored and it was found that NO-donor treatments enhanced protein S-nitrosylation in wild-type plants. Overexpression of GSNOR, however, alleviated the extent of this posttranslational modification, with the increased level much lower than that seen in wild-type plants, supporting the importance of this enzyme in modulating protein S-nitrosylation.(2) The physiological consequence of GSNOR overexpression was investigated with respect to the regulatory aspects of nitrogen assimilation. When fed with a single ^<15>N-labeled nitrogen source such as nitrate or NO_2, transgenic plants overexpressing GSNOR incorporated and assimilated more nitrate-and NO_2-derived ^<15>N than did the wild-type plants. Because GSNOR is catalytically not related to the assimilatory process of inroganic nitrogen, these results suggest the possible involvement of GSNOR, and hence S-nitrosylation of proteins, in the regulation of nitrogen assimilation. Less