Fate of nitrated proteins: denitration or degradation?

近年来，蛋白质的硝化作用与许多疾病的病理生理有关。硝化蛋白形成背后的化学机制通常被很好地理解，并涉及自由基反应。蛋白质酪氨酸硝化可以是位点特异性的，并导致结构和功能的巨大变化。值得注意的是，这些蛋白质的命运是完全未知的。由于以前研究硝化蛋白质命运的尝试都是使用将氧化修饰与蛋白质硝化结合的粗制剂，因此对于硝化蛋白质的命运无法给出明确的答案，尽管指出了两个主要途径：脱硝或降解。我们以前的一些结果表明存在脱硝酶活性。由于我们两个实验室的研究专长的结合，我们将能够解决和回答这个问题。R. Radi在蛋白质的靶向和分子内选择性化学硝化方面经验丰富，而T. Grune在研究修饰蛋白质在细胞中的命运方面经验丰富。因此，我们将生产位点特异性硝化蛋白，并将其暴露于组织和细胞匀浆中，以研究这些修饰蛋白的清除能力。同时，将建立一种HPLC/MS-based法来跟踪和定量脱硝，以简化进一步的生化分析。在这些生化研究中，要区分脱硝和降解。详细的研究将测试这一机制，并确定参与这一过程的酶。这包括参与脱硝的酶以及参与细胞内降解的蛋白质。将进行广泛的分析，以便进一步测试脱硝是如何精确地调节的，以及脱硝和降解是如何相互关联的。许多研究将集中在谷氨酰胺合成酶的硝化和反硝化上，这将作为硝化蛋白质的“案例研究”，具有重大的生物学意义。作为这个项目的结果，我们将对哺乳动物细胞中硝化蛋白的命运给出答案。

During recent years the nitration of proteins was connected to the pathophysiology of a number of diseases. The chemical mechanism behind the formation of nitrated proteins is in general well understood and involves free radical reactions. Protein tyrosine nitration can be site-specific and leads to dramatic changes in both structure and function. To note, the fate of such proteins is absolutely unknown. Since all previous attempts to study the fate of nitrated proteins used crude preparations combining oxidative modification with nitration of proteins, no clear answers could be given for the fate of nitrated proteins, although two principal pathways were indicated: denitration or degradation. Some of our previous results indicate the existence of a denitrase activity. Due to the combination of the research expertise of our two laboratories we will be able to address and answer this question. R. Radi is experienced in the targeted and intramolecular selective chemical nitration of proteins, whereas T. Grune has large experience in studying the fate of modified proteins in cells. Therefore, we will produce site-specific nitrated proteins and expose them to tissue and cell homogenates in order to study the clearing-capacity for these modified proteins. In parallel, a HPLC/MS-based assay to follow and quantitate denitration will be established, in order to simplify further biochemical analyses. In these biochemical investigations it will be discriminated between denitration and degradation. Detailed studies will test for the mechanism and identify the enzymes involved in this process. This includes the enzymes involved in the denitration as well as the proteins involved in the intracellular degradation. Extensive analyses will be performed in order to test further, how exactly both denitration is regulated and denitration and degradation are related to each other. A number of studies will focus on the nitration and denitration of glutamine synthetase, which will serve as a 'case study' nitrated protein, with large biological implications. As a result of the proposed project the answer will be given to the fate of nitrated proteins in mammalian cells.