Fate of nitrated proteins: denitration or degradation?

• Applicant: Professor Dr. Tilman Grune
• Subject Area: Nutritional Sciences ; Biogerontology and Geriatric Medicine
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 257921205

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.

• DFG Programme: Research Grants
• International Connection: Uruguay
• Participating Person: Professor Dr. Rafael Radi