A "fluorescence switch" technique increases the sensitivity of proteomic detection and identification of S-nitrosylated proteins.

Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.

The N-terminal region of Nurr1 (a.a 1-31) is essential for its efficient degradation by the ubiquitin proteasome pathway.

NURR1/NR4A2 is an orphan nuclear receptor that is critical for the development and maintenance of mesencephalic dopaminergic neurons and regulates transcription of genes involved in the function of dopaminergic neurons directly via specific NGFI-B response elements (NBRE).and substantial data support a possible role of Nurr1 in the pathogenesis of Parkinson's disease (PD). Here we show that Nurr1 is degraded by the ubiquitin-proteasome pathway and determined that N-terminal region (a.a 1-31) of Nurr1 is essential for an efficient targeting of Nurr1 to degradation in the cell. Nurr1 Δ1-31 has a much longer half-life, and as a consequence its steady-state protein levels were higher, than full-length Nurr1 in the cell. Nurr1 Δ1-31 was as potent as Nurr1 full length in transcriptional luciferase reporter assays after normalization with the corresponding steady-state protein expression levels, either in trans-activation of NBRE or trans-repression of iNOS (inducible NO synthase) reporters. These results suggest that Nurr1 Δ1-31, because of longer persistence in the cell, can be a good candidate for gene and cell therapies in the treatment of PD.