Peroxynitrite-Mediated Dimerization of 3-Nitrotyrosine: Unique Chemistry along the Spectrum of Peroxynitrite-Mediated Nitration of Tyrosine.

Peroxynitrite (ONOO-, PN) has long been considered a potent nitrating agent implicated in numerous inflammation-mediated diseases. The current work highlights an unexplored oxidation chemistry initiated under conditions of sustained PN exposure. Impetus for this investigation developed from mass spectral results that suggested dimerization of a model peptide with a single tyrosine residue that was first nitrated following extended exposure to PN generated in situ. In attempts to substantiate this dimerization event and divulge the possible mode of linkage between the tyrosine derivatives of the peptide monomers, 3-nitrotyrosine (3-NT) was exposed to sustained fluxes of PN in a two-component PN-generating platform developed in this laboratory. Such exposure afforded products with tandem mass spectrometry and fluorescence spectroscopy profiles indicative of C-O coupling between 3-NT moieties. Synthesis and comparative analysis of the C-C coupled 3-NT isomer corroborated these findings. Most notably, the mass spectral data of the C-C coupled 3-NT dimer displayed a 226.80 m/z peak following exposure to high collision energy, corresponding to symmetric cleavage of the parent dimer peak (m/z = 453) along with a fragmentation product at m/z = 180.04 (-NO2 species). This fragmentation profile was distinct from the C-O coupled 3-NT dimer that exhibited a predominant 209.14 m/z peak with a small secondary 226.15 m/z peak indicative of asymmetric cleavage of the parent dimer. Results of this study indicate that formation of C-O coupled 3-NT dimer is promoted by elevated levels of 3-NT formed under high and sustained flux of PN.

Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity.

Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF~0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 × 10-3), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.

Tyrosine nitration in peptides by peroxynitrite generated in situ in a light-controlled platform: effects of pH and thiols.

Peroxynitrite has been shown to play a critical role in inflammation and affords 3-nitrotyrosine as the hallmark product. The reported methods of generating this reactive nitrogen species in situ often fails to provide a high and steady flux of peroxynitrite resulting in poor yields of 3-nitrotyrosine. Herein we report a two-component peroxynitrite-generating platform in which this anion is produced in a biomimetic fashion and under the control of visible light. Incorporation of the nitric oxide- and superoxide-generating components in polymer matrices allows easy alterations of pH in the reaction wells of this platform. We have demonstrated very efficient nitration of tyrosine by peroxynitrite at different pH values and with varying concentrations of carbonate. In addition to tyrosine, a set of tyrosine-containing peptides was also studied. Presence of glutathione in the reaction wells increases the extent of tyrosine nitration in such peptide substrates presumably by raising the lifetime of nitric oxide in the reaction medium. When a cysteine residue was included in the sequence of the peptide, the extent of nitration of the tyrosine residue was found to depend on the position of the cysteine residue with respect to tyrosine. The extent of tyrosine nitration is strongly attenuated when the cysteine residue is directly adjacent to the tyrosine. This effect has been attributed to an intramolecular radical transfer mechanism. Taken together, results of this study demonstrate the potential of this light-controlled platform as a convenient bioanalytical tool in studying the reactions of peroxynitrite under widely varying conditions.