Significance: Nitric oxide (NO) classical and less classical signaling mechanisms (through interaction with soluble guanylate cyclase and cytochrome c oxidase, respectively) operate through direct binding of NO to protein metal centers, and rely on diffusibility of the NO molecule. S-Nitrosylation, a covalent post-translational modification of protein cysteines, has emerged as a paradigm of nonclassical NO signaling. Recent Advances: Several nonenzymatic mechanisms for S-nitrosylation formation and destruction have been described. Enzymatic mechanisms for transnitrosylation and denitrosylation have been also studied as regulators of the modification of specific subsets of proteins. The advancement of modification-specific proteomic methodologies has allowed progress in the study of diverse S-nitrosoproteomes, raising clues and questions about the parameters for determining the protein specificity of the modification. Critical Issues: We propose that S-nitrosylation is mainly a short-range mechanism of NO signaling, exerted in a relatively limited range of action around the NO sources, and tightly related to the very controlled regulation of subcellular localization of nitric oxide synthases. We review the nonenzymatic and enzymatic mechanisms that support this concept, as well as physiological examples of mammalian systems that illustrate well the precise compartmentalization of S-nitrosylation. Future Directions: Individual and proteomic studies of protein S-nitrosylation-based signaling should take into account the subcellular localization in order to gain further insight into the functional role of this modification in (patho)physiological settings.

< 0.001. Two-tailed Student's t-test (last normoxia time versus hypoxia times in KO + pNCLX; d, e): & P < 0.05, && P < 0.01. In f, h, statistical comparisons are shown only for normoxia versus 0-10 groups. AU, arbitrary units; NS, not significant.

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Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

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The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.

Polyacrylamide (PAM) hydrogels are commonly used as substrates for cell mechanical and mechanobiological studies because of their tunable stiffness and ease of handling. The dependence of bulk rheological and local elastic properties (assessed by Atomic Force Microscopy, or AFM) of PAM hydrogels on its composition and polymerization temperature has been extensively studied. PAM hydrogels swell when immersed in media, but the influence of swelling on local elastic properties is poorly characterized. Direct measurements of the effect of swelling on PAM elastic properties are scarce. We report here, for the first time, the direct measurements of volumetric swelling and local elastic properties of PAM gels throughout the post-polymerization swelling process until equilibrium. First, local and global elastic properties (measured by rheology), were obtained during polymerization in the absence of swelling, and showed good agreement with each other. Four PAM hydrogel compositions were characterized thus, with corresponding storage shear moduli (as measured immediately after polymerization) of 4,530 Pa (termed stiffest), 2,900 Pa (stiff), 538 Pa (soft), and 260 Pa (softest). Next, all compositions were subjected to swelling in phosphate buffered saline. Swelling ratios and local elastic moduli were measured at 0, 3, 6, 9, 12, and 24 h post-polymerization for the soft and softest compositions, and once daily till 6 days post-polymerization for all four compositions. For the stiffest and stiff gels, swelling ratio, and local elastic modulus changed negligibly with time, while for the soft and softest gels, substantial changes between Day 0 and Day 1 were found for both swelling ratio (increased by 21.6 and 133%, respectively), and local elastic modulus decreased (by 33.7 and 33.3%, respectively), substantially. Experimental data were analyzed by a model that combined ideal elastomer mechanics and poroelastic swelling kinetics model. Model predictions confirmed the validity of present measurements with respect to past studies where swelling and elastic properties were not measured simultaneously. The present study underlines the important effect swelling can have on PAM elastic properties and provides detailed quantitative data to guide the duration taken to reach equilibrium-a useful information for cell mechanics experiments. In addition, the simultaneous measurements of swelling and local elastic moduli provide novel data for the validation of theoretical models.

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