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Nitric oxide (NO) is a free radical signaling molecule that plays a crucial role in modulating physiological homeostasis across multiple biological systems. NO dysregulation is linked to the pathogenesis of multiple diseases; therefore, its quantification is important for understanding pathophysiological processes. The detection of NO is challenging, typically limited by its reactive nature and short half-life. Additionally, the presence of interfering analytes and accessibility to biological fluids in the native tissues make the measurement technically challenging and often unreliable. Here, a bio-inspired peptide-based NO sensor is developed, which detects NO-derived oxidants, predominately peroxynitrite-mediated nitration of tyrosine residues. It is demonstrated that these peptide-based NO sensors can detect peroxynitrite-mediated nitration in response to physiological shear stress by endothelial cells in vitro. Using the peptide-conjugated fluorescent particle immunoassay, peroxynitrite-mediated nitration activity with a detection limit of ≈100 × 10−9m is detected. This study envisions that the NO detection platform can be applied to a multitude of applications including monitoring of NO activity in healthy and diseased tissues, localized detection of NO production of specific cells, and cell-based/therapeutic screening of peroxynitrite levels to monitor pronitroxidative stress in biological samples.

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. Antioxid. Redox Signal. 19, 1220-1235. Spanish Government [CSD2007-00020, CP07/00143, PS09/00101, SAF2009-7520, PI10/02136]; Spanish-Portuguese Integrated Action Grant [PRI-AIBPT-2011-1015/E-10/12]; Foundation for Science and Technology (FCT, Portugal) [PTDC/SAU-NEU/102612/2008, PTDC/SAU-NMC/112183/2009, PEst-OE/EQB/LA0023/2011]; COST action [BM1005] info:eu-repo/semantics/publishedVersion

Protein tyrosine nitration (PTN) is a post-translational modification of proteins associated with a number of inflammatory diseases. While PTN is rather selective (not all proteins are modified and within a protein, only certain tyrosines are subject to nitration), no consensus sequence has been identified. Since PTN is a low-abundance post-translational modification, it is necessary to enrich modified proteins and/or to detect them with high selectivity and sensitivity. Until now this has been mostly accomplished with anti-nitrotyrosine antibodies in combination with two-dimensional gel electrophoresis and mass spectrometry. We propose a chemical labeling approach designed to allow enrichment of tyrosine-nitrated peptides independent of the sequence context, which is a potential shortcoming of antibody-based approaches. In this procedure, all amines are blocked by acetylation followed by conversion of nitrotyrosine to aminotyrosine and biotinylation of aminotyrosine. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes thereby reducing sample loss. Free biotin is subsequently removed with a strong cation exchanger, the labeled pepticles are enriched on an immobilized avidin column and the enriched peptides analyzed by LC-MS/MS. As a proof of concept, this method was successfully applied to the enrichment of tyrosine-nitrated angiotensin 11 in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics. (C) 2009 Elsevier B.V. All rights reserved.

Tyrosine nitration is a hallmark for nitrosative stress caused by the release of reactive oxygen and nitrogen species by activated macrophages and neutrophilic granulocytes at sites of inflammation and infection. In the first part of the study, we used an informative host¿parasite animal model to describe the differential contribution of macrophages and neutrophilic granulocytes to in vivo tissue nitration. To this purpose common carp (Cyprinus carpio) were infected with the extracellular blood parasite Trypanoplasma borreli (Kinetoplastida). After infection, serum nitrite levels significantly increased concurrently to the upregulation of inducible nitric oxide synthase (iNOS) gene expression. Tyrosine nitration, as measured by immunohistochemistry using an anti-nitrotyrosine antibody, dramatically increased in tissues from parasite-infected fish, demonstrating that elevated NO production during T. borreli infection coincides with nitrosative stress in immunologically active tissues. The combined use of an anti-nitrotyrosine antibody with a panel of monoclonal antibodies specific for several carp leukocytes, revealed that fish neutrophilic granulocytes strongly contribute to in vivo tissue nitration most likely through both, a peroxynitrite- and an MPO-mediated mechanism. Conversely, fish macrophages, by restricting the presence of radicals and enzymes to their intraphagosomal compartment, contribute to a much lesser extent to in vivo tissue nitration. In the second part of the study, we examined the effects of nitrosative stress on the parasite itself. Peroxynitrite, but not NO donor substances, exerted strong cytotoxicity on the parasite in vitro. In vivo, however, nitration of T. borreli was limited if not absent despite the presence of parasites in highly nitrated tissue areas. Further, we investigated parasite susceptibility to the human anti-trypanosome drug Melarsoprol (Arsobal), which directly interferes with the parasite-specific trypanothione anti-oxidant system. Arsobal treatment strongly decreased T. borreli viability both, in vitro and in vivo. All together, our data suggest an evolutionary conservation in modern bony fish of the function of neutrophilic granulocytes and macrophages in the nitration process and support the common carp as a suitable animal model for investigations on nitrosative stress in host¿parasite interactions. The potential of T. borreli to serve as an alternative tool for pharmacological studies on human anti-trypanosome drugs is discussed.

Nitric oxide (NO) has long been recognized as a multifaceted participant in brain physiology. Despite the knowledge that was gathered over many years regarding the contribution of NO to neuronal plasticity, for example the ability of the brain to change in response to new stimuli, only in recent years have we begun to understand how NO acts on the molecular and cellular level to orchestrate such important phenomena as synaptic plasticity (modification of the strength of existing synapses) or the formation of new synapses (synaptogenesis) and new neurons (neurogenesis). Post-translational modification of proteins by NO derivatives or reactive nitrogen species is a non-classical mechanism for signalling by NO. S-nitrosation is a reversible post-translational modification of thiol groups (mainly on cysteines) that may result in a change of function of the modified protein. S-nitrosation of key target proteins has emerged as a main regulatory mechanism by which NO can influence several levels of brain plasticity, which are reviewed in this work. Understanding how S-nitrosation contributes to neural plasticity can help us to better understand the physiology of these processes, and to better address pathological changes in plasticity that are involved in the pathophysiology of several neurological diseases. Linked ArticlesThis article is part of a themed section on Pharmacology of the Gasotransmitters. To view the other articles in this section visit FEDER funds via Programa Operacional Factores de Competitividade (COMPETE); COST action [BM1005]; Foundation for Science and Technology (FCT, Portugal) [PTDC/SAU-OSD/0473/2012, PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014]; Spanish-Portuguese Integrated Action grant [PRI-AIBPT-2011-1015/E-10/12]; FCT [SFRH/BD/77903/2011]; I3SNS programme (ISCIII, Spanish Government)

Nitrite (NO(2)-), N (G)-hydroxy-L-arginine (NOHA), and hydroxylamine (NH(2)OH) are products of nitric oxide synthase (NOS) activity and can also be formed by secondary reactions of nitric oxide (NO). These compounds are commonly considered to be rather stable and as such to be dosimeters of NO biosynthesis. However, each can be converted via metal-catalyzed reactions into either NO or other reactive nitrogen oxide species (RNOS), such as nitrogen dioxide (NO(2)) and nitroxyl (HNO), which have biologic activities distinct from those of the parent molecules. Consequently, certain aspects of tissue regulation controlled by RNOS may be dictated to a significant extent by metal-dependent reactions, thereby offering unique advantages for cellular and tissue regulation. For instance, because many metal-catalyzed reactions depend on the redox and oxygen status of the cellular environment, such reactions could serve as redox indicators. Formation of RNOS by metal-mediated pathways would confine the chemistry of these species to specific cellular sites. Additionally, such mechanisms would be independent both of NO and NOS, thus increasing the lifetime of RNOS that react with NO. Thus metal-mediated conversion of nitrite, NOHA, and NH(2)OH into biologically active agents may provide a unique signaling mechanism. In this review, we discuss the biochemistry of such reactions in the context of their pharmacologic and biologic implications.