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Cysteine (Cys) residues, especially those located on protein surface, are considered to be important for metal coordination, catalysis, protein structure by forming disulfide bonds and protein function regulation. Crucial Cys residues can be involved in the modulation of protein activity and signalling events via reactions of their thiol groups. These reactions can take several forms, such as redox events, chelation of transition metals or S-nitrosylation. In several cases, these disparate reactions can compete with each other for the same thiol group on a single Cys residue, forming a molecular switch composed of possible redox, NO or Zn(2+) modifications to control protein function. This work investigates the basis for these molecular Cys switches in spermine oxidase (SMO), an enzyme involved in the polyamine homeostasis of animal cells, which oxidizes spermine (Spm) to produce spermidine (Spd), 3-aminopropanal and H2O2. The murine SMO enzyme contains 14 Cys (correspond to 2.5% amino acid content), mainly localized on the protein surface. We carried out an in vitro chemical modifications of Cys residues to analyze their effect on the biochemical features of SMOWT (containing 14 Cys) and SMOC263-C429 mutant (containing only 2 Cys) recombinant proteins. Regarding the mutant protein, the rationale was that of mutating all the surface Cys residues into non reactive and isosteric Ser residues, while leaving the Cys263 and Cys429 residues which potentially play an important structural role. We used the NO-generating donor NOR-3 for S-nitrosylation, glutathione (GSH) for S-glutathionylation and S-nitrosoglutathione (GSNO) for both S-glutathionylation and S-nitrosylation. We could observed that treatment with NOR-3, but not with GSH and GSNO, decreased both SMOWT and SMOC263-C429 enzymatic activity.

The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of "functional silencing" of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.

Cystein residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitro sative/oxidative stress. MT from rabbit liver was treated with different concentrations of GSNO, diamide plus GSH or H2O2 plus GSH. Parallel sets of samples were treated with 10 mM DTT for 30 min at 37 degrees C to reduce mixed disulphides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell extracts. Structural aspects of S-glutathionylation of MTs were also examined. Treatment with GSNO, diamide/GSH or H2O2/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulphides. Structural analysis of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance. (C) 2007 Elsevier Inc. All rights reserved.

We present here a brief description of the relationships among metals, nitric oxide metabolism, and ageing. In particular, we will discuss the interactions occurring between redox (copper, iron) and non-redox (zinc) metals and nitric oxide, the metal- and nitric oxide-catalyzed formation of thiol adducts (nitrosothiols, mixed disulphides) and the possible involvement of these species in the ageing process. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the Cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1beta in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1beta, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1beta has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

The action of copper on the nitric oxide (NO) pathway was investigated in rat C6 glioma cells expressing both inducible and constitutive NO synthase (NOS) isoforms, The inducible NOS-II-mediated NO synthesis (i.e., nitrite production induced by LPS plus IFN gamma) was found to be increased upon copper uptake by cells, this effect being attributable to NOS-II mRNA transcriptional over-expression. On the other hand, the constitutive neuronal isoform (NOS-I) was inhibited after copper uptake, as revealed by the decrease of basal intracellular cGMP levels in C6 cells. Consistently, in vitro experiments showed that copper selectively blocked the catalytic activity of NOS-I, but not of NOS-II, The observed modulation of NOS isoforms by copper in C6 cells is in line with the previous hypothesis that selective inhibition of NOS-I leads to enhanced NO production through transcriptional activation of NOS-II. (C) 2000 Academic Press.

Nitric oxide (NO), produced in different cell types through the conversion of L-arginine into L-citrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NO-reactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acid-base catalysed Cys beta 93-nitrosylation and Cys beta 93NO-denitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites -1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for Cys-NO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the -2 position has been observed in some NO-reactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri- or tetra-peptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et at, Neuron (1997) 18, 691-696]. Here, we analyse the three-dimensional structure of several proteins containing NO-reactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the Cys-Sy atomic structural microenvironment rather than on the tri- or tetra-peptide sequence consensus motif.

Nitric oxide (NO) is a polypotent regulatory molecule involved in a variety of activities, such as the modulation of the catalytic activity of cysteine-containing enzymes. The present study reports the modulation of the HIV-1 reverse transcriptase activity by NO, released by the NO donors 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), 4-(phenylsulfonyl)-3-((2-(dimethylamino) ethyl)thio)furoxan oxalate (SNO-102), and sodium nitroprusside (SNP). NO inhibits dose-dependently the HIV-1 reverse transcriptase activity, Likely due to oxidation of Cys residue(s). Present results, representing a new insight into the modulation mechanism of the HIV-1 reverse transcriptase activity, may be relevant to develop new strategies for inhibition of HIV-1 replication. (C) 1999 Academic Press.

Current evidence suggests that integrating diverse warning systems at different biological levels may not only increase the probability of detecting threats but also mitigate their impact. Here, we propose the use of both molecular and morphological descriptors at different biological levels in sessile bivalves (a suitable biological model in monitoring programs) to collect information on the ecosystem health of coastal marine habitats. In this context, studies may be implemented on biomarkers to exploit some population features, with the aim to propose an actual monitoring program that predictively would provide possible scenarios on the species fitness and ecosystem changes. Thus, the use of quality biotic elements may provide an objective environmental monitoring method and facilitate the development of sanitary, economic, and social strategies related to sustainable exploitation.