Simona Frosali

Thiolation and nitrosation of cysteines in biological fluids and cells

Summary. Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (–OH or –NH2), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH.When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation

AbstractWe previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, β-actin, tubulin-α-1-chain and enolase-α. Interestingly, all carbonylated proteins except calreticulin and enolase-α showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

Glutathione recycling and antioxidant enzyme activities in erythrocytes of term and preterm newborns at birth.

We previously demonstrated a high susceptibility of neonatal red blood cells (RBC) to oxidative stress at birth. The aim of this study was to compare the RBC antioxidant capacity and redox cycle enzyme activities as well as glutathione (GSH) recycling in full-term and preterm infants at birth and in normal adults. GSH and GSH disulfide (GSSG) concentrations, GSH/GSSG ratio, and the activities of glucose-6-phosphate dehydrogenase (G-6-PDH), GSH peroxidase, GSH reductase (GR), catalase (CAT), superoxide dismutase (SOD), and hexokinase (HK) were measured in RBC of 25 healthy adults and 56 newborns (23 term, 33 preterm) at birth. The GSH recycling was measured in adult and newborn RBC exposed to oxidative stress (1 mM tert-butylhydroperoxide). The RBC of term and preterm babies showed higher GSH, GSSG, G-6-PDH, GR, and HK levels/activities and lower GSH/GSSG ratios and higher GSH-recycling rates than those of adults. In preterm babies significant correlations were found between G-6-PDH and CAT, GSH, GSH/GSSG ratio, and GSSG (r = –0.67, r = 0.71, r = –0.66, p < 0.01; r = 0.71, p < 0.05, respectively). In term newborns, statistically significant correlations were observed between G-6-PDH and CAT, SOD, and GSH (r = –0.65, r = –0.65, r = –0.69, p < 0.01, respectively). The results indicate the central role of the G-6-PDH activity in antioxidant defenses. We speculate that preterm babies have prompter involvement of antioxidant defenses than term babies.

Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model.

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb‐SS‐X+RSH↔Alb‐SS‐R+XSH) or dethiolation (Alb‐SS‐X+XSH↔Alb‐SH+XSSX), depending on the different pKa values of thiols involved in protein–thiol mixed disulfides (Alb‐SS‐X). It appeared in these reactions that the compound with lower pKa in mixed disulfide was a good leaving group and that the pKa differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb‐TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb‐SS‐X (peaks at 0.25–1 min). In turn, Alb‐SS‐X were dethiolated by the excess nonprotein SH groups because of the lower pKa value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb‐SS‐X was accompanied by formation of XSSX and Alb‐SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.

The role of protein sulfhydryl groups and protein disulfides of the platelet surface in aggregation processes involving thiol exchange reactions.

Blood platelets are central to haemostasis and platelet aggregation is considered to be a direct index of platelet function. Although protein disulfides (PSSP) are structural components of most proteins, current evidence suggests that PSSP work together with protein SH groups (PSH) to activate various platelet functions in dynamic processes involving thiol/disulfide exchange reactions. Based on these assumptions, we performed experiments to demonstrate how PSH and PSSP are involved in platelet aggregation and how modifications of PSH and PSSP concentrations on the platelet surface by N-ethylmaleimide (NEM) (a PSH-blocking reagent) and dithiothreitol (DTT) (a PSSP-reducing reagent), respectively, may condition platelet susceptibility in protein rich plasma and washed platelets and integrin αIIbβ3 conformation. Our data strongly suggest that the PSH blockage and the PSSP reduction of the platelet surface are deeply involved in aggregation processes evoked in protein rich plasma and washed platelets by ADP and collagen; that endogenous thiols (e.g. GSH) may interfere with NEM actions; that NEM and DTT, acting on preexisting PSH and PSSP of active platelets have opposite conformational changes on integrin αIIbβ3 conformation. Although the precise mechanism and the populations of specific PSH and PSSP involved remain unresolved, our data support the notion that PSH and PSSP of the platelet surface are involved in platelet activation by thiol exchange reactions. A plausible molecular mechanism of PSH and PSSP recruitment during thiol exchange reactions is here proposed.

Role of intracellular calcium and S-glutathionylation in cell death induced by a mixture of isothiazolinones in HL60 cells

Previously we reported that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) shifts the cells into a state of oxidative stress that induces apoptosis and necrosis. In this study, flow cytometric analysis showed that CMI/MI induces early perturbation of calcium homeostasis, increasing cytosolic and mitochondrial calcium and depleting the intracellular endoplasmic reticulum (ER) stores. The calcium chelator BAPTA-AM reduced necrosis and secondary necrosis, the loss of DeltaPsim and S-glutathionylation induced by necrotic doses of CMI/MI, but did not protect against CMI/MI-induced apoptosis, mitochondrial calcium uptake and mitochondrial hyperpolarization. This indicates that increased cytoplasmic calcium does not have a causal role in the induction of apoptosis, while cross-talk between the ER and mitochondria could be responsible for the induction of apoptosis. GSH-OEt pretreatment, which enhances cellular GSH content, reduced S-glutathionylation and cytosolic and mitochondrial calcium levels, thus protecting against both apoptosis and necrosis shifting to apoptosis. Therefore, the degree of GSH depletion, paralleled by the levels of protein S-glutathionylation, may have a causal role in increasing calcium levels. The mitochondrial calcium increase could be responsible for apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathionylation of specific proteins acts as a molecular linker between calcium and redox signalling.

A structurally driven analysis of thiol reactivity in mammalian albumins

Understanding the structural basis of protein redox activity is still an open question. Hence, by using a structural genomics approach, different albumins have been chosen to correlate protein structural features with the corresponding reaction rates of thiol exchange between albumin and disulfide DTNB. Predicted structures of rat, porcine, and bovine albumins have been compared with the experimentally derived human albumin. High structural similarity among these four albumins can be observed, in spite of their markedly different reactivity with DTNB. Sequence alignments offered preliminary hints on the contributions of sequence-specific local environments modulating albumin reactivity. Molecular dynamics simulations performed on experimental and predicted albumin structures reveal that thiolation rates are influenced by hydrogen bonding pattern and stability of the acceptor C34 sulphur atom with donor groups of nearby residues. Atom depth evolution of albumin C34 thiol groups has been monitored during Molecular Dynamic trajectories. The most reactive albumins appeared also the ones presenting the C34 sulphur atom on the protein surface with the highest accessibility. High C34 sulphur atom reactivity in rat and porcine albumins seems to be determined by the presence of additional positively charged amino acid residues favoring both the C34 S⁻ form and the approach of DTNB.

The control of S-thiolation by cysteine via gamma-glutamyltranspeptidase and thiol exchanges in erythrocytes and plasma of diamide-treated rats

Protein thiol modifications including cysteinylation (CSSP) and glutathionylation (GSSP) in erythrocytes of rat treated with diamide have been reported, but mechanism and origin of CSSP formation are unknown. Experiments were performed to relate CSSP formation to GSH hydrolysis via gamma-glutamyltranspeptidase (gamma-GT) and know whether cysteine may act as deglutathionylation factor. Time-dependent variations of redox forms of glutathione and cysteine were investigated in erythrocytes, plasma, liver and kidney of diamide-treated rats (0.4 mmol/kg by infusion for 45 min followed by 135 min of washout) in the presence and absence of acivicin (10 mg/kg administered twice 1 h before diamide) a known gamma-GT inhibitor. Diamide-treated rats showed decreased concentrations of erythrocyte GSH and increased levels of GSSP and CSSP. The rate of CSSP formation was slower than that of GSSP. Besides the entity of CSSP accumulation of erythrocytes was high and equivalent to approximately 3-fold of the normal plasma content of total cysteine. The result was paradoxically poorly related to gamma-GT activity because the gamma-GT inhibition only partially reduced erythrocyte CSSP. After gamma-GT inhibition, a large concentration fluctuation of glutathione (increased) and cysteine (decreased) was observed in plasma of diamide-treated rats, while little changes were seen in liver and kidney. There were indications from in vitro experiments that the CSSP accumulation in erythrocytes of diamide-treated rats derives from the coexistence of GSH hydrolysis via gamma-GT and production of reduced cysteine via plasma thiol exchanges. Moreover, reduced cysteine was found to be involved in deglutathionylation processes. Mechanisms of protein glutathionylation by diamide and deglutathionylation by cysteine were proposed.

Lycopene phytocomplex, but not pure lycopene, is able to trigger apoptosis and improve the efficacy of photodynamic therapy in HL60 human leukemia cells.

We compared the ability of pure lycopene (Lyco) versus lycopene phytocomplex (LycoC) to induce apoptosis in vitro. We found that LycoC, but not Lyco, was able to trigger apoptosis in HL60 cells, as documented by subdiploid DNA content and phosphatidylserine exposure. LycoC-induced apoptosis was associated with reactive oxygen species (ROS) generation and loss of mitochondrial transmembrane potential, suggesting that LycoC triggered apoptosis via a mitochondrial pathway. We also verified the redox state of cells by measuring glutathione (GSH) content, but only a small percentage of cells showed GSH depletion, suggesting that the loss of GSH may be a secondary consequence of ROS generation. Moreover, LycoC pretreatment effectively increased apoptosis induced by photodynamic therapy (PDT), a mode of cancer treatment using a photosensitizer and visible light. LycoC pretreatment was even more potent in improving PDT than pretreatment with ascorbic acid or alpha-tocopherol (or the two combined). Our results demonstrate that LycoC has a stronger cytotoxic effect than Lyco and is a better source of agents able to trigger apoptosis in HL60 cells and improve the efficacy of PDT in vitro.

In vitro inhibition of human and rat platelets by NO donors, nitrosoglutathione, sodium nitroprusside and SIN-1, through activation of cGMP-independent pathways.

Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug. SNP, GSNO and SIN-1 (100 μM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (α(IIb)β(3)) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured. Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC(50) values, μM): (a) in human, GSNO=0.52±0.09, SNP=2.83 ± 0.53, SIN-1=2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1=108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to α(IIb)β(3) through cGMP-dependent pathways. A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.