Erythrocyte glutathione transferase. A sensitive Up-Down biomarker of environmental and industrial pollution.

Erythrocyte glutathione transferase is a well-known biomarker of environmental pollution. Examination of the extensive scientific literature discovers an atypical and very interesting property of this enzyme which may reveal a chronic exposition to many contaminants but in some cases even an acute and short-term dangerous contamination. This review also underlines the peculiar molecular and kinetic properties of this enzyme which makes it unique in the panorama of enzymes used as biomarker for environmental contamination.

Oxidative Folding of Proteins: The "Smoking Gun" of Glutathione.

Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found "not guilty". Now, new surprising evidence repeats its request to be the "smoking gun" which reopens the criminal trial revealing the crucial involvement of this tripeptide.

New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures.

Protein cysteines often play crucial functional and structural roles, so they are emerging targets to design covalent thiol ligands that are able to modulate enzyme or protein functions. Some of these residues, especially those involved in enzyme mechanisms-including nucleophilic and reductive catalysis and thiol-disulfide exchange-display unusual hyper-reactivity; such a property is expected to result from a low pKa and from a great accessibility to a given reagent. New findings and previous evidence clearly indicate that pKa perturbations can only produce two-four-times increased reactivity at physiological pH values, far from the hundred and even thousand-times kinetic enhancements observed for some protein cysteines. The data from the molten globule-like structures of ribonuclease, lysozyme, bovine serum albumin and chymotrypsinogen identified new speeding agents, i.e., hydrophobic/electrostatic interactions and productive complex formations involving the protein and thiol reagent, which were able to confer exceptional reactivity to structural cysteines which were only intended to form disulfides. This study, for the first time, evaluates quantitatively the different contributions of pKa and other factors to the overall reactivity. These findings may help to clarify the mechanisms that allow a rapid disulfide formation during the oxidative folding of many proteins.

Ultra-Rapid Glutathionylation of Ribonuclease: Is this the Real Incipit of its Oxidative Folding?

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40-50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5'-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease.

Animal Biomonitoring for the Surveillance of Environment Affected by the Presence of Slight Contamination by ß-HCH.

The aim of this study was to evaluate the influence of hidden environmental pollution on some blood parameters of sheep to detect susceptible biomarkers able to reveal slight contamination. Four dairy sheep farms, two with semi-extensive and two with intensive type systems were involved in this study. Two farms in different systems were chosen as properly located in a southern area of Latium (Italy), close to the Sacco River, in which contamination with ß-hexachlorocyclohexane (ß-HCH) occurred in the past due to industrial waste. A recent study established the presence of low but detectable residual contamination in these areas. The other two farms were outside the contaminated area. Erythrocyte glutathione transferase (e-GST) and oxidative stress parameters were monitored as well as some immune response and metabolic profile parameters throughout the investigated period of four months. The present study showed a relevant and significant increase in e-GST (+63%) in the extensive farming system of the contaminated area, whereas some immune response biomarkers, i.e., white blood cells, neutrophils, lymphocytes, and lysozyme resulted within the physiological range. In all farms, oxidative stress and acute phase response parameters were also within the physiological range. Our results suggest that e-GST is a very effective alarm signal to reveal "hidden" persistent contamination by ß-HCH, and reasonably, by many other different dangerous pollutants.

Trypsinogen and chymotrypsinogen: the mysterious hyper-reactivity of selected cysteines is still present after their divergent evolution.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD  = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

A Pilot Study of a Natural Food Supplement as New Possible Therapeutic Approach in Chronic Kidney Disease Patients.

The identification of natural bioactive compounds, able to counteract the abnormal increase of oxidative stress and inflammatory status in chronic degenerative non-communicable diseases is useful for the clinical management of these conditions. We tested an oral food supplement (OFS), chemically characterized and evaluated for in vitro and in vivo activity. Vitamin C, analyzed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), was 0.19 mg/g in rosehip dry extract and 15.74 mg/capsule in the OFS. The identification of polyphenols was performed by HPLC-DAD; the total antioxidant capacity was assessed by Folin-Ciocalteu test. Total polyphenols were 14.73 mg/g gallic acid equivalents (GAE) for rosehip extract and 1.93 mg/g GAE for OFS. A total of 21 chronic kidney disease (CKD) patients and 10 healthy volunteers were recruited. The evaluation of routine laboratory and inflammatory parameters, erythrocyte glutathione transferase (e-GST), human oxidized serum albumin (HSAox), and assessment of body composition were performed at two different times, at baseline and after 5 weeks of OFS assumption. In the study, we highlighted a significant decrease of traditional inflammatory biomarkers (such as C-reactive protein, erythrocyte sedimentation rate, platelet to lymphocyte ratio) and other laboratory parameters like e-GST, azotaemia, and albuminuria after OFS treatment in CKD patients. Moreover, we demonstrated a lipid profile improvement in CKD patients after OFS supplementation.

Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

Glutathione Transferase P1-1 an Enzyme Useful in Biomedicine and as Biomarker in Clinical Practice and in Environmental Pollution.

Glutathione transferase P1-1 (GSTP1-1) is expressed in some human tissues and is abundant in mammalian erythrocytes (here termed e-GST). This enzyme is able to detoxify the cell from endogenous and exogenous toxic compounds by using glutathione (GSH) or by acting as a ligandin. This review collects studies that propose GSTP1-1 as a useful biomarker in different fields of application. The most relevant studies are focused on GSTP1-1 as a biosensor to detect blood toxicity in patients affected by kidney diseases. In fact, this detoxifying enzyme is over-expressed in erythrocytes when unusual amounts of toxins are present in the body. Here we review articles concerning the level of GST in chronic kidney disease patients, in maintenance hemodialysis patients and to assess dialysis adequacy. GST is also over-expressed in autoimmune disease like scleroderma, and in kidney transplant patients and it may be used to check the efficiency of transplanted kidneys. The involvement of GSTP in the oxidative stress and in other human pathologies like cancer, liver and neurodegenerative diseases, and psychiatric disorders is also reported. Promising applications of e-GST discussed in the present review are its use for monitoring human subjects living in polluted areas and mammals for veterinary purpose.

Hemodialysis biomarkers: total advanced glycation end products (AGEs) against oxidized human serum albumin (HSAox).

AIMS: Nephropathic patients show higher levels of advanced glycation end products (AGEs) and oxidized human serum albumin (HSAox) compared to healthy subjects. These two classes of compounds are formed as the result of oxidative insults; for this reason, they can be useful oxidative stress biomarkers. The present study examines the variation of AGEs and HSAox in hemodialysis (HD) patients before and after dialysis session, evaluating the impact of different dialytic techniques and filters on their removal. METHODS: A total of 50 healthy subjects (control group) and 130 HD patients were enrolled in the study. Hemodialysis patients were subdivided based on dialytic techniques: 109 in diffusive technique and 22 in convective technique. We monitored HSAox, AGEs and other laboratory parameters at early morning in healthy subjects and in HD patients before and after the dialysis procedures. RESULTS: The level of HSAox decreases after a single dialytic session (from 58.5 ± 8.8% to 41.5 ± 11.1%), but the concentration of total AGEs increases regardless of adopted dialytic techniques (from 6.8 ± 5.2 µg/ml to 9.2 ± 4.4 µg/ml). In our study, levels of HSAox and total AGEs are similar in diabetic and non-diabetic HD patients. The increase in total AGEs after dialysis was only observed using polysulfone filters but was absent with polymethacrylate filters. CONCLUSIONS: HSAox is a simple and immediate method to verify the beneficial effect of a single dialysis session on the redox imbalance, always present in HD patients. Total AGEs assayed by ELISA procedure seem to be a less reliable biomarker in this population.

The impact of ionizing irradiation on liver detoxifying enzymes. A re-investigation.

By looking at many studies describing the impact of ionizing irradiations in living mice on a few key detoxifying enzymes like catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione transferase, we noted conflicting evidences: almost all papers finalized to demonstrate the protective effects of natural or synthetic drugs against the damage by irradiations, described also a relevant inactivation of these enzymes in the absence of these compounds. Conversely, no inactivation and even enhanced activity has been noted under similar irradiation modality in all studies supporting the "adaptive response". Motivated by these curious discrepancies, we performed irradiation experiments on living mice, explanted mouse livers and liver homogenates observing that, in all conditions the activity of all these enzymes remained almost unchanged except for a slight increase found in explanted livers. Our results put a question about many previous scientific reports in this field.

Thiol disulfide exchange reactions in human serum albumin: the apparent paradox of the redox transitions of Cys34.

Human serum albumin (HSA) is characterized by 17 disulfides and by only one unpaired cysteine (Cys34 ), which can be free in the reduced albumin or linked as a mixed disulfide with cysteine, or in minor amount with other natural thiols, in the oxidized albumin. In healthy subjects, the level of the oxidized form is about 35%, but it rises up to 70% after oxidative insults or in patients with kidney diseases. Oxidized albumin is therefore considered a short-term biomarker of oxidative stress as its level may increase or decrease under appropriate redox inputs in discrete temporal spans. This paper defines, for the first time, the kinetic properties of reduced and oxidized Cys34 of HSA in their reactions with natural disulfides and thiols. Kinetic constants support the evidence that the Cys34 redox oscillations observed in vivo are mainly due to the interaction with cysteine and cystine without the involvement of any enzymatic support. This study gives also a plausible explanation for the absence of involvement of the 17 disulfides naturally present in HSA in these redox transitions. This inert behavior toward cysteine is marginally due to solvent accessibility or flexibility factors of these bonds but mainly to their strong thermodynamic stability, which is caused essentially by a proximity effect. A similar mechanism is likely at play in the many proteins that maintain disulfide bridges in a reducing medium like the cytosol.

The extreme hyper-reactivity of Cys94 in lysozyme avoids its amorphous aggregation.

Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pKa of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (KD = 0.3 mM), causes a fast glutathionylation of this residue (t1/2 = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.

Erythrocyte glutathione transferase in kidney transplantation: a probe for kidney detoxification efficiency.

Erythrocyte glutathione transferase (e-GST) is overexpressed in case of increased blood toxicity and its level correlates with the kidney disease progression. Thus, it represents a probe of kidney efficiency against circulating toxins. We measured the activity of e-GST in patients with transplant kidney from living and cadaver donors, correlated its level to biochemical parameters of kidney function, and measured the level of oxidized albumin as a probe of oxidative stress using a new simple procedure. Interestingly, the activity of e-GST in transplant patients from cadaver donors (N = 153) is very high (11.7 U/gHb) compared to healthy subjects (N = 80) ( 5.6 U/gHb). Lower values were observed in transplant patients with kidney from living donors (N = 16) (9.8 U/gHb). Except for steroids, no correlation has been found with the immunosuppressive therapies and routine clinical and laboratory parameters. Also serum oxidized albumin, which reveals oxidative stress, is significantly higher in transplant patients from cadaver donors (53%) compared to that from living donors (36%). Overall, these data indicate that most of transplant kidneys from cadavers lost part of the detoxifying power against circulating toxins and suffer a relevant oxidative stress compared to those coming from living donors. A case report suggests that e-GST could represent a very early marker of incipient graft rejection. In conclusion, e-GST may be used to check the decline or maintenance of the kidney detoxification competence during post-transplantation course.

Structure of a PEGylated protein reveals a highly porous double-helical assembly.

PEGylated proteins are a mainstay of the biopharmaceutical industry. Although the use of poly(ethylene glycol) (PEG) to increase particle size, stability and solubility is well-established, questions remain as to the structure of PEG-protein conjugates. Here we report the structural characterization of a model ß-sheet protein (plastocyanin, 11.5 kDa) modified with a single PEG 5,000. An NMR spectroscopy study of the PEGylated conjugate indicated that the protein and PEG behaved as independent domains. A crystal structure revealed an extraordinary double-helical assembly of the conjugate, with the helices arranged orthogonally to yield a highly porous architecture. Electron density was not observed for the PEG chain, which indicates that it was disordered. The volume available per PEG chain in the crystal was within 10% of the calculated random coil volume. Together, these data support a minimal interaction between the protein and the synthetic polymer. Our work provides new possibilities for understanding this important class of protein-polymer hybrids and suggests a novel approach to engineering protein assemblies.