A new Coronavirus strain, named SARS-CoV-2, suddenly emerged in early December 2019. SARS-CoV-2 resulted in being dramatically infectious, with thousands of people infected. In this scenario, and ...without effective vaccines available, the importance of an immediate tool to support patients and against viral diffusion becomes evident. In this study, we exploit the molecular docking approach to analyze the affinity between different viral proteins and several inhibitors, originally developed for other viral infections. Our data show that, in some cases, a relevant binding can be detected. These findings support the hypothesis to develop new antiviral agents against COVID-19, on the basis of already established therapies.
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.
Human serum albumin shows 17 disulfides and one unpaired Cys34 which can be free in the reduced albumin or linked as a mixed disulfide in the oxidized albumin. 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.
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 p
and from a great accessibility to a given reagent. New findings and previous evidence clearly indicate that p
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 p
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.
Lactoferrin, a multifunctional iron-binding protein containing 16 disulfides, is actively studied for its antibacterial and anti-carcinogenic properties. However, scarce information is nowadays ...available about its oxidative folding starting from the reduced and unfolded status. This study discovers unusual properties when this protein is examined in its reduced molten globule-like conformation. Using kinetic, CD and fluorescence analyses together with mass spectrometry, we found that a few cysteines display astonishing hyper-reactivity toward different thiol reagents. In details, four cysteines (i.e. 668, 64, 512 and 424) display thousands of times higher reactivity toward GSSG but normal against other natural disulfides. The formation of these four mixed-disulfides with glutathione probably represents the first step of its folding in vivo. A widespread low pKa decreases the reactivity of other 14 cysteines toward GSSG limiting their involvement in the early phase of the oxidative folding. The origin of this hyper-reactivity was due to transient lactoferrin-GSSG complex, as supported by fluorescence experiments. Lactoferrin represents another disulfide containing protein in addition to albumin, lysozyme, ribonuclease, chymotrypsinogen, and trypsinogen which shows cysteines with an extraordinary and specific hyper-reactivity toward GSSG confirming the discovery of a fascinating new feature of proteins in their nascent phase.
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.
The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role ...of the correct disulfide in the attainment of the native structure. CD analysis, mass spectrometry, fluorescence spectroscopy and enzyme activity indicate that native disulfides drive the formation of the secondary and tertiary structures that cannot be entirely formed in their absence. This opposes a common opinion that these structures are first formed and then stabilized by the native disulfides. Our results also indicate that a spontaneous re-oxidation of a reduced RNase cannot produce a complete recovery of activity, as described by many textbooks; this can be obtained only in the presence of a reshuffling solution such as GSH/GSSG.
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.
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.
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 pK
(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 pK
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.
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