Extracellular Thiol Research Articles

Deteriorated thiol-disulphide and oxidized-reduced glutathione status in blood in Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is a steadily advancing neurodegenerative condition, the occurrence and prevalence of which are on the rise in various populations. Suspected factors contributing to its development encompass the buildup of amyloid β (Aβ) plaques, the formation of neurofibrillary tangles induced by tau proteins, and heightened oxidative stress. In this study, we aimed to evaluate intra-cellular glutathione status and extracellular thiol-disulphide status in patients with AD. MethodsAdult patients (>60 years old) diagnosed with AD based on DSM-IV diagnostic criteria were included in the study. Patients were divided into 3 groups as mild, moderate and severe according to Mini Mental Status Examination (MMSE) and clinical findings. Extracellular thiol-disulfide and intracellular oxidized-reduced glutathione status parameters for patient and control groups were analyzed before and after reduction procedures by using reaction of thiol groups with DTNB. ResultsThe reduced forms of both balances (native thiol (NT) and reduced glutathione (GSH)) were significantly lower in the patient group than the control group (p = 0.031 and <0.001, respectively), while oxidized forms (disulphide (SS) and oxidized glutathione (GSSG)) and SS/NT and GSSG/GSH percent ratios were significantly higher (p < 0.05 for all). The disease duration and oxidative stress were significantly higher in the severe group of AD. There was a shift in intracellular and extracellular thiol balances towards the oxidized side, along with correlations between MMSE and these balances (rho = −0.412 for SS/NT and rho = −0.488 for GSSG/GSH), with GSSG/GSH identified as a significant predictive factor (odds ratio (95 % confidence interval): 1.352 (1.136–1.610) for the moderate group and 1.829 (1.451–2.305) for the severe group. ConclusionsThese findings suggest that blood redox balance is disrupted in AD.

Erythrocytic Reduced/Oxidized Glutathione and Serum Thiol/Disulfide Homeostasis in Patients with Opioid Use Disorder.

This study aimed to evaluate oxidative damage by measuring erythrocytic reduced/oxidized glutathione as an intracellular thiol pool and serum thiol/disulfide homeostasis as an extracellular thiol pool in patients with opioid use disorder. In this prospective cross-sectional study, 33 male patients diagnosed with opioid use disorder and 30 healthy male controls were included. Sociodemographic characteristics and psychometric analyzes were performed and addiction characteristics (duration and amount of heroin use, usage methods) were recorded. For the evaluation of oxidative balance, intracellular reduced-oxidized glutathione (reduced glutathione and oxidized glutathione), and extracellular thiol-disulfide (native thiol and disulfide) levels were measured. There was a decrease in reduced glutathione and native thiol levels and an increase in GSSG and SS levels. Similarly, while oxidized/reduced glutathione, oxidized/total glutathione%, and disulfide/native thiol % ratios increased, the ratio of reduced glutathione/total glutathione% and native thiol/total thiol% decreased. Moreover, a positive correlation was found between the level of both intracellular and extracellular oxidant molecules and the duration and amount of opioid use. Impaired intracellular reduced glutathione/oxidized glutathione and extracellular disulfide/native thiol homeostasis were found in patients with opioid use disorder. The intracellular and extracellular oxidative stress may cause complications related to chronic opioid use.

A comprehensive assessment of redox balance in small for gestational age newborns and their mothers.

The objective of this study was to assess oxidative stress in small for gestational age (SGA) newborns and their mothers by evaluating intra- and extracellular thiol homeostasis and the quantification of major oxidants and antioxidants. A total of 75 mothers and their 75 newborns (43 SGA) were enrolled in this study. Thiol-disulfide homeostasis, serum myeloperoxidase, catalase, total oxidant, and antioxidant status were analyzed. Additionally, erythrocytic glutathione (GSH) homeostasis was measured. Although native and total thiol levels were decreased, disulfide levels were increased in SGA groups. Additionally, myeloperoxidase activity and total oxidant status levels were significantly elevated whereas total antioxidant status levels and enzymatic antioxidant systems were diminished in SGA groups. Similarly, intra-erythrocytic GSH homeostasis was shifted in favor of oxidants in SGA groups. Our results demonstrate that insufficient antioxidant systems in mothers and a robust source of oxidative stress in SGA might contribute to the pathophysiology of SGA births.

A ratiometric fluorescent probe for the detection of biological thiols based on a new supramolecular design

A new ratiometric fluorescent probe is designed and prepared based on the concept of supramolecular encapsulation and dye competition. This supramolecular probe is based on two commercially-available dyes, one common guest and a simple-to-synthesize host. Fluorescence spectroscopy confirms that the supramolecular probe is capable of detecting thiols quantitatively with a broad linear region in phosphate buffered saline or fetal bovine serum. Mechanistic study shows a reaction between thiol specie and the guest to alter the distribution of encapsulated dyes. The supramolecular probes are demonstrated to quantitatively detect extracellular biological thiols by plate reader, which shows it keeps its effectiveness in complex buffered systems.

Coupling fluorescent probes to characterize S-containing compounds in a sulfate reducing bacteria involved in Hg methylation

The microbial methylation of inorganic mercury Hg(II) is governed by S-containing compounds such as thiols (RSH) and sulfides (S2−). Various S-containing molecules in an environmental or culture medium can be difficult to assess because of the complexity of the medium, poor stability, and low concentration ranges of sulfide and thiol compounds. Here, we applied two fluorescence spectroscopy-based methods using α, β-unsaturated ethanoylcoumarin fluorophore (DHC) for the quantification of sulfides, and monobromo (trimethylammonio) bimane (qBBr) to quantify total thiol concentrations (in extracellular and bacterial cell fractions). The potential interferences of both organic and inorganic compounds from the matrix were evaluated. In the presence of Hg species, both methods allowed the quantification of free sulfides or thiols (not forming complexes with Hg). The two methods were highly sensitive, with detection limits of 100 nM and 20 nM for thiols and sulfides, respectively. They also exhibited high selectivity for the detection of thiols or sulfides against other tested matrix compounds. Finally, both methods were applied to characterize S-containing compounds in a culture of Pseudodesulfovibrio hydrargyri strain BerOc1, a methylating sulfate-reducing bacterium (SRB) exposed to 0.1 mM of cysteine. During bacterial growth, we used (i) DHC probe to quantify sulfide concentration in the bulk fraction, (ii) qBBr for total extracellular thiols and total thiols adsorbed on the cells, and (iii) liquid chromatography-tandem mass spectrometry to track cysteine degradation and characterize other thiols. The time series until the end of BerOc1 growth showed biodegradation of cysteine, and biosynthesis of sulfides and other thiol compounds.

The relationship between rehabilitation outcomes and extracellular thiol-disulphide and intracellular oxidized-reduced glutathione homeostasis in patients with subacute stroke

Background &amp; Objective: To evaluate the relationship between the clinical outcome of subacute stroke patients and extracellular thiol-disulphide (SH-SS) and intracellular oxidized-reduced glutathione (GSSG-GSH) homeostasis and the effect of the rehabilitation program on these homeostasis. Methods: In this prospective observational study, outcome assessments (National Institutes of Health Stroke Scale Scores (NIHSS), modified Rankin Scale (mRS), and Barthel Daily Living Activities Index (BI)) and SH-SS and GSSG-GSH homeostasis parameters were investigated from 42 patients with subacute stroke before and after 4-week rehabilitation treatment protocol. Also, SH-SS and GSSG-GSH homeostasis parameters were measured from 35 healthy volunteers. Results: SS/SH and GSSG/GSH ratios were significantly higher in the patient group at baseline and post-rehabilitation than the control group (p&lt;0.05). SS/SH ratio, GSSG/GSH ratio, NIHSS and mRS values significantly decreased with the rehabilitation (p&lt;0.05). The baseline SS/SH and GSSG/GSH ratios were correlated with baseline and post-rehabilitation NIHSS, mRS and BI scores (p&lt;0.05). Conclusions: The better outcomes of patients with higher baseline antioxidant thiol groups suggest that active thiol groups may play a role in achieving better outcomes in subacute stroke. In addition, rehabilitation treatment increased clinical scores and additionally shifted intracellular and extracellular thiol groups to the antioxidant side.

Thermoactive metallo-keratinase from Bacillus sp. NFH5: Characterization, structural elucidation, and potential application as detergent additive

In recent times, robust green technological developments have advanced the goal of a circular economy by minimizing waste generation. The study was undertaken to explore the keratinolytic activity of chicken feather-degrading bacteria from South African soil. Isolates coded as SSN-01 and HSN-01 were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM and their sequences were deposited in GenBank, with accession numbers MW165830.1 and MW165831.1, respectively. Extracellular enzyme production and thiol group generation by Bacillus sp. NFH5 peaked at 120 h with 1879.09 ± 88.70 U/mL and 9.49 ± 0.78 mM, respectively. Glutamic acid (4.44%), aspartic acid (3.50%), arginine (3.23%), glycine (2.61%), serine (2.08%), and proline (2.08%) were relatively higher in concentration. Keratinase (KerBAN) activity was highest at pH 8.0 and 90 °C but was inhibited by both EDTA and 1,10-phenanthroline. In addition, the keratinase-encoding gene (kerBAN) accessioned OK033360 had 362 amino acid residues, with molecular weight and theoretical isoelectric point of 39 kDa and 8.81, respectively. Findings from this study highlight the significance of Bacillus sp. NFH5 in the bio-recycling of recalcitrant keratinous wastes to protein hydrolysates – potential dietary supplements for livestock feeds. The properties of KerBAN underscore its application potential in green biotechnological processes.

Mechanistic insights into bio-stabilization of lead (II) in flue gas by a sulfate-reducing bioreactor

This study investigated bio-stabilization of divalent lead (Pb2+) in the simulated flue gas by a sulfate-reducing bioreactor (SRBR). The SRBR achieved Pb2+ bio-removal effectively (95.7–98.7%). Metagenomic and metaproteomic analyses revealed that bacterial heavy metal resistance, sulfate bio-reduction/dephosphorylation-driven Pb2+ bioprecipitation, Pb2+ bio-complexation by extracellular polymeric substances (EPS) and thiols, Pb2+ biosorption by cell wall and cell membrane, as well as Pb2+ bio-expulsion by bacterial efflux and secretion system synergistically actualized flue gas Pb2+ biodetoxification. Lead speciation analysis by sequential extraction processes followed by inductively coupled mass spectrometry and further characterization by a series of spectroscopic and spectrometric techniques comprehensively verified that well-crystalized Galena (PbS) and Pyromorphite (Pb5(PO4)3Cl) cell surface-deposited microparticles in the form of residual lead (Res-Pb, 53.4–73.7%) were the major products in Pb2+ bio-stabilization. Besides, organic matter-bound lead (OM-Pb, 4.1–12.6%) and humic acids-bound lead (HA-Pb, 9.0–23.1%) were the second major products. Flue gas Pb2+ mostly bioprecipitated with biogenic sulfides and phosphates to form PbS and Pb5(PO4)3Cl, partly complexed by humic acids, EPS and thiols to form HA-Pb and OM-Pb, and was partly immobilized by cell wall and cell membrane. The weakly stable OM-Pb and HA-Pb may thermodynamically convert into the less mobile, highly chemically stable and less bioavailable products: PbS and Pb5(PO4)3Cl via long-term equilibration. Therefore, SRBR effectively achieved bio-removal, bio-stabilization and bio-recovery of Pb2+ in flue gas, which may provide a feasible and environment-friendly biotechnique for flue gas Pb2+ decontamination.

Intra- and Extra-cellular Thiol–Disulfide Homeostasis in Blood of Patients with Alcohol Use Disorder

Intra- and Extra-cellular Thiol–Disulfide Homeostasis in Blood of Patients with Alcohol Use Disorder

Značaj tiol-disulfidne ravnoteže u SARS-CoV-2 infekciji

Studies of the molecular mechanisms regarding interaction of different viruses with receptors on the host cell surface have shown that the viral entry depends on the specific relationship between free thiol (SH) groups and disulfides on the virus surface, as well as the thiol disulfide balance on the host cell surface. The presence of oxidizing compounds or alkylating agents, which disturb the thiol-disulfide balance on the surface of the virus, can also affect its infectious potential. Disturbed thiol-disulfide balance may also influence protein-protein interactions between SARS-CoV-2 protein S and ACE2 receptors of the host cell. This review presents the basic mechanisms of maintaining intracellular and extracellular thiol disulfide balance and previous experimental and clinical evidence in favor of impaired balance in SARS-CoV-2 infection. Besides, the results of the clinical application or experimental analysis of compounds that induce changes in the thiol disulfide balance towards reduction of disulfide bridges in proteins of interest in COVID-19 infection are presented.

Thiol and Glutathione Homeostasis Parameters as Plasma Biomarkers of Oxidative Stress in Age-Related Macular Degeneration

Purpose: To determine extracellular thiol homeostasis and intracellular glutathione homeostasis as a plasma biomarker for oxidative stress and to compare these parameters in non-exudative/exudative AMD patients and healthy individuals. Method: 30 non-exudative AMD, 28 exudative AMD, and 36 age-matched healthy control subjects enrolled to the study. Extracellular total thiol, native thiol, disulphide amounts and intracellular oxidized/reduced glutathione levels of subjects were determined, and disulphide/thiol and oxidized/reduced glutathione percent ratios were calculated. Results: In comparison with the control group both non-exudative and exudative AMD patients had higher plasma disulfide levels (20.5(4.8) vs. 4(3.1), p<0.001 and 22.5(7.5) vs. 15.4(3.1), p<0.001; respectively) and higher disulphide/thiol (6.64(2.57) vs. 5.4(1.9), p=0,002 and 7.05(3.14) vs. 5.4(1.9), p<0.001; respectively), in addition to higher oxidized glutathione levels (64.6(40.8) vs. 27.3(21.9), p=0.015 and 73.9(44.1) vs. 27.3(21.9), p=0.002; respectively) and oxidized/reduced glutathione ratio(6.48(8.35) vs. 3.14(3.31), p=0,034 and 10.21(10.28) vs. 3.14(3.31), p=0,003; respectively). Although there was no significant difference between groups in term of total thiol (361.5(61.6), 355.1(87.7) and 340.9(72.4), respectively, p=0,585); native thiol (318.8(62.4), 307.1(73.7) and 299.3(79.2), respectively, p=0,382); total reduced glutathione (986.3(282.1), 871.5(271.6) and 881.8(290.9), respectively, p=0.344) and native reduced glutathione (873.4(367.6), 723.7(379.0) and 797.2(307.5), respectively, p=0,113). However, there was no significant difference between non-exudative and exudative AMD groups in terms of both extracellular thiol homeostasis and intracellular glutathione homeostasis. Conclusion: Greater extent of both extracellular disulphide and intracellular oxidized glutathione production occurred in AMD patients compared to age-matched healthy controls indicates the role of increased oxidative stress in AMD development. Further studies are needed to confirm the pathophysiologic role of homeostasis in these buffer systems in AMD.

The Relationship between Choroidal Thickness and Intracellular Oxidised-reduced Glutathione and Extracellular Thiol–disulfide Homeostasis at Different Stages of Diabetic Retinopathy

ABSTRACT Purpose: To evaluate the relationship between diabetic retinopathy and oxidative damage by measuring intracellular and extracellular thiol levels, and to compare intracellular and extracellular thiol levels. Method: In this prospective, cross-sectional, and comparative study, 25 healthy control participants (group 1), a total of 25 diabetic macular edema (DME) patients with non-proliferative diabetic retinopathy (DRP) and without DME (group 2), and 25 DME patients with non-proliferative DRP and with DME (group 3) were included. Choroidal thickness (ChT) and central macular thickness (CMT) were measured by spectral domain optic coherence tomography. For the evaluation of antioxidant/oxidant balance, intracellular GSH (reduced glutathione) and GSSG (oxidized glutathione), extracellular SH (thiol) and SS (disulfide) levels were measured and recorded. Results: Comparing intracellular and extracellular thiol levels between groups, intracellular GSSG level and GSSG/GSH percent ratio, and extracellular disulfide and SS/SH percent ratio values were higher in diabetic patients than healthy participants. Choroidal thicknesses were significantly thinner in DRP groups compared to the healthy population. When the relationship between choroidal thicknesses and thiol levels was investigated, there were significant relationships between choroidal thicknesses and thiol levels in group 3. Conclusion: Oxidative stress and impaired intracellular GSH/GSSG and serum SH/SS balances were observed to have an effect on DRP and DME pathogenesis. In addition, in groups with and without DME, thinning in choroidal thicknesses and the relationship between these thicknesses and intra/extracellular oxidative stress indicators can also be explained.

Thiol switches in membrane proteins - Extracellular redox regulation in cell biology.

Redox-mediated signal transduction depends on the enzymatic production of second messengers such as hydrogen peroxide, nitric oxide and hydrogen sulfite, as well as specific, reversible redox modifications of cysteine-residues in proteins. So-called thiol switches induce for instance conformational changes in specific proteins that regulate cellular pathways e.g., cell metabolism, proliferation, migration, gene expression and inflammation. Reduction, oxidation and disulfide isomerization are controlled by oxidoreductases of the thioredoxin family, including thioredoxins, glutaredoxins, peroxiredoxins and protein dsisulfide isomerases. These proteins are located in different cellular compartments, interact with substrates and catalyze specific reactions. Interestingly, some of these proteins are released by cells. Their extracellular functions and generally extracellular redox control have been widely underestimated. Here, we give an insight into extracellular redox signaling, extracellular thiol switches and their regulation by secreted oxidoreductases and thiol-isomerases, a topic whose importance has been scarcely studied so far, likely due to methodological limitations. We focus on the secreted redox proteins and characterized thiol switches in the ectodomains of membrane proteins, such as integrins and the metalloprotease ADAM17, which are among the best-characterized proteins and discuss their underlying mechanisms and biological implications.

Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells.

Simple SummaryMetastasis is one of the most devastating aspect of cancer progression and involves biochemical and physical interactions between cancer cells and surrounding microenvironment. In particular, cancer cells adhesion to endothelium and their subsequent transendothelial migration represent important steps in the metastatic process in target organs. In this study we characterized the functional role of Protein Disulfide Isomerase A1 (PDIA1) in breast cancer cells adhesion and transendothelial migration. We identified the full repertoire of protein disulfide isomerases in endothelial cells as well as in breast cancer cells. We provided insight into the mechanisms involved in cancer-endothelial cells interactions and suggested that PDIA1 regulates the adhesion and transendothelial migration of breast cancer cells by disulphide exchange involving most likely the activation of integrins. Our results suggest that the inhibition of extracellular PDIA1 or other PDIs represents an interesting target for anti-metastatic treatment. Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that β1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer–endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.

Impact of combined MRP1 inhibition and mutant p53-targeting compound APR-246.

e14712 Background: The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. Mutant p53 protein is often expressed at high levels and accompanied with gain-of-function activities that promote tumor development and resistance towards conventional treatment. APR-246 is a mutant p53-reactivating small molecule undergoing a Phase III clinical study in myelodysplastic syndrome (MDS), and several phase II studies. APR-246 is non-enzymatically converted to its active product methylene quinuclidinone (MQ) which binds to cysteine residues in p53. This stimulates proper folding of p53's DNA-binding core domain, leading to cell death. APR-246 also exhibits pro-oxidant activity as the electrophile MQ binds and inactivates important antioxidants such as glutathione and thioredoxin reductase, which both are essential for cellular defense against oxidative and electrophilic stress. Methods: All results are in vitro experiments by LC-MS as well as experiments in cultured cells, including 14C-APR-246/MQ detection, cell viability measurements, LC-MS analysis, enzyme recycling measurements and Western blotting. Results: We have utilized 14C-labelled APR-246 to investigate the effect of APR-246 and its active moiety MQ in tumor cell cultures upon inhibition of efflux transporter multidrug resistance protein 1 (MRP1) or cystine-glutamate transporter (xCT). Transient downregulation or small molecule inhibitors (MK-571, reversan, sulfasalazin) targeting MRP1 or xCT increased drug content and shifted intra- and extracellular thiol status. Missense mutant TP53-carrying cells exhibited higher sensitivity to APR-246 and combination therapies. MRP1 inhibition showed strong synergy with APR-246 and increased intracellular levels of MQ conjugated to glutathione (GS-MQ). We found that GS-MQ conjugate formation is reversible, and suggest that the intrinsic reversibility of MQ adduct formation is an important aspect of the mechanism of action of APR-246. This reversibility may also in part account for the benign safety profile reported from clinical studies with APR-246. Conclusions: Sensitivity to APR-246 is affected by MRP1 efflux activity and the redox status, reflected in ratios of cysteine/cystine and reduced/oxidized glutathione (GSH/GSSG).

Integrin-mediated cell adhesion requires extracellular disulfide exchange regulated by protein disulfide isomerase

Cell adhesion to extracellular matrix, mediated by integrin receptors, is crucial for cell survival. Receptor-ligand interaction involves conformational changes in the integrin by a mechanism not fully elucidated. In addition to several direct evidence that there is disulfide re-arrangement of integrins, we previously demonstrated a role for extracellular thiols and protein disulfide isomerase (PDI) in integrin-mediated functions using platelets as model system. Exploring the possible generality of this mechanism, we now show, using three different nucleated cells which depend on adhesion for survival, that non-penetrating blockers of free thiols inhibit α2β1 and α5β1 integrin-mediated adhesion and that disulfide exchange takes place in that process. Inhibiting extracellular PDI mimics thiol blocking. Transfection with WT or enzymatically inactive PDI increased their membrane expression and enhanced cell adhesion, suggesting that PDI level is a limiting factor and that the chaperone activity of the enzyme contributes to adhesion. Exogenously added PDI also enhanced adhesion, further supporting the limiting factor of the enzyme. These data indicate that: a) Dependence on ecto-sulfhydryls for integrin-mediated adhesion is not exclusive to the platelet; b) PDI is involved in integrin-mediated adhesion, catalyzing disulfide bond exchange; c) PDI enhances cell adhesion by both its oxidoreductase activity and as a chaperone.

Can the effect of cold physical plasma-derived oxidants be transported via thiol group oxidation?

PurposeIntra- and intercellular redox-signaling processes where found responsible in various physiological and pathological processes with cellular thiol groups as important signal transducers. Using cold atmospheric plasma (CAP), a similar oxidation pattern of thiol groups can be achieved. Hence, it must be clarified which role extracellular thiol groups play in mediating CAP effects and whether or not the effects of short-lived reactive species can be preserved in a molecule like cysteine. MethodsPhysiological buffer solutions containing the amino acid cysteine were treated by an MHz argon plasma jet with molecular gas admixtures (kINPen) and transferred to cultured human keratinocytes. Cell proliferation, migratory activity, and metabolism were investigated. High-resolution mass spectrometry was used to estimate the impact of plasma generated species on thiol groups. ResultsWhile treated physiologic cysteine concentrations showed no impact on cell behavior, artificially high concentrations decreased proliferation, migration and lactate secretion. GSH levels inside cells were stabilized. ConclusionExtracellular thiol groups scavenge plasma-generated species and form a multitude of covalent modifications. Unexpectedly, human keratinocytes show only small functional consequences for treated physiologic cysteine concentrations. Results for high concentrated cysteine solutions indicate an improved cytostatic/cytotoxic impact by plasma treatment suggesting a potential application as a “preserving agent” of the chemical energy of plasma-derived species.

Identification of PDI Substrates by Mechanism-Based Kinetic Trapping.

Protein disulphide isomerase (PDI) is secreted by activated platelets and endothelial cells and is required for thrombus formation upon vascular injury. PDI catalyzes the reduction, oxidation, or isomerization of disulphide bonds in its substrate proteins. The specific substrates of PDI during thrombus formation have largely remained elusive, in part due to the transient nature of the PDI-substrate interaction.To overcome this challenge we have adapted and developed a kinetic substrate trapping strategy to identify extracellular substrates of PDI. By substitution of selected amino acids in the PDI active sites, we have generated PDI variants that form stable complexes with their substrates for subsequent isolation and identification. We here describe the substrate trapping methodology in detail, including generation and characterization of PDI variants, kinetic trapping experiments, and isolation and identification of bound substrates. The protocol can be adapted for most any biological fluid or sample, and can be applied to other extracellular thiol isomerases.

Enhanced uptake in 2D- and 3D- lung cancer cell models of redox responsive PEGylated nanoparticles with sensitivity to reducing extra- and intracellular environments

In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG (polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhancing cell internalization of the particles and their drug cargo.The new co-polymers, in which PEG and PLGA were linked by ‘anchiomeric effector’ dithiylethanoate esters, were synthesized by a combination of ring-opening polymerization and Michael addition reactions and employed to prepare NPs. Non redox-responsive nanoparticles (nRR-NPs) based on related PLGA-PEG copolymers were also prepared as comparators. Spherical NPs of around 120 nm diameter with a low polydispersity index and negative zeta potential as well as good drug loading of docetaxel were obtained. The NPs showed prolonged stability in relevant simulated biological fluids and a high ability to penetrate an artificial mucus layer due to the presence of the external PEG coating. Stability, FRET and drug release studies in conditions simulating intracellular reductive environments demonstrated a fast disassembly of the external shell of the NPs, thus triggering on-demand drug release.FACS measurements and confocal microscopy showed increased and faster uptake of RR-NPs in both 2D- and 3D- cell culture models of lung cancer compared to nRR-NPs. In particular, the ‘designed-in’ reductive instability of RR-NPs in conditioned cell media, the fast PEG release in the extracellular compartment, as well as a diminution of uptake rate in control experiments where extracellular thiols were neutralized, suggested a partial extracellular release of the PEG fringe that promoted rapid internalization of the residual NPs into cells.Taken together, these results provide further evidence of the effectiveness of PEGylated reducible nanocarriers to permeate mucus layer barriers, and establish a new means to enhance cancer cell uptake of drug carriers by extra-and intra-cellular cleavage of protein- and cell-shielding hydrophilic blocks.

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis