Search Results (116)

Thiol isomerases are a family of enzymes that participate in oxidative protein folding. They contain highly reactive vicinal thiols in a CXXC motif within their catalytic domains to mediate thiol-disulfide switching as part of their reductase, oxidase, and isomerase activity. In addition, they participate in chaperone function by binding to partially folded or misfolded proteins and preventing aggregation, thereby facilitating correct protein folding. The CXXC motif is conducive to oxidative influence based on the sulfur nucleophilicity. Redox modification of the CXXC motif may influence the enzymatic function. In this review we briefly discuss the family of thiol isomerases as it relates to thrombotic disorders. We then discuss the chemical mechanisms of making and breaking disulfides by the enzymes. Enzymatic and chemical models of oxidizing the CXXC motif are proposed. Lastly, we highlight evidence that natural galloylated polyphenols can inhibit both the coronavirus main protease Mpro and thiol isomerases, supporting a therapeutic strategy for COVID-19-associated coagulopathy and thrombosis by targeting the CXXC motif with these anti-oxidative compounds. Full article

Background and Clinical Significance: Cystinuria is the most common genetic cause of pediatric nephrolithiasis, characterized by impaired renal cystine reabsorption and resulting in increased urinary cystine excretion. Due to the poor solubility of cystine at normal urine pH, increased urinary cystine excretion leads to urine supersaturation and precipitation of cystine, resulting in nephrolithiasis. Case Presentation: Here, we report two cases of female patients diagnosed with cystinuria caused by SLC7A9 mutations. Both patients were initially managed with conservative treatments to minimize stone recurrence including increased oral fluid intake, a low-salt/low-protein diet, and potassium citrate supplementation with the goal of reducing urinary cystine levels and minimizing stone recurrences. Due to persistent stone formation, the patients were started on two distinct cystine-binding thiol medications. One patient was initiated on tiopronin, and the other on D-penicillamine. Tiopronin and D-penicillamine are both used in the treatment of pediatric cystinuria, although tiopronin is often preferred due to its more favorable side effect profile. However, due to insurance constraints, D-penicillamine was initiated for one patient in place of tiopronin. Since the initiation of these two distinct cystine-binding thiol medications, both patients have demonstrated reduced urinary cystine excretion and minimal to no recurrence of kidney stones. Conclusions: Cystine-binding thiols, including tiopronin and D-penicillamine, can both be used in the management of cystinuria in pediatric patients. Full article

Background: A comparative analysis was conducted between two immunisation protocols using different amounts of protein extracts from adult Haemonchus contortus worms, purified by thiol-Sepharose chromatography (625 μg/animal vs. 200 μg/animal). These protocols involved either five or two inoculations of the immunogen, respectively. Methods: To evaluate the level of immunoprotection, animals were challenged with L3 of H. contortus two weeks after the last inoculation of the immunogen and humanely sacrificed at 8 weeks post-infection. Parasitological, biopathological, and serological parameters were monitored through the experiment. Parasite burden, abomasal-specific antibody responses, and histopathological changes were determined at the end of the trial. Results: The immunisation protocols resulted in similar reductions in cumulative faecal egg counts (60.5–64.9%) and the total worm burden (47.5–50%) compared to non-immunized (control) animals. Overall, these parasitological data showed an early recovery of the haematocrit (PCV) after challenge in the immunised groups relative to control. Similarly, levels of H. contortus-specific IgG and IgA antibodies increased in both the serum and gastric mucus of immunised groups. Conclusions: These findings represent a further step towards the potential application of this type of immunogen under field conditions, as protective responses (associated with a reduction in faecal egg output) were achieved using a simplified protocol, with lower immunogen doses and fewer inoculations required to induce immunoprotection, thereby mitigating the pathological effects of the parasite and reducing its ability to spread and infect susceptible hosts. Full article

Background/Objectives: Microcolins A–M are cytotoxic marine lipopeptides produced by the cyanobacterium Moorena producens, also known as Lyngbya majuscula. Recent studies have shown that two compounds in the series, microcolins B and H, can form covalent complexes with phosphatidylinositol transfer proteins α and β (PITPα/β) upon the reaction of their α,β-unsaturated ketone group with the thiol group of a key cysteine residue of PITP. These observations prompted us to compare the binding of all microcolins and a few related derivatives (VT01454 and (deoxy)majusculamide D) to PITP to delineate structure–binding relationships. Methods: A molecular docking analysis led to the identification of microcolin E as the potentially best PITPα binder in the series, followed by microcolins B and H and analog VT01454. The computational data agree well with the published experimental results. Results: The binding of microcolin H into a large cavity of PITPα positions its reactive electrophilic α,β-unsaturated ketone close to the thiol of Cys95, enabling the facile formation of a covalent C-S linkage. A similar bonding can occur with the Cys94 of PITPβ. Molecular models of microcolins bound to PITP were compared to identify structural elements chiefly implicated in the recognition process. Conclusions: This computational study provides guidance in the design of microcolin derivatives targeting PITPα/β considered targets for cancer and inflammatory pathologies. Full article

With the goal of fast and accurate diagnosis of infectious diseases, this study presents a novel electrochemical biosensor that employs a refined aptamer (C9t) for the detection of spike (S) protein SARS-CoV-2 variants in a flexible multielectrode aptasensor array with PoC capabilities. Two aptamer modifications were employed: removing the primer binding sites and including two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 h and increasing the stability and sparseness for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without a reduction in aptamer density. The biosensor fabrication, optimization, and detection were verified in detail by electrochemistry, QCM-D, SPR, and XPS. The analyte–receptor binding was further confirmed spectroscopically at the level of individual molecules by AFM-IR. The aptasensor possesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 signal per concentration decade), and a wide dynamic detection range (8.0 fg/mL–38 ng/mL) in nasopharyngeal samples, covering the clinically relevant range. Furthermore, the C9t aptasensor showed high selectivity for SARS-CoV-2 S proteins over biomarkers for MERS-CoV, RSV, and Influenza. Even more, it showed a three times higher sensitivity for the Omicron in comparison to the Wuhan strain (wild type), alpha, and beta variants of the SARS-CoV-2 virus. Those results demonstrate the creation of an affordable and variant-selective refined C9t aptasensor that outperformed current rapid diagnosis tests. Full article

Ovarian cancer (OC) must be detected in its early stages when the mortality rate is the lowest to provide patients with the best chance of survival. Lysophosphatidic acid (LPA) is a critical OC biomarker since its levels are elevated across all stages and increase with disease progression. This paper presents an LPA assay based on a thickness shear mode acoustic sensor with dissipation monitoring that involves a new thiol molecule 3-(2-mercaptoethanoxy)propanoic acid (HS-MEG-COOH). HS-MEG-COOH is an antifouling linker that provides (a) antifouling properties for gold substrates and (b) linking ability via its terminal carboxylic acid functional group. The antifouling ability of HS-MEG-COOH was tested in whole human serum. The new molecule was applied to the LPA assay in conjunction with a spacer molecule, 2-(2-mercaptoethoxy)ethan-1-ol (HS-MEG-OH), in a 1:1 v/v ratio. HS-MEG-COOH was covalently linked to gelsolin–actin, a protein complex probe that dissociates due to LPA-binding. LPA was detected in phosphate-buffered saline and undiluted human serum and achieved a low limit of detection (1.0 and 0.7 μM, respectively) which was below the concentration of LPA in healthy individuals. The antifouling properties of HS-MEG-COOH and the detection of LPA demonstrate the ability of the sensor to successfully identify the early-stage OC biomarker in undiluted human serum. Full article

Mitochondrial holocytochrome c synthase (HCCS) is an essential protein in assembling cytochrome c (cyt c) of the electron transport system. HCCS binds heme and covalently attaches the two vinyls of heme to two cysteine thiols of the cyt c CXXCH motif. Human HCCS recognizes both cyt c and cytochrome c₁ of complex III (cytochrome bc₁). HCCS is mutated in some human diseases and it has been investigated recombinantly by mutational, biochemical, and reconstitution studies in the past decade. Here, we employ structural prediction programs (e.g., AlphaFold 3) on HCCS and its two substrates, heme and cytochrome c. The results, when combined with spectroscopic and functional analyses of HCCS and variants, provide insights into the structural basis for heme binding, apocyt c binding, covalent attachment, and release of the holocyt c product. Results from in vitro reconstitution of purified human HCCS using cyt c and cyt c₁ peptides as acceptors are consistent with the structural modeling of substrate binding. Reconstitution of HCCS and cyt c₁ provides an approach to studying cyt c₁ assembly, which has been refractile to recombinant in vivo reconstitution (unlike HCCS and cyt c). We propose a structural basis for release of the holocyt c product from HCCS based on in vitro studies and on cryoEM structures of the bacterial cyt c synthase (CcsBA) active site. We analyze the kinetoplastid mitochondrial synthase (KCCS), and hypothesize a molecular evolutionary path from mitochondrial endosymbiosis to the current HCCS. Full article

Human serum albumin (HSA) is an endogenous inhibitor of angiotensin I-converting enzyme (ACE) and, thus, plays a key role in the renin–angiotensin–aldosterone system (RAAS). However, little is known about the mechanism of interaction between these proteins, and the structure of the HSA–ACE complex has not yet been obtained experimentally. The purpose of the presented work is to apply computer modeling methods to study the interaction of HSA with ACE in order to obtain preliminary details about the mechanism of their interaction. Ten possible HSA–ACE complexes were obtained by the procedure of macromolecular docking. Based on the number of steric and polar contacts between the proteins, three leading complexes were selected, the stabilities of which were then tested by molecular dynamics (MD) simulation. Based on the results of MD simulation, the two most probable conformations of the HSA–ACE complex were selected. The analysis of these conformations revealed that the processes of oxidation of the thiol group of Cys34 of HSA and the binding of albumin to ACE can reciprocally affect each other. Known point mutations in the albumin molecules Glu82Lys, Arg114Gly, Glu505Lys, Glu565Lys and Lys573Glu can also affect the interaction with ACE. According to the result of MD simulation, the known ACE mutations, albeit associated with various diseases, do not affect the HSA–ACE interaction. A comparative analysis was performed of the resulting HSA–ACE complexes with those obtained by AlphaFold 3 as well as with the crystal structure of the HSA and the neonatal Fc receptor (FcRn) complex. It was found that domains DI and DIII of albumin are involved in binding both ACE and FcRn. The obtained results of molecular modeling outline the direction for further study of the mechanisms of HSA–ACE interaction in vitro. Information about these mechanisms will help in the design and improvement of pharmacotherapy aimed at modulation of the physiological activity of ACE. Full article

Pterostilbene (PTS), which is abundant in blueberries, is a dimethyl derivative of the natural polyphenol resveratrol (RES). Several plant species, including peanuts and grapes, also produce PTS. Although RES has a wide range of health benefits, including anti-cancer properties, PTS has a robust pharmacological profile that includes a better intestinal absorption and an increased hepatic stability compared to RES. Indeed, PTS has a higher bioavailability and a lower toxicity compared to other stilbenes, making it an attractive drug candidate for the treatment of various diseases, including diabetes, cancer, cardiovascular disease, neurodegenerative disorders, and aging. We previously reported that RES serves as a substrate for tyrosinase, producing an o-quinone metabolite that is highly cytotoxic to melanocytes. The present study investigated whether PTS may also be metabolized by tyrosinase, similarly to RES. PTS was oxidized as a substrate by tyrosinase to form an o-quinone, which reacted with thiols, such as N-acetyl-L-cysteine, to form di- and tri-adducts. We also confirmed that PTS was taken up and metabolized by human tyrosinase-expressing 293T cells in amounts several times greater than RES. In addition, PTS showed a tyrosinase-dependent cytotoxicity against B16BL6 melanoma cells that was stronger than RES and also inhibited the formation of melanin in B16BL6 melanoma cells and in the culture medium. These results suggest that the two methyl groups of PTS, which are lipophilic, increase its membrane permeability, making it easier to bind to intracellular proteins, and may therefore be more cytotoxic to melanin-producing cells. Full article

Cys is one of the least abundant amino acids in proteins. However, it is often highly conserved and is usually found in important structural and functional regions of proteins. Its unique chemical properties allow it to undergo several post-translational modifications, many of which are mediated by reactive oxygen, nitrogen, sulfur, or carbonyl species. Thus, in addition to their role in catalysis, protein stability, and metal binding, Cys residues are crucial for the redox regulation of metabolism and signal transduction. In this review, we discuss Cys post-translational modifications (PTMs) and their role in plant metabolism and signal transduction. These modifications include the oxidation of the thiol group (S-sulfenylation, S-sulfinylation and S-sulfonylation), the formation of disulfide bridges, S-glutathionylation, persulfidation, S-cyanylation S-nitrosation, S-carbonylation, S-acylation, prenylation, CoAlation, and the formation of thiohemiacetal. For each of these PTMs, we discuss the origin of the modifier, the mechanisms involved in PTM, and their reversibility. Examples of the involvement of Cys PTMs in the modulation of protein structure, function, stability, and localization are presented to highlight their importance in the regulation of plant metabolic and signaling pathways. Full article

The rapid and accurate detection of SARS-CoV-2, particularly its spike receptor-binding domain (S-RBD), was crucial for managing the COVID-19 pandemic. This study presents the development and optimization of two types of aptasensors: quartz crystal microbalance (QCM) and electrochemical sensors, both employing thiol-modified DNA aptamers for S-RBD detection. The QCM aptasensor demonstrated exceptional sensitivity, achieved by optimizing aptamer concentration, buffer composition, and pre-treatment conditions, with a limit of detection (LOD) of 0.07 pg/mL and a linear range from 1 pg/mL to 0.1 µg/mL, and a significant frequency change was observed upon target binding. The electrochemical aptasensor, designed for rapid and efficient preparation, utilized a one-step modification process that reduced the preparation time to 2 h while maintaining high sensitivity and specificity. Electrochemical impedance spectroscopy (EIS) enabled the detection of S-RBD concentrations as low as 132 ng/mL. Both sensors exhibited high specificity, with negligible non-specific interactions observed in the presence of competing proteins. Additionally, the QCM aptasensor’s functionality and stability were verified in biological fluids, indicating its potential for real-world applications. This study highlights the comparative advantages of QCM and electrochemical aptasensors in terms of preparation time, sensitivity, and specificity, offering valuable insights for the development of rapid, sensitive, and specific diagnostic tools for the detection of SARS-CoV-2 and other viruses. Full article

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a “dual anchor” mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors. Full article

The post-harvest processing of coffee beans leads to a wide range of reactions involving proteins. The formation of crosslinks between proteins and phenolic compounds present in high concentrations of coffee beans represents one of the most challenging and still not fully characterized reactions. The aim of this work was to assess the presence of products from such reactions in coffee samples, focusing on the adducts between cysteine and chlorogenic acids (CQAs). For this purpose, 19 green and 15 roasted coffee samples of the Coffea arabica, Coffea canephora, and Coffea liberica varieties were selected for this study and basically characterized. Then, targeted liquid chromatography mass spectrometry (LC-MS/MS) methods were developed to assess the formation of adducts between CQA and cysteine, glutathione, and N-acetylcysteine as the amino acid and peptide models, and quantified such adducts in coffee samples. The results of the characterization showed a heterogeneous distribution of the protein content (8.7–14.6%), caffeine (0.57–2.62 g/100 g), and antioxidant capacity (2–4.5 g ascorbic acid/100 g) in Arabica, Canephora, and Liberica samples. Glutamic acid, arginine, and proline were found to be the major amino acids, while 5-CQA (38–76%), 3-CQA (4–13%), and 4-CQA (4–13%) were the most abundant CQA derivatives of all coffee varieties. The model experiments for adduct formation demonstrated that cysteine binds to CQA via thiol groups and 5-CQA initially isomerizes to 3- and 4-CQA, depending on the conditions, allowing cysteine to bind to two different sites on 3-, 4- or 5-CQA molecules, thus, forming six different Cys-CQA adducts with m/z 476. The reaction was more favored at pH 9, and the adducts proved to be stable up to 90 °C for 10 min and up to 28 days at room temperature. The relative quantification of adducts showed peak area values ranging from 1100 to 3000 in green coffee bean samples, while no adducts were detected in roasted coffee beans. Overall, this work was the first attempt to demonstrate the presence of Cys-CQA adducts in coffee beans and paves the way for further investigations of such adduct formation at the protein level. Full article

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl⁻ and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation. Full article

In view of the increasing occurrence of anthelmintic-resistant strains of gastrointestinal nematodes in ruminants, various alternative control strategies have been investigated, such as those based on the induction of protective immune responses by immunisation with parasite antigens. In this study, the protective activity of somatic antigens from adult worms of Teladorsagia circumcincta purified by affinity chromatography on thiol-sepharose was analysed in goats. After challenge, the enriched products induced a slight reduction in the cumulative faecal egg counts (21%) and in the number of worms (23.3%), with a greater effect on female worms, which also showed a reduction in parameters related to their fertility. These parasitological findings were associated with a Th2 immune response, with a prominent local humoral response and an eosinophilic infiltrate in the gastric mucosa (negatively associated with the fertility of female worms and the number of worms, respectively), as well as an infiltration of MCHII+, CD4+, IgG+ and IgA+ cells. However, several analyses showed an increase in CD8+ cells in the mucosa, as well as IL-2 expression in the gastric lymph nodes, which may have been associated with inhibition of protective responses or with the development of mixed Th1/Th2 responses, a finding that should be analysed in future studies. Full article