-
1.
An Evaluation of Oxidative Stress With Thiol/Disulfide Homeostasis in Patients With Persistent Allergic Rhinitis.
Göker, AE, Alagöz, MH, Kumral, TL, Karaketir, S, Yilmazer, AB, Tutar, B, Ahmed, EA, Biçer, C, Uyar, Y
Ear, nose, & throat journal. 2022;(1):NP13-NP17
-
-
Free full text
-
Abstract
BACKGROUND We evaluated the efficacy of medical treatment on thiol-disulfide balance despite ongoing allergic stimulation. METHODS The research design was a prospective observational study that included 35 persistent allergic rhinitis (AR) patients. All patients who were diagnosed with persistent AR were included. A skin prick test was applied to all patients, and the Sino-nasal Outcome Test-22 was used to evaluate sinonasal symptoms. Thiol/disulfide homeostasis balance parameters were measured using a novel automatic and spectrophotometric method and compared statistically. Serum total thiol (TT), native thiol (SH), disulphide (SS), disulphide/native thiol (SS/SH), disulphide/total thiol (SS/TT), and native thiol/total thiol (SH/TT) ratios were measured after the second month of the treatment. RESULTS The 35 patients included 20 (58%) females and 15 (42%) males. The mean age of the patients was 33.17 ± 9.9 years. Disulphide, SS/SH, and SS/TT ratios decreased significantly after the treatment (P < .05), while SH and SH/TT increased significantly (P < .05). The mean SH measurement increased significantly in the second month (P = .001), but TT mean measurements showed no difference after the treatment (P = .058). The mean SS measurements, on the other hand, decreased significantly in the second month (P = .003). CONCLUSION Thiol/disulfide homeostasis may be used as a marker to evaluate the efficacy of persistent AR treatments. After the treatment, the increase in SH levels suggested the decrease in oxidative stress, even though allergen exposure continued.
-
2.
Dual Enkephalinase Inhibitors and Their Role in Chronic Pain Management.
Southerland, WA, Gillis, J, Kuppalli, S, Fonseca, A, Mendelson, A, Horine, SV, Bansal, N, Gulati, A
Current pain and headache reports. 2021;(5):29
Abstract
PURPOSE OF REVIEW Dual enkephalinase inhibitors (DENKIs) are pain medications that indirectly activate opioid receptors and can be used as an alternative to traditional opioids. Understanding the physiology of enkephalins and their inhibitors and the pharmacology of these drugs will allow for proper clinical application for chronic pain patients in the future. RECENT FINDINGS DENKIs can be used as an alternative mode of analgesia for patients suffering from chronic pain by preventing the degradation of endogenous opioid ligands. By inhibiting the two major enkephalin-degrading enzymes (neprilysin and aminopeptidase N), DENKIs can provide analgesia with less adverse effects than nonendogenous opioids. The purpose of this paper is to review the current literature investigating DENKIs and explore their contribution to chronic pain management.
-
3.
Computational design and experimental substantiation of conformationally constrained peptides from the complex interfaces of transcriptional enhanced associate domains with their cofactors in gastric cancer.
Zhang, D, Wu, H, Zhao, J
Computational biology and chemistry. 2021;:107569
Abstract
Transcriptional enhanced associate domains (Teads) are the downstream effectors of the hippo signaling pathway and have been recognized as attractive druggable targets of gastric cancer. The biological function of Teads is regulated by diverse cofactors. In this study, the intermolecular interactions of Teads with their cognate cofactors were systematically characterized at structural, thermodynamic and dynamic levels. The Teads possess a double-stranded helical hairpin that is surrounded by three independent structural elements β-sheet, α-helix and Ω-loop of cofactor proteins and plays a central role in recognition and association with cofactors. A number of functional peptides were split from the hairpin region at Tead-cofactor complex interfaces, which, however, cannot maintain in native conformation without the support of protein context and would therefore incur a considerable entropy penalty upon competitively rebinding to the interfaces. Here, we further used disulfide and hydrocarbon bridges to cyclize and staple the hairpin and helical peptides, respectively. The chemical modification strategies were demonstrated to effectively constrain peptide conformation into active state and to largely reduce peptide flexibility in free state, thus considerably improving their affinity. Since the cyclization and stapling only minimize the indirect entropy cost but do not influence the direct enthalpy effect upon peptide binding, the designed conformationally constrained peptides can retain in their native selectivity over different cofactors. This is particularly interesting because it means that the cyclized/stapled, affinity-improved peptides can specifically compete with their parent Teads for the cofactor arrays as they share consistent target specificity.
-
4.
Electrostatic interactions contribute to the control of intramolecular thiol-disulfide isomerization in a protein.
Maag, D, Putzu, M, Gómez-Flores, CL, Gräter, F, Elstner, M, Kubař, T
Physical chemistry chemical physics : PCCP. 2021;(46):26366-26375
Abstract
The roles of structural factors and of electrostatic interactions with the environment on the outcome of thiol-disulfide exchange reactions were investigated in a mutated immunoglobulin domain (I27*) under mechanical stress. An extensive ensemble of molecular dynamics trajectories was generated by means of QM/MM simulations for a total sampling of 5.7 μs. A significant number of thiol-disulfide exchanges were observed, and the Cys32 thiolate preferred to attack Cys55 over Cys24, in agreement with previous experimental and computational studies. The structural features as well as electronic structures of the thiol-disulfide system along the reaction were analyzed, as were the electrostatic interactions with the environment. The previous findings of better accessibility of Cys55 were confirmed. Additionally, the reaction was found to be directed by the electrostatic interactions of the involved sulfur atoms with the molecular environment. The relationships of atomic charges, which stem from the electrostatic interactions, lead to the kinetic preference of the attack on Cys55. Further, QM/MM metadynamics simulations of thiol-disulfide exchange in a small model system with varied artificial external electric potentials revealed changes in reaction kinetics of the same magnitude as in I27*. Therefore, the electrostatic interactions are confirmed to play a role in the regioselectivity of the thiol-disulfide exchange reactions in the protein.
-
5.
Amyloid-β induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation.
Dilna, A, Deepak, KV, Damodaran, N, Kielkopf, CS, Kagedal, K, Ollinger, K, Nath, S
Biochimica et biophysica acta. Molecular basis of disease. 2021;(12):166246
Abstract
Alzheimer's disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-β1-42 oligomers (oAβ) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oAβ induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oAβ propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oAβ-induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-staining dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of Ca2+. The massive endocytosis eventually caused accumulation of internalized oAβ in Lamp1 positive multivesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three-dimensional quantitative confocal imaging, structured illumination superresolution microscopy, and flowcytometry quantifications revealed that oAβ induces activation of phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, the formation of TNT-like conduits was inhibited by preventing PAK1-dependent internalization of oAβ using the small-molecule inhibitor IPA-3, a highly selective cell-permeable auto-regulatory inhibitor of PAK1. The present study reveals that the TNT-like conduits are probably instigated as a consequence of oAβ induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium.
-
6.
Effects of antiviral drug therapy on dynamic thiol/disulphide homeostasis and nitric oxide levels in COVID-19 patients.
Mete, AÖ, Koçak, K, Saracaloglu, A, Demiryürek, S, Altınbaş, Ö, Demiryürek, AT
European journal of pharmacology. 2021;:174306
-
-
Free full text
-
Abstract
The novel coronavirus disease 2019 (COVID-19) has led to a serious global pandemic. Although an oxidative stress imbalance occurs in COVID-19 patients, the contributions of thiol/disulphide homeostasis and nitric oxide (NO) generation to the pathogenesis of COVID-19 have been poorly identified. Therefore, the aim of this study was to evaluate the effects of antiviral drug therapy on the serum dynamics of thiol/disulphide homeostasis and NO levels in COVID-19 patients. A total of 50 adult patients with COVID-19 and 43 sex-matched healthy control subjects were enrolled in this prospective study. Venous blood samples were collected immediately on admission to the hospital within 24 h after the diagnosis (pre-treatment) and at the 15th day of drug therapy (post-treatment). Serum native thiol and total thiol levels were measured, and the amounts of dynamic disulphide bonds and related ratios were calculated. The average pre-treatment total and native thiol levels were significantly lower than the post-treatment values (P < 0.001 for all). We observed no significant changes in disulphide levels or disulphide/total thiol, disulphide/native thiol, or native thiol/total thiol ratios between pre- and post-treatments. There was also a significant increase in serum NO levels in the pre-treatment values when compared to control (P < 0.001) and post-treatment measurements (P < 0.01). Our results strongly suggest that thiol/disulphide homeostasis and nitrosative stress can contribute to the pathogenesis of COVID-19. This study was the first to show that antiviral drug therapy can prevent the depletion in serum thiol levels and decrease serum NO levels in COVID-19 patients.
-
7.
Harnessing the potential of bacterial oxidative folding to aid protein production.
Slater, SL, Mavridou, DAI
Molecular microbiology. 2021;(1):16-28
-
-
Free full text
-
Abstract
Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine-containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision-making workflow for exploiting disulfide bond formation for the expression of any given protein of interest.
-
8.
Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway.
Robinson, PJ, Bulleid, NJ
Cells. 2020;(9)
Abstract
Disulphide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulphide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulphide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulphide bond formation is reviewed with emphasis on events that occur during ER entry. Disulphide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulphide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulphide bond formation and identifies key questions that need to be addressed to further understand this fundamental process.
-
9.
C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning.
Banaś, AM, Bocian-Ostrzycka, KM, Plichta, M, Dunin-Horkawicz, S, Ludwiczak, J, Płaczkiewicz, J, Jagusztyn-Krynicka, EK
PloS one. 2020;(3):e0230366
Abstract
Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.
-
10.
Thioredoxin inhibitor PX-12 induces mitochondria-mediated apoptosis in acute lymphoblastic leukemia cells.
Ehrenfeld, V, Fulda, S
Biological chemistry. 2020;(2):273-283
Abstract
Imbalances in redox homeostasis have been described to be involved in the development, progression and relapse of leukemia. As the thioredoxin (Trx) system, one of the major cellular antioxidant networks, has been implicated in acute lymphoblastic leukemia (ALL), we investigated the therapeutic potential of Trx inhibition in ALL. Here, we show that the Trx inhibitor PX-12 reduced cell viability and induced cell death in a dose- and time-dependent manner in different ALL cell lines. This antileukemic activity was accompanied by an increase in reactive oxygen species (ROS) levels and enhanced PRDX3 dimerization. Pre-treatment with the thiol-containing ROS scavenger N-acetylcysteine (NAC), but not with non-thiol-containing scavengers α-tocopherol (α-Toc) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP), significantly rescued PX-12-induced cell death. Furthermore, PX-12 triggered activation of BAK. Importantly, knockdown of BAK reduced PX-12-stimulated ROS production and cell death. Similarly, silencing of NOXA provided significant protection from PX-12-mediated cell death. The relevance of mitochondria-mediated, caspase-dependent apoptosis was further supported by data showing that PX-12 triggered cleavage of caspase-3 and that addition of the broad-range caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD.fmk) potently blocked cell death upon PX-12 treatment. This study provides novel insights into the mechanisms of PX-12-induced cell death in ALL and further highlights the therapeutic potential of redox-active compounds in ALL.