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1.
Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach.
Akbarzadeh, A, Pourzardosht, N, Dehnavi, E, Ranaei Siadat, SO, Zamani, MR, Motallebi, M, Nikzad Jamnani, F, Aghaeepoor, M, Barshan Tashnizi, M
International journal of biological macromolecules. 2018;(Pt B):1572-1580
Abstract
EndoglucanaseII (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 1.3 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant EndoglucanaseII may be increased the flexibility of protein, to form a suitable E-S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 °C were increased 2.4 and 2.34 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications.
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2.
Dynamic thiol/disulphide homeostasis in acute pancreatitis.
Köseoğlu, H, Alışık, M, Başaran, M, Tayfur Yürekli, Ö, Solakoğlu, T, Tahtacı, M, Ersoy, O, Erel, Ö
The Turkish journal of gastroenterology : the official journal of Turkish Society of Gastroenterology. 2018;(3):348-353
Abstract
BACKGROUND/AIMS: The dynamic thiol/disulfide homeostasis plays pivotal roles in many physiological mechanisms in an organism. We aimed to investigate whether dynamic thiol/disulfide homeostasis changes among patients with acute pancreatitis. MATERIALS AND METHODS This prospective trial contained 45 patients with acute pancreatitis and 45 sex-and age-matched healthy volunteers as control group. Thiol/disulfide homeostasis parameters were measured by a novel and automated assay, and detected results were compared between the two groups. RESULTS Disulfide/total thiol percent ratio and disulfide/native thiol percent ratios were significantly higher in acute pancreatitis group; besides the native thiol, total thiol levels and native thiol/total thiol percent ratios were significantly lower (for all p < 0.001). CONCLUSION The thiol/disulfide homeostasis is impaired in acute pancreatitis with a shift toward the oxidative status, and this deficiency might be a pathogenic factor in acute pancreatitis. The correction of this thiol/disulfide imbalance may be a new target in the management of acute pancreatitis.
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3.
Homogeneous antibody-drug conjugates via site-selective disulfide bridging.
Forte, N, Chudasama, V, Baker, JR
Drug discovery today. Technologies. 2018;:11-20
Abstract
Antibody-drug conjugates (ADCs) constructed using site-selective labelling methodologies are likely to dominate the next generation of these targeted therapeutics. To this end, disulfide bridging has emerged as a leading strategy as it allows the production of highly homogeneous ADCs without the need for antibody engineering. It consists of targeting reduced interchain disulfide bonds with reagents which reconnect the resultant pairs of cysteine residues, whilst simultaneously attaching drugs. The 3 main reagent classes which have been exemplified for the construction of ADCs by disulfide bridging will be discussed in this review; bissulfones, next generation maleimides and pyridazinediones, along with others in development.
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4.
The Effect of Different Intraabdominal Pressures on Thiol/Disulfide Homeostasis in Children Who Underwent Ambulatory Laparoscopic Surgery: A Prospective Randomized Study.
Ozgunay, SE, Ustundag, Y, Karasu, D, Uguz, I, Erel, O, Korfali, G, Kaya, M
Journal of laparoendoscopic & advanced surgical techniques. Part A. 2018;(9):1142-1147
Abstract
BACKGROUND Thiol/disulfide homeostasis is a significant parameter in determining the oxidative stress response after ischemia and reperfusion. We aimed to investigate the effects of applying different intraabdominal pressure (IAP) on thiol/disulfide homeostasis, ischemia-modified albumin (IMA) levels, and hemodynamics in pediatric laparoscopic surgery. MATERIALS AND METHODS Blood samples were collected from 36 pediatric patients who were planned to undergo laparoscopic surgery for nonpalpable testis or varicocele under general anesthesia, immediately after intubation as the baseline and 5 minutes after abdominal desufflation for determining the thiol/disulfide, and IMA levels. The patients were divided into two groups; group 1 received a pneumoperitoneum pressure of 8 mm Hg (n = 18), and group 2 received 12 mm Hg (n = 18). The clinical characteristics and thiol/disulfide homeostasis and IMA levels of the patients were compared. RESULTS No difference was detected regarding the clinical features between the groups. The comparison after intubation and after desufflation in group 1 demonstrated lower native thiol (453 ± 67 versus 422 ± 57 μmol/L, P = .059) and total thiol (497 ± 73 versus 466 ± 62 μmol/L, P = .061) levels, which was statistically insignificant. The serum native thiol level was found lower than baseline in group 2 where a 12 mm Hg IAP was applied, this difference was not statistically significant (429 ± 47 versus 412 ± 53 μmol/L, P = .078). The comparison of serum IMA levels after desufflation with the baseline (0.505 ± 0.018 versus 0.632 ± 0.022) in group 2 was found statistically significantly high (P = .031). The comparison of the perioperative heart rate and SpO2 levels with before induction was found statistically insignificant. CONCLUSIONS Neither of 8 nor 12 mm Hg IAPs in pediatric laparoscopic surgery caused any changes in novel indicators of thiol/disulfide homeostasis parameters; however, 12 mm Hg IAP increased the levels of IMA.
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5.
An alternative method for measuring oxidative stress in intrahepatic cholestasis of pregnancy: thiol/disulphide homeostasis.
Sanhal, CY, Daglar, K, Kara, O, Yılmaz, ZV, Turkmen, GG, Erel, O, Uygur, D, Yucel, A
The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians. 2018;(11):1477-1482
Abstract
PURPOSE The aim of our study was to evaluate the oxidative stress (OS) in pregnant women with intrahepatic cholestasis of pregnancy (ICP) by evaluating thiol/disulphide homeostasis using an alternative technique. METHODS A total of 57 pregnant women with ICP were compared with 50 gestational age and body mass index matched controls. A recently defined method was used for the measurement of plasma native-total thiol and disulphide levels. The independent two-sample t test, Mann-Whitney-U test, Chi-square test, binary logistic regression with backward elimination and receiver operating characteristic (ROC) curve was performed for statistical analyses. RESULTS Pregnant women with ICP (n = 57) versus controls (n = 50) had significantly lower serum levels of native thiol (233.8 ± 47.4 μmol/L vs. 308.5 ± 51.7 μmol/L, p < .001), total thiol (258.4 ± 46.5 μmol/L vs. 328.0 ± 52.0 μmol/L, p < .001) and higher levels of disulphide (12.3 ± 3.6 μmol/L vs. 9.7 ± 3.4 μmol/L, p < .001). Binary logistic regression showed that the most important variables related to ICP were native thiol and total thiol. According to the ROC curve, the optimal cut-off level for native thiol was 280.0 μmol/L (sensitivity: 86%, specificity: 84.2%, area under the curve (AUC):0.896, 95% CI: 0.831-0.962, p < .001), and the optimal cut-off level for total thiol was 300.0 μmol/L (sensitivity: 86%, specificity: 80.7%, AUC: 0.883, 95% CI: 0.815-0.951, p < .001). CONCLUSIONS To our knowledge, this is the first study in the literature exploring thiol/disulphide balance in ICP. We found that thiol/disulphide balance indicate OS in pregnant woman with ICP.
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6.
Peptide Sequence and Solvent as Levers to Control Disulfide Connectivity in Multiple Cysteine Containing Venom Toxins.
Sajeevan, KA, Roy, D
The journal of physical chemistry. B. 2018;(22):5776-5789
Abstract
Judicious choice of solvent, temperature, and strategic mutations along a peptide backbone can minimize formation of non-native disulfide bond isoforms in chemical synthesis of multiple cysteine containing venom toxins. By exploiting these controls, one can drive the population distribution in favor of a particular isoform. Some chosen ionic liquids (ILs), like 1-ethyl-3-methyl-imidazolium acetate, [Im21][OAc], have proven efficient in favoring the native globular isoform in some conotoxins. To comprehend such a preference, we report an explicit solvent replica exchange molecular dynamics (REMD) study of two conotoxins, AuIB and GI, solvated in either neat water or ∼50% (v/v) mixture of water-[Im21][OAc]. Our simulations indicate that compared to neat water, the probability of obtaining native globular isoform of AuIB significantly increases in a water-IL mixture at 305 K. Strikingly, and aligned with experimental observations, peptide GI does not favor the native connectivity in the water-IL mixture. In presence of IL, strong solvent mediated fluctuations of the GI backbone are observed in our simulations. Uneven ion accumulation along the backbone owing to strong H-bonding interactions of some GI residues with IL ions, especially the anion OAc-, restricts conformational freedom of the peptide. Estimation of backbone entropy and Helmholtz free energy corroborates the lack of conformational freedom in GI as compared to AuIB, especially in the presence of IL. In line with prior experiments, simulations of GI mutants indicate that one could possibly force a given pair of Cys residues to come closer by strategically mutating GI residues with glycine and/or alanine, resulting in the breakage/formation of helix-like motifs.
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7.
Effects of Hemodialysis on Thiol-Disulphide Homeostasis in Critically Ill Pediatric Patients with Acute Kidney Injury.
Ayar, G, Sahin, S, Yazici, MU, Neselioglu, S, Erel, O, Bayrakcı, US
BioMed research international. 2018;:1898671
Abstract
AIM: To evaluate thiol/disulphide homeostasis as a new indicator of oxidative stress in AKI patients and to determine the effect of HD on antioxidant balance and oxidative stress through plasma thiols. METHODS This study was performed in patients aged between 12 months and 18 years prospectively who underwent hemodialysis due to AKI and were followed up for a year in a 22-bed tertiary pediatric intensive care unit. 20 patients and 39 controls were included. RESULTS No difference was present between the groups in terms of age and gender. Median values of plasma native thiol, total thiol, and percent thiol were significantly lower in AKI group both before and after dialysis when compared to control group. The median dynamic disulphide values were significantly lower in the AKI group of predialysis compared to the controls. When pre- and postdialysis values were compared, disulphide values were statistically higher after dialysis. When pre- and postdialysis native thiol, dynamic disulphide, total thiol, and percent thiol median values were compared, postdialysis values were significantly higher than the predialysis values. There was a positive correlation between albumin, total thiol, and native thiol values before dialysis in the patient group. CONCLUSION AKI patients have low levels of thiol species showing the presence of oxidative stress and hemodialysis has a positive effect on thiol/disulphide balance. This new method may be an inexpensive and simple tool suitable for clinical studies and can be used in routine screening as a useful indicator to show oxidative stress.
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8.
Is early cord clamping, delayed cord clamping or cord milking best?
Vatansever, B, Demirel, G, Ciler Eren, E, Erel, O, Neselioglu, S, Karavar, HN, Gundogdu, S, Ulfer, G, Bahadir, S, Tastekin, A
The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians. 2018;(7):877-880
Abstract
PURPOSE To compare the antioxidant status of three cord clamping procedures (early clamping, delayed clamping and milking) by analyzing the thiol-disulfide balance. PATIENTS AND METHODS This randomized controlled study enrolled 189 term infants who were divided into three groups according to the cord clamping procedure: early clamping, delayed clamping and milking. Blood samples were collected from the umbilical arteries immediately after clamping, and the thiol/disulfide homeostasis was analyzed. RESULTS The native and total thiol levels were significantly (p < .05) lower in the early cord clamping group compared with the other two groups. The disulfide/total thiol ratio was significantly (p = .026) lower in the delayed cord clamping and milking groups compared with the early clamping groups. Early cord clamping causes the production of more disulfide bonds and lower thiol levels, indicating that oxidation reactions are increased in the early cord clamping procedure compared with the delayed cord clamping and milking procedures. CONCLUSION The oxidant capacity is greater with early cord clamping than with delayed clamping or cord milking. Delayed cord clamping or milking are beneficial in neonatal care, and we suggest that they be performed routinely in all deliveries.
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9.
Effects of an Interchain Disulfide Bond on Tropomyosin Structure: A Molecular Dynamics Study.
Koubassova, NA, Bershitsky, SY, Tsaturyan, AK
International journal of molecular sciences. 2018;(11)
Abstract
Tropomyosin (Tpm) is a coiled-coil actin-binding dimer protein that participates in the regulation of muscle contraction. Both Tpm chains contain Cys190 residues which are normally in the reduced state, but form an interchain disulfide bond in failing heart. Changes in structural and functional properties of Tpm and its complexes with actin upon disulfide cross-linking were studied using various experimental methods. To understand the molecular mechanism underlying these changes and to reveal the possible mechanism of the involvement of the cross-linking in heart failure, molecular dynamics (MD) simulations of the middle part of Tpm were performed in cross-linked and reduced states. The cross-linking increased bending stiffness of Tpm assessed from MD trajectories at 27 °C in agreement with previous experimental observations. However, at 40 °C, the cross-linking caused a decrease in Tpm stiffness and a significant reduction in the number of main chain hydrogen bonds in the vicinity of residues 133 and 134. These data are in line with observations showing enhanced thermal unfolding of the least stable part of Tpm at 30⁻40 °C and accelerated trypsin cleavage at residue 133 at 40 °C (but not at 27 °C) upon cross-linking. These results allow us to speculate about the possible mechanism of involvement of Tpm cross-linking to heart failure pathogenesis.
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10.
Disulfide bond formation in prokaryotes.
Landeta, C, Boyd, D, Beckwith, J
Nature microbiology. 2018;(3):270-280
Abstract
Interest in protein disulfide bond formation has recently increased because of the prominent role of disulfide bonds in bacterial virulence and survival. The first discovered pathway that introduces disulfide bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in disulfide bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote disulfide bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with disulfide bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of disulfide-bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development.