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1.
Vitamin D deficiency is associated with an oxidized plasma cysteine redox potential in critically Ill children.
Alvarez, JA, Grunwell, JR, Gillespie, SE, Tangpricha, V, Hebbar, KB
The Journal of steroid biochemistry and molecular biology. 2018;:164-169
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Abstract
Critically ill populations incur high levels of oxidative stress and commonly present with vitamin D deficiency. This study aimed to investigate the relationship between vitamin D status and plasma markers of glutathione (GSH) and cysteine (Cys) redox and immunity in critically ill children. This was a cross-sectional study of n=50 PICU patients. Subjects were categorized according to their plasma 25-hydroxyvitamin D [25(OH)D] concentrations: (<20, 20-30, and ≥30ng/dL). Plasma GSH, glutathione disulfide (GSSG), Cys, and cystine (CySS) were measured with high-performance liquid chromatography, and their associated redox potentials determined (EhGSSG and EhCySS, respectively). Plasma LL-37, an indicator of innate immune function, was assayed with ELISA. Data were analyzed using general linear regression before and after adjustment for age, sex, and race. Results showed that EhCySS was more reduced in subjects with plasma 25(OH)D concentrations ≥30ng/mL compared to those with 25(OH)D concentrations <20ng/mL (P=0.009). Plasma GSH, GSSG, and total GSH decreased with increasing 25(OH)D category (P=0.06, 0.03, and 0.01, respectively), and plasma glutamine levels were lowest in subjects with plasma 25(OH)D concentrations ≥30ng/mL (P=0.004). Plasma LL-37 concentrations did not significantly differ by vitamin D status (P=0.08). In conclusion, vitamin D sufficiency was associated with more reduced plasma EhCySS, indicative of lower oxidative stress in critically ill children. Plasma GSH, GSSG, and glutamine, however, were lower in the vitamin D sufficient group. The role of vitamin D in maintaining redox status during pediatric critical illness requires further study.
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Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport.
Scalise, M, Pochini, L, Console, L, Pappacoda, G, Pingitore, P, Hedfalk, K, Indiveri, C
International journal of molecular sciences. 2018;(3)
Abstract
The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2.
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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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4.
Food Overconsumption in Healthy Adults Triggers Early and Sustained Increases in Serum Branched-Chain Amino Acids and Changes in Cysteine Linked to Fat Gain.
Elshorbagy, AK, Samocha-Bonet, D, Jernerén, F, Turner, C, Refsum, H, Heilbronn, LK
The Journal of nutrition. 2018;(7):1073-1080
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Abstract
BACKGROUND Plasma concentrations of branched-chain amino acids (BCAAs) and the sulfur-containing amino acid cysteine are associated with obesity and insulin resistance. BCAAs predict future diabetes. OBJECTIVE We investigated amino acid changes during food overconsumption. METHODS Forty healthy men and women with a body mass index (mean ± SEM) of 25.6 ± 0.6 were overfed by 1250 kcal/d for 28 d, increasing consumption of all macronutrients. Insulin sensitivity and body composition were assessed at baseline (day 0) and day 28. Fasting serum amino acids were measured at days 0, 3, and 28. Linear mixed-effects models evaluated the effect of time in the total group and separately in those with low and high body fat gain (below compared with at or above median fat gain, 1.95 kg). At days 0 and 28, insulin-induced suppression of serum amino acids during a hyperinsulinemic-euglycemic clamp test and, in a subset (n = 20), adipose tissue mRNA expression of selected amino acid metabolizing enzymes were assessed. RESULTS Weight increased by 2.8 kg. High fat gainers gained 2.6 kg fat mass compared with 1.1 kg in low fat gainers. Valine and isoleucine increased at day 3 (+17% and +22%, respectively; P ≤ 0.002) and remained elevated at day 28, despite a decline in valine (P = 0.019) from day 3 values. Methionine, cystathionine, and taurine were unaffected. Serum total cysteine (tCys) transiently increased at day 3 (+11%; P = 0.022) only in high fat gainers (P-interaction = 0.043), in whom the cysteine catabolic enzyme cysteine dioxygenase (CDO1) was induced (+26%; P = 0.025) in adipose tissue (P-interaction = 0.045). Overconsumption did not alter adipose tissue mRNA expression of the BCAA-metabolizing enzymes branched-chain keto acid dehydrogenase E1α polypeptide (BCKDHA) or branched-chain amino transferase 1 (BCAT1). In the total population at day 0, insulin infusion decreased all serum amino acids (-11% to -47%; P < 0.01), except for homocysteine and tCys, which were unchanged, and glutathione, which was increased by 54%. At day 28, insulin increased tCys (+8%), and the insulin-induced suppression of taurine and phenylalanine observed at day 0, but not that of BCAAs, was significantly impaired. CONCLUSIONS These findings highlight the role of nutrient oversupply in increasing fasting BCAA concentrations in healthy adults. The link between cysteine availability, CDO1 expression, and fat gain deserves investigation. This trial was registered at www.clinicaltrials.gov as NCT00562393.
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Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase.
Kostevich, VA, Sokolov, AV
Biochemical and biophysical research communications. 2018;(3):2146-2151
Abstract
Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called "halogenating cycle". Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated.
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SVM-SulfoSite: A support vector machine based predictor for sulfenylation sites.
Al-Barakati, HJ, McConnell, EW, Hicks, LM, Poole, LB, Newman, RH, Kc, DB
Scientific reports. 2018;(1):11288
Abstract
Protein S-sulfenylation, which results from oxidation of free thiols on cysteine residues, has recently emerged as an important post-translational modification that regulates the structure and function of proteins involved in a variety of physiological and pathological processes. By altering the size and physiochemical properties of modified cysteine residues, sulfenylation can impact the cellular function of proteins in several different ways. Thus, the ability to rapidly and accurately identify putative sulfenylation sites in proteins will provide important insights into redox-dependent regulation of protein function in a variety of cellular contexts. Though bottom-up proteomic approaches, such as tandem mass spectrometry (MS/MS), provide a wealth of information about global changes in the sulfenylation state of proteins, MS/MS-based experiments are often labor-intensive, costly and technically challenging. Therefore, to complement existing proteomic approaches, researchers have developed a series of computational tools to identify putative sulfenylation sites on proteins. However, existing methods often suffer from low accuracy, specificity, and/or sensitivity. In this study, we developed SVM-SulfoSite, a novel sulfenylation prediction tool that uses support vector machines (SVM) to identify key determinants of sulfenylation among five feature classes: binary code, physiochemical properties, k-space amino acid pairs, amino acid composition and high-quality physiochemical indices. Using 10-fold cross-validation, SVM-SulfoSite achieved 95% sensitivity and 83% specificity, with an overall accuracy of 89% and Matthew's correlation coefficient (MCC) of 0.79. Likewise, using an independent test set of experimentally identified sulfenylation sites, our method achieved scores of 74%, 62%, 80% and 0.42 for accuracy, sensitivity, specificity and MCC, with an area under the receiver operator characteristic (ROC) curve of 0.81. Moreover, in side-by-side comparisons, SVM-SulfoSite performed as well as or better than existing sulfenylation prediction tools. Together, these results suggest that our method represents a robust and complementary technique for advanced exploration of protein S-sulfenylation.
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Comparative effects of acute-methionine loading on the plasma sulfur-amino acids in NAC-supplemented HIV+ patients and healthy controls.
Burini, RC, Borges-Santos, MD, Moreto, F, Yu, YM
Amino acids. 2018;(5):569-576
Abstract
In this study, an acute overloading of methionine (MetLo) was used to investigate the trassulfuration pathway response comparing healthy controls and HIV+ patients under their usual diet and dietary N-acetyl-L-cysteine (NAC) supplementation. MetLo (0.1 g Met/kg mass weight) was given after overnight fasting to 20 non-HIV+ control subjects (Co) and 12 HIV+ HAART-treated patients. Blood samples were taken before and after the MetLo in two different 7-day dietary situations, with NAC (1 g/day) or with their usual diet (UD). The amino acids (Met, Hcy, Cys, Tau, Ser, Glu and Gln) and GSH were determined by HPLC and their inflow rate into circulation (plasma) was estimated by the area under the curve (AUC). Under UD, the HIV+ had lower plasma GSH and amino acids (excepting Hcy) and higher oxidative stress (GSSG/GSH ratio), similar remethylation (RM: Me/Hcy + Ser ratio), transmethylation (TM; Hcy/Met ratio) and glutaminogenesis (Glu/Gln ratio), lower transsulfuration (TS: Cys/Hcy + Ser ratio) and Cys/Met ratio and, higher synthetic rates of glutathione (GG: GSH/Cys ratio) and Tau (TG: Tau/Cys ratio). NAC supplementation changed the HIV pattern by increasing RM above control, normalizing plasma Met and TS and, increasing plasma GSH and GG above controls. However, plasma Cys was kept always below controls probably, associatively to its higher consumption in GG (more GSSG than GSH) and TG. The failure of restoring normal Cys by MetLo, in addition to NAC, in HIV+ patients seems to be related to increased flux of Cys into GSH and Tau pathways, probably strengthening the cell-antioxidant capacity against the HIV progression (registered at http://www.clinicaltrials.gov , NCT00910442).
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Effects of the Usage of l-Cysteine (l-Cys) on Human Health.
Clemente Plaza, N, Reig García-Galbis, M, Martínez-Espinosa, RM
Molecules (Basel, Switzerland). 2018;(3)
Abstract
This review summarizes recent knowledge about the use of the amino acid l-Cysteine (l-Cys) through diet, nutritional supplements or drugs with the aim to improve human health or treat certain diseases. Three databases (PubMed, Scopus, and Web of Science) and different keywords have been used to create a database of documents published between 1950 and 2017 in scientific journals in English or Spanish. A total of 60,885 primary publications were ultimately selected to compile accurate information about the use of l-Cys in medicine and nutritional therapies and to identify the reported benefits of l-Cys on human health. The number of publications about the use of l-Cys for these purposes has increased significantly during the last two decades. This increase seems to be closely related to the rise of nutraceutical industries and personalized medicine. The main evidence reporting benefits of l-Cys usage is summarized. However, the lack of accurate information and studies based on clinical trials hampers consensus among authors. Thus, the debate about the role and effectiveness of supplements/drugs containing l-Cys is still open.
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A Prospective Comparison of 18F-Sodium Fluoride PET/CT and PSMA-Targeted 18F-DCFBC PET/CT in Metastatic Prostate Cancer.
Harmon, SA, Bergvall, E, Mena, E, Shih, JH, Adler, S, McKinney, Y, Mehralivand, S, Citrin, DE, Couvillon, A, Madan, RA, et al
Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 2018;(11):1665-1671
Abstract
The purpose of this study was to compare the diagnostic performance of 18F-DCFBC PET/CT, a first-generation 18F-labeled prostate-specific membrane antigen (PSMA)-targeted agent, and 18F-NaF PET/CT, a sensitive marker of osteoblastic activity, in a prospective cohort of patients with metastatic prostate cancer. Methods: Twenty-eight prostate cancer patients with metastatic disease on conventional imaging prospectively received up to 4 PET/CT scans. All patients completed baseline 18F-DCFBC PET/CT and 18F-NaF PET/CT scans, and 23 patients completed follow-up imaging, with a median follow-up interval of 5.7 mo (range, 4.2-12.6 mo). Lesion detection was compared across the 2 PET/CT agents at each time point. Detection and SUV characteristics of each PET/CT agent were compared with serum prostate-specific antigen (PSA) levels and treatment status at the time of baseline imaging using nonparametric statistical testing (Spearman correlation, Wilcoxon rank). Results: Twenty-six patients had metastatic disease detected on 18F-NaF or 18F-DCFBC at baseline, and 2 patients were negative on both scans. Three patients demonstrated soft tissue-only disease. Of 241 lesions detected at baseline, 56 were soft-tissue lesions identified by 18F-DCFBC only and 185 bone lesions detected on 18F-NaF or 18F-DCFBC. 18F-NaF detected significantly more bone lesions than 18F-DCFBC (P < 0.001). Correlation of PSA with patient-level SUV metrics was strong in 18F-DCFBC (ρ > 0.5, P < 0.01) and poor in 18F-NaF (ρ < 0.3, P > 0.1). When PSA levels were combined with treatment status, patients with below-median levels of PSA (<2 ng/mL) on androgen deprivation therapy (n = 11) demonstrated more lesions on 18F-NaF than 18F-DCFBC (P = 0.02). In PSA greater than 2 ng/mL, patients on androgen deprivation therapy (n = 8) showed equal to or more lesions on 18F-DCFBC than on 18F-NaF. Conclusion: The utility of PSMA-targeting imaging in metastatic prostate cancer appears to depend on patient disease course and treatment status. Compared with 18F-NaF PET/CT, 18F-DCFBC PET/CT detected significantly fewer bone lesions in the setting of early or metastatic castrate-sensitive disease on treatment. However, in advanced metastatic castrate-resistant prostate cancer, 18F-DCFBC PET/CT shows good concordance with NaF PET/CT.
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Cysteine Linkages Accelerate Electron Flow through Tetra-Heme Protein STC.
Jiang, X, Futera, Z, Ali, ME, Gajdos, F, von Rudorff, GF, Carof, A, Breuer, M, Blumberger, J
Journal of the American Chemical Society. 2017;(48):17237-17240
Abstract
Multi-heme proteins have attracted much attention recently due to their prominent role in mediating extracellular electron transport (ET), but one of their key fundamental properties, the rate constants for ET between the constituent heme groups, have so far evaded experimental determination. Here we report the set of heme-heme theoretical ET rate constants that define electron flow in the tetra-heme protein STC by combining a novel projector-operator diabatization approach for electronic coupling calculation with molecular dynamics simulation of ET free energies. On the basis of our calculations, we find that the protein limited electron flux through STC in the thermodynamic downhill direction (heme 1→4) is ∼3 × 106 s-1. We find that cysteine linkages inserting in the space between the two terminal heme pairs 1-2 and 3-4 significantly enhance the overall electron flow, by a factor of about 37, due to weak mixing of the sulfur 3p orbital with the Fe-heme d orbitals. While the packing density model, and to a higher degree, the pathway model of biological ET partly capture the predicted rate enhancements, our study highlights the importance of the atomistic and chemical nature of the tunneling medium at short biological tunneling distances. Cysteine linkages are likely to enhance electron flow also in the larger deca-heme proteins MtrC and MtrF, where heme-heme motifs with sub-optimal edge-to-edge distances are used to shuttle electrons in multiple directions.