We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

The antioxidant activity and chemical stability of 6-amino-6-deoxy-L-ascorbic acid (D1) and N-methyl-6-amino-6-deoxy-L-ascorbic acid (D2) were examined with ABTS and DPPH assays and compared with the reference L-ascorbic acid (AA). In addition, the optimal storing conditions, as well as the pH at which the amino derivatives maintain stability, were determined using mass spectrometry. Comparable antioxidant activities were observed for NH-bioisosteres and AA. Moreover, D1 showed higher stability in an acidic medium than the parent AA. In addition, AA, D1, and D2 share the same docking profile, with wild-type human peroxiredoxin as a model system. Their docking scores are similar to those of dithiothreitol (DTT). This suggests a similar binding affinity to the human peroxiredoxin binding site.

The pathogenesis of ischemia-reperfusion (I/R) injuries is based on oxidative stress caused by a sharp increase in the concentration of free radicals, reactive oxygen species (ROS) and secondary products of free radical oxidation of biological macromolecules during reperfusion. Application of exogenous antioxidants lowers the level of ROS in the affected tissues, suppresses or adjusts the course of oxidative stress, thereby substantially reducing the severity of I/R injury. We believe that the use of antioxidant enzymes may be the most promising line of effort since they possess higher efficiency than low molecular weight antioxidants. Among antioxidant enzymes, of great interest are peroxiredoxins (Prx1-6) which reduce a wide range of organic and inorganic peroxide substrates. In an animal model of bilateral I/R injury of kidneys (using histological, biochemical, and molecular biological methods) it was shown that intravenous administration of recombinant typical 2-Cys peroxiredoxins (Prx1 and Prx2) effectively reduces the severity of I/R damage, contributing to the normalization of the structural and functional state of the kidneys and an almost 2-fold increase in the survival of experimental animals. The use of recombinant Prx1 or Prx2 can be an efficient approach for the prevention and treatment of renal I/R injury.

The equilibrium hetero-association of NADH oxidase and peroxiredoxin was characterized by means of independently conducted measurements of composition-gradient sedimentation equilibrium and composition-gradient static light scattering. Results obtained from both experiments were quantitatively accounted for by a model according to which a dimer of NADH oxidase forms a 1:1 equilibrium complex with a decamer of peroxiredoxin under the conditions of these experiments. The best-fit equilibrium constants for heteroassociation of the two proteins obtained from the two measurements were found to be identical to well within the uncertainty of estimate of each of the two methods. The relative virtues of each of the methods are discussed.

Babesia microti is an emerging human pathogen and the primary causative agent of human babesiosis in many regions of the world. Although the peroxiredoxins (Prxs) or thioredoxin peroxidases (TPx) enzymes of this parasite have been sequenced and annotated, their biological properties remain largely unknown. Prxs are a family of antioxidant enzymes that protect biological molecules against metabolically produced reactive oxygen species (ROS) and reduce hydrogen peroxide (H2O2) to water in both eukaryotes and prokaryotes. In this study, TPx-1 cDNA was cloned from B. microti (designated BmTPx-1). Recombinant BmTPx-1 (rBmTPx-1) was expressed in Escherichia coli as a histidine fusion protein and purified using Ni-NTA His bind resin. To test the defense capacity of enzymatic antioxidants against the effect of ROS, a mixed-function oxidation system was utilized with the recombinant BmTPx-1 protein. A decreased ability of rBmTPx-1 to donate electrons to the thioredoxin (Trx)/TrxR reductase system was clarified by reaction with H2O2. These results suggest that BmTPx-1 has a great impact on protecting parasites from oxidative stress in the erythrocytic stage.

BACKGROUND: The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors.
METHODS: Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters.
CONCLUSIONS: Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments.
CONCLUSIONS: Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways.

Members of the peroxiredoxin (Prx) family of antioxidant enzymes are inactivated via hyperoxidation of the active site cysteine by the substrate H2O2 and are reactivated via an ATP-consuming process catalyzed by sulfiredoxin (Srx). PrxIII is reversibly inactivated by H2O2 produced by cytochrome P450 11B1 (CYP11B1) in mitochondria during corticosterone synthesis in the adrenal gland of mice injected with adrenocorticotropic hormone (ACTH). Inactivation of PrxIII triggers a sequence of events including accumulation of H2O2, activation of p38 mitogen-activated kinase (MAPK), inhibition of cholesterol transfer, and suppression of corticosterone synthesis. Srx expression is significantly induced by ACTH injection. The coupling of CYP11B1 activity to PrxIII inactivation and Srx induction provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis. Furthermore, the PrxIII-Srx regulatory pathway is critical for the circadian rhythm of corticosterone production. Although adrenocortical tumor cell lines such as Y-1 and H295R have been used extensively for studying the mechanism of steroidogenesis, those clonal cells were found to be unsuitable as an in vitro model for redox signaling because the amount of Srx in the cell lines is much higher than that in mouse adrenal gland and not affected by ACTH stimulation. Furthermore, the levels of PrxIII in the clonal cells are greatly reduced compared to that in the adrenal gland, and ACTH does not induce PrxIII hyperoxidation in the clonal cells. Primary adrenocortical cells isolated from the mouse adrenal gland were also found to be an invalid model because Srx levels are increased, along with decreased levels of hyperoxidized PrxIII, soon after isolation of these cells. Organ culture system is, however, appropriate for studying the PrxIII-Srx regulatory function as the levels of hyperoxidized PrxIII and Srx in the adrenal glands maintained overnight in culture medium are not changed.