Inhibitory Peptide of Soluble Guanylyl Cyclase/Trx1 Interface Blunts the Dual Redox Signaling Functions of the Complex.

Soluble guanylyl cyclase (GC1) and oxido-reductase thioredoxin (Trx1) form a complex that mediates two NO signaling pathways as a function of the redox state of cells. Under physiological conditions, reduced Trx1 (rTrx1) supports the canonical NO-GC1-cGMP pathway by protecting GC1 activity from thiol oxidation. Under oxidative stress, the NO-cGMP pathway is disrupted by the S-nitrosation of GC1 (addition of a NO group to a cysteine). In turn, SNO-GC1 initiates transnitrosation cascades, using oxidized thioredoxin (oTrx1) as a nitrosothiol relay. We designed an inhibitory peptide that blocked the interaction between GC1 and Trx1. This inhibition resulted in the loss of a) the rTrx1 enhancing effect of GC1 cGMP-forming activity in vitro and in cells and its ability to reduce the multimeric oxidized GC1 and b) GC1's ability to fully reduce oTrx1, thus identifying GC1 novel reductase activity. Moreover, an inhibitory peptide blocked the transfer of S-nitrosothiols from SNO-GC1 to oTrx1. In Jurkat T cells, oTrx1 transnitrosates procaspase-3, thereby inhibiting caspase-3 activity. Using the inhibitory peptide, we demonstrated that S-nitrosation of caspase-3 is the result of a transnitrosation cascade initiated by SNO-GC1 and mediated by oTrx1. Consequently, the peptide significantly increased caspase-3 activity in Jurkat cells, providing a promising therapy for some cancers.

Structure, chaperone-like activity and allergenicity profile of bovine caseins upon peroxynitrite modification: New evidences underlying mastitis pathomechanisms.

Mastitis, an inflammatory reaction frequently develops in response to intra-mammary bacterial infection||, may induce the generation of peroxynitrite (PON)| which is a highly potent reactive oxygen and nitrogen species. Caseins as the intrinsically unfolded proteins seem feasible substrates to react with PON. Therefore, in the current study, structural and functional aspects of both ß-casein (ß-CN) and whole casein fraction (WCF) were evaluated after PON modification, using a variety of techniques. Modification of the bovine caseins with PON results in an important enhancement in the carbonyl, nitrotryptophan, nitrotyrosine and dityrosine content of these proteins|. The results of fluorescence and far UV-CD assessments suggested significant structural alteration of caseins upon PON-modification. The chaperone-like activity of ß-casein was significantly altered after PON modification. The results of scanning electron microscopy suggest that bovine caseins display unique morphological features after treatment with PON. Also, the PON-modified caseins preserved their allergenicity profile and displayed partial resistance against digestion by the pancreatic proteases. Ascorbic acid, an important antioxidant component of milk, was also capable to significantly prevent the PON-induced structural damages in bovine milk caseins. In conclusion, our results suggest that PON may have significant role in the structural and functional alteration of milk caseins. Also, the PON-induced structural damaging effects of caseins might be effectively prevented by a sufficient level of milk antioxidant components particularly by ascorbic acid.

Evaluation of Structure, Chaperone-Like Activity and Allergenicity of Reduced Glycated Adduct of Bovine ß-casein.

Milk has a potent reducing environment with an important quantity of sugar levels. In the current study ß-casein was glycated in the presence of D-glucose and sodium cyanoborohydride as a reducing agent. Then, the reduced glucitol adduct of ß-casein was used for the structural and functional analyses using different spectroscopic techniques. The results of fluorescence and far ultraviolet circular dichroism assessments suggest important structural alteration upon non-enzymatic glycation of ß-casein. In addition, the chaperone activity, micellization properties and antioxidant activity of this protein were altered upon glucose modification. Also, as a result of reduced glycation, the allergenicity profile of this protein remained largely unchanged. Additional to its energetic and nutritional values, ß-casein has important functional properties. The native structure of this protein is important to perform accurately its biological functions. Non-enzymatic glycation under reducing state was capable to alter both structural and functional aspects of ß-casein. Due to effective reducing environment and significant quantity of reducing sugar of human milk, similar structural and functional alterations are most likely to occur upon reducing glycation of ß-casein in vivo. Also, these changes might be even intensified during chronic hyperglycemia in diabetic mothers.