Oxidative stress induced orexins misfolding

Orexins, small neuropeptides produced in the lateral hypothalamus, are involved in various physiological functions and energy homeostasis, regulating arousal, wakefulness, and appetite [1]. Two highly conserved isoforms of orexins were described: orexin A, a 33-amino-acid peptide with an N-terminal pyroglutamyl residue and Cterminal amidation, and orexin B, a 28-amino-acid, C-terminally amidated linear peptide. In particular, orexin A bears two intramolecular disulfide bridges making it susceptible to structural misfolding [2]. Orexins misfunction has been associated to sleep, mood and apetite disorders, as well as with neurodegenerative disorders [3]. A possible hypothesis involves oxidative stress to be implicated in orexin malfunction. During oxidative stress conditions reactive oxygen species (ROS), such as the superoxide anion (O2 •–) , hydroxyl radical (OH• ), are generated in cells. Additionally, lipid peroxidation can arise in vivo during redox imbalance situations, such as inflammation or environmental toxicity, generating many different oxidation products including most reactive aldehydes and α,β-unsaturated moieties that can form covalent adducts with proteins and nucleic acids [4]. The conformational behaviour of orexin A and B peptides was investigated under physiological conditions and under oxidative stress conditions, either by incubation with commercially available lipid peroxidation by-products or by addition of NO radical generated from commercially available products such as DEA NONOate, in buffer or in membrane-mimetic environment. While orexin B appears to exhibit greater stability to the effects of radicals, orexin A is extremely sensitive to these, as confirmed by substantial change in conformation observed by circular dichroism and by adducts formation detected by MALDI-TOF mass spectrometry