Deep blue autofluorescence reflects the oxidation state of human transthyretin

Human transthyretin (TTR) is a tetrameric protein transporting thyroid hormones and retinol. TTR is a neuroprotective factor and sensor of oxidative stress which stability is diminished due to mutations and aging, leading to amyloid deposition. Adverse environmental conditions, such as redox and metal ion imbalances, induce destabilization of the TTR structure. We have previously shown that the stability of TTR was disturbed by Ca(2+) and other factors, including DTT, and led to the formation of an intrinsic fluorophore(s) emitting blue light, termed deep blue autofluorescence (dbAF). Here, we show that the redox state of TTR affects the formation dynamics and properties of dbAF. Free thiols lead to highly unstable subpopulations of TTR and the frequent ocurrence of dbAF. Oxidative conditions counteracted the destabilizing effects of free thiols to some extent. However, strong oxidative conditions led to modifications of TTR, which altered the stability of TTR and resulted in unique dbAF spectra. Riboflavin and/or riboflavin photoproducts bound to TTR and crosslinked TTR subunits. Riboflavin-sensitized photooxidation increased TTR unfolding, while photooxidation, either in the absence or presence of riboflavin, increased proteolysis and resulted in multiple oxidative modifications and dityrosine formation in TTR molecules. Therefore, oxidation can switch the role of TTR from a protective to pathogenic factor.