Peroxiredoxin-6-interacting proteins in rat olfactory epithelium

Abstract

Peroxiredoxin 6 (Prx6) belongs to the family of thiol-dependent peroxidases that catalyze the reduction of hydrogen peroxide, organic peroxides, and peroxynitrite. Peroxiredoxins (Prxs) are increasingly recognized as a multi-functional proteins involved in various cellular processes. Accordingly, individual Prxs have been found to interact with multiple partners. Although the list of Prxs-binding proteins is rapidly growing, interactions reported so far show very limited overlap with each other. Our earlier studies indicated that Prx6 is a major cytosolic protein of rat olfactory epithelium and an important component of antioxidant defense system in this tissue. Here we used a combination of biochemical methods including pull-down assay, chemical cross-linking with a tri-functional cross-linker sulfo-SBED, co-immunoprecipitation, and mass spectrometry-based detection to conduct a random search for the Prx6-binding partners in water-soluble extracts from rat olfactory epithelium. Our findings showed that recombinant Prx6 formed complexes with peroxiredoxin 1, cytoskeletal proteins actin and tubulin, metabolic protein glyceraldehyde-3-phosphate dehydrogenase, heat shock proteins 70 and 90, and native Prx6. A common property of the identified proteins is the presence of conserved redox-sensitive Cys residue in their molecules. Novel interactions of rat Prx6 were validated by several independent methods, thus eliminating technical false-positives. However, the conventional methodological approaches to capture reproducibly real physical interactions have intrinsic limitations in distinguishing between randomly and functionally associated proteins, since not all of the naturally occurring protein complexes necessarily bear functional significance. We hypothesize that protein-protein interactions of Prx6 in vivo might induce conformational changes in its molecule, thus increasing the accessibility of the active-site Cys to general cellular reducing agents, such as thioredoxin or glutathione.