The impact of physiological oxidants on triggering amyloid formation of the cell cycle regulator protein p16INK4A.

Abstract

The tumor suppressor protein p16^INK4a plays a key role in cell cycle control and is frequently inactivated in human cancers. We previously discovered that oxidation of its single cysteine residue induces a structural transition from the functional monomeric form into inactive amyloid fibrils. Here, we investigate whether five physiologically relevant oxidants can trigger the conformational switch of the protein. Using time-resolved SDS-PAGE, thioflavin-T fluorescence assays, mass spectrometry, and electron microscopy, we show that peroxymonocarbonate and hypothiocyanous acid (HOSCN) efficiently induce cysteine-dependent dimerization with subsequent amyloid formation of recombinant p16. In contrast, hydrogen peroxide, hypochlorous acid, and taurine chloramine are less efficient and predominantly lead to the formation of amorphous aggregates. These findings demonstrate that physiological oxidants differ in their ability to induce amyloid transitions in p16, with potential implications for oxidative stress-mediated inactivation of this cell-cycle regulator protein.