Determination of the self-cleavage mechanism of selenoprotein K

Abstract

Selenoprotein K (selenok) is membrane protein that is a resident of the endoplasmic reticulum. Bearing the twenty-first amino acid selenocysteine, it is a member of the selenoprotein family, a group of proteins known for their antioxidant behavior. In the literature, selenok is described as a participant in endoplasmic reticulum-associated degradation of proteins, indirect modulation of calcium flux, and proper function of the immune system. Because it is intrinsically disordered and membrane-bound, selenok is a challenging protein to study.

Structurally, selenok is made of a nineteen residue N-terminal peptide, a twenty-three-residue alpha helix, and a disordered C-terminus. The disordered region is the majority of the total protein length. The disordered region of selenok is rich in short linear motifs, or sequences of peptides typically ten residues or fewer in length. These motifs are recognized by protein domains and thus form the basis of many protein-protein interactions. These interactions can also be further regulated through post-translational modifications of the short linear motif, such as phosphorylation. The enrichment of selenok with a diverse group of short linear motifs is potentially why it has so many described functions.

Coexistent with its multifunctionality, selenok exhibits non-enzymatic cleavage of itself. We describe it as non-enzymatic because point residue mutations cannot halt or significantly slow it and cleavage is not specific to any residue or type of residue. In contrast, twenty cleavages have been seen in the disordered region, and cleavage is accelerated by centrifugal filter-based concentration instead of treatment with a protease. We determine the mechanism of this self-cleavage by identifying cleavages by mass spectrometry, assessing the secondary and quaternary structures of variants of selenok, and probing the preferred cleavage feedstock of these variants. We propose that the N-terminal region of selenok preferentially cleaves the disordered region and that selenok must oligomerize through its alpha helix to accelerate self-cleavage.

Together with the fact that cleavage was previously observed in vivo, self-cleavage of selenok may be an autoregulatory process. Specifically, it may determine interactors and localization of selenok by modifying its short linear motif profile. A rigorous understanding of the self-cleavage mechanism will then help explain how it participates in its multiple functions.