Identification of selenoprotein O substrates using a biotinylated ATP analog

Abstract

Selenoprotein O is one of 25 human selenoproteins that incorporate the 21st amino acid selenocysteine. Recent studies have revealed a previously undocumented mechanism of redox regulation by which SelO protects cells from oxidative damage. SelO catalyzes the covalent addition of AMP from ATP to the hydroxyl side chain of protein substrates in a post translational modification known as AMPylation. Although AMPylation was discovered over 50 years ago, methods to detect and enrich substrates are limited. Here, we describe protocols to clone, purify, and identify the substrates of bacterial SelO using a biotinylated ATP analog. Identification of SelO substrates and the functional consequences of AMPylation will illuminate the significance of this evolutionarily conserved selenoprotein.

Keywords: AMPylation; Adenylylation; Mitochondria; Oxidative stress; Pseudokinase; SelenoO; ydiU.

Figure 1.. E.coli SelO binds ATP in the active site to AMPylate protein substrates.

(A) Surface rendering of Pseudomonas syringae SelO demonstrates the orientation of ATP bound in the active site of SelO. (B) E.coli SelO catalyzes the transfer of AMP from ATP to serine, threonine and tyrosine residues of protein substrates. Figure adapted from (Sreelatha et al., 2018). (C) Structure of Bio-17-ATP, which allows for biotin labeling of substrates in an in vitro AMPylation reaction with E.coli SelO. Figure adapted from (Sreelatha et al., 2018).

Figure 2:. Expression and purification of E.coli SelO.

(A) SDS-PAGE and Coomassie staining of the following samples. Lane 1: uninduced bacterial cell lysate. Lane 2: induced bacterial lysate. Lane 3: soluble protein. Lane 4: insoluble and unbroken cells. Lane 5: unbound protein/flowthrough. Lane 6: wash. Lane 7: eluted protein. Lane 8: eluted protein cleaved with Ulp1. (B) Size exclusion chromatography trace (upper) and SDS-PAGE and Coomassie staining (lower) of E.coli SelO after treatment with Ulp1.

Figure 3:. Optimization of in vitro AMPylation assay using Bio-17-ATP.

Varying concentrations of bacterial lysate were used in an AMPylation reaction with recombinant E.coli SelO or the inactive mutant, D256A. Biotinylated reaction products were detected using streptavidin-HRP following SDS-PAGE and transfer to nitrocellulose membrane. The Ponceau stained membrane is shown.

Figure 4:. E.coli SelO AMPylates proteins in bacterial lysate.

In vitro AMPylation assay with bacterial lysate, Bio-17-ATP and recombinant E.coli SelO or the inactive mutant, D256A. Biotinylated reaction products were detected using streptavidin-HRP following SDS-PAGE and transfer to nitrocellulose membrane. The Ponceau stained membrane is shown. Figure adapted from (Sreelatha et al., 2018).