Regulation of Actin Dynamics by SelR

Abstract

Regulation of actin polymerization and disassembly by the Mical/SelR enzyme pair has been shown to play an important role in abscission, the terminal step of cytokinesis, helping to prevent multiploidy. Oxidation of filamentous actin by the Mical family of monooxygenases promotes its disassembly, and reduction of actin by methionine sulfoxide reductase B (MsrB/SelR) promotes actin repolymerization. However, this process is poorly understood mechanistically. For instance, it is not known which forms of actin preferentially interact with SelR. Likewise, little is known about the enzymatic reaction of Mical-oxidized (Mox-) actin reduction catalyzed by SelR. In this study, SelR-C124S, a catalytically dead mutant, was created and validated as a tool for studying SelR binding to Mox-actin. The expression and purification protocols for recombinant SelR proteins were developed and optimized. This study provided the first insights on SelR binding to different forms of actin, assessed in analytical ultracentrifugation, native gel electrophoresis, and high-speed pelleting assays. The effectiveness of two reducing agents in supporting the catalytic activity of SelR in vitro were evaluated. Both reducing reagents were shown to be effective at lower concentrations than originally expected; tris(2-carboxyethyl)phosphine (TCEP) was shown to be a more efficient reductant compared to Dithiothreitol (DTT). These findings provided new insights on SelR interaction with actin.