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. 2018 Jan 17;8:937. doi: 10.1038/s41598-017-17943-5

Figure 3.

Figure 3

Each human MICAL family member is activated by actin filaments, oxidizing the Met44 residue of actin to induce F-actin disassembly. (a) The enzymatic activity of each of the human (h) MICALs, similar to Drosophila (d) Mical, notably increases in the presence of F-actin. Enzyme activity was determined by consumption (conversion of NADPH to NADP+) of MICAL’s co-enzyme NADPH, which was monitored by recording the light absorbance at 340 nm wavelength/time. [MICALs] = 600 nM; [NADPH] = 200 μM. (b) Each of the human MICALs oxidizes the Met44 residue of actin, as determined using an antibody that specifically recognizes Mical-oxidized actin (actinMetO44 16). Similar amounts of actin (lower panel) are present in all experiments. Note, as described in the methods, actin was polymerized to generate F-actin and 600 nM of each of the human MICALs and 200–300 µM of NADPH were added to 1.15 μM of F-actin at room temperature for 2 hours. (c) Each of the human MICALs requires actin’s Met44 and Met47 residues to disassemble F-actin. Drosophila Mical oxidizes the Met44 and Met47 residues of actin13 and so we substituted as previously described a chemically related leucine residue for the methionine 44 and 47 residues in actin (M4447L)13 to determine if each of the human MICALs also requires these residues to disassemble F-actin. Sedimentation assays reveal that each of the human MICALs robustly disassembles filaments composed of wild-type (WT) actin but not M4447L actin. [Actin] = 1.15 μM; [MICALs-1, 1DG, 2, and 3] = 600 nM; [NADPH] = 200–300 μM. (d) The stereospecific methionine sulfoxide reductase SelR/MsrB restores the polymerization properties of actin treated with each of the human MICALs. Pyrene-actin assays, where the fluorescence is higher in the polymerized state, reveal that SelR restores the polymerization of human MICAL-1 (hM-1), human MICAL-2 (hM-2), and human MICAL-3 (hM-3) treated actin. Buffer (buffer that SelR is stored in), n.u. (normalized units between the 2 graphs). [MICAL-1] = 100 nM, [dMical and MICALs-2, 3] = 600 nM, [NADPH] = 100 μM, [Actin] = 1.15 μM. Unprocessed original scans of gels/blots are shown in Supplementary Fig. 11.