Supplementary Figure 3: Further characterization of SelR effects on Mical-modified actin.

a, Schematic of SelR’s reducing activity and catalytic action– and how SelR can be subsequently converted from an oxidized state to a reduced state. SelR converts Met(R)-O in proteins to Met and this reduction reaction generates oxidized SelR. In particular, previous results characterizing SelR have revealed that SelR employs a catalytic cysteine (Cys)-124 thiolate, which directly interacts with methionine sulfoxide, resulting in a methionine–SelR^Cys−124 sulfenic acid intermediate (SelR-S-OH; ⁶). A subsequent reaction of this intermediate with SelR^Cys−69 generates an intramolecular disulfide (SelR-S-S; ⁶). In vitro (1), DTT can serve as a reducing agent to reduce the oxidized state of SelR ⁶. In vivo (2), one possible means to regenerate the oxidized state of SelR to a reduced state is through the thioredoxin (Trx)/thioredoxin reductase (TrxR) system ^6,7. Thioredoxins/thioredoxin reductases are also reducing enzymes that have been implicated in regulating the properties of actin in response to oxidation ^8,9,10 and also as being involved in Semaphorin/Plexin signaling ¹¹. Other enzymes also exist that may reduce SelR ^7,12,13. b, SelR, but not DTT nor thioredoxin (Trx)/thioredoxin reductase (TrxR)/NADPH alone, restores Mical-treated actin polymerization. Consistent with a catalytic requirement for SelR in restoring the polymerization properties of Mical-modified actin, it should also be noted as seen on this gel that SelR utilizes both DTT and thioredoxin/thioredoxin reductase to restore the polymerization properties of Mical-modified actin. Actin monomers/G-actin in supernatant (S); actin polymers/F-actin in pellet (P). Sedimentation/Coomassie staining assay. See also Figure S3g for uncropped gel. c, H₂O₂ alters actin polymerization over time when added in high concentrations (40 mM; ^14,15,16) but neither SelR (2.4 μM; purple dots) nor MsrA (2.4 μM; blue dots) nor both together (1.2 μM of SelR and 1.2 μM of MsrA; grey dots) restores normal polymerization to H₂O₂-treated actin. d, SelR (green dots) but not SelR^C124S (blue dots) induces polymerization of pyrene actin that has been treated with Mical/NADPH and purified. Pyrene actin assay. e-g, Uncropped Coomassie-stained gels for Figure 2b (e [red box]), Figure 3d (f), and Figure S3b (g).