A peptide-crosslinking approach identifies HSPA8 and PFKL as selective interactors of an actin-derived peptide containing reduced and oxidized methionine

Abstract

The oxidation of methionine to methionine sulfoxide occurs under conditions of cellular oxidative stress, and modulates the function of a diverse array of proteins. Enzymatic systems that install and reverse the methionine sulfoxide modifications have been characterized, however, little is known about potential readers of this oxidative modification. Here, we apply a peptide-crosslinking approach to identify proteins that are able to differentially interact with reduced and oxidized methionine-containing peptides. Specifically, we generated a photo-crosslinking peptide derived from actin, which contains two sites of methionine oxidation, M44 and M47. Our proteomic studies identified heat shock proteins, including HSPA8, as selective for the reduced methionine-containing peptide, whereas the phosphofructokinase isoform, PFKL, preferentially interacts with the oxidized form. We then demonstrate that the favored interaction of PFKL with oxidized methionine is also observed in the full-length actin protein, suggesting a role of methionine oxidation in regulating the actin-PFKL interaction in cells. Our studies demonstrate the potential to identify proteins that can differentiate between reduced and oxidized methionine and thereby mediate downstream protein functions under conditions of oxidative stress. Furthermore, given that numerous sites of methionine oxidation have now been identified, these studies set the stage to identify putative readers of methionine oxidation on other protein targets.

Fig. 1. Photo-crosslinking peptide probes to identify readers of methionine oxidation. (A) Structures of actin probes 1 and 2. (B) Probes 1 and 2 label MCF7 lysates in a concentration-dependent manner and show distinct labeling patterns. Uncropped gel image provided in Fig. S5 (ESI†). (C) Workflow used to identify proteins that interact selectively with oxidized or reduced methionine. 50 μM probes 1 or 2 are crosslinked in isotopically heavy or light MCF7 lysates. After UV crosslinking, equal amounts of heavy and light lysate are combined. Proteins that are bound to the probe are enriched on streptavidin agarose beads, trypsinized, and subjected to LC–MS/MS analysis. (D) 2D plot showing SILAC R_L/H for each protein in the “forward” (x-axis) and “reversed” (y-axis) experiments. Proteins that interact preferentially with probe 1 are in the upper right portion of the plot and preferential interactors of probe 2 are in the bottom left portion.

Fig. 2. Validation of HSPA8 and PFKP as selective binders for Met and Met sulfoxide-containing peptide probes. (A) Detection of endogenous PFKL and HSPA8 in MCF7 lysates enriched with probe 1 or 2. MCF7 lysates were crosslinked with 75 μM of the indicated probe. An aliquot of the labeled lysate was taken and run as the “input” fraction. The labeled proteins were enriched on streptavidin-agarose beads. The bound proteins were eluted by heating and treatment with 50 μM biotin to obtain the “enriched” fraction. Each fraction was analyzed by western blot using antibodies for HSPA8 and PFKL. Streptavidin-HRP was used to detect probe-labeled proteins. Uncropped gel images provided in Fig. S6 (ESI†). (B) 12 μM of the indicated probe was combined with 1 μM purified HSPA8 and (C) PFKL in vitro. Each lane is a biological replicate performed with a new preparation of protein. Uncropped gel images provided in Fig. S7 (ESI†). (D) Mean densitometry measurements of 3 biological replicates shown in B and C. Band intensities from the streptavidin blot were normalized to the intensity of the HSPA8 or PFKL band and then normalized intensity of the most intense replicate, such that the intensity of the most intense replicate is set to 1. Replicates were statistically analyzed by Student's t test (*p < 0.005). Error bars represent the standard deviation of the 3 measurements.

Fig. 3. PFKL preferentially interacts with oxidized full-length actin. (A) Immunoprecipitation of ACTB from MCF7 lysates was followed by western blotting of the input (I) and bead-bound (E) fractions with anti-HSPA8, PFKL and GAPDH antibodies. Uncropped gel images provided in Fig. S8 (ESI†). (B) 25 μg biotin-phalloidin was added to MCF7 lysates and then incubated with streptavidin-agarose beads. The input (I) and bead bound (E) fractions were analyzed by western blot with anti-HSPA8, PFKL and GAPDH antibodies. Uncropped gel images are provided in Fig. S9 (ESI†). (C) 50 μg reduced or Mical oxidized G-actin was labeled with biotin-NHS then bound to streptavidin-agarose beads. 6 μM of recombinant HSPA8 and PFKL were combined and incubated with the immobilized actin. The bead bound fractions for reduced (M), oxidized (M(O)), and no actin (−) was analyzed by western blot using anti-PFKL and HA (HSPA8) antibodies. A representative of 3 biological replicates is shown. Uncropped gel images shown in Fig. S10 (ESI†). (D) Mean densitometry measurements of 3 biological replicates of the experiment shown in replicates were statistically analyzed by Student's t test (*p < 0.05, **p < 0.005). Error bars represent the standard deviation of the 3 measurements.