Alkylation damage by lipid electrophiles targets functional protein systems

Abstract

Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.

Fig. 1.

THP-1 and RKO cells have markedly different GSH content and THP-1 cells are deficient in GSH-dependent electrophile detoxication. A, RKO cells contain millimolar levels of GSH, which is depleted by treatment with both aHNE and aONE. B, Differentiation of THP-1 monocytes to THP-1 macrophages with PMA reduces GSH content from ∼1 mm to ∼0.02 mm. Loss of GSH appears to result from loss of cellular glutatione equivalents, rather than formation of mixed disulfides, as reduction with 12.5 mm dithiothreitol (+PMAr and -PMAr) did not increase measured GSH. C, Treatment of THP-1 macrophages with aHNE and aONE did not further decrease GSH. D, RKO cells (circles), but not THP-1 cells (squares) efficiently detoxify aHNE by conjugation with GSH. aHNE-GS conjugates were measured in cells (filled symbols) and culture medium (open symbols) after treatment with 100 μm aHNE.

Fig. 2.

aHNE and aONE alkylated distinct sets of protein targets in THP-1 and RKO cell proteomes. A, B, aHNE and aONE together alkylated approximately half of the THP-1 reference proteome and approximately half of the RKO reference proteome. C, D, aHNE and aONE targeted distinct sets of proteins in both THP-1 and RKO cells, together with substantial overlapping sets modified by both electrophiles and termed “dual targets.” E, Overlap of the dual target proteomes identified a “core target proteome,” consisting of protein targets of both electrophiles in both cell types.

Fig. 3.

Concentration-dependent accumulation of protein adducts reveals three alkylation susceptibility classes. A, Comparison of THP-1 cell proteins significantly alkylated at 5, 10 and 20 μm aHNE indicates three protein target classes. Class III proteins are significantly alkylated at all exposure concentrations, whereas Class II proteins are significantly alkylated only at 10 and 20 μm aHNE and Class I proteins are alkylated only at the 20 μm aHNE aHNE. The concentration-dependence of adduct accumulation for the three classes is illustrated in the accompanying representations. B, Heat maps illustrate dramatic differences in the distribution of electrophile target proteins between Classes I, II and III between THP-1 and RKO cells. The majority of aHNE and aONE targets in THP-1 cells are in Class III, which includes proteins with the highest susceptibility to alkylation. In contrast, most electrophile protein targets in RKO are in Class I, which has the lowest susceptibility to alkylation.

Fig. 4.

Electrophile alkylation occurs selectively across protein interaction networks. A, Proteins in a protein-protein interaction network are placed in a linear order together with the hierarchical modular organization of the network. Alternating bar colors (green and orange) are used to distinguish neighboring modules. Preferentially adducted network modules are indicated by red and blue asterisks, and the latter is expanded in (B). The proteins in the core target proteome are visualized by black bars in the “Core targets” track. Network adduction data are visualized as heat maps with each row representing a concentration. Increasing darkness of red in the heat maps symbolizes increased level of adduction, whereas gray represents proteins not detected in the reference proteomes. Extensive alkylation in THP-1 cells at all concentrations contrasts with a clear concentration-dependent accumulation of adducts in RKO cells. B, Detailed view of a network module involved in RNA splicing. C, Link-node diagram of the RNA splicing module. Node color and node border color represent data for RKO/aHNE and THP-1/aHNE respectively, with the following color key: gray (not in reference proteomes), light yellow (nontarget), light red (Class I), medium red (Class II) and dark red (Class III).

Fig. 5.

Electrophiles target distinct functional protein systems in protein alkylation susceptibility classes. GO biological groups at right were based on shared proteins and functional similarity (GO biological processes significantly enriched for Class I, II, and III protein targets are listed in supplemental Tables S21 and S22). Numerals in the boxes indicating numbers of distinct GO biological processes within the groups are represented within each susceptibility class. Class II/III proteins were significantly enriched for GO biological processes involved in cytoskeletal regulation in both THP-1 and RKO cells. Class I/II proteins were significantly enriched for GO biological processes involved in protein synthesis and turnover in both THP-1 and RKO cells.