Study of protein targets for covalent modification by the antitumoral and anti‐inflammatory prostaglandin PGA₁: focus on vimentin

Abstract

Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.

Prostaglandins antagonistically control Bax activation during apoptosis

“…[14][15][16][17] Bax-induced release of mitochondrial cytochrome c is often related to its oligomerization through the BH3 domain and the exposure of the Ha present in its C-terminal end. 3 However, as PGE 2 did not trigger Bax oligomerization (Figure 1d) and the deletion of the Ha9 of Bax did not impair its ability to interact with PGE 2 (Figure 1e), we conclude that these domains are not fundamental for PGE 2 Given the widely reported affinity of cyclopentenone prostaglandins for cysteine residues, 18,19 we questioned the ability of cysteine reagents, such as the competitive inhibitor N-acetylcysteine (NAC) or the cysteine blocker iodoacetamide, to interfere with the PGE 2 -Bax interaction. As shown in Figure 2a, both reagents inhibit the interaction suggesting that the free access to the regions including one or both cysteine residues of Bax was crucial for the physical interaction with PGE 2 .…”

Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody

“…C328 in human vimentin has been shown to be the target of electrophilic lipids that include cyclopentenone prostaglandins (cyPG), which are reactive lipids that are generated under conditions of inflammation and oxidative stress (49,81,82). C328 is also subject to covalent addition of the reactive aldehyde HNE, which is similar to MDA (48).…”

“…CML was previously shown to modify lysines in the exposed linker regions of vimentin after UV treatment (44), and although we detected CML-modified vimentin by IP-Western, our proteomic screen failed to identify the location of these modifications. Importantly, at the cellular level, the modification of vimentin by these oxidation-associated modifications (cyPG, HNE, and CML) has been shown to result in the disruption of the intermediate filament network and the generation of intracellular aggresomes (44,48,82).…”

Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAK1)→Signal Transducer and Activator of Transcription-3 (STAT3) Pathway by Direct Inhibition of JAK1 and STAT3

“…The cyclopentenone prostaglandins also contain an a,h-unsaturated carbonyl moiety and react with the InB kinase h Cys 179 residue (38). However, few targets of the cyclopentenone prostaglandins have been identified to date, even when using an unbiased proteomic approach (39). Nonetheless, the findings that the cyclopentenone prostaglandins react with H-Ras (40), heat shock protein 90 (39), and estrogen receptor a (41) provide potential leads for additional targets of the synthetic oleanane triterpenoids.…”