Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal.

Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.

ω-Alkynyl lipid surrogates for polyunsaturated fatty acids: free radical and enzymatic oxidations.

Lipid and lipid metabolite profiling are important parameters in understanding the pathogenesis of many diseases. Alkynylated polyunsaturated fatty acids are potentially useful probes for tracking the fate of fatty acid metabolites. The nonenzymatic and enzymatic oxidations of ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were compared to that of linoleic and arachidonic acid. There was no detectable difference in the primary products of nonenzymatic oxidation, which comprised cis,trans-hydroxy fatty acids. Similar hydroxy fatty acid products were formed when ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were reacted with lipoxygenase enzymes that introduce oxygen at different positions in the carbon chains. The rates of oxidation of ω-alkynylated fatty acids were reduced compared to those of the natural fatty acids. Cyclooxygenase-1 and -2 did not oxidize alkynyl linoleic but efficiently oxidized alkynyl arachidonic acid. The products were identified as alkynyl 11-hydroxy-eicosatetraenoic acid, alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid, and alkynyl prostaglandins. This deviation from the metabolic profile of arachidonic acid may limit the utility of alkynyl arachidonic acid in the tracking of cyclooxygenase-based lipid oxidation. The formation of alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid compared to alkynyl prostaglandins suggests that the ω-alkyne group causes a conformational change in the fatty acid bound to the enzyme, which reduces the efficiency of cyclization of dioxalanyl intermediates to endoperoxide intermediates. Overall, ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid appear to be metabolically competent surrogates for tracking the fate of polyunsaturated fatty acids when looking at models involving autoxidation and oxidation by lipoxygenases.

Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins.

Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences. We now report the identification and verification of an electrically-active form of modified proteins recognized by a group of small molecules commonly used to interact with DNA. This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators. Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified N(ε)-pyrrolelysine as a key adduct. Unexpectedly, the pyrrolation conferred an electronegativity and electronic properties on the proteins that potentially constitute an electrical mimic to the DNA. In addition, we found that the pyrrolated proteins indeed triggered an autoimmune response and that the production of specific antibodies against the pyrrolated proteins was accelerated in human systemic lupus erythematosus. These findings and the apparent high abundance of N(ε)-pyrrolelysine in vivo suggest that protein pyrrolation could be an endogenous source of DNA mimic proteins, providing a possible link connecting protein turnover and immune disorders.

Identification of a lipid peroxidation product as the source of oxidation-specific epitopes recognized by anti-DNA autoantibodies.

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.