Molecular Structures of Isolevuglandin-Protein Cross-Links

Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS–PAGE with Western blotting. <i>N</i>-Acetyl-glycyl-lysine methyl ester and β-amyloid(11–17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]­LGE₂. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]­LGE₂ with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]­LGE₂. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]­LGE₂. It is tempting to speculate that the EVHHQKL peptide–pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11–17) tends to spontaneously oligomerize

Detection and Biological Activities of Carboxyethylpyrrole Ethanolamine Phospholipids (CEP-EPs)

Oxidation of docosahexaenoate phospholipids produces 4-hydroxy-7-oxo-hept-5-eonyl phospholipids (HOHA-PLs) that react with protein lysyl ε-amino residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives, endogenous factors that induce angiogenesis in the retina and tumors. It seemed likely, but remained unproven, that HOHA-PLs react with ethanolamine phospholipids (EPs) <i>in vivo</i> to generate CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold higher levels in age-related macular degeneration plasma than in normal plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube formation by human umbilical vein endothelial cells by activating Toll-like receptor 2. CEP-EP levels may be a useful biomarker for clinical assessment of AMD risk and CEP-associated tumor progression and a tool for monitoring the efficacy of therapeutic interventions