Functional implications of electrophilic protein adducts

Abstract

Oxidative stress is a contributing factor in a number of chronic diseases, including cancer, atherosclerosis, and neurodegenerative diseases. Lipid peroxidation that occurs during periods of oxidative stress and decomposition of these oxidized lipids results in the formation of lipid electrophiles. 4-Hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) are two lipid aldehydes which are generated as a result of lipid peroxidation, and both can adduct nucleophilic side chains of amino acids in proteins. A large number of protein targets have been identified for HNE and ONE, consisting of an array of adduct structures Here, we show that these adducts have distinct functional implications on the activity and regulation of the target protein. CDK2, a key cell cycle kinase which regulates the G1/S-phase transition, is adducted by HNE for up to 16 h. The adduction of CDK2 inhibits kinase activity in vitro and in cells and delays cell cycle progression into S-phase following HNE treatment. PIN1 is a cis/trans isomerase, which plays a key role in regulating of a number of cell signaling pathways. PIN1 is modified by 4-oxo-2-nonenal ONE at the active-site Cys and forms a cross-link with a nearby Lys, thereby inactivating the protein. Using site-specific incorporation of deuterium in ONE, we were able to determine a mechanism of cross-link formation and definitively show that nucleophilic attack occurs at the third carbon of ONE. Histone proteins have also been shown to be preferential targets for ONE modification, and these proteins have a direct effect on the regulation of gene expression and chromatin structure. We have developed a method to selectively isolate regions of DNA associated with these adducted histones using click-chemistry. The method, coupled with next generation DNA sequencing, termed Click-Seq, shows few regions of enrichment, suggesting that ONE broadly adducts chromatin. Furthermore, the levels of these adducts are two orders of magnitude lower than the canonical histone modifications. Together, these data show that the lipid electrophile HNE and ONE can have a significant impact on enzyme activity, alterations in cell signaling pathway, and regulation of gene expression.