Abstract 3050: Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury
Authors: Mont Stacy , Davies Sean S. , Roberts L Jackson , Mernaugh Raymond L. , McDonald W Hayes , Segal Brahm H. , Kropski Jonathan A. , Blackwell Timothy S. , Sekhar Konjeti R. , Galligan James J. , Marnett Lawrence J. , Freeman Michael L. 
Published: July 2016

The development of radiation induced lung injury (RiPI) is a major barrier that limits the dose that can be administered for controlling locally advanced lung cancer. Although progress has been made toward identifying the pathophysiological events responsible for RiPI, there is still a substantial gap in knowledge. It is well established that oxidative stress is central to the progression of RiPR. In addition, reoccurring cellular injury appears to be a critical event for promotion of radiation-induced fibrosis. Yet, the exact mechanism by which reactive oxygen species (ROS) promotes injury and the nature of the reoccurring injury remain to be fully elucidated. Isolevuglandins are generated by free radical peroxidation of phospholipid-esterified arachidonic acid. Isoketalation, defined as covalent adduction of isolevuglandins to the ϵ-amino group of protein lysine residues, is emerging as a novel mechanism by which ROS contributes to the genesis of some diseases. Although isoketalation promotes proteotoxicity and cytotoxicity that can contribute to disease progression, there is currently a lack of knowledge concerning its role in RiPI, the identity of susceptible proteins or whether the process can be genetically regulated. We used a multifaceted approach that included wild type, Nrf2 and p47 null mice, confocal microscopy, and LC-MS/MS analysis of affinity purified proteins to address these questions. Mass spectrometry and gene ontology analysis identified several potential protein targets involved in cytoskeletal regulation, Wnt signaling, integrin signaling, chemokine and cytokine signaling and histone biology. Genetic evidence linking oxidant challenge to protein adduction was provided from the mouse knockout studies where it was shown that Nrf2 expression suppressed, while NADPH oxidase activity promoted isoketalation. We found that when isoketalation exceeded a critical level, cells underwent apoptosis. We identified the cell types in mouse lung that are susceptible to adduction. In a C57BL/6j murine model of radiation induced pulmonary injury we found that ionizing radiation increased the level of adduction concomitant with the development of a chronic apoptotic phenotype. We used human idiopathic pulmonary fibrotic (IPF) tissue as a surrogate for radiation-induced pulmonary fibrotic tissue, which is very hard to obtain. Human IPF and radiation-induced lung injury share a common phenotype that includes slow/chronic development and a prominent ROS component. We found an abundance of adducted protein in human IPF compared to control lung tissue, identified collagen 1α1 as one of the highly adducted proteins and show that adduction impairs collagen degradation by MMP1. In summary, these data support the hypothesis that radiation-induced oxidant stress promotes pulmonary injury, in part, by a hitherto, unrecognized mechanism: isoketalation. Supported in part by NIH/NHLBI Grant RO1HL112286.

Citation Format: Stacy Mont, Sean S. Davies, L Jackson Roberts, Raymond L. Mernaugh, W Hayes McDonald, Brahm H. Segal, Jonathan A. Kropski, Timothy S. Blackwell, Konjeti R. Sekhar, James J. Galligan, Lawrence J. Marnett, Michael L. Freeman. Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3050.