Chromic inflammation, as a result of continuous oxidative stress, has been shown to be a contributing factor to the development of cancer by both experimental and epidemiological studies. Inflammation can contribute to tumorigenesis by a number of different pathways, including promotion of genomic instability, alterations of epigenetic markers, increases in proliferation, and resistance to apoptosis. While reactive oxygen species (ROS) alone are capable of inducing these different pathways to promote transformation, the high reactivity of ROS results in reactions near the site of production. Polyunsaturated lipids within membrane bilayers, most notably mitochondrial membranes, are key targets of ROS. Reactions between these lipids and ROS result in lipid peroxidation and, in some cases, can generate small, reactive lipid electrophiles. 4-Hydroxynonenal (HNE) is the most commonly studied lipid electrophile. Due to its lower reactivity relative to ROS, HNE can diffuse throughout the cell and covalently modify both DNA and proteins;modification of cellular proteins can result in altered function. Previous proteomic data have shown a number of proteins susceptible to modification by HNE, one of which is CDK2. Cyclin-dependent kinase 2 (CDK2) is a cell cycle protein responsible for the G1/S transition and for maintaining progression through S-phase. Interestingly, microarray studies have shown down-regulation of genes associated with S-phase following HNE treatment;many of these genes are controlled upstream by CDK2 kinase activity. Together, these data suggest that HNE-modification of CDK2 could alter its function, resulting in protein inactivation and cell cycle arrest. Our preliminary data hve shown that CDK2 is modified by HNE on a number of histidine and lysine residues. Treatment of synchronized cells with HNE causes a delay in progression into S-phase. Most notably, treatment of cells treated with HNE results in a decrease in CDK2 kinase activity. Given these data, we hypothesize that HNE covalently modifies CDK2 and inhibits its kinase activity, thereby resulting in G1-phase arrest until HNE-modified CDK2 is degraded. I propose two aims to test this hypothesis: 1) Assess the functional impact of HNE modification of CDK2 and 2) Quantify the extent of CDK2 modification by HNE in RKO cells. The research proposed in this application will assess the functional significance of HNE modification on a main cell cycle regulator and kinase, thereby increasing our understanding of altered protein function as a result of HNE modification.
The aims proposed will provide a mechanism for how small changes in protein structure from the adduction by HNE can significantly disrupt interactions within the cell.

Public Health Relevance

Reactive oxygen species are generated during normal cellular processes, but can be increased as a result of inflammation, exposure to exogenous toxicants, and loss of antioxidant responses within the cell. Reactive oxygen species readily react with membrane lipids to generate lipid electrophiles that can modify cellular proteins and alter their function. The studies proposed will expand our knowledge of how protein damage resulting from modification by lipid electrophiles can be inhibit cell growth by observing how protein modification alters the structure-function relationship of a cell cycle kinase.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Predoctoral Individual National Research Service Award (F31)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-F09A-L (20))
Program Officer
Vallejo-Estrada, Yolanda
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Vanderbilt University Medical Center
Schools of Medicine
United States
Zip Code
Camarillo, Jeannie M; Ullery, Jody C; Rose, Kristie L et al. (2017) Electrophilic Modification of PKM2 by 4-Hydroxynonenal and 4-Oxononenal Results in Protein Cross-Linking and Kinase Inhibition. Chem Res Toxicol 30:635-641
Camarillo, Jeannie M; Rose, Kristie L; Galligan, James J et al. (2016) Covalent Modification of CDK2 by 4-Hydroxynonenal as a Mechanism of Inhibition of Cell Cycle Progression. Chem Res Toxicol 29:323-32
Aluise, Christopher D; Camarillo, Jeannie M; Shimozu, Yuki et al. (2015) Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal. Chem Res Toxicol 28:817-27