It is widely appreciated that reactive oxygen species (ROS) play a major role in the initiation of cancer, and they are also implicated in many cancer therapies, such as ionizing radiation, cisplatin and taxanes. More recently, it has been discovered that cancer cells produce ROS as signaling molecules that promote proliferation. Unfortunately, the molecular details of how redox regulation affects cell signaling events are far from clear. New experimental and computational technologies that we have developed are uniquely suited to identifying the molecular targets that are modified by ROS, either as a result of ROS damage or ROS signaling. With the reagents and methods that we have recently developed, we can now evaluate the """"""""redox profile"""""""" of cell populations by targeting uniquely reactive cysteine sulfenic acid (Cys-SOH) groups, the initial intermediates generated following reaction of activated protein thiolate groups with hydrogen peroxide and peroxynitrite (and perhaps other ROS). In this R33 application, our labeling technology will be further developed for quantification and multiplex analysis, so that it will have broad applicability in: 1) the investigation of basic mechanisms of ROS damage and ROS signaling;2) molecular profiling to stratify patients with cancers that are sensitive to ROS-generating therapies;and 3) the development of novel cancer therapies based on the inhibition of ROS-dependent proliferative signaling. The following Specific Aims are proposed: 1) to develop reagents and methods of use for additional new, multicolor fluorescently-labeled Cys-SOH reagents for multiplex analysis of samples;2) to develop quantitative mass spectrometry methods, which have some major advantages over gel-based methods (including direct readout of protein identity and numerous posttranslational modifications);and 3) to use the new quantitative methods to detect and identify Cys-SOH modified proteins generated during ROS-dependent signaling in HEK-293 cells and ovarian cancer cells. Taken together, the approaches developed in Specific Aims 1 and 2 will provide new tools for the research community to use to study the mechanisms of redox regulation and signaling.
In Specific Aim 3, these tools will be used to determine the targets of ROS in the regulation of cell proliferation and apoptosis. First we will continue our study of NF-?B regulation in HEK-293 cells in response to cytokine (TNF-?) and tumor promoter (TPA) stimulation. Second, we will use ovarian cancer cells treated with cisplatin or taxane to determine which protein oxidations are critical to regulating survival and apoptosis. Besides providing specific information about the mechanism of redox regulation and signaling, these biological experiments will allow us to further refine our reagents and methods to make them most useful to the cancer biology community. These approaches to detecting functional oxidative modifications to cellular proteins hold promise in identifying specific protein targets that mediate the actions of anticancer drugs, e.g. through their effects on cell cycle arrest, cell division or apoptosis. An outgrowth of these studies could also be the development of new anticancer drugs and the ability to predict efficacy of a given drug in the treatment of individual patients.
|Klomsiri, Chananat; Rogers, LeAnn C; Soito, Laura et al. (2014) Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling. Free Radic Biol Med 71:49-60|
|Furdui, Cristina M; Poole, Leslie B (2014) Chemical approaches to detect and analyze protein sulfenic acids. Mass Spectrom Rev 33:126-46|
|Reisz, Julie A; Bechtold, Erika; King, S Bruce et al. (2013) Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids. FEBS J 280:6150-61|
|Qian, Jiang; Wani, Revati; Klomsiri, Chananat et al. (2012) A simple and effective strategy for labeling cysteine sulfenic acid in proteins by utilization of Î²-ketoesters as cleavable probes. Chem Commun (Camb) 48:4091-3|
|Michalek, Ryan D; Crump, Katie E; Weant, Ashley E et al. (2012) Peroxiredoxin II regulates effector and secondary memory CD8+ T cell responses. J Virol 86:13629-41|
|Salsbury Jr, Freddie R; Poole, Leslie B; Fetrow, Jacquelyn S (2012) Electrostatics of cysteine residues in proteins: parameterization and validation of a simple model. Proteins 80:2583-91|
|Crump, Katie E; Juneau, Daniel G; Poole, Leslie B et al. (2012) The reversible formation of cysteine sulfenic acid promotes B-cell activation and proliferation. Eur J Immunol 42:2152-64|
|Kaplan, Nihal; Urao, Norifumi; Furuta, Eiji et al. (2011) Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis. Free Radic Res 45:1124-35|
|Wani, Revati; Qian, Jiang; Yin, Leimiao et al. (2011) Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species. Proc Natl Acad Sci U S A 108:10550-5|
|Qian, Jiang; Klomsiri, Chananat; Wright, Marcus W et al. (2011) Simple synthesis of 1,3-cyclopentanedione derived probes for labeling sulfenic acid proteins. Chem Commun (Camb) 47:9203-5|
Showing the most recent 10 out of 17 publications