The association of reactive oxygen species (ROS) with the initiation and progression of cancer, including stimulation of tumor growth and metastasis, is well established;paradoxically, ROS are also important players in many anti-cancer treatments involving ionizing radiation and chemotherapies. Yet we have only a limited appreciation for the molecular mechanisms involved in the many normal and disease-associated functional roles played by ROS, largely due to the limited tools available for studying the molecular targets of ROS. Our research team at Wake Forest University has pioneered the development of highly specific chemical probes, with previous support from the IMAT program, which enable detection and identification of oxidized proteins, targeting the initial sulfenic acid(-SOH) product of cysteine thiols undergoing oxidation. While these probes have been used successfully to identify targets of oxidation within specific proteins such as Akt2 (in the context of PDGF signaling) and specific lipid raft-associated protein tyrosine phosphatases involved in angiogenesis, they have not yet proven amenable to wide-scale identification of such sites using high- throughput mass spectrometry (MS) analysis. As demonstrated in our preliminary data, factors which interfere with MS have been identified and circumvented with new probe designs;for example, acid-base properties of these 1,3-dicarbonyl probes which interfere with the charge states needed for MS detection can be blocked by post-labeling cyclization of the products, and new linear probes exhibiting much higher reactivity with the low abundance sulfenic acids have been generated. This application describes additional new strategies to overcome the remaining issues that limit detection and analysis of the oxidized proteome.
The first aim describes new chemical probes for more efficient trapping of electrophilic and nucleophilic sulfenic acids. With the second aim we will investigate new imaging and MS technologies to visualize selective protein -SOH modification in situ and identify sulfenic acid sites in endogenously expressed proteins. Successful completion of this project will have high impact, enabling a much deeper understanding of redox-controlled intracellular processes involved in normal and cancer signaling, angiogenesis and metastasis, as well as chemotherapeutic and radiation-based treatments. In the long term, it may enable the design of selective agonists or antagonists to modulate the activity of target proteins in tumors.

Public Health Relevance

Reactive oxygen species (ROS) are important factors both in the development of cancer and in the response of cancer to many therapies. We propose to develop chemical reagents and methods to find the proteins in cells that are directly modified by ROS and their precise sites of modification by using state-of-the-art imaging and mass spectrometry technologies. This information can then be used to gain a better overall understanding of processes integral to cancer, including growth, blood vessel development and tumor metastasis, to reveal opportunities for more informed interventions for treating human cancers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
1R33CA177461-01
Application #
8547235
Study Section
Special Emphasis Panel (ZCA1-SRLB-Q (M1))
Program Officer
Knowlton, John R
Project Start
2013-09-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
1
Fiscal Year
2013
Total Cost
$342,076
Indirect Cost
$92,952
Name
Wake Forest University Health Sciences
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Baez, Nelmi O Devarie; Reisz, Julie A; Furdui, Cristina M (2015) Mass spectrometry in studies of protein thiol chemistry and signaling: opportunities and caveats. Free Radic Biol Med 80:191-211
Poole, Leslie B (2015) The basics of thiols and cysteines in redox biology and chemistry. Free Radic Biol Med 80:148-57
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
Poole, Thomas H; Reisz, Julie A; Zhao, Weiling et al. (2014) Strained cycloalkynes as new protein sulfenic acid traps. J Am Chem Soc 136:6167-70
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