? While reactive oxygen species, including H2O2, have been shown to modulate a number of kinases, phosphatases, redox-sensitive transcription factors, and genes involved in cellular events including apoptosis and cancer, little information is available as to the proteins directly targeted by H2O2. The primary target of H2O2-linked protein modification is likely to be cysteine residues that are converted first to cysteine sulfenic acids (Cys-SOH), then either directly re-reduced or further modified to form disulfide bonds or other oxidized species. Due to the unstable nature of the sulfenic acid intermediate, identification of proteins containing this species has been difficult. To provide a tool to identify and monitor redox sensitive proteins, an assay will be developed and validated to monitor Cys-SOH appearance and disappearance in vivo after stimulation with reactive oxygen species. With this method in hand, we will be able to identify which proteins are modified, to determine the reactive cysteines in each of these proteins, and to determine if different stimulants, including a representative group of peptide growth factors, cytokines, and peroxides, mediate Cys-SOH formation on different proteins. Finally, the last specific aim will focus on how Cys-SOH formation could be specific to a limited number of proteins by investigating whether or not a low pKa value for the reactive cysteine is critical to the formation of this intermediate. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM074537-02
Application #
7119637
Study Section
Special Emphasis Panel (ZRG1-F04B (20))
Program Officer
Marino, Pamela
Project Start
2005-09-01
Project End
2007-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
2
Fiscal Year
2006
Total Cost
$48,796
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
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Soito, Laura; Williamson, Chris; Knutson, Stacy T et al. (2011) PREX: PeroxiRedoxin classification indEX, a database of subfamily assignments across the diverse peroxiredoxin family. Nucleic Acids Res 39:D332-7
Nelson, Kimberly J; Knutson, Stacy T; Soito, Laura et al. (2011) Analysis of the peroxiredoxin family: using active-site structure and sequence information for global classification and residue analysis. Proteins 79:947-64
Klomsiri, Chananat; Nelson, Kimberly J; Bechtold, Erika et al. (2010) Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins. Methods Enzymol 473:77-94
Nelson, Kimberly J; Klomsiri, Chananat; Codreanu, Simona G et al. (2010) Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins. Methods Enzymol 473:95-115
Poole, Leslie B; Nelson, Kimberly J (2008) Discovering mechanisms of signaling-mediated cysteine oxidation. Curr Opin Chem Biol 12:18-24
Nelson, Kimberly J; Parsonage, Derek; Hall, Andrea et al. (2008) Cysteine pK(a) values for the bacterial peroxiredoxin AhpC. Biochemistry 47:12860-8
Nelson, Kimberly J; Day, Amanda E; Zeng, Bu-Bing et al. (2008) Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pK(a) values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem 375:187-95