The main objective of this project is to elucidate the effects of alkylation on the structure and biological functions of proteins that contain the """"""""thioredoxin fold"""""""". Upon conjugation with glutathione (GSH) the 1,2-dihaloethanes, 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE, become alkylating agents via the episulfonium ion of the respective S-(2-chloroethyl) glutathione (CEG) and S-(2-bromoethyl)glutathione (BEG). These conjugates have been shown to alkylate proteins at about 50-100 times the rate of nucleic acid alkylation. In vitro studies with CEG have shown that the Cys32 position of recombinant E.coli thioredoxin is preferentially alkylated compared to any other amino acid residue, whereas the alkylation of hemoglobulin is over one order of magnitude less alkylated under the same conditions. In this proposal, we hypothesize that the thioredoxin fold, Cys-X-Y-Cys, present in the active site of several proteins of the """"""""thioredoxin fold"""""""" family, including thioredoxin (TRX), thioredoxin reductase (TR), and protein disulfide isomerase (PDI) are targets for alkylation by the glutathione conjugates of DCE and DBE and that the biological functions of the proteins are highly compromised after alkylation at their active sites. PDI is highly involved in protein folding during protein synthesis in the endoplasmic reticulum. Sites of alkylation will be established for recombinant mammalian TRX, TR and PDI as this laboratory has accomplished with E. coli thioredoxin. Alkylating agents besides CEG include 1-chloro-2,4-dinitrobenzene (DNCB) and iodoacetamide. Recombinant human macrophage-colony stimulating factor beta (rhm-CSFbeta), a cytokine that is being used as a model protein for protein folding studies, will be investigated to determine the effects of CEG alkylation on protein folding and biological activity. Effects on protein folding, activation on transcription factors, and redox modifications will be investigated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Program Projects (P01)
Project #
5P01ES000040-35
Application #
6105991
Study Section
Project Start
1998-12-01
Project End
1999-11-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
35
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Oregon State University
Department
Type
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
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