Disulfide bonds critical to the native structure of secreted proteins, and to the regulation of a range of biochemical processes, however their biosynthesis in multicellular organisms remains poorly understood. This proposal continues investigation of a newly-recognized flavoenzyme family (the Quiescin-sulfhydryl oxidase, QSOX, family) formed from the fusion of two thioredoxin domains with a 4-helix bundle FAD-binding domain. Thioredoxin and flavin-binding domains cooperate to ensure the facile insertion of disulfide bonds into unfolded reduced proteins with the reduction of oxygen to hydrogen peroxide.
Three aims of this proposal use the non-recombinant avian sulfhydryl oxidase as the prototypical member of this new flavoenzyme family. The cooperation between protein disulfide isomerase (PDI) and QSOX in oxidative folding will be investigated by steady-state and rapid-reaction kinetics. The existence of a binding site on QSOX for peptides and unfolded reduced proteins will be assessed by kinetic and spectroscopic experiments. The inhibition of QSOX during turnover in the presence of arsenic(Ill) compounds or transition metals will be examined for insights into ways to modulate sulfhydryl oxidase activity in vivo.
The fourth aim, heterologous expression of both avian and human QSOX enzymes, will permit a mutational dissection of the roles of the three CxxC motifs in catalysis. Expression of a human QSOX (QSOX- 1) will allow the first detailed characterization of its catalytic specificity towards small and large thiol substrates. The fifth and sixth aims extend this work to the enzyme's smaller, stand-alone, flavoprotein cousins: human recombinant ALR (augmenter of liver regeneration) and Erv2p (the closest functional counterpart of QSOX in yeast). ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026643-28
Application #
7105460
Study Section
Special Emphasis Panel (ZRG1-BPC-B (02))
Program Officer
Preusch, Peter C
Project Start
1979-07-01
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
28
Fiscal Year
2006
Total Cost
$288,637
Indirect Cost
Name
University of Delaware
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
059007500
City
Newark
State
DE
Country
United States
Zip Code
19716
Hudson, Devin A; Caplan, Jeffrey L; Thorpe, Colin (2018) Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging. Biochemistry 57:1178-1189
Yu, Tiantian; Laird, Joanna R; Prescher, Jennifer A et al. (2018) Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding. Protein Sci 27:1509-1517
Fass, Deborah; Thorpe, Colin (2018) Chemistry and Enzymology of Disulfide Cross-Linking in Proteins. Chem Rev 118:1169-1198
Foster, Celia K; Thorpe, Colin (2017) Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase. Free Radic Biol Med 108:741-749
Zhang, Han; Trout, William S; Liu, Shuang et al. (2016) Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation. J Am Chem Soc 138:5978-83
Hudson, Devin A; Thorpe, Colin (2015) Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity. Arch Biochem Biophys 579:1-7
Sapra, Aparna; Ramadan, Danny; Thorpe, Colin (2015) Multivalency in the inhibition of oxidative protein folding by arsenic(III) species. Biochemistry 54:612-21
Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin (2015) Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum. Free Radic Biol Med 80:171-82
Israel, Benjamin A; Jiang, Lingxi; Gannon, Shawn A et al. (2014) Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase. Free Radic Biol Med 69:129-35
Schaefer-Ramadan, Stephanie; Thorpe, Colin; Rozovsky, Sharon (2014) Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein augmenter of liver regeneration. Arch Biochem Biophys 548:60-5

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