Although disulfide bonds are critical to the structure of many secreted proteins, and to the regulation of a range of biochemical processes, their biosynthesis in multicellular organisms remains surprisingly cryptic. This application deals with several evolutionarily-related FAD-dependent sulfhydryl oxidases: members of the Quiescin-sulfhydryl oxidase (QSOX) family of flavoproteins, and a representative of the smaller single-domain Erv-like oxidases, augmenter of liver regeneration (ALR). The QSOX enzymes introduce disulfide bonds directly into unfolded reduced proteins, but have also been identified as growth factors in vertebrates (e.g. bone-derived growth factor, placental-derived prostrate growth factor, and erythroid cell stimulating factor). QSOX1 is strongly up-regulated in a number of human cancers (most notably of prostrate and pancreas) and may be involved in the remodeling of the extracellular matrix. ALR shares the same FAD-binding domain as QSOX and is found in a long form (lfALR) in the intermembrane space of the mitochondrion and in a short form (sfALR) functioning in a variety of cellular and extracellular locales. The first of three specific aims of this application explores the molecular mechanism by which two diverse QSOX enzymes (human QSOX1 and the simpler QSOX from the protozoan parasite Trypanosoma brucei) catalyze the efficient oxidation of unfolded reduced protein substrates.
The second aim i s to search for inhibitors of these enzymes by quantitative high-throughput screening and to continue the design of arsenical inhibitors targeting CxxC motifs in biology.
The third aim deals with short and long forms of ALR. We will extend our crystallographic investigations of sf- and lfALR and probe the reductive and oxidative halves of lfALR catalysis by rapid reaction techniques. Finally, we intend to reconstitute oxidative protein folding pathways driven by lfALR and examine their kinetic competence in vitro. Overall, these three aims will contribute to a better understanding of the redox-enzymology of oxidative protein folding in higher eukaryotes.

Public Health Relevance

This research studies a family of poorly understood enzymes that play diverse roles in protein folding, in the formation and remodeling of the extracellular matrix, and in the regeneration of liver tissue. Some of these proteins are tissue growth factors that are over-expressed in prostrate and pancreatic cancer. A better understanding of the mechanism of these important proteins may help in the design of chemotherapeutic agents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function E Study Section (MSFE)
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Anderson, Vernon
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University of Delaware
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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
Israel, Benjamin A; Kodali, Vamsi K; Thorpe, Colin (2014) Going through the barrier: coupled disulfide exchange reactions promote efficient catalysis in quiescin sulfhydryl oxidase. J Biol Chem 289:5274-84
Winther, Jakob R; Thorpe, Colin (2014) Quantification of thiols and disulfides. Biochim Biophys Acta 1840:838-46
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
Schaefer-Ramadan, Stephanie; Gannon, Shawn A; Thorpe, Colin (2013) Human augmenter of liver regeneration: probing the catalytic mechanism of a flavin-dependent sulfhydryl oxidase. Biochemistry 52:8323-32
Schaefer, Stephanie A; Dong, Ming; Rubenstein, Renee P et al. (2013) (77)Se enrichment of proteins expands the biological NMR toolbox. J Mol Biol 425:222-31
Sapra, Aparna; Thorpe, Colin (2013) An arsenical-maleimide for the generation of new targeted biochemical reagents. J Am Chem Soc 135:2415-8
Daithankar, Vidyadhar N; Wang, Wenzhong; Trujillo, Joliene R et al. (2012) Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover. Biochemistry 51:265-72
Kodali, Vamsi K; Gannon, Shawn A; Paramasivam, Sivakumar et al. (2011) A novel disulfide-rich protein motif from avian eggshell membranes. PLoS One 6:e18187

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