Generating Disulfides in Multicellular Organisms: Emerging Roles for a New Flavoprotein Family

Abstract

Peptides and proteins destined for secretion in multicellular organisms usually contain disulfide bonds, from small peptides to massive extracellular matrix (ECM) 2The abbreviations used are: ECM, extracellular matrix; ALR, augmenter of liver regeneration; Erv, a protein essential for respiration and vegetative growth; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; QSOX, quiescin-sulfhydryl oxidase. 2The abbreviations used are: ECM, extracellular matrix; ALR, augmenter of liver regeneration; Erv, a protein essential for respiration and vegetative growth; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; QSOX, quiescin-sulfhydryl oxidase. proteins with hundreds of disulfide bridges. Disulfides are important to the structure, stability, and regulation of many proteins having at least one extracellular domain; they are critical to the formation and remodeling of the ECM and other disulfide networks, and they are crucial elements in various redox signaling pathways. However, the pathways for their biosynthesis in multicellular organisms remain surprisingly cryptic. We do not really know how a single protein disulfide bond is introduced in any metazoan, green plant, or protist.Why is our understanding of oxidative folding in so rudimentary a state? One reason is the very reactivity of thiolate nucleophiles and the degeneracy of pathways for the interconversion of thiols and disulfides. A second factor is the facile non-enzymatic oxidation of thiols by a number of potential cellular oxidants including GSSG (1Chakravarthi S. Jessop C.E. Bulleid N.J. EMBO Rep. 2006; 7: 271-275Crossref PubMed Scopus (320) Google Scholar). A third issue is the common misperception that oxygen is a facile oxidant of juxtaposed thiols, a reaction that is spin-forbidden and strongly catalyzed by traces of redox-active transition metal ions (notably copper and iron). Finally, multicellular organisms have additional pathways for disulfide bond formation that are not shared with the genetically tractable yeast systems.ScopeA key issue in this Minireview is the identity of the oxidizing catalysts for disulfide bond formation in multicellular organisms. Although we identify likely candidates, it is important to recognize that there may be major routes to disulfide generation that remain to be uncovered. A second issue is the involvement of the protein disulfide isomerases (PDIs) (for representative reviews see Refs. 2Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (167) Google Scholar, 3Ellgaard L. Ruddock L.W. EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (620) Google Scholar, 4Wilkinson B. Gilbert H.F. Biochim. Biophys. Acta. 2004; 1699: 35-44Crossref PubMed Scopus (491) Google Scholar, 5Gruber C.W. Cemazar M. Heras B. Martin J.L. Craik D.J. Trends Biochem. Sci. 2006; 31: 455-464Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) in addressing incorrectly paired cysteine partners, and the phasing of PDI’s cooperation with the other components needed for the successful exit of a mature protein from the quality control system of the ER (6van Anken E. Braakman I. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 191-228Crossref PubMed Scopus (164) Google Scholar, 7Marciniak S.J. Ron D. Physiol. Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (774) Google Scholar). Again, the precise roles of PDIs in this critical aspect of oxidative folding are still uncertain, in part because of the difficulties inherent with systems in which thiols and disulfides are in complex and rapid flux. Although many aspects of these fascinating proteins remain to be resolved, there is one feature of PDI that can be addressed definitively. PDIs are not “oxidases,” and they are not enzymes showing “oxidase activity.” Oxidases, according to the Enzyme Commission, use the electrons abstracted from one substrate to reduce molecular oxygen. PDIs, in their oxidoreductase mode, just exchange one disulfide for another, thereby shifting the burden of the disposal of pairs of reducing equivalents elsewhere. Calling PDI an “oxidase” tends to divert attention from this critical aspect of oxidative folding: what to do with the pairs of electrons liberated with every disulfide made. Sulfhydryl oxidases accomplish this task with the stoichiometry:2R–SH + O2 → R–S–S–R + H2O2. This essentially irreversible reaction can only proceed rapidly using a cofactor that can communicate facilely both with thiols and molecular oxygen. In eukaryotes the best understood of these sulfhydryl oxidases are flavin-linked.Perspectives: Making Protein Disulfide Bonds in Prokaryotes and YeastAn important perspective for all oxidative folding comes from investigations on the Escherichia coli periplasm (for example, see Refs. 8Nakamoto H. Bardwell J.C. Biochim. Biophys. Acta. 2004; 1694: 111-119Crossref PubMed Scopus (150) Google Scholar, 9Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (444) Google Scholar, 10Messens J. Collet J.F. Int. J. Biochem. Cell Biol. 2006; 38: 1050-1062Crossref PubMed Scopus (132) Google Scholar, 11Rozhkova A. Stirnimann C.U. Frei P. Grauschopf U. Brunisholz R. Grutter M.G. Capitani G. Glockshuber R. EMBO J. 2004; 23: 1709-1719Crossref PubMed Scopus (108) Google Scholar, 12Inaba K. Murakami S. Suzuki M. Nakagawa A. Yamashita E. Okada K. Ito K. Cell. 2006; 127: 789-801Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). These studies have identified the net oxidant for disulfide bond insertion, the requirement for an isomerase system to correct disulfide connectivities, and the challenge of minimizing futile cycles between multiple components carrying redox-active dithiols. Ultimately, oxidative folding is driven by an integral membrane protein, DsbB, that relays reducing equivalents to coenzyme Q and thence to the respiratory chain, neatly avoiding the need to interact with oxygen directly (Fig. 1A). Emerging studies on pathways for generating disulfide bonds in the mitochondrial intermembrane space, a compartment with distant evolutionary lineage to the bacterial periplasm, suggest interesting parallels (13Farrell S.R. Thorpe C. Biochemistry. 2005; 44: 1532-1541Crossref PubMed Scopus (137) Google Scholar).Disulfide bond generation pathways in the yeast ER appear entirely different. Here, the generally accepted view is depicted in Fig. 1B. For simplicity the arrows depict a unidirectional flow of pairs of reducing equivalents toward molecular oxygen. A disulfide bond is inserted into the folding substrate with reduction of a CXXC disulfide in a PDI. (There are multiple PDI or PDI-like proteins in all eukaryotes (3Ellgaard L. Ruddock L.W. EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (620) Google Scholar, 4Wilkinson B. Gilbert H.F. Biochim. Biophys. Acta. 2004; 1699: 35-44Crossref PubMed Scopus (491) Google Scholar, 5Gruber C.W. Cemazar M. Heras B. Martin J.L. Craik D.J. Trends Biochem. Sci. 2006; 31: 455-464Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 14Norgaard P. Westphal V. Tachibana C. Alsoe L. Holst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (106) Google Scholar), and the one here is suggested to be PDI1p.) Reduced PDI is then believed to be the immediate substrate for either of two FAD-dependent oxidases, Ero1p (15Tu B.P. Weissman J.S. Mol. Cell. 2002; 10: 983-994Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 16Gross E. Kastner D.B. Kaiser C.A. Fass D. Cell. 2004; 117: 601-610Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) or Erv2p (17Gross E. Sevier C.S. Vala A. Kaiser C.A. Fass D. Nat. Struct. Biol. 2002; 9: 61-67Crossref PubMed Scopus (162) Google Scholar, 18Sevier C.S. Cuozzo J.W. Vala A. Aslund F. Kaiser C.A. Nat. Cell Biol. 2001; 3: 874-882Crossref PubMed Scopus (153) Google Scholar, 19Gerber J. Muhlenhoff U. Hofhaus G. Lill R. Lisowsky T. J. Biol. Chem. 2001; 276: 23486-23491Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), prior to the final reduction of molecular oxygen. Studies from the Fass and Kaiser laboratories have revealed the key architecture and likely mechanism of both flavoproteins. In both, the thiols of the donor substrate (here reduced PDI, Fig. 1B) do not communicate with the flavin center directly but by a series of disulfide exchanges. This is illustrated in Fig. 2 for Erv2p (17Gross E. Sevier C.S. Vala A. Kaiser C.A. Fass D. Nat. Struct. Biol. 2002; 9: 61-67Crossref PubMed Scopus (162) Google Scholar). The penultimate arrow involves 2-electron reduction of the flavin cofactor by a pair of thiols: one (C57) poised to interact with the cofactor directly and the other (C54) positioned to reform a disulfide bond as the pair of electrons is transferred to the flavin. The final catalytic step is the reoxidation of reduced cofactor to generate hydrogen peroxide (see below).FIGURE 2Flow of reducing equivalents during the oxidation of dithiol substrates by Erv2p. Note the disulfide exchange between gray and violet subunits of the homodimer. ALR has a similar-fold (62Wu C.K. Dailey T.A. Dailey H.A. Wang B.C. Rose J.P. Protein Sci. 2003; 12: 1109-1118Crossref PubMed Scopus (102) Google Scholar) and may function as a cytochrome c reductase in the mitochondrial intermembrane space (13Farrell S.R. Thorpe C. Biochemistry. 2005; 44: 1532-1541Crossref PubMed Scopus (137) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The QSOX Family of Sulfhydryl OxidasesBefore Erv1p or Erv2p were discovered to be flavin-dependent sulfhydryl oxidases, a larger protein incorporating an Erv-like domain, first called Quiescin Q6 (20Coppock D.L. Kopman C. Scandalis S. Gillerman S. Cell Growth & Differ. 1993; 4: 483-493PubMed Google Scholar), was found to be a sulfhydryl oxidase (21Hoober K.L. Glynn N.M. Burnside J. Coppock D.L. Thorpe C. J. Biol. Chem. 1999; 274: 31759-31762Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The substrate specificity has been best characterized for a sulfhydryl oxidase isolated from avian egg white. Although the enzyme oxidizes small thiols like GSH, unfolded reduced proteins are much better substrates (22Hoober K.L. Sheasley S.S. Gilbert H.F. Thorpe C. J. Biol. Chem. 1999; 274: 22147-22150Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Here, typical kcat values are ∼1000 protein disulfides introduced per min with Km values of ∼150 μm (on a per–SH basis). This direct and facile oxidation of a seemingly unlimited array of reduced unfolded proteins and peptides (22Hoober K.L. Sheasley S.S. Gilbert H.F. Thorpe C. J. Biol. Chem. 1999; 274: 22147-22150Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) is in clear distinction to the yeast and human Ero oxidases and yeast Erv2p. These oxidases rely on PDI to mediate the flow of reducing equivalents between client reduced proteins and molecular oxygen (Fig. 1B) (18Sevier C.S. Cuozzo J.W. Vala A. Aslund F. Kaiser C.A. Nat. Cell Biol. 2001; 3: 874-882Crossref PubMed Scopus (153) Google Scholar, 23Kulp M.S. Frickel E.M. Ellgaard L. Weissman J.S. J. Biol. Chem. 2006; 281: 876-884Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 24Benham A.M. Cabibbo A. Fassio A. Bulleid N. Sitia R. Braakman I. EMBO J. 2000; 19: 4493-4502Crossref PubMed Google Scholar, 25Pagani M. Fabbri M. Benedetti C. Fassio A. Pilati S. Bulleid N.J. Cabibbo A. Sitia R. J. Biol. Chem. 2000; 275: 23685-23692Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar).Sequencing of the avian egg white (21Hoober K.L. Glynn N.M. Burnside J. Coppock D.L. Thorpe C. J. Biol. Chem. 1999; 274: 31759-31762Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and the rat seminal vesicle sulfhydryl oxidases (26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) showed that they were founding members of a new family of multidomain sulfhydryl oxidases (schematically depicted in Fig. 3). These QSOXs are found in all metazoans and in those plants and protists for which genomes have been sequenced (21Hoober K.L. Glynn N.M. Burnside J. Coppock D.L. Thorpe C. J. Biol. Chem. 1999; 274: 31759-31762Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar). In humans the sequences have been frequently annotated as PDI-like or given names that are suggestive of biological significance such as “cell growth-inhibiting factor” or “bone-derived growth factor” (26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar). Humans have two QSOX paralogs QSOX1 (QSCN6) and QSOX2 (QSCN6L1, SOXN) showing about 35% identity over 740 amino acids (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar, 29Wittke I. Wiedemeyer R. Pillmann A. Savelyeva L. Westermann F. Schwab M. Cancer Res. 2003; 63: 7742-7752PubMed Google Scholar).FIGURE 3Schematic domain structure of QSOXs. Metazoans have two thioredoxin domains, whereas plants and protists have one. Shown here are one of the two human (Hs) and Arabidopsis (At) QSOXs and the single Trypanosoma brucei (Tb) QSOX. Redox-active CXXC motifs are depicted by the yellow bars. This motif is absent in the Trx2 domain. FAD is shown closest to the proximal disulfide (equivalent to C54–57 in Fig. 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)QSOX Domain Structure and MechanismIn metazoans, two thioredoxin domains follow a typical N-terminal signal sequence (Fig. 3). The first thioredoxin domain (Trx1) has a WCGHC motif typical of many PDIs whereas Trx2 appears redox-inactive. Plants and protists lack the second thioredoxin domain but retain a highly helical “spacer” module fused to the Erv/ALR flavin-binding domain (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar). The C-terminal region varies widely among species but invariably ends with a single transmembrane span that can serve as a membrane anchor (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar). This C-terminal region is frequently spliced out, leading to the most common short form of the enzyme (604 amino acids for the human enzyme). The longer form has been detected in brain (30Radom J. Colin D. Thiebault F. Dognin-Bergeret M. Mairet-Coello G. Esnard-Feve A. Fellmann D. Jouvenot M. Biochim. Biophys. Acta. 2006; 1759: 225-233Crossref PubMed Scopus (14) Google Scholar). Conventional ER retention sequences are absent in QSOXs, yet the enzyme is observed in the ER (see later).Fig. 2 provides important insight into the likely flow of reducing equivalents within the Erv/ALR domain of QSOX (17Gross E. Sevier C.S. Vala A. Kaiser C.A. Fass D. Nat. Struct. Biol. 2002; 9: 61-67Crossref PubMed Scopus (162) Google Scholar). What, then, are the roles of the thioredoxin domains in catalysis? Partial proteolysis of the avian egg white QSOX (31Raje S. Thorpe C. Biochemistry. 2003; 42: 4560-4568Crossref PubMed Scopus (64) Google Scholar) and site-directed mutagenesis of human QSOX1 3E. Heckler and C. Thorpe, unpublished observations. show that the first CXXC disulfide is critical for effective catalysis of protein oxidation and is the site of entry of reducing equivalents. Hence the general flow of reducing equivalents in QSOX1 is clear as shown below. Reduced client protein → Trx1 domain → Erv/ALR domain→ oxygen This ancient fusion of thioredoxin and Erv/ALR domains (21Hoober K.L. Glynn N.M. Burnside J. Coppock D.L. Thorpe C. J. Biol. Chem. 1999; 274: 31759-31762Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 32Coppock D.L. Cina-Poppe D. Gilleran S. Genomics. 1998; 54: 460-468Crossref PubMed Scopus (93) Google Scholar) provides an important catalytic advantage over an isolated Erv domain in the oxidation of protein substrates (31Raje S. Thorpe C. Biochemistry. 2003; 42: 4560-4568Crossref PubMed Scopus (64) Google Scholar). Although one might assume that the single domain flavoprotein Erv2p was an evolutionary precursor to QSOX, the phylogeny seems to point to the opposite conclusion. Thus yeast/fungi have Erv2p, but not QSOX, although these organisms apparently diverged from branches of the eukaryotic tree that have QSOX but not Erv2p (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar).Multiple issues remain to be resolved concerning the topology and interplay between QSOX domains, the function of the spacer domain, and the reasons for the apparent involvement of a chain of 3 CXXC disulfides in catalysis (31Raje S. Thorpe C. Biochemistry. 2003; 42: 4560-4568Crossref PubMed Scopus (64) Google Scholar). For example, does the common rate-limiting step observed for a range of thiol substrates for avian QSOX1 (33Hoober K.L. Thorpe C. Biochemistry. 1999; 38: 3211-3217Crossref PubMed Scopus (63) Google Scholar) reflect communication between thioredoxin and Erv/ALR domains; does the second thioredoxin domain and/or the spacer module play roles in peptide or client protein binding; and why is QSOX so much more effective with unfolded reduced proteins than the isolated Erv/ALR domains (31Raje S. Thorpe C. Biochemistry. 2003; 42: 4560-4568Crossref PubMed Scopus (64) Google Scholar)?Although we do not know the physiological substrate for any QSOX, its prodigious, if somewhat unmodulated, ability to insert disulfide bonds into unfolded proteins suggests a likely physiological role for these enzymes (22Hoober K.L. Sheasley S.S. Gilbert H.F. Thorpe C. J. Biol. Chem. 1999; 274: 22147-22150Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar). Another clue comes from the relative abundance of QSOX1 in cell types with a heavy secretory load or those that accumulate protein disulfides intracellularly as a result of terminal differentiation (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar, 34Tury A. Mairet-Coello G. Esnard-Feve A. Benayoun B. Risold P.Y. Griffond B. Fellmann D. Cell Tissue Res. 2006; 323: 91-103Crossref PubMed Scopus (31) Google Scholar).QSOX Location and Possible Physiological RolesHuman QSOX1 appears generally more abundant than either QSOX2 or Ero1-α and -β in a range of expression profiling, serial analysis of gene expression, and expressed sequence tag frequency compilations (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar). Expression of QSOX1 is especially prominent in differentiated tissues generating high levels of disulfide-containing proteins. For example, Fig. 4 (A–C) shows human epidermis, sebaceous gland, and hair follicle. QSOX1 is also abundant in the seminal vesicle (Fig. 4D), in the syncytiotrophoblastic layer of the placenta (Fig. 4E), and the eccrine gland (Fig. 4F). In addition, plasma cells that express high levels of immunoglobulins consistently show strong QSOX1 staining (for an example see Fig. 7D in Ref. 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar). Detailed analyses of the distribution of QSOX1 in the developing rat brain (35Mairet-Coello G. Tury A. Fellmann D. Risold P.Y. Griffond B. J. Comp. Neurol. 2005; 484: 403-417Crossref PubMed Scopus (20) Google Scholar) and in rat peripheral tissues (34Tury A. Mairet-Coello G. Esnard-Feve A. Benayoun B. Risold P.Y. Griffond B. Fellmann D. Cell Tissue Res. 2006; 323: 91-103Crossref PubMed Scopus (31) Google Scholar) have appeared.FIGURE 4QSOX1 is abundant in cells with a heavy secretory load and tissues that are disulfide-rich after terminal differentiation. QSOX1 distribution is shown by brown staining. A, epidermis; B, sebaceous gland; C, hair follicle; D, seminal vesicle; E, placenta; F, eccrine gland.View Large Image Figure ViewerDownload Hi-res image Download (PPT)QSOX1 was initially identified as a protein secreted from quiescent human fibroblasts (20Coppock D.L. Kopman C. Scandalis S. Gillerman S. Cell Growth & Differ. 1993; 4: 483-493PubMed Google Scholar) and is present in chicken egg white (36Hoober K.L. Joneja B. White H.B. II I Thorpe C. J. Biol. Chem. 1996; 271: 30510-30516Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), mammalian seminal fluid (26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 34Tury A. Mairet-Coello G. Esnard-Feve A. Benayoun B. Risold P.Y. Griffond B. Fellmann D. Cell Tissue Res. 2006; 323: 91-103Crossref PubMed Scopus (31) Google Scholar, 37Ostrowski M.C. Kistler W.S. Biochemistry. 1980; 19: 2639-2645Crossref PubMed Scopus (84) Google Scholar), Chinese hamster ovary epithelial cell supernatants (26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and blood serum (38Zanata S.M. Luvizon A.C. Batista D.F. Ikegami C.M. Pedrosa F.O. Souza E.M. Chaves D.F. Caron L.F. Pelizzari J.V. Laurindo F.R. Nakao L.S. Redox Rep. 2005; 10: 319-323Crossref PubMed Scopus (20) Google Scholar). However, QSOXs have also been found in the ER, Golgi, and secretory granules and are located at the cell surface (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar, 29Wittke I. Wiedemeyer R. Pillmann A. Savelyeva L. Westermann F. Schwab M. Cancer Res. 2003; 63: 7742-7752PubMed Google Scholar, 35Mairet-Coello G. Tury A. Fellmann D. Risold P.Y. Griffond B. J. Comp. Neurol. 2005; 484: 403-417Crossref PubMed Scopus (20) Google Scholar, 39Tury A. Mairet-Coello G. Poncet F. Jacquemard C. Risold P.Y. Fellmann D. Griffond B. J. Endocrinol. 2004; 183: 353-363Crossref PubMed Scopus (28) Google Scholar). The long form of QSOX, with its transmembrane span, may secure the oxidase at the plasma membrane surface (29Wittke I. Wiedemeyer R. Pillmann A. Savelyeva L. Westermann F. Schwab M. Cancer Res. 2003; 63: 7742-7752PubMed Google Scholar, 30Radom J. Colin D. Thiebault F. Dognin-Bergeret M. Mairet-Coello G. Esnard-Feve A. Fellmann D. Jouvenot M. Biochim. Biophys. Acta. 2006; 1759: 225-233Crossref PubMed Scopus (14) Google Scholar). In considering potential redox roles for surface-bound QSOX, there is now considerable evidence for functionally important thiol/disulfide interconversions at the outer face of the plasma membrane. These often involve reductions mediated by protein disulfide isomerases (for example see Refs. 40Essex D.W. Li M. Curr. Drug Targets. 2006; 7: 1233-1241Crossref PubMed Scopus (34) Google Scholar, 41Matthias L.J. Hogg P.J. Antioxid. Redox Signal. 2003; 5: 133-138Crossref PubMed Scopus (41) Google Scholar, 42Jordan P.A. Gibbins J.M. Antioxid. Redox Signal. 2006; 8: 312-324Crossref PubMed Scopus (129) Google Scholar, 43Turano C. Coppari S. Altieri F. Ferraro A. J. Cell. Physiol. 2002; 193: 154-163Crossref PubMed Scopus (401) Google Scholar), and so these effects could be moderated or reversed by surface sulfhydryl oxidases.The abundance of QSOX1 in bone, the correlations suggesting coordinate expression of QSOX with certain collagens and other extracellular matrix components (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 28Thorpe C. Hoober K. Raje S. Glynn N. Burnside J. Turi G. Coppock D. Arch. Biochem. Biophys. 2002; 405: 1-12Crossref PubMed Scopus (176) Google Scholar), and emerging data from Caenorhabditis elegans QSOX deletion mutants (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 44Birnie A. Molecular and Cellular Biology of Helminth Parasites Conference. 2005; : 6-11Google Scholar) are suggestive of roles for these oxidases in the maturation of collagen networks. QSOXs may also be important in the elaboration of disulfide-rich materials such as those involving keratin-associated proteins (in some cases comprising >33% cysteine; for example Fig. 4, A and C).In addition to roles in the biosynthesis of structural disulfides, secreted or membrane-bound QSOX enzymes may generate hydrogen peroxide in the extracellular space for antimicrobial effects (26Benayoun B. Esnard-Fève A. Castella S. Courty Y. Esnard F. J. Biol. Chem. 2001; 276: 13830-13837Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 37Ostrowski M.C. Kistler W.S. Biochemistry. 1980; 19: 2639-2645Crossref PubMed Scopus (84) Google Scholar) and for cellular signaling (27Coppock D.L. Thorpe C. Antioxid. Redox Signal. 2006; 8: 300-311Crossref PubMed Scopus (81) Google Scholar, 45Mairet-Coello G. Tury A. Esnard-Feve A. Fellmann D. Risold P.Y. Griffond B. J. Comp. Neurol. 2004; 473: 334-363Crossref PubMed Scopus (32) Google Scholar). Finally, QSOX2 expression in neuroblastoma cells is a key step in maintaining their apoptotic response to cytostatic drugs (29Wittke I. Wiedemeyer R. Pillmann A. Savelyeva L. Westermann F. Schwab M. Cancer Res. 2003; 63: 7742-7752PubMed Google Scholar).The Interaction between Flavin-dependent Oxidases and PDIsThe pioneering work of the Fass, Kaiser, Lisowsky, and Weissman laboratories on yeast Erv2p and Ero1p and the extension of the work to the mammalian proteins raise a number of basic questions. A key issue for Erv2p is the identity of its physiological reductant. Although yeast PDI1p has been identified as a candidate by disulfide cross-linking (18Sevier C.S. Cuozzo J.W. Vala A. Aslund F. Kaiser C.A. Nat. Cell Biol. 2001; 3: 874-882Crossref PubMed Scopus (153) Google Scholar), it is reported to be a poor substrate of Erv2p in vitro (46Vala A. Sevier C.S. Kaiser C.A. J. Mol. Biol. 2005; 354: 952-966Crossref PubMed Scopus (31) Google Scholar). It will be interesting to test the comparable prediction from Fig. 1B that reduced PDI1p would be a preferred substrate of Ero1p. In the case of Erv2p, Vala et al. (46Vala A. Sevier C.S. Kaiser C.A. J. Mol. Biol. 2005; 354: 952-966Crossref PubMed Scopus (31) Google Scholar) have suggested that other unidentified redox proteins in the yeast ER may be the preferred substrate in vivo or that the in vitro experiments lack an important factor. Logical additional features might include the extreme macromolecular crowding within the lumen of the ER and the possibility of transitory multienzyme complexes promoting disulfide bond formation and isomerization.However, an efficient, unregulated oxidation of reduced PDIs by these flavoprotein oxidases would be highly damaging for the cell. In the mammalian ER, PDI is largely reduced and present at a concentration approaching millimolar (4Wilkinson B. Gilbert H.F. Biochim. Biophys. Acta. 2004; 1699: 35-44Crossref PubMed Scopus (491) Google Scholar, 47Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (215) Google Scholar, 48Jessop C.E. Chakravarthi S. Watkins R.H. Bulleid N.J. Biochem. Soc. Trans. 2004; 32: 655-658Crossref PubMed Scopus (63) Google Scholar, 49Wilkinson B. Xiao R. Gilbert H.F. J. Biol. Chem. 2005; 280: 11483-11487Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). A rapid accumulation of oxidized PDI would certainly favor protein disulfide bond formation but deprive the ER of reduced PDI that is essential for the isomerization of incorrectly placed disulfide bridges. Fu