Production of H₂O₂ in the endoplasmic reticulum promotes in vivo disulfide bond formation.

AIMS Oxidative protein folding in the luminal compartment of endoplasmic reticulum (ER) is thought to be accompanied by the generation of H₂O₂, as side-product of disulfide bond formation. We aimed to examine the role of H₂O₂ produced in the lumen, which on one hand can lead to redox imbalance and hence can contribute to ER stress caused by overproduction of secretory proteins; on the other hand, as an excellent electron acceptor, H₂O₂ might serve as an additional pro-oxidant in physiological oxidative folding. RESULTS Stimulation of H₂O₂ production in the hepatic ER resulted in a decrease in microsomal GSH and protein-thiol contents and in a redox shift of certain luminal oxidoreductases in mice. The oxidative effect, accompanied by moderate signs of ER stress and reversible dilation of ER cisternae, was prevented by concomitant reducing treatment. The imbalance also affected the redox state of pyridine nucleotides in the ER. Antibody producing cells artificially engineered with powerful luminal H₂O₂ eliminating system showed diminished secretion of mature antibody polymers, while incomplete antibody monomers/dimers were accumulated and/or secreted. INNOVATION Evidence are provided by using in vivo models that hydrogen peroxide can promote disulfide bond formation in the ER. CONCLUSION The results indicate that local H₂O₂ production promotes, while quenching of H₂O₂ impairs disulfide formation. The contribution of H₂O₂ to disulfide bond formation previously observed in vitro can be also shown in cellular and in vivo systems

The course of major developmental events in the late larval (in late 3^rd instar larva) and prepupal salivary glands illustrated by staining with antibodies to highlight appropriate structures.

<p>(<b>a</b>) At -12 hr prior to pupariation, when Sgs glue proteins and secretory granules are synthesized, a dense “reticulate”meshwork forms from cytoskeletal components inside cells; myosin II (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>b</b>) During metamorphic pulse of ecdysteroids at 7 hrs prior to pupariation (-7 hr), the larval salivary glands start to release the accumulated secretory granules into the lumen by exocytosis; transcription factor BR-C (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>c</b>) At -3 hr prior to pupariation (-3 hr), exocytosis is complete and the salivary gland undergoes glue solvatation, increasing the diameter of the lumen. This solvatation will facilitate the expectoration of the glue at the pupariation; myosin II (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>d</b>) About +2 hr APF, the salivary gland cells become highly vacuolized by membrane recycling due to massive endocytosis, a consequence of exocytosis; BR-C (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>e</b>) The process of vacuolization and membrane recycling is consolidated by +7 hr APF, shortly prior to the next secretion; BR-C (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>f</b>) At +8 hr APF, the salivary glands are showing an early phase of release of myosin II, p127^l(2)gl and filamentous actin into the centrally located lumen. fb in (<b>a</b>), (<b>b</b>), (<b>d</b>)  =  piece of adherent fat body. All confocal images 400×.</p

Evidence for apocrine secretion of undegraded proteins and the presence of intact genomic DNA in nuclei, and for the release of mitochondria into lumen.

<p>Panels a and b show western blots of secreted proteins isolated from the lumen. (<b>a</b>) Rab11 protein was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion (lane 3). (<b>b</b>) The transcription factor BR-C Z1 was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion from +9–10 hr APF (lane 3). (<b>c</b>) In +8–8.5 hr APF prepupae, ribosomal protein Rp40 (green) and β-tubulin (red) are detectable in the lumen of the salivary glands, while the signal for DNA remains nuclear. (<b>d</b>) In +9 hr APF prepupae, the ribosomal protein Rp21 (green) and transcription factor E74 (red) are detected in the lumen, while the signal for DNA remains nuclear. (<b>e</b>) In +10 hr APF prepupae, both the ribosomal protein p127 (green) and the transcription factor BR-C (red) are detected in the lumen, while the signal for DNA remains nuclear throughout the entire salivary gland, including its columnar, transitional and corpuscular cells; confocal images 80×. (<b>f</b>, <b>g</b>) Mitochondria are released by apocrine secretion into the lumen as evidenced by chasing a vital Rhodamine 123 signal. In larval as well as early prepupal salivary glands, intact living mitochondria are visible only inside cells (<b>f</b>), whereas in +8–10 hr APF prepupae they also can be detected inside the lumen (<b>g</b>); both confocal images 630×. This is also consistent with detection of more than dozen of various mitochondrial proteins listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t001" target="_blank">Tables 1</a> through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t004" target="_blank">4</a>. In addition, <i>in situ</i> hybridization with a mitochondrial genome-specific DNA probe (3'-OH end of mt cytochrome c oxidase I, entire coding sequence of mt tRNA-Leu, and 5'-OH end of mt cytochrome c oxidase II) confirmed the presence of mitochondrial DNA in the secretory material in +9 hr APF prepupae (<b>h</b>, <b>i</b>, (green)) along with F-actin (<b>h</b>, <b>j</b>, (blue)). Although nuclear proteins are released by an apocrine mechanism into the lumen, nuclear DNA was never detected in the secretion. When <i>in situ</i> hybridization was performed in +9 hr APF prepupae with a probe for a nuclear gene <i>Doa</i> locus, signal was found only in nuclei (<b>k</b>, <b>n</b>, (red)) together with Hoechst 33258 staining DNA (<b>k</b>, <b>l</b>, (green)), while F-actin was detectable in the lumen (<b>k</b>, <b>m</b>, (blue)). Remaining confocal images 400×. L in (<b>f</b>), (<b>g</b>), (<b>h</b>) and (<b>k</b>)  =  lumen.</p

List of proteins released by apocrine secretion and detected by antibodies using immunostaining.

<p>This table shows 47 proteins identified using laser confocal or fluorescence microscopy of antibody-stained salivary glands. Proteins are listed alphabetically with the corresponding gene name, molecular weight (in kDa), function and predominant cellular localization. The rightmost columns describe the detection method and predominant time of their release into lumen.</p

A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by <i>lacZ</i>-containing <i>P</i>-element insertion stocks.

<p>We consistently used 9–10 hr old prepupal salivary glands for these types of detection. (<b>a</b>) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF₄₈₈-phalloidin for F-actin. (<b>b</b>) Similarly to (<b>a</b>), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). (<b>c</b>) Tumor suppressor protein p127, the product of <i>l(2)gl</i> gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 (<b>d</b>). Examples of proteins monitored via <i>lacZ</i>-fusion include the transcription factor Ttk (<b>e</b>), the dual-specific LAMMER kinase Doa (<b>f</b>), the D subunit of the vacuolar H⁺ vATPase Vha36-1 (<b>g</b>) and the transcription factor Fkh (<b>h</b>).</p

List of proteins released by apocrine secretion and detected by fluorescent tagging.

<p>Table shows 32 proteins identified using GFP-/EYFP-/RFP-constructs, as mentioned also in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#s2" target="_blank">Materials and Methods</a> section. Also here proteins are listed alphabetically with the corresponding gene name, molecular weight (in kDa), function and predominant cellular localization. The rightmost columns describe not only the detection method but also predominant time of their release into lumen and whenever possible also genotype reference.</p><p>*non-FBti and non-FBal References related to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t003" target="_blank">3</a>.</p><p>{a} Flytrap (<a href="http://flytrap.med.yale.edu/" target="_blank">http://flytrap.med.yale.edu/</a>).</p><p>Morin X, Daneman R, Zavortink M and Chia W (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in <i>Drosophila</i>. Proc. Natl. Acad. Sci. USA 98: 15050–15055.</p><p>{b} Gavdos Protein trap.</p><p>(<a href="http://biodev.obs-vlfr.fr/gavdos/protrap.htm" target="_blank">http://biodev.obs-vlfr.fr/gavdos/protrap.htm</a>) Alain Debec; Biologie du Développement, UMR 7009, CNRS/Université Pierre et Marie Curie, Observatoire Océanologique, Villefranche sur mer, 06230, France.</p><p>{c} Kanesaki T, Edwards CM, Schwarz US and Grosshans J (2011) Dynamic ordering of nuclei in syncytial embryos: a quantitative analysis of the role of cytoskeletal networks. Integr. Biol. (Camb.) 3: 1112–1119.</p><p>{d} Edwards KA, Demsky M, Montague RA, Weymouth N and Kiehart DP (1997) GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in <i>Drosophila</i>. Dev. Biol. 191: 103–117.</p><p>{e} Costantino BF, Bricker DK, Alexandre K, Shen K, Merriam JR, Antoniewski C, Callender JL, Henrich VC, Presente A and Andres AJ (2008) A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of <i>Drosophila</i>. PLoS Genet. 4: e1000102.</p