Abstract
Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.
Highlights
Cyclophilins catalyze cis-trans isomerization of peptidyl-prolyl bonds
We examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B
Combined siRNA-mediated depletion of CypB and CypC unexpectedly accelerated oxidative folding and secretion of albumin. This prompted an examination of proteindisulfide isomerases (PDIs) family members and we discovered that all enzymes tested had shifted to a more oxidized state, and the ER was hyperoxidized as exemplified by a dramatic increase in oxidized to total glutathione ratio
Summary
Cyclophilins catalyze cis-trans isomerization of peptidyl-prolyl bonds. Results: Depletion of cyclophilins B and C results in endoplasmic reticulum (ER) hyperoxidation including more oxidized protein-disulfide isomerase enzymes and elevated oxidized:total glutathione ratio. The large number of PDI family members appears to reflect distinct roles in the formation or reduction of disulfides during folding and ERAD, respectively, as well as differences in specificity for the diversity of folding proteins they encounter Both PDI and ERp57 have well established roles in the oxidative folding of proteins [3,4,5,6], whereas ERdj functions primarily as a reductase in cooperation with the ER Hsp BiP to remove. Combined siRNA-mediated depletion of CypB and CypC unexpectedly accelerated oxidative folding and secretion of albumin This prompted an examination of PDI family members and we discovered that all enzymes tested had shifted to a more oxidized state, and the ER was hyperoxidized as exemplified by a dramatic increase in oxidized to total glutathione ratio. Neither Ero, PRDX4, VKOR, nor QSOX1 were responsible for the hyperoxidation, suggesting the existence of an additional oxidative pathway that is modulated by ER cyclophilins
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