Periodic Report Summary - ER AND METABOLISM (Oxidative protein folding and pathogenesis of metabolic disorders) The ability of secreted proteins to arrive at their correct three-dimensional structure is dependent on the formation of correctly placed disulfide bonds between cysteine residues. This reaction is termed oxidative protein folding, because it entails abstraction of electrons from the protein (two for every disulfide bond formed) and their donation to a terminal electron acceptor - usually molecular oxygen.This process is catalyzed by an enzymatic apparatus located in the lumen of the endoplasmic reticulum, an intracellular organelle charged with the handling of newly-synthesized proteins destined for secretion.In this project we examined the impact of interfering genetically with the function of the aforementioned enzymatic apparatus in live mice and in cells explanted from them. The objective was to gain insight into the functional significance of oxidative protein folding in a complex mammal and to explore the consequences of interfering with it.Genetically-modified mice with compound deficiency in the enzymes mediating disulfide bond formation were produced through the techniques of germline gene manipulation. Compound mutant mice deficient in Ero1l (encoding the dithiol oxidase ERO1a), Ero1lb (encoding the dithiol oxidase ERO1b) and Prdx4 (encoding an ER-localized peroxiredoxin) were produced and analyzed. Achievement: These mice are now available for the larger research community as research tool.Unexpectedly, we found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1a, ERO1b, and PRDX4 was NOT incompatible with cell survival.Achievement: This finding indicates that the process of disulfide bond formation in the secretory pathway is highly redundant and suggests broad latitudes for manipulating this parameter to therapeutic needs - should the utility of such manipulation be clarified by further research.Unexpectedly, combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1a, ERO1b, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with a 5-fold lower procollagen 4-hydroxy- proline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Indicating a defect in the normal maturation of collagen, a major constituent of tissues. Ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2-generating system. Achievement: This study indicates that compromised ER disulfide relay exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy. Thus uncovering an unexpected link between oxidative protein folding and metabolism of an essential human nutrient.
