title: Role of ascorbate in oxidative protein folding.
abstract: Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.
authors.affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Bánhegyi
authors.forename: Miklós
authors.initials: M
authors.lastname: Csala
authors.forename: András
authors.initials: A
authors.lastname: Szarka
authors.forename: Marianne
authors.initials: M
authors.lastname: Varsányi
authors.forename: Angelo
authors.initials: A
authors.lastname: Benedetti
authors.forename: József
authors.initials: J
authors.lastname: Mandl
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000502:physiology; D001205:Ascorbic Acid / Q000502:physiology*; D001419:Bacteria; D001692:Biological Transport; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004579:Electron Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015003:Yeasts
chemical_list: D000975:Antioxidants; D004220:Disulfides; D005609:Free Radicals; D013438:Sulfhydryl Compounds; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid