Role of ascorbate in oxidative protein folding

Area of Science:

• Biochemistry
• Cell Biology

Background:

• Disulfide bond formation is crucial for protein folding and occurs in the endoplasmic reticulum (ER) of eukaryotic cells.
• This process requires an oxidant to accept electrons, as spontaneous formation is not feasible.
• Oxygen is generally ineffective for direct protein thiol oxidation, necessitating a membrane-permeable electron carrier.

Purpose of the Study:

• To investigate the role of the ascorbate/dehydroascorbate redox couple in oxidative protein folding.
• To elucidate the mechanism by which ascorbate facilitates disulfide bond formation in the ER.

Main Methods:

• Utilized rat liver microsomes to study ascorbate-driven protein thiol oxidation.
• Investigated the transport of dehydroascorbate and glutathione (GSH) across microsomal membranes.
• Assessed the activity of protein disulfide isomerase in the reduction of dehydroascorbate.

Main Results:

• Ascorbate addition or in situ synthesis promoted protein thiol and GSH oxidation in microsomes.
• Demonstrated facilitated diffusion of dehydroascorbate across the microsomal membrane, likely via a GLUT-type transporter.
• Showed that protein disulfide isomerase catalyzes dehydroascorbate reduction in the ER lumen, with GSH or protein thiols as electron donors.
• Observed intraluminal accumulation of ascorbate and GSSG, correlating with dehydroascorbate and GSH uptake.

Conclusions:

• Ascorbate effectively promotes protein disulfide formation in an in vitro system.
• A three-component system involving a microsomal metalloenzyme, dehydroascorbate transport, and protein disulfide isomerase facilitates oxidative folding.
• Further research is needed to confirm the role of ascorbate and other antioxidants in vivo.