Disulfide Bond Formation: Identifying Roles of PDI Family Thiol Oxidoreductases and ER Oxidant Pathways

Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.

Department dc:contributor.department

Biochemistry

Year dc:date.issued

2012

Author dc:creator

• Rutkevich, Lori Ann

Advisor dc:contributor.advisor

• Williams, David

• Protein Disulfide Isomerase
• Endoplasmic reticulum
• biogenesis & biodegradation
• protein folding
• chaperone
• disulfide bonds

Statement dc:rights

• Attribution-NonCommercial-NoDerivs 2.5 Canada

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc-nd/2.5/ca/

Language dc:language.iso

en_ca

Handle dc:identifier.uri

http://hdl.handle.net/1807/34871

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/34871