Abstract 15319: Thioredoxin Interacting Protein Interacts with Protein Disulfide Isomerases and increases Endoplasmic Reticulum Stress
Introduction: The alpha arrestin Thioredoxin interacting protein (Txnip) is an inhibitor of the antioxidant thioredoxin and regulates the endothelial response to shear stress as well as metabolism in many tissues. However, the mechanism of Txnip's effects is incompletely defined.
Hypothesis: We hypothesized that Txnip may interact with proteins other than thioredoxin to regulate cellular behavior.
Methods: We screened for potential binding partners through affinity purification of Txnip and subsequent mass spectrometry analysis of purified protein complexes. Initial hits were validated by co-immunoprecipitation and further characterized.
Results: We identified and validated protein disulfide isomerase family A, member 6 (PDIA6) as a binding partner of Txnip. Since PDIA6 contains two thioredoxin-like domains, we hypothesized that Txnip interacts at the same site with PDIA6 and thioredoxin. Indeed, a C247S mutant form of Txnip that does not bind to thioredoxin did not interact with either exogenous or endogenous PDIA6. Additional co-immunoprecipitation experiments with truncated forms of PDIA6 showed that Txnip interacted with both the N-terminal as well as the C-terminal thioredoxin domain. Among the alpha arrestins (Arrestin Domain Containing 1 (ARRDC1), ARRDC2, ARRDC3, ARRDC4, and Txnip), only Txnip interacted with PDIA6. Since PDIA6 contains thioredoxin folds with a WCGHC motif, we hypothesized that Txnip is a regulator of other family members of the PDI family containing that motif. Further pulldown experiments revealed that Txnip also interacts with PDI, PDIA3, PDIA4, PDIA13, and PDIA15. Protein disulfide isomerases play an important role for protein folding in the endoplasmic reticulum, and protein misfolding leads to endoplasmic reticulum (ER) stress. Overexpression of Txnip in HEK293F cells exposed to the ER stress inducer L-Azetidine-2-carboxylic acid led to a significant increase in mRNA levels of sXBP1, GRP78, EDEM1, ATF4 and CHOP, thus indicating an increase in ER stress.
Conclusions: These results reveal Txnip as a novel regulator of protein disulfide isomerases and endoplasmic reticulum stress.
- © 2011 by American Heart Association, Inc.