Abstract
The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress.
Highlights
The endoplasmic reticulum (ER) is an organelle that participates in metabolic pathophysiologies including insulin resistance and type 2 diabetes (Hotamisligil, 2010)
A Relative transcript levels of X-box binding protein 1 (Xbp1) measured by qPCR normalized to 18S in mouse embryonic fibroblasts (MEFs) from wild-type (WT) and Thioredoxin-interacting protein (Txnip)-null (KO) mice treated with increasing concentrations of tunicamycin for 2 h (n = 4). 0 lg/ml: ***P = 0.00004 versus WT; 0.5 lg/ml: ***P = 0.00001 versus WT; 1 lg/ml: ***P = 0.0005 versus WT
Anti-Txnip antibody for Western blot analysis was from MBL International (JY2), anti-VDUP1 antibody for immunofluorescence was from Invitrogen, anti-PDIA6 antibody was from Abnova (3B4), anti-FLAG antibody was from SigmaAldrich (M2), anti-HA antibody was from Covance (16B12), antiPDI antibody was from Novus Biologicals (RL77), anti-ubiquitin antibody was from Enzo Life Sciences (FK2), anti-Xbp1 antibody was from Santa Cruz, and anti-actin antibody was from SigmaAldrich. 4-Phenylbutyric acid and tauroursodeoxycholic acid were obtained from EMD Chemicals
Summary
The endoplasmic reticulum (ER) is an organelle that participates in metabolic pathophysiologies including insulin resistance and type 2 diabetes (Hotamisligil, 2010). Upon ER stress, three different response pathways are activated that are collectively called the ‘unfolded protein response’ (UPR). This short-term adaptive system is necessary to maintain balance in the protein folding machinery by activating protein modification and gene expression programs designed to inhibit protein translation, increase the production of protein chaperones, and stimulate protein degradation. Chronic UPR activation by ER stress can be detrimental and may lead to maladaptive changes in cellular signaling that contribute to disease processes including insulin resistance and type 2 diabetes (Hotamisligil, 2010; Samuel & Shulman, 2012)
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