Abstract
Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip.
Highlights
Thioredoxin-interacting protein (Txnip/VDUP1/TBP-2) was originally discovered as a vitamin D3-inducible gene [1] but has gained recent interest for being involved in diabetes, hyperlipidemia, carcinogenesis, cardiac function, angiogenesis, and inflammation [2]
A promiscuous biotin ligase from E. coli (BirA∗) with an aminoterminal myc epitope was fused to human Txnip
We chose to generate an aminoterminal fusion protein because BirA∗ is sized to green fluorescence protein (GFP, ∼35 and 27 kDa, resp.) and ectopic expression of GFP-Txnip retained proper localization [23] as well as apoptotic function [24]
Summary
Thioredoxin-interacting protein (Txnip/VDUP1/TBP-2) was originally discovered as a vitamin D3-inducible gene [1] but has gained recent interest for being involved in diabetes, hyperlipidemia, carcinogenesis, cardiac function, angiogenesis, and inflammation [2]. Txnip is involved in several prominent biological processes including proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis [2]. Txnip plays a major role in glucose homeostasis. As part of a negative-feedback loop, Txnip inhibits glucose uptake and promotes caspase-3 cleavage, contributing to glucosedependent β-cell death [7]. Txnip regulates proinflammatory gene expression by inflammasome activation via NLRP3 binding [8]. We do not understand precise mechanisms governing differential Txnip signaling, it is clear that several of these pathways are linked by alterations in redox homeostasis
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