Identification of redox‐ and glucose‐sensitive Txnip interacting proteins using BioID (1153.14)

Abstract

Many cell signaling processes are dependent on redox‐dependent interactions through the thioredoxin (Trx) family. Thioredoxins regulate the reduction‐oxidation state of protein substrates through antioxidant enzymatic activities. Thioredoxins have a conserved catalytic site that undergoes reversible oxidation to cysteine disulfide through the transfer of reducing equivalents from the catalytic site cysteine residues to a disulfide substrate. While Txnip has been identified as a critical modulator of several disease pathologies, including β‐cell death during hyperglycemia, cardiac hypertrophy, and bronchopulmonary dysplasia, the molecular mechanisms underlying these processes are relatively unknown. The objective of these studies was to establish a cell model to identify Txnip interacting proteins in an effort to understand molecular events underlying the aforementioned disease pathologies. A proximity‐based biotin‐dependent labeling system (called BioID) was fused to Txnip and stably‐transfected into HEK293 cells to identify novel interacting proteins. Proteomic analysis of BioID lysates identified 50 interacting proteins and mutation of a redox‐senstive cysteine (C247S) in Txnip disrupted several interactions. Furthermore, we demonstrate that this cell model can identify dynamic Txnip interactions through treatment with endogenous Txnip regulators such as hyperglycemia. HEK293 cells treated with increasing glucose concentration upregulated Txnip expression in a dose‐dependent manner. Comparative BioID analysis revealed several glucose‐dependent Txnip interacting proteins such as its E3 ubiquitin ligase, ITCH. Continued studies will focus on understanding the biological relevance of the identified interactions to better understand cellular alterations resulting in disease pathologies.Grant Funding Source: Supported by NIH/NIGMS (1P20GM103620) and NSF/REU (1262744)