Acetylation regulates Thioredoxin Reductase activity and oligomerization
Abstract
The Thioredoxin (Trx) system provides the cell with robust defense against oxidative stress and regulates the function of nearly every cellular process through the reduction-oxidation (redox) regulation of proteins. The Trx system is involved in the development of many diseases ranging from cancer to cardiovascular disorders. Thioredoxin reductase (TrxR) is the key enzyme in the Trx system and contains the 21st genetically encoded amino acid, selenocysteine (Sec). There were multiple experimentally identified TrxR acetylation sites with an unknown effect on TrxR activity. My thesis tested the hypothesis that programmed protein acetylation will enhance the activity of TrxR1. I used genetic code expansion (GCE) to produce Sec-containing and site-specifically acetylated TrxR variants to biochemically characterize their effect TrxR1 activity.
First, I review GCE and the function of the Trx system and relevance to disease. I combined two GCE systems to produce pure Sec-containing, site-specifically acetylated human TrxR variants from Escherichia coli and demonstrate that acetylation increases TrxR activity by reducing the formation of low activity TrxR oligomers. In oxidizing conditions, the TrxR population shifted towards low activity TrxR oligomers resulting from covalent linkages between non-productive TrxR subunits. I demonstrated that site-specific acetylation protects TrxR from oxidative inactivation by reducing the formation of low activity TrxR oligomers and drastically reducing oxidation productions that covalently linked TrxR subunits. I also demonstrate TrxR can be non-enzymatically acetylated by aspirin, which does not stimulate TrxR activity but does protect TrxR1 from oxidation. I produced and purified Sec-containing TrxR fused to a cell penetrating peptide (CPP). I showed the CPP-linked TrxR was efficiently delivered to mammalian cells. A fluorescent TrxR-specific activity reporter demonstrated cytosolic activity from the CPP-tagged TrxR1. The approach provides a novel route to use GCE in bacteria, and study site-specifically modified human proteins in the homologous context of mammalian cells.