Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article


Doctor of Philosophy




Patrick O'Donoghue


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.

Summary for Lay Audience

Many people have heard of the beneficial effects of antioxidants for health. Antioxidants protect your cells against highly reactive molecules called reactive oxygen species (ROS). ROS can be bad for your health because they ‘steal’ electrons from proteins and deoxyribonucleic acid (DNA) in your cells, causing damage to these important molecules. ROS are generated as your cells produce energy, and can also be generated by other factors, such as exposure to sunlight. Damage caused by ROS can contribute to various diseases such as cancer and heart disease. Antioxidants prevent this damage by providing electrons to ROS before they have a chance to ‘steal’ electrons from your DNA and proteins, and provide electrons to molecules in your body that have been damaged by losing an electron to ROS to repair the damage caused by ROS.

Most people think antioxidants come from healthy foods such as fruit and vegetables, but your cells also produce antioxidants of their own. Our cells have a network of proteins that defend the cell against ROS and repairs damage caused by ROS. One of these naturally occurring antioxidant systems in your cells is the thioredoxin (Trx) system, which is largely dependent on a protein called Thioredoxin Reductase (TrxR). The Trx system transfers electrons to ROS to prevent damage to your cells, and also transfer electrons to protein and DNA molecules damaged by ROS to repair them.

When the TrxR and the Trx system are not functioning properly, damage from ROS can accumulate, leading to the development of many diseases. TrxR and the Trx system is regulated by complex cellular processes to fine-tune the Trx system to defend against ROS. My thesis focuses on understanding how protein modifications regulate TrxR to control its activity and ability to defend itself and the cell against ROS. My thesis developed a new method to produce the TrxR protein in differently active forms, and I efficiently delivered the TrxR protein to human cells to study the enzyme in its native environment.

Included in

Biochemistry Commons