Thioredoxin and its Target Proteins: Thioredoxin Expression under Different Oxygen Conditions
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Thioredoxin is an antioxidant protein that performs multiple functions in the intracellular and extracellular environment of cells. Thioredoxin is highly expressed in cancer cells, especially more metastatic and aggressive cancers. Previous studies have demonstrated a functional role for thioredoxin in cancer cell invasion, however little information is currently available regarding the role of thioredoxin in the invasive process. In order to perform these and other functions, thioredoxin interacts with several different protein partners. The primary aim of this project was to identify previously unknown binding partners for thioredoxin, particularly those involved in the cancer cell invasion process, on the cell surface of breast cancer cells. In order to identify the binding partners for thioredoxin, a kinetic trapping protocol was employed. Constructs that expressed a thioredoxin mutant wherein the second cysteine of the active site (Cys35) is changed to a serine, were utilized to perform trapping experiments. The trapping mutants remain covalently linked to the target protein (because they lack the second cysteine to resolve the mixed disulfide bond formed), allowing for purification of resulting thioredoxin–substrate complexes. One of the known protein partners for thioredoxin is Methionine Sulfoxide Reductase A (MsrA). Kinetic trapping experiments were performed to determine which of the four cysteine residues within the MsrA protein directly bound to thioredoxin. The experiments were performed using various MsrA cysteine mutants and determined that thioredoxin can bind to either the 3rd or 4th cysteine residue within the MsrA sequence. The kinetic trapping protocol was also used to attempt to identify previously unknown binding partners for thioredoxin on the surface of MDA-MB-231 breast cancer cells. Cell-surface trapping resulted in a single band visualized on anti-thioredoxin western blots, which indicates the recovery of a protein partner. However, the high number of cells used to get a positive result made it unrealistic to attempt even higher cell numbers which would be required in order to trap enough protein to identify the unknown protein partner.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Item Access Status
Methionine Sulfoxide Reductase A