Redox properties and partners of the yeast thioredoxin reductase TRR1

The College of Wooster received a $316,797 Project Grant from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), effective September 1, 2025, through August 31, 2028. This award supports fundamental research investigating the redox properties and protein interactions of yeast thioredoxin reductase (TRR1), an enzyme critical to cellular oxidative stress defense mechanisms. The project aims to characterize specific cysteine residues within thioredoxin reductase and identify potential protein partners beyond thioredoxin itself, with particular focus on a conserved C-terminal cysteine residue that may serve as an alternative site for disulfide bond exchange. By elucidating the structural and mechanistic properties of this microbial enzyme, the research establishes foundational knowledge that could inform the development of novel antimicrobial therapeutic targets, given that the microbial thioredoxin reductase differs significantly from its mammalian counterpart.

As a private liberal arts institution with established research capabilities, The College of Wooster will conduct this research at its Ohio facilities, leveraging its emphasis on undergraduate student involvement in scientific research. The project deliverables will include experimental characterization of enzyme function, mechanistic insights into redox-active cysteine modifications, and identification of alternative protein interaction partners—outcomes that advance fundamental understanding of biological processes and contribute to the broader scientific knowledge base supporting future disease diagnosis, treatment, and prevention innovations.

Abstract

Reactive oxygen species produced during the innate immune response slow pathogen growth and potentially contribute to pathogen killing. However, pathogenic microbes counter this oxidative burst by using numerous oxidant defense enzymes that detoxify reactive oxygen species, often by forming disulfide bonds. The enzyme thioredoxin and its partner protein thioredoxin reductase ensure appropriate disulfide bond reduction in these oxidized proteins, carrying out coupled reactions that are ultimately dependent on the reductant nadph. While thioredoxin is highly conserved across biological domains, the thioredoxin reductase in bacteria and unicellular eukaryotes differs significantly on a structural and mechanistic level from its counterpart found in mammals, making IT a potential antimicrobial target. The microbial thioredoxin reductase is dimeric and employs a conserved pair of cysteine residues in its active site that enable its interaction with and reduction of oxidized thioredoxin. In addition, another cysteine, located near the C-terminus of the protein and positioned directly across from the active site in the other subunit of the dimer, is subject to modification by thiol-modifying oxidants in vitro and a thiol-reactive electrophile that commonly targets redox-active cysteines in vivo. Notably, this C- terminal cysteine is conserved in fungal and gram-negative bacteria but is less highly conserved in gram- positive bacteria. Because yeast lacking the cytosolic thioredoxin reductase grow more slowly than yeast lacking the corresponding thioredoxins (which themselves have decreased growth rates), we propose that thioredoxin reductase may partner with other proteins beside thioredoxin, perhaps through involving this conserved C- terminal cysteine as an alternate site of disulfide exchange. In this proposal, we aim to (1) characterize which cysteine residues in thioredoxin reductase are redox-active and how they contribute to its cellular function, and (2) determine whether additional proteins besides thioredoxin partner with thioredoxin reductase. To accomplish the proposed work, undergraduate students and I will use a combination of protein biochemistry and cell biology, the latter being performed in baker's yeast. Funding of this project will lead to a better understanding of the fundamental biochemistry of thioredoxin reductase and may provide a new therapeutic avenue (or avenues) for treating microbial infections by exploiting the unique features of the microbial protein. The award will also provide meaningful opportunities for undergraduate students who have a long-term interest in biomedical research and health-related disciplines.