Grant: Discovery Grants Program - Individual

One of the main successful uses of biologically active (bioactive) peptides is as inhibitors of enzyme activities involved in critical cellular functions. Glutathione peroxidase 4 (GPX4) is an enzyme with high specificity for glutathione, which is then used to neutralize toxic peroxides that normally would cause damages to the cells. Therefore, under normal cell conditions, the catalytic activity of GPX4 is crucial for maintaining cell survival. However, under aberrant growth conditions, especially excessive cell proliferation, high or normal functioning of GPX4 maintains a low level of lipid peroxides and prevents free radicals-dependent non-apoptotic cell death, which is also called ferroptosis. Thus, a recently (2012) proposed therapeutic approach is the use of GPX4 inhibitors to enhance cellular contents of toxic free radicals and promote ferroptosis that can prevent or treat excessive cell proliferation. The long-term objective of my research program is to determine the structure-function relationships of food protein-derived peptides that could be used as nutritional tools to prevent or alleviate metabolic disorders and restore homoestasis. Specifically, the proposed work will study the mechanism of action of peptides as GPX4 inhibitors, and confirm cellular potency by measuring their ability to create a toxic environment for ferroptosis to occur. The work will use wild rice, hemp seed and whey proteins because of their sulfur levels, which will enhance release of sulfur containing peptides that are excellent GPX4 inhibitors. Protein hydrolysis will be conducted with several enzymes as a means of producing a wide spectrum of peptides suitable for structure-function studies. Once enzymatic digests with potent inhibition of GPX4 have been achieved, they will be subjected to separation and purification using reverse-phase high performance HPLC system. The amino acid sequence of identified peptides will be determined using tandem mass spectrometry and this information will be coupled with the GPX4 inhibition potency to build a database that will be analyzed by partial least regression (PLS). The PLS data will provide useful information to describe how the type and arrangement of amino acids influence potency of peptides against GPX4. Mechanism of enzyme inhibition will be determined using Lineweaver-Burk plots as well as spectroscopic determination of the structural conformations of GPX4-peptide complexes. Finally, active peptides will be tested for ability to reduce cellular activity of GPX4 and create a toxic intracellular environment filled with free radicals. The decrease in cell proliferation will be correlated with content of free radicals and peptide dose to determine potential use as nutritional supplements. This work will lead to the training of two MSc and two PhD students in addition to three postdoctoral fellows all acquiring critical skills in the areas of food chemistry, metabolic nutrition, statistics and spectroscopy.