Elucidation of Therapeutic Peptide Binding Partners from Isolated Mitochondria.

Abstract

CT20p is a protein derived from the C-terminus of Bax. It has selective cytotoxicity for cancer cells, such as the sensitive triple-negative MDA-MB-231 breast adenocarcinoma cells, but not normal cells like the resistant MCF-10A epithelial breast cells. To understand the reason for the peptide’s selective toxicity, a "pull-down" experiment with biotinylated CT20p (biotin-CT20p) and whole-cell protein lysates from breast cancer and normal cells were performed. These studies revealed that CT20p binds to a cytosolic protein called chaperonin-containing TCP-1 (CCT), a molecular chaperone that folds actin and tubulin. However, this method could not detect possible rare interactions made by CT20p with mitochondrial proteins. To determine whether CT20p is associated with mitochondrial proteins as part of the mechanism by which it induces cell death, mitochondrial protein lysates from MDA-MB-231 and MCF-10A cells were isolated and a streptavidin-agarose pulldown procedure using biotin-CT20p was performed. Protein interactions were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The results of the experimental procedure showed that biotin-CT20p did not "pull down" any observable mitochondrial proteins from the sensitive MDA-MB-231 cells, indicating that the peptide may not interact with mitochondrial proteins in breast cancer cells. Rather, the interactions observed with biotin-CT20p were with mitochondrial proteins derived from resistant MCF-10A cells, indicating that these interactions were not driving the cancer-selective cell death process. The absence of CT20p-associated proteins from the mitochondrial lysates of MDA-MB-231 breast cancer cells supports the hypothesis that CT20p, unlike the parent protein, Bax, exerts its cytotoxic effects via a cytosolic protein.