detection-of-protease-activity-by-fluorescent-peptide-zymography

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Ohio State University

Epithelial cancer cells can undergo an epithelial-mesenchymal transition (EMT), a complex genetic program that enables cells to break free from the primary tumor, breach the basement membrane, invade through the stroma and metastasize to distant organs. Myoferlin (MYOF), a protein involved in plasma membrane function and repair, is overexpressed in several invasive cancer cell lines. Depletion of myoferlin in the human breast cancer cell line MDA-MB-231 (MDA-231) reduced migration and invasion and caused the cells to revert to an epithelial phenotype. To test if this mesenchymal-epithelial transition was durable, MDA-231 cells were treated with TGF-β1, a potent stimulus of EMT. After 48 hr with TGF-β1, MDA-231 cells underwent an EMT. TGF-β1 treatment also decreased directional cell motility toward more random migration, similar to the highly invasive control cells. To probe the potential mechanism of MYOF function, we examined TGF-β1 receptor signaling. MDA-MB-231 growth and survival has been previously shown to be regulated by autocrine TGF-β1. We hypothesized that MYOF depletion may result in the dysregulation of TGF-β1 signaling, thwarting EMT. To investigate this hypothesis, we examined production of endogenous TGF-β1 and observed a decrease in TGF-β1 protein secretion and mRNA transcription. To determine if TGF-β1 was required to maintain the mesenchymal phenotype, TGF-β receptor signaling was inhibited with a small molecule inhibitor, resulting in decreased expression of several mesenchymal markers. These results identify a novel pathway in the regulation of autocrine TGF-β signaling and a mechanism by which MYOF regulates cellular phenotype and invasive capacity of human breast cancer cells.

Myoferlin regulates epithelial cancer cell plasticity and migration through autocrine TGF-β1 signaling.

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

Jessica L. Weist

Comprehensive Cancer Center,
James Cancer Hospital and Solove Research Center,
Comprehensive Cancer Center, James Cancer Hospital and Solove Research Center

Ohio State University

Detection of Protease Activity by Fluorescent Peptide Zymography

Jessica L. Weist

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Comprehensive Cancer Center,James Cancer Hospital and Solove Research Center,Comprehensive Cancer Center, James Cancer Hospital and Solove Research Center

Ohio State University

Detection of Protease Activity by Fluorescent Peptide Zymography

Comprehensive Cancer Center,
James Cancer Hospital and Solove Research Center,
Comprehensive Cancer Center, James Cancer Hospital and Solove Research Center