Researchers at the Scripps Research Institute and the Université Louis Pasteur have jointly developed high-throughput assays to characterize proteases and their substrates.
Proteases have their enzymatic fingers in a lot of biological pies. Their activity contributes to the post-translational modification of many proteins—so many, in fact, that a high-throughput assay was needed to rapidly screen for protease activities and their substrates. This need prompted Jennifer Harris, a biologist from The Scripps Research Institute, and Nicolas Winssinger, a chemist from the Université Louis Pasteur, to join forces. In two recent papers in Chemistry & Biology they describe techniques to isolate active proteases from cell lysates and determine their substrate specificities.
Both approaches use fluorescent tetrapeptide libraries bar coded with a peptide nucleic acid (PNA) tag. To isolate active cysteine proteases from a sample, Harris et al. used a peptide library designed to mechanistically inhibit these proteases, and incubated it with a cell lysate. They then separated peptides that covalently bound active proteases from those that did not and hybridized these peptides via their PNA tag to a DNA microarray, thus easily identifying the inhibitors' sequences. By coupling the most potent inhibitor to biotin, they could isolate the active proteases and identify them by mass spectrometry (Harris et al., 2004)
Winssinger et al. modified this approach as a means to identify the substrate specificity of a particular protease. They incubated purified proteases with a library of PNA-encoded fluorescent substrate peptides; upon cleavage of a peptide, its fluorescence intensity increased and could easily be detected after hybridization of the PNA tags to the microarray (Winssinger et al., 2004). Once the 'substrate fingerprint' of a protease has been established, complex mixtures can be screened for the presence of the active protease.
Winssinger says these techniques could also be adapted for other enzymes, such as kinases, or even to receptors and their ligands. Most of all, he emphasizes his interdisciplinary collaboration with Harris as the main strength of their work: "It has been the combination of my chemistry and Jennifer Harris' enzymology background that has made this project work. Without each other we wouldn't have been able to make it work."
