| Serine proteases acting at the cell surface are a unique class of enzymes that carry out important cell signaling functions. By acting upstream of proteolytic cascades, growth factor signaling, and extracellular matrix remodeling, these proteases are being increasingly recognized for their relevance to cancer progression and metastasis. However, as enzymes, their mere expression in a given cell, tissue, or tumor is less relevant to biology than the enzymatic activity of each protease. As such, technologies for specifically monitoring active serine proteases at the cell surface are needed to better understand the contributions these proteases make to cell signaling and cancer progression. To identify which specific serine proteases are expressed at the message level, a high-throughput TaqMan array was constructed to profile mRNA expression of every extracellular serine protease in the mouse genome. To quantitatively detect active serine proteases at the cell surface of living cells, activity-based probes (ABPs) based on a peptide diphenylphosphonate scaffold were developed, characterized, and structurally evaluated. Substrate specificity profiling was used to characterize a newly identified cancer-associated transmembrane protease, TMPRSS2/epitheliasin, and novel antibody-based inhibitors of this protease were identified using phage display technology. Lastly, nanoscale devices were designed to measure proteolytic activity with single-molecule sensitivity. Collectively, these technologies expand the enzymologist's "toolbox" to further the understanding of serine proteases and their roles in cancer progression, metastasis, and cell signaling. |
