Quantitative profiling of protease specificity

Proteases comprise an important class of enzymes, whose activity is
central to many physiologic and pathologic processes. Detailed knowledge
of protease specificity is key to understanding their function. Although
many methodologies have been developed to profile specificities of
proteases, few have the diversity and quantitative grasp necessary to
fully define specificity of a protease both in terms of substrate numbers
and their catalytic efficiencies. We have developed a concept of
“selectome”, which defines the set of substrates that uniquely represents
specificity of a protease. We applied it to two closely related members of
the Matrixin family – MMP-2 and MMP-9 by using substrate phage display
coupled with Next Generation Sequencing and information theory-based data
analysis. We have also derived a quantitative measure of substrate
specificity, which accounts for both the numbers and relative catalytic
efficiencies of substrates. Using these advances greatly facilitates
uncovering selectivity between closely related members of protease
families and provides insight into to the degree of contribution of
catalytic cleft specificity to protein substrate recognition, thus
providing basis to overcoming two of the major challenges in the field of
proteolysis: 1) development of highly selective activity probes and
inhibitors for studying proteases with overlapping specificities, and 2)
distinguishing targeted proteolysis from bystander proteolytic events.