Protease Proteomics: Scott L. Diamond PhDDue to their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases also participate in cellular homeostasis, inflammation, tissue remodeling, and coagulation and play an important roles in the pathogenicity and progression of many diseases such as viral infection or replication. Proteases comprise one of the largest protein families in organisms from E. coli to humans. Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets. To properly function, proteases must preferentially cleave their
target substrates in the presence of other proteins. While many
factors impact protease substrate selection, one of the key aspects
is the complementarity of the enzyme active site with the residues
surrounding the cleaved bond in the substrate. As such, determination
of the residues that comprise the preferred cleavage site of a protease
provides critical information regarding substrate selection. Furthermore,
determination of substrate specificity also provides a framework
for the design of potent and selective inhibitors. We have used
solution-phase substrate nanodroplet microarrays, in which fluorogenic
substrates suspended in glycerol droplets are treated with aerosolized
aqueous enzyme solutions, to provide protease substrate specificity
profiles. These arrays allow high throughput characterization of
the preferred residues on the P side of the substrate in a highly
parallel and miniaturized format. We have studied the use of these
arrays here to map the substrate specificity of 24 serine and cysteine
proteases in a rapid and efficient manner. The combination of substrate
mapping and compound screening provides critical information for
training of Structure-Activity Relation (SAR) models.
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