Proteases play a central role in a wide variety of crucial biological functions to sustain life including food digestion, immune response, and cell apoptosis. As a consequence of their contribution to the regulation of fundamental intra- and extracellular processes, abnormal regulation of protease activity has been associated with diverse human conditions. Despite their importance in these fundamental physiological processes relatively little is known or understood about the rules that govern proteases' individual specificities.

In the work presented here, the substrate specificity of the serine protease kallikrein-7 (KLK7) is elucidated by bacterial peptide display. Sequential rounds of cell sorting for bacteria displaying substrates that exhibit favorable cleavage kinetics revealed a preferred substrate motif that was remarkably similar to the hydrophobic core of the amyloid-beta (Abeta) peptide that is implicated in the pathogenesis of Alzheimer's disease. KLK7 was found to cleave specifically within the hydrophobic core of Abeta peptide that is largely responsible for its aggregation propensity, thereby slowing amyloid fibril formation and promoting the degradation of preformed fibrils. Further work demonstrated that Abeta oligomer preparations treated with KLK7 afforded a significant increase in neuronal survival compared to untreated preparations in an in vitro model.

To better understand the substrate:protease interactions that determine the specificity of KLK7, site directed mutagenesis was applied. Active site residue N189 that was previously proposed to determine the specifity of the enzyme was randomized, resulting in five active KLK7 variants, one of which increased its selectivity from a tyrosine motif towards a phenylalanine motif that is present within the Abeta hydrophobic core. Furthermore, W215 of KLK7 was further implicated to be a primary determinant of its substrate specificity and subsequent turnover by participating in a putative cation-pi interaction with basic residues of peptidyl substrates. The results presented here establish for the first time that KLK7 has an intrinsic Abeta degrading capacity with novel specificity determinants that could possibly be exploited in future therapeutic applications.