Sequence and Conformational Specificity in Substrate Recognition

Abstract

Background: Mesotrypsin targets protease inhibitors as substrates; substrate recognition depends on sequence and conformational motifs. Results: Mesotrypsin cleaves three human Kunitz domains as specific substrates, but other similarly shaped substrates are cleaved less efficiently. Conclusion: Substrate conformation aids recognition by mesotrypsin but is not sufficient for efficient cleavage. Significance: Mesotrypsin cleavage of human Kunitz domains may contribute to cancer progression.

Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy

“…The mesotrypsin S 2 ′ subsite is defined by the atypical Arg-193 residue (a highly conserved Gly in other trypsins), responsible for steric interactions that disfavor bulky P 2 ′ substrate or inhibitor residues [29]. Arg-193 has been found to adopt multiple conformations depending on the P 2 ′ residue of a bound inhibitor [17, 29]. In the structure of mesotrypsin bound to APPI WT [PDB ID: 3L33; [32]], Arg-193 is pushed upward by the inhibitor P 2 ′ residue Met-17 into a cleft between the two beta-barrels of mesotrypsin (Figure 5A).…”

“…While treatment with mesotrypsin directly promoted an invasive cellular phenotype, neither cationic trypsin nor a non-catalytic mesotrypsin variant could similarly drive this invasive phenotype, suggesting that the promotion of invasion depends on the specific proteolytic activity of mesotrypsin [2]. Indeed, a clue to the role played by mesotrypsin in metastasis may be found in the enhanced catalytic capability of mesotrypsin to hydrolyze canonical trypsin inhibitors that are highly abundant in the tumor microenvironment, such as human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2) and others [16, 17]. Cleavage and inactivation of these inhibitors as physiological substrates by mesotrypsin may plausibly contribute to its significant role in the mechanism of metastasis enhancement [16, 17].…”

“…Indeed, a clue to the role played by mesotrypsin in metastasis may be found in the enhanced catalytic capability of mesotrypsin to hydrolyze canonical trypsin inhibitors that are highly abundant in the tumor microenvironment, such as human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2) and others [16, 17]. Cleavage and inactivation of these inhibitors as physiological substrates by mesotrypsin may plausibly contribute to its significant role in the mechanism of metastasis enhancement [16, 17]. In the present work, we have engineered a human Kunitz domain inhibitor that is remarkably resistant to mesotrypsin proteolysis, which we expect will block mesotrypsin’s metastasis-promoting activity.…”

“…A particular challenge in inhibitor development derives from the inability of mesotrypsin to form tight complexes with protein inhibitors, due to the presence of distinctive active site mutations [11, 13–15]. Furthermore, mesotrypsin also cleaves and inactivates many protein protease inhibitors as physiological substrates [12, 16, 17]. An additional challenge lies the inherent difficulty of obtaining selective inhibitors, since mesotrypsin shows high sequence homology and structural similarity with the major digestive trypsins (cationic and anionic trypsin) as well as with other serine proteases, including kallikreins and coagulation factors [18, 19].…”

“…Initial attempts to engineer a potent polypeptide inhibitor of mesotrypsin by using bovine pancreatic trypsin inhibitor (BPTI) as a Kunitz domain scaffold that is relatively resistant to cleavage by mesotrypsin [29] revealed a number of drawbacks: BPTI exhibits a low affinity for mesotrypsin [15], a lack of target specificity [29], and a high potential for immune stimulation [30], and it may promote renal dysfunction [31]. Kunitz domains of human origin are likely to be less immunogenic, but they are much more susceptible than BPTI to cleavage and inactivation by mesotrypsin [16, 17]. However, if such liabilities could be overcome, an engineered human Kunitz domain possessing mesotrypsin affinity, enhanced proteolytic stability, and target selectivity could offer a promising avenue for cancer therapy.…”