Masae Futakami

In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH

The aspartic protease cathepsin E has been shown to induce apoptosis in cancer cells under physiological conditions. Therefore, cathepsin E-activity-enhancing peptides functioning in the physiological pH range are valuable potential cancer therapeutic candidates. Here, we have used a general in vitro selection method (evolutionary rapid panning analysis system (eRAPANSY)), based on inverse substrate-function link (SF-link) selection to successfully identify cathepsin E-activity-enhancing peptide aptamers at neutral pH. A successive enrichment of peptide activators was attained in the course of selection. One such peptide activated cathepsin E up to 260%, had a high affinity (KD; ∼300 nM), and had physiological activity as demonstrated by its apoptosis-inducing reaction in cancerous cells. This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method.

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E‐inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E‐activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module‐finding, module‐shuffling, and module‐pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. Copyright

Peptide Aptamer-Based ELISA-Like System for Detection of Cathepsin E in Tissues and Plasma

ELISA (enzyme-linked immunosorbent assay), a highly sensitive and powerful molecular detection tool, has been constructed based on antibodies, as its name denotes. However, it is not so easy to prepare antibodies for any target molecule and there are still problems in their cost and stability. In this study, peptide-based ELISA-like systems (pep- ELISA) were first constructed and shown to be effective. In particular, if the target is an enzyme, such as cathepsin E, that can generate a fluorescent product, the construct of pep-ELISA can be very simple, as its name indicates; i.e., Enzyme-on-Peptide. These constructs, together with peptide-based Sandwich ELISA-like (Sandwich pep-ELISA), were actually constructed and examined using the tissues and blood specimens extracted from cathepsin E-normal/knockout rats. Through these experiments, the sufficient sensitivity (~10µg/ml) and methodological convenience of pep-ELISA were demonstrated.