Discovery of the Cysteine Protease Cathepsin B as a Drug Target for Alzheimer’s Disease - Proteinases as Drug Targets - page 149

b-secretase site of APP is relevant to the majority of AD patients because
they express WT APP.
6.2.1 Specificity of Peptide Substrate Cleavage by Proteases
The specificity for peptide substrate cleavage by proteases involves the active
site and subsites, using the model and nomenclature of Schechter and
Berger. 23,24 This model describes interactions of the protease with the substrate
amino acids flanking the cleavage site (Figure 6.2b.i). The amino acids forming
the cleaved peptide bond are termed P1- k P1 0 . Residues at the N-terminal and
C-terminal sides of the cleavage site are indicated as P1, P2 ...and P1 0 ,P2 0 ...,
respectively. The protease active site domain is viewed as a series of subsites
(S1, S2 . . . and S1 0 ,S2 0 . . .) each accommodating one amino acid residue of the
substrate. Protease subsites interact with the side chains of amino acids of the
substrate.
Multiple points of interactions at several subsites are essential for obtaining
the high association constants necessary for ecient proteolysis. Within the
active site, certain subsites (close or remote from the catalytic site) play a major
role in determining specificity. 23,25 For example, the S1 subsite of trypsin has a
clear preference for binding to basic P1 residues consisting of lysine or argi-
nine. 26 In papain 24 and the cysteine cathepsins, 27 specificity is determined
mainly by the hydrophobic S2 subsite that prefers binding to hydrophobic P2
residues. The specificity and biological activity of caspases are determined by
both S1 and S4 remote from the catalytic site. 28 Generally, the P1 to P3 residues
are important for protease selectivity for cleavage sites, based on interaction
with S1 to S3 subsites of the protease. Therefore, cleavage-site-specific assays
often incorporate the native P1 to P3 residues, or longer extensions of the
peptide substrate.
6.2.2 Design of Wild-Type b-Secretase Peptide Substrate, Based
on the P2-P1 Residues at the Wild-Type b-Secretase Site
of APP
At the WT b-secretase site of APP, the -P3-P2-P1- k P1 0 -P2 0 -P3 0 - residues are
Val-Lys-Met- k Asp-Ala-Glu- (Figure 6.2b.ii). This WT b-secretase site is
expressed by the majority of AD patients and, therefore, represents a key
cleavage event for production of neurotoxic Ab. Interestingly, a mutant
b-secretase site is expressed in an extended Swedish family 13 and results in
elevated production of Ab in these patients. Transgenic mice expressing the
human Swedish mutant APP show elevated levels of Ab. 29 The Swedish mutant
P2-P1 residues consist of Asn-Leu that substitute for the WT P2-P1 residues of
Lys-Met. It is important to realize that the amino acids at the Swedish mutant
compared to the WT b-secretases site differ in chemical properties. A change
in the chemical properties of the amino acid side chains may define substrate
preferences by the protease.