Discovery of the Cysteine Protease Cathepsin B as a Drug Target for Alzheimer’s Disease - Proteinases as Drug Targets - page 151

regulated compared to constitutive secretory pathways. 32,36-38 Therefore, it is
important to identify the proteases for b-secretase in the regulated secretory
vesicles that provides the majority of secreted, extracellular Ab that accumu-
lates in AD.
A model neuronal-like cellular system that produces Ab in regulated secre-
tory vesicles can advantageously facilitate identification of Ab-producing
proteases. Studies of neuronal chroman cells (in primary culture) from in vivo
nervous tissue reveal that they naturally produce Ab in the regulated secretory
pathway that provides the majority of secreted Ab. 15 These regulated secretory
vesicles can be purified in high quantity from sympathoadrenal medullary
tissue (bovine) that allows purification and biochemical characterization. These
secretory vesicles have been used as a model for studies of neurotransmitter
synthesizing enzymes that function in brain. 39,40 These secretory vesicles con-
tain Ab40 and Ab42 with full-length APP (with WT b-secretase site), indicating
the presence of secretases for production of Ab peptides from APP. Therefore,
these model Ab-producing secretory vesicles were utilized for identification of
b-secretease protease(s).
6.3.2 Wild-Type b-Secretase Substrate Leads to Identification of
Cathepsin B in Ab-Producing Secretory Vesicles
Proteolytic activity detected with the WT b-secretase site substrate, Z-Val-Lys-
Met- k MCA, in the Ab-containing secretory vesicles is a cysteine protease,
based on its labeling by the activity probe DCG-04, a biotinylated form of the
cysteine protease inhibitor E64c (Figure 6.3a). The purified b-secretase activity
is inhibited by E64c and CA074; CA074 is a selective inhibitor of cathepsin B
(Figure 6.3b). 41 Peptide sequencing of the anity-labeled enzyme protein by
tandem mass spectrometry indicated its identity as cathepsin B (Figure 6.3c).
To confirm that cathepsin B is colocalized with Ab peptides in these secretory
vesicles, immunoelectron microscopy of their localization was conducted.
Results showed the colocalization of cathepsin B with Ab40 and Ab42 in
purified Ab secretory vesicles (from bovine adrenal medullary chroman cells)
(Figure 6.3d). 41 This finding indicates the secretory vesicle as a new subcellular
compartment for cathepsin B function.
6.3.3 Distinct Cleavage Specificity of Cathepsin B for the
Wild-Type b-Secretase Site of APP, But Not the
Swedish Mutant b-Secretase Site
Cathepsin B cleavage of the WT b-secretase site of the model substrate Z-Val-
Lys-Met- k MCA was compared to its activity with the Swedish mutant
b-secretase site with the substrate Z-Val- Asn-Leu - k MCA (mutant residues are
underlined) that mimics the Swedish mutant site. Significantly, cathepsin B
shows a clear preference for cleaving the WT b-secretase substrate, with