Biomedical Engineering Reference
In-Depth Information
extended family.
13
The Swe mutation prompted the field to search for
b-secretase using APP substrate with the Swe mutation and assessing Ab in the
conditioned media, representing constitutive secretion; these studies led to
identification of BACE1.
16-19,22
These data showed that although BACE1 readily cleaves the Swe b-secretase
site, it has poor eciency for cleaving the WT b-secretase site. BACE1 activity
for the WT b-secretase substrate is extremely low, cleaving it with only low k
cat
/
K
m
values of 40-60 M
-1
s
-1
,
19,52
whereas proteases acting on relevant substrates
have k
cat
/K
m
values that range between tens of thousands to a few millions.
53
In
order for BACE1 to obtain a k
cat
/K
m
that approaches that of other proteases
for their substrates, seven out of eight amino acid residues in the WT b-
secretase peptide substrate must be modified.
54
On the other hand, cathepsin B
has a k
cat
/K
m
for cleaving WT b-secretase site substrates of 317 000 M
-1
s
-1
.
40,42
These kinetic properties demonstrate the low effectiveness of BACE1 for
cleaving the WT b-secretase site, compared to the highly effective cathepsin B
cleavage of WT b-secretase site substrates.
The difference in effectiveness of BACE1 to cleave the Swe mutant compared
to the WT b-secretase sites is consistent with knowledge of the importance of
amino acid residues adjacent to protease cleavage sites. The Swe mutation
substitutes the neutral asparagine residue for the positively charged lysine
residue at the key P2 protease-substrate recognition site. The different
uncharged residue compared to charged residue at the P2 position is likely to be
critical for recognition and cleavage site by proteases.
55-57
Such protease
cleavage properties can explain the preference of BACE1 for the Swe mutant
b-secretase site compared to the WT site.
6.5.2 BACE1 Knockout in Swedish APP-Expressing Mice
Since BACE1 cleaves the Swe mutant APP, investigators have utilized trans-
genic mice expressing human Swe mutant APP as an AD model for studies of
BACE1 gene knockout. Indeed, a study by Luo and colleagues
20
showed that
knockout of BACE1 reduced brain Ab and CTFb, indicating involvement of
BACE1 as b-secretase for the Swedish mutant APP. These BACE1 studies,
however, do not rule out a role for cathepsin B in production of Ab from
WT APP.
6.5.3 b-Secretase Activity in BACE1 Knockout Mice
Apparently, convincing evidence for BACE1 as the brain b-secretase by a study
by Roberds et al.
21
found that BACE1 knockout mice completely lack brain b-
secretase activity, but a closer reading of the study indicates that the b-secretase
assays contained E64, a potent inhibitor of cathepsin B. Thus, the assay con-
dition with the E64 inhibitor made it impossible to detect the cysteine protease
activity of cathepsin B. Therefore, the assay conditions by Roberds do not rule
out cathepsin B as representing b-secretase activity.
