192
Alzheimer’s Disease
polypeptides that rapidly undergo endo-
proteolysis within the cytoplasmic loop,
generating a 30-kD N-terminal fragment
(NTF) and a 20-kD C-terminal fragment
(CTF). The NTF and CTF form a sta-
ble complex, which appears to be the
functional form of presenilins. PS endo-
proteolysis is tightly regulated such that
overexpression of PS leads to the accumu-
lation of a full-length protein, but does
n
o
tr
e
s
u
l
ti
na
ni
n
c
r
e
a
s
ei
nt
h
ef
r
a
g
-
ments. The NTF/CTF complex, but not
the holoprotein, are components of a high
molecular weight complex, estimated at
250 kD or even larger. This high molec-
ular weight complex is believed to be the
γ
-secretase complex.
Many lines of evidence indicate that
PS1
is the catalytic component of the
γ
-
secretase. The two membrane-embedded
aspartyl residues in
PS1
(D257 in TM6
and D385 in TM7) are essential for
γ
-
secretase activity. The mutation of either
residue abolishes
γ
-secretase processing
of APP and Notch. The knockout of the
PS genes blocks the A
β
production and
Notch function. While in
PS1
single K/O
cells, there is residual A
β
production, in
PS1/2 double knockout cells, there is no
detectable A
β
, demonstrating that there
is no PS-independent
γ
-secretase activity.
Inhibitors designed to be transition-state
analogs of the
γ
-secretase bind to pre-
senilin fragments. Recently, a family of
signal peptide peptidases was discovered,
which are apparently
bona fde
intramem-
branous aspartyl proteases, with similar
active site motifs and similar topology to
the presenilins. This ±nding further sup-
ports the hypothesis that presenilin is an
aspartyl protease.
3.4.2
Cofactors
Nicastrin is a large type I transmembrane
glycoprotein
that
was
immunopuri±ed
from PS1 immunoprecipitates. It has no
signi±cant sequence homology to other
functionally characterized proteins. Nicas-
trin is a 709 amino acid protein containing
a putative signal peptide, a long N-terminal
hydrophilic domain with numerous glyco-
sylation sites, a TM domain and a short
hydrophilic C-terminal tail of 20 residues.
The mature protein migrates at
150 kD
owing to heavy glycosylation. There is ev-
idence suggesting that the association of
nicastrin with
γ
-secretase is tightly reg-
ulated via glycosylation. Nicastrin binds
to presenilins and the active
γ
-secretase
complex. Nicastrin de±ciency in
Drosophila
abolishes Notch signaling and
γ
-secretase
cleavage of APP. In addition, presenilin
stability was compromised in the absence
of nicastrin. All these data support that
nicastrin is a component of the
γ
-secretase
complex and an important regulator of the
γ
-secretase activity.
Genetic screens in
C. elegans
identi±ed
two proteins, APH-1 and PEN-2, which in-
teract with worm homolog of
PS1
and
nicastrin. Loss-of-function mutations of
either protein produces phenotypes that
resemble that of null mutants in nicas-
trin and presenilins. APH-1 is an integral
protein containing seven predicted TM do-
mains. There are two mammalian
APH-1
genes (
mAPH-1a
and
mAPH-1b
).
mAPH-
1a
has at least two splice variants:
mAPH-
1a
L
has seven exons and encodes a longer
protein of 265 residues; mAPH-1a
S
has six
exons and encodes a shorter protein of 247
residues. All three variants are widely ex-
pressed, although their abundance seems
to be coordinately regulated. To date, no
functional difference among these variants
has been documented. PEN-2 is a small
protein of 101 amino acids containing two
TM domains. Topology study suggests that
both the N- and C-terminals of the protein
face the lumen of the ER.
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