Length and overall sequence of the PEN-2 C-terminal domain determines its function in the stabilization of presenilin fragments

Article · August 2005with31 Reads
DOI: 10.1111/j.1471-4159.2005.03165.x · Source: PubMed
Abstract
Gamma-secretase is an aspartyl protease complex that catalyzes the intramembrane cleavage of a subset of type I transmembrane proteins including the beta-amyloid precursor protein (APP) implicated in Alzheimer's disease. Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2) constitute the active gamma-secretase complex. PEN-2, the smallest subunit, is required for triggering PS endoproteolysis. Stabilization of the resultant N- and C-terminal fragments, which carry the catalytically active site aspartates, but not endoproteolysis itself, requires the C-terminal domain of PEN-2. To functionally dissect the C-terminal domain we created C-terminal deletion mutants and mutagenized several evolutionary highly conserved residues. The PEN-2 mutants were then probed for functional complementation of a PEN-2 knockdown, which displays deficient PS1 endoproteolysis and impaired NCT maturation. Progressive truncation of the C-terminus caused increasing loss of function. This was also observed for an internal deletion mutant as well as for C-terminally tagged PEN-2 with a twofold elongated C-terminal domain. Interestingly, only simultaneous, but not individual substitution of the highly conserved D90, F94, P97 and G99 residues with alanine interfered with PEN-2 function. All loss of function mutants identified allowed PS1 endoproteolysis, but failed to stably associate with the resultant PS1 fragments, which like the PEN-2 loss of function mutants underwent proteasomal degradation. However, complex formation of the PEN-2 mutants with PS1 fragments could be recovered when proteasomal degradation was blocked. Taken together, our data suggest that the PS-subunit stabilizing function of PEN-2 depends on length and overall sequence of its C-terminal domain.
4 Figures
Length and overall sequence of the PEN-2 C-terminal domain
determines its function in the stabilization of presenilin fragments
Stefan Prokop, Christian Haass and Harald Steiner
Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer’s and Parkinson’s Disease Research,
Ludwig-Maximilians-University, Munich, Germany
Abstract
c-Secretase is an aspartyl protease complex that catalyzes
the intramembrane cleavage of a subset of type I transmem-
brane proteins including the b-amyloid precursor protein
(APP) implicated in Alzheimer’s disease. Presenilin (PS),
nicastrin (NCT), anterior pharynx defective (APH-1) and
presenilin enhancer-2 (PEN-2) constitute the active c-secret-
ase complex. PEN-2, the smallest subunit, is required
for triggering PS endoproteolysis. Stabilization of the resultant
N- and C-terminal fragments, which carry the catalytically
active site aspartates, but not endoproteolysis itself, requires
the C-terminal domain of PEN-2. To functionally dissect the
C-terminal domain we created C-terminal deletion mutants
and mutagenized several evolutionary highly conserved resi-
dues. The PEN-2 mutants were then probed for functional
complementation of a PEN-2 knockdown, which displays
deficient PS1 endoproteolysis and impaired NCT maturation.
Progressive truncation of the C-terminus caused increasing
loss of function. This was also observed for an internal dele-
tion mutant as well as for C-terminally tagged PEN-2 with a
twofold elongated C-terminal domain. Interestingly, only sim-
ultaneous, but not individual substitution of the highly con-
served D90, F94, P97 and G99 residues with alanine
interfered with PEN-2 function. All loss of function mutants
identified allowed PS1 endoproteolysis, but failed to stably
associate with the resultant PS1 fragments, which like the
PEN-2 loss of function mutants underwent proteasomal de-
gradation. However, complex formation of the PEN-2 mutants
with PS1 fragments could be recovered when proteasomal
degradation was blocked. Taken together, our data suggest
that the PS-subunit stabilizing function of PEN-2 depends on
length and overall sequence of its C-terminal domain.
Keywords: Alzheimer’s disease, anterior pharynx defective-1,
c-secretase, nicastrin, presenilin, presenilin enhancer-2.
J. Neurochem. (2005) 94, 57–62.
c-Secretase is a multisubunit aspartyl protease complex,
which catalyzes the regulated intramembrane proteolysis of
the Alzheimer’s disease-associated b-amyloid precursor
protein (APP) and an increasing number of other type I
transmembrane proteins (Haass 2004). Genetic and bio-
chemical studies have identified four integral membrane
proteins, presenilin (PS), nicastrin (NCT), anterior pharynx
defective-1 (APH-1) and presenilin enhancer-2 (PEN-2),
which are necessary and sufficient for c-secretase activity
(Francis et al. 2002; Edbauer et al. 2003; Kimberly et al.
2003; Takasugi et al. 2003; Hayashi et al. 2004). In human
cells, two PS (PS1 and PS2) and two APH-1 [APH-1a (two
splice variants) and APH-1b] homologues have been iden-
tified that are part of distinct c-secretase complexes (Shiro-
tani et al. 2004). Distinct c-secretase complexes were also
identified in mice (Hebert et al. 2004). A large body of
evidence suggests that PS is the catalytic subunit of the
complex harboring the two active site aspartates in trans-
membrane domains 6 and 7 (Wolfe et al. 1999; Esler et al.
2000; Li et al. 2000; Steiner et al. 2000). These aspartates
are located in the N- and C-terminal fragment (NTF, CTF) of
PS (Thinakaran et al. 1996), which are likely generated by
autoproteolysis (Wolfe et al. 1999; Edbauer et al. 2003;
Hayashi et al. 2004). Apart from their essential requirement
Received December 2, 2004; revised manuscript received February 17,
2005; accepted February 17, 2005.
Address correspondence and reprint requests to Harald Steiner
or Christian Haass, Adolf-Butenandt-Institute, Ludwig-Maximilians-
University Munich, Department of Biochemistry, Schillerstr. 44, 80336
Munich, Germany.
E-mail: hsteiner@med.uni-muenchen.de (HS)
or chaass@med.uni-muenchen.de (CH)
Abbreviations used: APH, anterior pharynx defective; APP, b-amyloid
precursor protein; CTF, C-terminal fragment; mH
6
,myc-hexahistidine;
NCT, nicastrin; NTF, N-terminal fragment; PEN, presenilin enhancer;
PS, presenilin; RNAi, RNA interference; wt, wild type.
Journal of Neurochemistry, 2005, 94, 57–62 doi:10.1111/j.1471-4159.2005.03165.x
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 57–62 57
during c-secretase complex assembly and maturation (Haass
2004), the function of the other subunits is unclear.
The 101-amino acid protein PEN-2 is the smallest
subunit of the c-secretase complex (Francis et al. 2002;
Steiner et al. 2002) and adopts a hairpin orientation in the
membrane with its N- and C-terminal domains facing the
luminal/extracellular space (Crystal et al. 2003; Bergman
et al. 2004). PEN-2 assembles as the last component during
c-secretase complex assembly and initiates PS endoprote-
olysis (Kim et al. 2003; Kimberly et al. 2003; Luo et al.
2003; Takasugi et al. 2003; Prokop et al. 2004). Within the
c-secretase complex, PS appears to be an important binding
partner of PEN-2, as in the absence of PS PEN-2 is
unstable and degraded by the proteasome (Bergman et al.
2004; Crystal et al. 2004). Furthermore, we recently
showed that PEN-2 is specifically required for the stabil-
ization of the NTF/CTF heterodimer following PS endo-
proteolysis and that this function requires the C-terminal
domain of PEN-2 (Prokop et al. 2004). Consistent with
these results, PEN-2 has also recently been found in
association with the PS1 NTF (Fraering et al. 2004).
Finally, PEN-2 has recently been proposed to enhance the
activity of c-secretase after PS endoproteolysis (Shiraishi
et al. 2004).
In this study, we have dissected the functionally critical
C-terminal domain of PEN-2 by extensive mutagenesis to
define the sequence requirements for its PS fragment-
stabilizing function.
Materials and methods
Antibodies
The polyclonal antibody against the PEN-2 N-terminus (1638), the
polyclonal antibodies against the APH-1aL C-terminus (433),
against the PS1 N-terminus (2953) and PS1 C-terminus (3027),
and the monoclonal antibody to the N-terminus (PS1N) were
described previously (see Steiner et al. 2002; Prokop et al. 2004;
Shirotani et al. 2004 and citations therein). The polyclonal antibody
against the C-terminus of NCT (N1660) was obtained from Sigma
(St Louis, MO, USA).
cDNA constructs
Mutant PEN-2 variants were generated by PCR-mediated mutagen-
esis and cloned into the pcDNA3.1/Zeo(+) vector (Invitrogen,
Carlsbad, CA, USA). Silencer mutations that do not alter the
encoded amino acid sequence were introduced in all PEN-2
constructs as described (Prokop et al. 2004) to escape RNA
interference (RNAi). C-terminally myc-hexahistidine (mH
6
)-epi-
tope-tagged PEN-2 has been described before (Prokop et al. 2004).
All constructs were verified by DNA sequencing.
Cell culture and cell lines
HEK 293 cells stably expressing Swedish mutant APP (HEK 293/sw)
and the stable PEN-2 knockdown cell line have been described
before (Prokop et al. 2004). This cell line was stably transfected
with the indicated wild type (wt) and mutant RNAi-resistant PEN-2
constructs as described (Prokop et al. 2004).
Protein analysis
Membrane fractions of HEK 293 cells were prepared and analyzed
for the presence and interaction of the c-secretase complex subunits
by direct immunoblotting or coimmunoprecipitation as described
(Prokop et al. 2004).
Results
As PEN-2 requires the C-terminus to stabilize the PS1
fragment heterodimer within the c-secretase complex (Pro-
kop et al. 2004), we sought to define the precise sequence
requirements for this function by a mutagenesis analysis.
Although no obvious protein–protein interaction motif(s)
were found by database searches, the C-terminal domain
contains several evolutionary highly conserved amino acids,
in particular the D90, F94, P97 and G99 residues (Fig. 1a).
We first constructed C-terminally truncated PEN-2 variants
lacking the last three (PEN-2 DC3), five (PEN-2 DC5) or
eight (PEN-2 DC8) amino acids thus stepwise removing
highly conserved residues including the G99 and P97 or F94
(Fig. 1b). Throughout the study, the mutant PEN-2 cDNAs
constructs created were stably transfected in our previously
described PEN-2 knockdown cell line (HEK 293/sw cells in
which PEN-2 expression is stably knocked down by RNAi)
PEN-2
1 2
Cytosol
L
umen
(a)
(b)
Wt
C3
C5
C8
90-94
D90A
F94A
P97A
G99A
4A
Hs
Mm
Rn
Dr
Tn
Xl
Dm
Ag
Cb
Ce
84 94 97 9990 101
Fig. 1 Schematic depiction of PEN-2 and its C-terminal domain. (a)
Sequence alignment of the C-terminal domain of human PEN-2 and its
homologues. The last 17 amino acids of PEN-2 (residues 84–101) are
shown, which are deleted in PEN-2 DC (Prokop et al. 2004). Residues
90, 94, 97 and 99 that are most highly conserved during evolution are
boxed in red. Hs, Homo sapiens; Mm, Mus musculus;Rn,Rattus
norwegicus;Dr,Danio rerio; Tn, Tetraodon nigroviridis;Xl,Xenopus
laevis; Dm, Drosophila melanogaster;Ag,Anopheles gambiae; Cb,
Caenorhabditis briggsae;Ce,Caenorhabditis elegans. (b) Schematic
representation of mutants generated in the C-terminal domain of hu-
man PEN-2. Deletions and substitutions are boxed in red.
58 S. Prokop et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 57–62
and tested for functional complementation of the PEN-2
deficiency (Prokop et al. 2004). As controls, we analyzed
PEN-2 knockdown cells stably expressing wt PEN-2 or the
previously described PEN-2 DC construct, in which the last
17 amino acids (residues 84–101, Fig. 1a) are deleted
(Prokop et al. 2004). As reported previously (Francis et al.
2002; Steiner et al. 2002; Luo et al. 2003; Hasegawa et al.
2004; Prokop et al. 2004), down-regulation of PEN-2 was
associated with an accumulation of the PS1 holoprotein,
reduced levels of PS1 fragments and impaired NCT matur-
ation, whereas levels of APH-1aL were not affected
(Fig. 2a). In agreement with our previous study (Prokop
et al. 2004), expression of wt PEN-2 allowed efficient
recovery of PS1 endoproteolysis and NCT maturation,
whereas expression of PEN-2 DC abrogated PEN-2 function
(Fig. 2a). Although expression of PEN-2 DC allowed PS1
endoproteolysis, the PS1 fragments were not recovered and
NCT maturation was still impaired (Fig. 2a). PEN-2 DC3
rescued the defect of the PEN-2 knockdown in PS1
endoproteolysis and considerably recovered NCT matur-
ation, whereas PEN-2 DC5 allowed only partial rescue of the
PEN-2 knockdown defect and PEN-2 DC8 did not show
rescuing activity. Similarly as observed for PEN-2 DC, PEN-
2DC5 and PEN-2 DC8 rescued the defect in PS1 endopro-
teolysis, but were defective in stabilizing the PS1 fragments
and in NCT maturation. Thus, progressive truncation of the
C-terminus was apparently associated with an increased loss
of function. As shown in Fig. 2b, PEN-2 function was also
abrogated in the same manner as observed for PEN-2 DC8 or
PEN-2 DC when we investigated the deletion mutant PEN-2
D90–94, which carries an internal deletion of the conserved
sequence block from D90 to F94 (Hasegawa et al. 2004)
while the terminal residues are left intact (Fig. 1b). The
C-terminally myc-hexahistidine-tagged PEN-2 mH
6
construct
(Prokop et al. 2004), in which the length of the luminal C-
terminal domain is roughly doubled by the tag, also did not
display normal PEN-2 function and behaved similar as PEN-
2DC or PEN-2 DC8 or PEN-2 D90–94 (Fig. 2b). Thus,
shortening of the C-terminus by progressive truncation or by
an internal deletion or elongation of the C-terminus by
epitope-tagging interferes with PEN-2 function.
Although the above results indicate that proper length of
the PEN-2 C-terminus is critical for PEN-2 function, the
results obtained with the deletion mutants PEN-2 DC, PEN-2
DC5, PEN-2 DC8 or PEN-2 D90–94 do not exclude the
possibility that the observed loss of function of these mutants
was due to the removal of one or more conserved residues.
To address this possibility we investigated the functional
importance of the most conserved C-terminal residues by
site-directed mutagenesis and constructed the PEN-2 D90A,
F94A, P97A, and G99A mutants (Fig. 1b). In addition, we
created the corresponding D90A/F94A/P97A/G99A quad-
ruple alanine mutant (PEN-2 4A) (Fig. 1b). Surprisingly, all
of the individual PEN-2 mutants restored PEN-2 function
(Fig. 3a) as judged by the recovery of normal PS1 fragment
levels and by the substantial restoration of NCT maturation
(Fig. 3a, see also individual PEN-2 G99A expressing cell
Control
-
wt
C
C3
C5
C8
(a)
(b)
PEN-2 mH6
Control
-
wt
90-94
mH6
PEN-2 KD
PEN-2 KD
NCTm
NCTim
PS1NTF
PEN-2
PS1holo
APH-1aL
NCTm
NCTim
PS1NTF
PEN-2
PS1holo
APH-1aL
Fig. 2 Length of the C-terminus is crucial for PEN-2 function. (a)
Membrane fractions of HEK 293/sw cells (control), PEN-2 knockdown
cells (PEN-2 KD) and PEN-2 knockdown cells stably transfected with
RNAi-resistant cDNA constructs encoding either wild type (wt) PEN-2
or the indicated C-terminal PEN-2 mutant variants were analyzed for
PEN-2 by immunoblotting with antibody 1638 (to the PEN-2 N-termi-
nus), for PS1 holoprotein and NTF by immunoblotting with antibody
PS1N (to the PS1 N-terminus) and for NCT and APH-1aL by immu-
noblotting with antibodies N1660 (to the NCT C-terminus) and 433
(to the APH-1aL C-terminus). (b) Membrane fractions of HEK 293/sw
cells, PEN-2 knockdown cells and PEN-2 knockdown cells stably
transfected with RNAi-resistant cDNA constructs encoding wt PEN-2
or the PEN-2 D90–94 or PEN-2 mH
6
mutants were analyzed for PEN-2,
PS1 holoprotein and NTF, NCT and APH-1aL as in (a).
PEN-2 C-terminal domain 59
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 57–62
clones in Fig. 3b). In contrast, the PEN-2 4A mutant allowed
PS1 endoproteolysis, but was still defective in stabilizing the
PS1 fragments and in NCT maturation and thus caused a
biochemical phenotype undistinguishable from that caused
by PEN-2 DC (Fig. 3a). Although these data showed that the
PEN-2 4A mutant is able to trigger PS1 endoproteolysis, it
remained possible that the expression level of the PEN-2 4A
mutant was too low to stabilize the PS1 fragments after PS1
cleavage (Fig. 3a). To address this concern we compared
three independent PEN-2 4A expressing cell clones (#3, #6,
#15) with selected independent cell clones expressing similar
low amounts of the rescuing mutant PEN-2 G99A (in
particular #4, #8). The independent PEN-2 4A and PEN-2
G99A expressing cell clones showed a different biochemical
behavior (Fig. 3b). The PEN-2 G99A mutants (#4, #7, #8),
but not the PEN-2 4A mutants (#3, #6, #15), stabilized the
PS1 fragments (Fig. 3b) and restored NCT maturation. These
data unambiguously show that the PEN-2 4A mutant is
defective both in PS1 fragment stabilization and NCT
maturation. Taken together, these data suggest that whereas
individual mutations of the most highly conserved residues
can be tolerated, simultaneous mutation of these residues is
detrimental for PEN-2 function.
To address the question whether the C-terminal PEN-2
loss of function mutants PEN-2 DC8, PEN-2 D90–94 and
PEN-2 4A identified above associated stably with the
c-secretase complex, we subjected CHAPSO-solubilized
membrane fractions to coimmunoprecipitation analysis with
an antibody to NCT. As shown in Fig. 4(a), robust amounts
of wt PEN-2 were coimmunoprecipitated, whereas consistent
with previous results (Prokop et al. 2004) only very minor
amounts of PEN-2 DC were coimmunoprecipitated. Simi-
larly, only tiny amounts of PEN-2 DC8, PEN-2 D90–94 and
PEN-2 4A were coimmunoprecipitated with the NCT
antibody (Fig. 4a). Compared to the deletion mutants, the
PEN-2 4A mutant was somewhat better recovered, indicating
very weak residual binding. As expected from our previous
results (Prokop et al. 2004), compared to wt PEN-2 expres-
sing cells, only very low levels of PS fragments, as
exemplified by the PS1 NTF, and predominantly immature
NCT were coimmunoprecipitated from the PEN-2 mutant
cells, whereas similar amounts of APH-1aL were coimmu-
noprecipitated from wt and mutant PEN-2 expressing cells
(Fig. 4a). Again, all C-terminal PEN-2 loss of function
mutants behaved very similar to the prototypic PEN-2 DC.
Since after PS1 endoproteolysis PEN-2 DC and the PS1
fragments fail to become stabilized within the c-secretase
complex and are therefore rapidly degraded by the protea-
some (Prokop et al. 2004), we asked whether the C-terminal
PEN-2 mutants could be found in association with the PS1
fragments when proteasomal degradation was inhibited. As
expected, inhibition of proteasomal degradation by MG132
treatment caused a robust accumulation of PEN-2 DC
(Fig. 4b). Similarly, the loss of function mutants PEN-2
DC8, PEN-2 D90–94 and PEN-2 4A accumulated under
these conditions. Furthermore, in cells expressing the
C-terminal loss of function mutants, the PS1 NTF and CTF
accumulated as well upon inhibition of proteasomal activity,
whereas due to their independent stabilization with APH-1
(Prokop et al. 2004), levels of NCT were not affected by the
inhibitor treatment (data not shown). Under these conditions,
the stabilized PEN-2 mutants could be coimmunoprecipitated
with the stabilized PS1 NTF and CTF (Fig. 4c). Thus, the
PEN-2 loss of function mutants and the PS1 NTF/CTF can
be coisolated within the c-secretase complex when they are
stabilized under conditions where their proteasomal degra-
dation is blocked.
Control
-
wt
C
D90A
F94A
P97A
G99A
4A
PEN-2 KD
APH-1a
L
(a)
(b)
PS1holo
PS1NTF
NCTim
NCTm
PEN-2
PS1holo
PS1NTF
NCTim
NCTm
PEN-2
Control
wt
# 3
# 6
# 15
C
# 4
# 7
4A G99A
PEN-2 KD
-
# 8
Fig. 3 Overall sequence of the C-terminal domain is crucial for PEN-2
function. (A) Membrane fractions of HEK 293/sw cells, PEN-2
knockdown cells and PEN-2 knockdown cells stably transfected with
RNAi-resistant cDNA constructs encoding wt PEN-2 or the indicated
mutant PEN-2 variants were analyzed for PEN-2, PS1 holoprotein and
NTF, NCT and APH-1aL as in Fig. 2. (b) Membrane fractions of
HEK 293/sw cells, PEN-2 knockdown cells and PEN-2 knockdown
cells stably transfected with RNAi-resistant cDNA constructs encoding
either wt PEN-2, PEN-2 DC, PEN-2 4A (independent single cell clones
#3, #6, #15) or PEN-2 G99A (independent single cell clones #4, #7,
#8) were analyzed for PEN-2, PS1 holoprotein and NTF and NCT as in
(a).
60 S. Prokop et al.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 57–62
Discussion
To further define the sequence requirements for PEN-2
function, we have dissected its functionally critical C-ter-
minal domain (Prokop et al. 2004) by probing a variety of
diverse mutants for functional complementation of PEN-2
deficiency. We had previously shown that C-terminally
epitope-tagged PEN-2 as well as C-terminally truncated
PEN-2 (PEN-2 DC) rescued the defect of PEN-2 deficiency
in PS1 endoproteolysis, but that these PEN-2 variants were
still defective in stabilizing the PS1 fragments after endo-
proteolysis has occurred (Prokop et al. 2004). In extension of
these results, we now demonstrate that both progressive
C-terminal truncation as well as an internal deletion causes
the loss of function phenotype described above. This
suggests that proper length of the C-terminal domain is
required for the PS fragment-stabilizing function of PEN-2.
The critical dependence of PEN-2 function on the length of
its C-terminus may suggest a tight spatial arrangement of the
PEN-2 subunit within the c-secretase complex and indicates a
critical requirement for spatial accessibility of the C-terminal
domain. This assumption is corroborated by the observation
that C-terminally GFP-tagged PEN-2 is not functional
(Francis et al. 2002) and by a mutagenesis study similar to
ours by Hasegawa et al. (2004) while this work was in
progress. Besides length, our mutagenesis study also sug-
gests that the overall sequence rather than individual
conserved residues of the C-terminus is a critical functional
determinant of PEN-2. This is supported by the observation
that individual substitutions of evolutionary highly conserved
C-terminal residues (D90, F94, P97 and G99, Fig. 1) with
alanine are functionally tolerated, whereas multiple substitu-
tions of the same residues with alanine are not. This
observation suggests that the highly conserved D, F, P, G
residues (in particular the D, F and P residues) constitute or
are part of a functionally important sequence segment whose
overall integrity and spatial accessibility is crucial for
mediating PS fragment-stabilizing interactions of PEN-2
within the c-secretase complex. Within such a sequence
segment, a single point mutation may be tolerated because
the remaining conserved residues are left intact and thus
remain available for stabilizing interactions, whereas the
stabilizing interactions are abrogated when several conserved
residues are mutated. Apparently, during evolution key
amino acid residues within this sequence segment were
preserved, probably because the entire set of these con-
served amino acid residues together allows optimal pro-
tein–protein interaction(s). This sequence segment in the
PEN-2 C-terminal domain may adopt a certain structure
(b)
(a)
IP PS1-N
Membranes IP NCT-C
NCTm
NCTim
APH-1aL
MG132
+++++
-----
PS1NTF
PEN-2
PS1CTF
(c)
MG132
+ ++++-----
wt
C
C8
4A
90-94
PS1NTF
PEN-2
PS1NTF
PEN-2
PS1CTF
wt
C
C8
4A
90-94
wt
C
C8
4A
90-94
wt
C
C8
4A
90-94
Fig. 4 Complex formation of C-terminal PEN-2 loss of function
mutants with the PS1 NTF/CTF heterodimer upon inhibition of pro-
teasomal degradation. (a) Membrane fractions of PEN-2 knockdown
cells stably expressing RNAi resistant wild type (wt) PEN-2 or the
indicated PEN-2 variants were solubilized with CHAPSO and analyzed
for c-secretase complex formation by immunoprecipitation with an
antibody N1660 to the NCT C-terminus. Membrane fractions (left
panel) and immunoprecipitates thereof (right panel) were analyzed by
immunoblotting as in Fig. 2. Minor amounts of the PS1 NTF (in
C-terminal PEN-2 loss of function mutants, lanes 2–5, right panel) that
are coimmunoprecipitated with the NCT antibody most likely represent
residual endogenous PS1 fragments (due to the incomplete knock-
down of PEN-2). (b) PEN-2 knockdown cells stably expressing RNAi-
resistant wt PEN-2 or the indicated PEN-2 variants were treated with
MG132 (5 l
M
) for 12 h. Membrane fractions were prepared as in (a)
and analyzed by immunoblotting with antibodies 1638, PS1N and
antibody 3027 (to the PS1 loop). (c) PEN-2 knockdown cells stably
expressing RNAi-resistant wt PEN-2 or the indicated PEN-2 variants
were treated with MG132 as in (b). Membrane fractions were solubi-
lized with CHAPSO and analyzed for c-secretase complex formation
by immunoprecipitation with antibody 2953 (to the PS1 NTF) and
immunoblotting as in (b).
PEN-2 C-terminal domain 61
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 57–62
required for stabilizing protein–protein interaction(s) within
the c-secretase complex. Alternatively, the sequence segment
in the PEN-2 C-terminal domain may be naturally unfolded
and stabilized by appropriate protein–protein interaction(s)
within the c-secretase complex.
Consistent with their failure to undergo their characteristic
PS fragment-stabilizing interactions, the PEN-2 C-terminal
loss of function mutants failed to stably associate with the
other c-secretase complex subunits. However, under condi-
tions when their proteasomal degradation and that of the PS1
fragments was blocked, the PEN-2 C-terminal loss of
function mutants could be found in stable association with
the PS1 fragments. Thus, the otherwise unstable interaction
of the C-terminal PEN-2 mutants with the PS1 fragments can
be recovered provided that degradation is blocked. These
data may therefore suggest that, within the c-secretase
complex, PEN-2 acts as a molecular clamp that holds
together the PS fragments (and thereby itself) in the
complex.
Acknowledgements
We thank Dr Ralph Nixon for the kind gift of monoclonal antibody
PS1N and Drs Keiro Shirotani, Christoph Kaether and Manuel Than
for helpful discussion. This work was supported by the Deutsche
Forschungsgemeinschaft (CH and HS, SFB 596 Molecular Mech-
anisms of Neurodegeneration), the European Union (CH, APOPIS)
and by a predoctoral fellowship (SP) from the Ludwig-Maximilians-
University of Munich (Fo¨FoLe).
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2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 57–62
    • In the absence of PEN2, the complex is degraded by the proteasome. The PS-subunit stabilizing function of PEN2 depends on: (i) the length and overall sequence of its C-terminal domain (Steiner et al. 2002; Prokop et al. 2004 Prokop et al. , 2005); and (ii) on the proximal two-thirds of the PEN2 transmembrane domain 1 (Kim and Sisodia 2005b). The 'NF' sequence within TM4 or PS1 is the minimal PS1 motif that is required for binding with PEN2 (Kim and Sisodia 2005a).
    [Show abstract] [Hide abstract] ABSTRACT: J. Neurochem. (2012) 120 (Suppl. 1), 84–88. The presenilin complex is composed of four core proteins (presenilin 1 or presenilin 2, APH1, nicastrin, and PEN2). Several endogenous proteins have been reported to selectively modulate the function of the presenilin complexes; these include transmembrane trafficking protein, 21-KD (TMP21), CD147 antigen (basigin), the γ-secretase-activating protein (gSAP), and the orphan G-protein-coupled receptor 3. Because the structure and assembly of these complexes underlies their activity, this review will discuss current work on the assembly of the complex and on presenilin-interacting proteins that regulate secretase activity.
    Article · Nov 2011
    • As these effects did not result from mislocalization of Pen-2, it was suggested that these residues – and the total length of the C-terminus – are critical for 1) its interaction with PS, 2) the subsequent maturation of PS and Nct, and 3) stabilization of the γ-secretase complex [128,131,132]. Moreover, the length and sequence of the C-terminus are critical for stabilizing the mature γ-secretase complex that is otherwise rapidly degraded by the proteasome; however, the Pen-2 C-terminus does not appear to regulate endoproteolysis [128,132]. Finally, a second point of interaction was found, where the N-terminal (luminal) two thirds of the first TMD of Pen-2 are necessary for interaction with PS [130]. Further studies with PS revealed that the " NF " motif at the cytoplasmic end of the PS's fourth TMD is involved in interactions with Pen-2, although the exact corresponding site on Pen-2 remains unidentified ([107,133]Fig.
    [Show abstract] [Hide abstract] ABSTRACT: In this review, we discuss the biology of gamma-secretase, an enigmatic enzyme complex that is responsible for the generation of the amyloid-beta peptide that constitutes the amyloid plaques of Alzheimer's disease. We begin with a brief review on the processing of the amyloid precursor protein and a brief discussion on the family of enzymes involved in regulated intramembrane proteolysis, of which gamma-secretase is a member. We then identify the four major components of the gamma-secretase complex - presenilin, nicastrin, Aph-1, and Pen-2 - with a focus on the identification of each and the role that each plays in the maturation and activity of the complex. We also discuss two new proteins that may play a role in modulating the assembly and activity of the gamma-secretase complex. Next, we summarize the known subcellular locations of each gamma-secretase component and the sites of gamma-secretase activity, as defined by the production of Abeta. Finally, we close by synthesizing all of the included topics into an overarching model for the assembly and trafficking of the gamma-secretase complex, which serves as a launching point for further questions into the biology and function of gamma-secretase in Alzheimer's disease.
    Full-text · Article · May 2008
    • After endoproteolysis, PS1 NTF and CTF are stabilised by interactions with the PEN2 C-terminus (Kim and Sisodia, 2005b). Interestingly, altering the length or integrity of either the luminal N-or C-terminus of PEN2 affects binding to other components and -secretase activity (Crystal et al., 2003; Hasegawa et al., 2004; Isoo et al., 2007; Prokop et al., 2005). PEN2 thus seems to use its 'hairpin' topology literally, hooking up and stabilizing the final -secretase complex.
    [Show abstract] [Hide abstract] ABSTRACT: gamma-Secretase is a promiscuous aspartyl protease responsible for the final intramembrane cleavage of various type I transmembrane proteins after their large ectodomains are shed. The vast functional diversity of its substrates, which are involved in cell fate decisions, adhesion, neurite outgrowth and synapse formation, highlights the important role gamma-secretase plays in development and neurogenesis. The most renowned substrates are the amyloid precursor protein and Notch, from which gamma-secretase liberates amyloid beta peptides and induces downstream signalling, respectively. gamma-Secretase is a multiprotein complex containing presenilin (which harbours the catalytic site), nicastrin, APH1 and PEN2. Its assembly occurs under tight control of ER-Golgi recycling regulators, which allows defined quantities of complexes to reach post-Golgi compartments, where gamma-secretase activity is regulated by multiple other factors. 3D-EM rendering reveals a complex with a translucent inner space, suggesting the presence of a water-filled cavity required for intramembrane proteolysis. Despite huge efforts, we are now only beginning to unravel the assembly, stoichiometry, activation and subcellular location of gamma-secretase.
    Full-text · Article · Mar 2008
    • One suggestion put forward by Hasegawa and colleagues [78] is that the C-terminus of pen-2 may be a linker/space molecule that maintains spatial interactions between proteins within the complex. However, recent evidence using pen-2 C-terminal loss of function mutations suggest that the C-terminus acts as a ''molecular clamp'' holding together the presenilin fragments and the whole c-secretase complex [81]. If this is the case then PEN-2 is an integral part of the catalytic process holding the complex together whilst the c-secretase products are generated.
    [Show abstract] [Hide abstract] ABSTRACT: The biogenesis and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterises Alzheimer's disease. The presenilins and its interacting proteins play a pivotal role in the generation of Abeta from the amyloid precursor protein (APP). In particular, three proteins (nicastrin, aph-1 and pen-2) interact with presenilins to form a large multi-subunit enzymatic complex (gamma-secretase) that cleaves APP to generate Abeta. Reconstitution studies in yeast and insect cells have provided strong evidence that these four proteins are the major components of the gamma-secretase enzyme. Current research is directed at elucidating the roles that each of these protein play in the function of this enzyme. In addition, a number of presenilin interacting proteins that are not components of gamma-secretase play important roles in modulating Abeta production. This review will discuss the components of the gamma-secretase complex and the role of presenilin interacting proteins on gamma-secretase activity.
    Full-text · Article · Apr 2007
    • Consistent with these findings, two deletions in the N-terminal region (3-9 and 10-16), two in the loop between TMD1 and TMD2 (40-46 and 52-60) and two in the C-terminal region (85-92 and 93-100) had no or very little effects on the endoproteolysis of PS1 and -secretase activity in contrast to the deletion of the whole C-terminal region (85- 101, including the conserved DYLSF motif at residues 90- 94), further supporting the critical function of the C-terminal hydrophilic region of PEN-2 for -secretase activity [120, 123] . The overall DYLSF motif is the critical functional determinant of PEN-2 as individual D90A and F94A mutations are functionally tolerated [123].
    [Show abstract] [Hide abstract] ABSTRACT: Alzheimer's disease is the most common form of neurodegenerative diseases in humans, characterized by the progressive accumulation and aggregation of amyloid-beta peptides (Abeta) in brain regions subserving memory and cognition. These 39-43 amino acids long peptides are generated by the sequential proteolytic cleavages of the amyloid-beta precursor protein (APP) by beta- and gamma-secretases, with the latter being the founding member of a new class of intramembrane-cleaving proteases (I-CliPs) characterized by their intramembranous catalytic residues hydrolyzing the peptide bonds within the transmembrane regions of their respective substrates. These proteases include the S2P family of metalloproteases, the Rhomboid family of serine proteases, and two aspartyl proteases: the signal peptide peptidase (SPP) and gamma-secretase. In sharp contrast to Rhomboid and SPP that function as a single component, gamma-secretase is a multi-component protease with complex assembly, maturation and activation processes. Recently, two low-resolution three-dimensional structures of gamma-secretase and three high-resolution structures of the GlpG rhomboid protease have been obtained almost simultaneously by different laboratories. Although these proteases are unrelated by sequence or evolution, they seem to share common functional and structural mechanisms explaining how they catalyze intramembrane proteolysis. Indeed, a water-containing chamber in the catalytic cores of both gamma-secretase and GlpG rhomboid provides the hydrophilic environment required for proteolysis and a lateral gating mechanism controls substrate access to the active site. The studies that have identified and characterized the structural determinants critical for the assembly and activity of the gamma-secretase complex are reviewed here.
    Article · Jan 2007 · Neurochemical Research
    • Pen-2 facilitates the cleavage of full-length PS into active NTF and CTF (Francis et al. 2002; Steiner et al. 2002). Both the sequence and length of the C-terminus of Pen-2 are critical for intermolecular interactions and function of presenilin complexes (Hasegawa et al. 2004; Kim and Sisodia 2005; Prokop et al. 2005). In particular, deletion of the last 12 amino acids of Pen-2 has been shown to abrogate the ability of Pen-2 to stabilize PS fragments and does not restore c-secretase activity in the background of Pen-2 knockdown by RNAi (Hasegawa et al. 2004; Kim and Sisodia 2005).
    [Show abstract] [Hide abstract] ABSTRACT: Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.
    Full-text · Article · Apr 2006
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