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J. Biol. Chem., Vol. 278, Issue 36, 33992-34002, September 5, 2003
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-Secretase" Complex
¶From the Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, Illinois 60637
Received for publication, June 3, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-secretase" processing of
-amyloid precursor protein (APP) and other transmembrane proteins,
including Notch, is mediated by a macromolecular complex consisting of
presenilins (PSs), nicastrin (NCT), APH-1, and PEN-2. We now demonstrate that
in cells coexpressing PS1, APH-1, and NCT, full-length PS1 accumulates to high
levels and is fairly stable. Upon expression of PEN-2, the levels of PS1
holoprotein are significantly reduced, commensurate with an elevation in
levels of PS1 fragments. These findings suggest that APH-1 and NCT are
necessary for stabilization of full-length PS1 and that PEN-2 is critical for
the proteolysis of stabilized PS1. In N2a and 293 cell lines that stably
overexpress PS1, APH-1, NCT, and PEN-2, PS1 fragment levels are elevated by up
to 10-fold over endogenous levels. In these cells, we find a marked
accumulation of the APP-CTF (AICD) fragment and a concomitant reduction
in levels of both APP-CTF and CTF
. Moreover, the production of
the -secretase-generated Notch S3/NICD derivative is modestly elevated.
However, we failed to observe a corresponding increase in levels of secreted
A peptides in the medium of these cells. These results lead us to
conclude that, although the PS1, APH-1, NCT, and PEN-2 are essential for
-secretase activity, the proteolysis of APP-CTF and Notch S2/NEXT are
differentially regulated and require the activity of additional cofactors that
promote production of AICD, NICD, and A. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-secretase" processing of -amyloid precursor protein
(APP), the developmental signaling receptor, Notch 1, and several additional
type 1 transmembrane proteins
(2,
3). Intramembranous proteolysis
of a set of membrane-tethered APP derivatives (APP-CTFs) leads to the
production and subsequent secretion of amyloid -peptides (As),
whereas proteolysis of a membrane-tethered Notch 1 derivative, termed S2/NEXT,
results in the release of the Notch intracellular
domain (S3/NICD). It has previously been observed that A and
S3/NICD production are eliminated in cells derived from mouse blastocysts with
compound deletions of PS1 and PS2
(4,
5), and PS1 and PS2 have been
demonstrated to photocross-link to transition-state isosteres that inhibit
-secretase activity
(6–8).
In addition, it has been found that two conserved aspartate residues within
predicted transmembrane domains 6 and 7 are critical for A and S3/NICD
production
(9–13).
These findings have led to the conclusion that PS are unusual diaspartyl
proteases that catalyze intramembranous proteolysis
(14).
Full-length PS1 and PS2 are subject to endoproteolysis and accumulate as
30-kDa N-terminal (NTF) and 20-kDa C-terminal fragments (CTF) in
vivo
(15–17).
The PS1-NTF and -CTF accumulate in a highly saturable manner
(15,
18,
19); overexpression of PS does
not elevate the levels of endoproteolytic derivatives
(15,
19), but, rather, the vast
majority of newly synthesized full-length molecules are rapidly degraded, with
only a small fraction of these "precursors" being stabilized as
endoproteolytic derivatives
(18,
20). Our interpretation of
these findings is that endoproteolysis of this fraction of PS precursors only
occurs following the association with cellular factors that are expressed at
limiting levels (18). PS1-NTF
and -CTF remain stably associated
(21,
22) along with several
additional polypeptides in high molecular mass complexes that range from
150 kDa to 2 MDa
(22–27).
Biochemical studies and genetic screening strategies in Caenorhabditis
elegans have led to the identification of three polypeptides that
interact with PS1, namely nicastrin (NCT), APH-1, and PEN-2
(28–30).
NCT is a type I membrane glycoprotein that associates in stoichiometric levels
with PS1 in high molecular weight complexes
(28,
31–35).
Furthermore, down-regulation of NCT expression by RNAi or genetic ablation
impairs -secretase cleavage of APP and Notch, commensurate with
reduction in the steady-state levels of PS fragments and diminished levels of
high molecular weight PS complexes
(24,
29,
36–39).
APH-1, proteins of 247–265 amino acids with seven predicted
membrane-spanning domains, physically interact with PS1 and NCT
(40), and RNAi-mediated
reduction in APH-1 levels leads to a reduction in PS1 levels, accumulation of
APP-CTFs, and reduced production of A and S3/NICD
(40,
41). Similarly,
down-regulation of PEN-2 leads to a reduction in levels of endogenous PS,
commensurate with compromised production of A and S3/NICD
(29,
30,
41,
42). On the other hand,
oligosaccharide maturation and trafficking of NCT are severely compromised and
levels of APH-1 and PEN-2 are reduced in cells with compound deficiencies of
PS1 and PS2
(24,
35,
42–45).
Finally, the demonstration that NCT, APH-1, PEN-2, and PS1 coexist in high
molecular weight assemblies
(24,
41,
42,
45) suggests that these
proteins are bona fide components of a complex required for
-secretase activity.
In this report, we show that, in stably transfected cells that coexpress
PS1, APH-1, NCT, and PEN-2, the levels of PS1 fragments hyperaccumulate
to levels 10-fold higher than endogenous levels. In these cell lines, we
observed robust enhancement in levels of an intracellular derivative of APP,
termed "CTF" (or APP intracellular
domain (AICD))
(46–48)
as well as a modest elevation in the production of Notch S3/NICD. However, we
failed to detect a corresponding increase in levels of secreted A
peptides. These results indicate that generation of AICD and A by
-secretase is differentially regulated and argue that additional
cellular components might be necessary to elicit these highly unusual
proteolytic events.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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s cDNAs were isolated using adult
human brain mRNA as a template. The cDNAs were epitope tagged with a sequence
encoding the last seven amino acids (RFLEERP) of APLP1, which is recognized by
CT11 antibody (50,
57). The sequences of each
cDNA were verified by sequencing.
Antibodies—Polyclonal antibodies against NCT (NCT54) were
generated against a GST fusion protein containing residues 242–546 of
human NCT. Polyclonal myc15 antibody was raised against a GST-hexa-myc fusion
protein. The following antibodies previously described were also used for the
study; PS1NT antibody is a polyclonal antibody that recognizes
residues 1–65 of PS1
(18); CT15 was generated
against a peptide corresponding to the last 15 amino acids of APP
(49); CT11 antibody
specifically recognizes the last 7 amino acids of APLP1
(50); polyclonal PNT-2
antibody was raised against a peptide corresponding to the N-terminal 26 amino
acids of PEN-2 (a kind gift from Dr. Gopal Thinakaran
(51)); 26D6 is a monoclonal
antibody that reacts with A residues 1–12
(52); and P2-1 is a monoclonal
antibody specific for epitopes in the human APP ectodomain
(53).
Cell Culture and Transfection—Mouse neuroblastoma N2a cells were maintained in 50% Dulbecco's modified Eagle's medium (DMEM) and 50% of Opti-MEM (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS). Human embryonic kidney (HEK) 293 cells and transformed fibroblasts derived from mouse embryo with homozygous deletions of PS1 (PS1–/–) were cultured in DMEM containing 10% FBS.
Cells were transiently transfected with plasmid DNA using LipofectAMINE Plus (Invitrogen). To generate stable cell lines, N2aSw.10 (58) or HEK293Sw.3 cells, which stably express myc epitope-tagged, Swedish variant of APP695 (APPswe), were cotransfected with 10 µg of transgenes and 100 ng of pIREShyg (Clontech) using a calcium phosphate method (54). Cells expressing transgenes were selected with 400 µg/ml hygromycin-B (Invitrogen).
Western Blot—Cells were lysed in immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate) supplemented with a protease inhibitors mixture (a mixture of AEBSF, pepstatin A, E-64, bestatin, leupeptin, and aprotinin; Sigma) (55). Cell lysates or conditioned medium were subject to Tris/glycine or 16.5% Tris/Tricine SDS-PAGE, and fractionated proteins were transferred to nitrocellulose membrane (Schleicher and Schuell) prior to incubation with selected antibodies. Immunoreactive bands were visualized using the enhanced chemiluminescence detection (ECL) system (PerkinElmer Life Sciences, Boston, MA).
Metabolic Labeling and Immunoprecipitation—Parallel dishes of stable N2a or HEK293 cells were labeled with 250 µCi/ml [35S]methionine (PerkinElmer Life Sciences) in methionine-free DMEM supplemented with 1% dialyzed FBS (Invitrogen) for 10 min or 4 h. Conditioned medium was collected and adjusted to 0.5 mM phenylmethylsulfonyl fluoride, and labeled cells were harvested as described above. Immunoprecipitation was carried out with respective antibodies at 4 °C overnight. The immune complexes were collected with Protein A-conjugated agarose beads (Pierce) and eluted by boiling for 5 min in Laemmli SDS sample buffer. After separation on 16.5% Tris/Tricine SDS-PAGE, radioactive bands were visualized and quantified by phosphorimaging (PhosphorImager, Amersham Biosciences).
For pulse-chase analysis of N
E processing, transiently transfected
N2a or HEK293 cells were starved in methionine-free DMEM and then labeled with
250 µCi/ml [35S]methionine for 20 min. At the end of the
labeling period, one dish of each cell line was immediately harvested in
immunoprecipitation buffer, as described above, whereas the other dish was
washed once and incubated for 2 h in DMEM containing 1% dialyzed FBS and 0.5
mM L-methionine (Invitrogen). When indicated, lactacystin (10
µM) was added to both pulse-labeling and pulse-chase media.
Detergent lysates were prepared and subject to immunoprecipitation analysis
with myc15 antibody, as described above.
Cytoplasmic RNA Isolation and RT-PCR—Cytoplasmic RNA was
isolated as described previously
(56). Briefly, after washing
with ice-cold phosphate-buffered saline, cells were lysed with lysis buffer
(140 mM NaCl, 1.5 mM MgCl2, 10mM
Tris, pH 8.6, 0.5% Nonidet P-40, 10 mM vanadylribonucleoside
complexes (New England BioLabs)). Cell extracts were centrifuged for 3 min at
10,000 x g at 4 °C to remove nuclei. The supernatant was
collected and diluted with an equal volume of 2x proteinase K buffer
(0.2 M Tris, pH 7.5, 25 mM EDTA, 300 mM NaCl,
2% SDS), and proteinase K was added to 200 µg/ml. After incubation at 37
°C for 30 min, the lysates were extracted with phenol-chloroform and
precipitated with ethanol. First strand cDNA was generated using the
Thermoscript cDNA kit (Invitrogen), and PCR was performed using human
APH-1s specific primers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, NCT, and PEN-2 Are
Coordinately Regulated—Our initial efforts focused on the effects
of APH-1, NCT, and PEN-2 on PS1 accumulation in transiently transfected mouse
neuroblastoma N2a cells. APH-1, NCT, and PEN-2 were epitope-tagged with
a sequence, RFLEERP, which is recognized by CT11 antibody
(57). We have verified that
the CT11-tagged NCT molecule can be coimmunoprecipitated with PS1 fragments in
a manner indistinguishable from untagged NCT (data not shown). Moreover, we
have successfully coimmunoprecipitated PS1 with CT11-tagged APH-1 or
CT11-tagged PEN-2 (data not shown), data consistent with previous findings
(40,
42,
51).
In transiently transfected N2a cells, we show that, in contrast to cells
expressing NCT without APH-1, the steady-state levels and,
particularly, the levels of mature, fully glycosylated NCT are significantly
increased in the cells coexpressing NCT and APH-1
(Fig. 1A, panel
I, compare lanes 4 and 6; 8 and 9).
Similar results were obtained for PEN-2; the steady-state levels of PEN-2 are
higher when coexpressed with APH-1 compared with settings in which only
PEN-2 is expressed (Fig.
1A, panel II, compare lanes 5 and
7; 8 and 9). Moreover, the accumulated levels of
APH-1 are higher when coexpressed with NCT compared with settings in
which APH-1 is expressed without NCT
(Fig. 1A, panel
III, compare lanes 3 and 6; 7 and
9).
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To assess whether APH-1, NCT, and/or PEN-2 are the limiting factor(s) for
stabilization of PS1 and could affect the levels of accumulated PS1 fragments,
we performed Western blot analysis using PS1NT antibody
(18). Although the levels of
full-length PS1 were elevated in all cases upon transfection of human PS1, the
level of PS1-NTF was not changed following coexpression with each component
individually (Fig. 1A,
panel IV, lanes 2-5). However, in cells that coexpress PS1 and
APH-1 with either NCT (Fig.
1A, panel IV, lane 6) or PEN-2
(Fig. 1A, panel
IV, lane 7), the levels of PS1-NTF were significantly elevated compared
with cells transfected with PS1 alone (Fig.
1A, panel IV, lane 2). Notably, the levels of
accumulated full-length PS1 were also much higher in cells expressing PS1,
APH-1, and NCT (Fig.
1A, panel IV, lane 6). Remarkably, when PEN-2
was expressed in addition to PS1, APH-1, and NCT, the levels of PS1
fragments were elevated even further, commensurate with a marked reduction in
levels of full-length PS1 (Fig.
1A, panel IV, lane 9). To verify that the
effects of expressing human PS1, NCT, APH-1, and PEN-2 in mouse neuroblastoma
cells could be reproduced in other cell types, we performed an identical
series of studies in HEK293 and immortalized fibroblasts from mice with
homozygous deletions of PS1 (PS1–/–). We
obtained essentially identical results in these cell lines to those observed
in N2a cells (Fig.
1B).
The finding that coexpression of PS1, APH-1, and NCT leads to the
elevated accumulation of PS1, together with increased levels of mature NCT and
APH-1, led us to conclude that these molecules assemble into a stable
complex and their steady-state levels are coordinately regulated. Among the
components, it would appear that APH-1 serves as a scaffold that stabilizes
the complex and, in the case of NCT, promotes (either directly or indirectly)
exit of underglycosylated molecules to late compartments where complex
oligosaccharide modification occurs. More importantly, the observation that
the highest levels of PS1 fragment accumulation are achieved under conditions
where PS1, APH-1, and NCT are coexpressed together with PEN-2 suggests
that PEN-2 is a critical component of the machinery necessary for PS1
endoproteolysis.
Stable Overexpression of PS1 with APH-1, NCT, and PEN-2—To
assay the effects of overexpressing components of the -secretase
complex on the production of A peptides and Notch S3/NICD, we generated
N2a and HEK293 cell lines that stably express PS1 and its complex components.
Equimolar amounts of cDNAs encoding human PS1, APH-1, CT11-tagged
PEN-2, and NCT were mixed and transfected into an N2a cell line, Sw.10
(55,
58), that constitutively
expresses APP695 harboring Swedish mutation with a C-terminal myc
epitope tag (APPswe). We obtained cell lines that express various combinations
of transgenes. For example, in one line (ANP.5), we detected high levels of
NCT, elevated levels of both full-length PS1 and PS1 fragments, but no PEN-2
(Fig. 2A, lane
4). In the absence of available antibodies, we cannot assay the levels of
APH-1 in the cell lines. However, RT-PCR analysis confirmed the
expression of human APH-1s mRNA in ANP.5 line
(Fig. 2A, panel
IV, lane 4). Hence, stable overexpression of NCT and APH-1 without PEN-2
in the N2aANP.5 line, led to the accumulation of full-length PS1 and
endoproteolytic derivatives, a result fully consistent with the transient
transfection assays (Fig. 1).
More importantly, the cell lines that stably express PS1, NCT, PEN-2, and
APH-1 exhibit marked elevation in accumulated PS1 fragments, and very
low levels of full-length PS1 (ANPP.1 and ANPP.7,
Fig. 2A, lanes
2 and 5). To confirm the observations in stable N2a cell lines,
we stably transfected cDNAs encoding PS1, APH-1, CT11-tagged PEN-2, and
NCT into an HEK293 cell line, 293Sw.3, that constitutively expresses APPswe.
We identified one line, ANPP.8, that hyperaccumulates PS1 fragments to
extremely high levels, in parallel with abundant levels of accumulated mature
NCT, PEN-2, and APH-1 (Fig.
2B, lane 2). In addition, line ANP.24,
expressing APH-1, NCT, and PS1 but not PEN-2, exhibited high levels of
both full-length PS1 and PS1 fragments
(Fig. 2B, lane
4), results similar to those obtained in transient transfection assays
(Fig. 1B) and in the
N2aANP.5 line (Fig.
2A). Interestingly, reprobing the blot with PNT-2
antibody, which recognizes epitopes within the N-terminal domain of PEN-2
(51), revealed that in PP.22
cells that overexpress PS1 and CT11-tagged PEN-2, expression of endogenous
PEN-2 was almost completely eliminated
(Fig. 2A, panel
IV, compare lanes 1 and 3). This
"replacement" phenomenon is similar to those reported for PS1
fragments, mature NCT, and APH-1
(15,
33,
45) and further supports the
notion that levels of these proteins are coregulated by association with other
"limiting" components of the complex. It should be noted that in
contrast to PP.22 cells, we did not observe replacement of endogenous PEN-2 in
ANPP.8 cells (Fig. 2B,
panel IV, lane 2). Our interpretation of this finding is that, in
ANPP cells that overexpress all components of the PS1 complex, the factors are
now not limiting and, hence, able to stabilize both endogenous and exogenous
PEN-2. Consistent with this interpretation, we observed that in ANP.24 lines
that overexpress APH-1, NCT, and PS1, endogenous PEN-2 accumulated to
even higher levels (Fig.
2B, panel IV, compare lanes 1 and
4). Thus, we posit that the stability and, hence, steady-state
accumulation of PEN-2 are in large part dependent on the coexpression of PS1,
APH-1, and NCT.
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Our demonstration that full-length PS1 accumulates in cells that
overexpress PS1 along with APH-1 and NCT suggested that APH-1
might serve as a scaffold that "primes" PS1 for endoproteolytic
cleavage, whereas expression of additional PEN-2 is necessary for promoting
endoproteolysis. One prediction from this model is that both full-length PS1
and endoproteolytic derivatives might be stable in cells overexpressing PS1,
APH-1, and NCT. To test this possibility, we treated HEK293 lines
ANPP.8 and ANP.24 with the protein synthesis inhibitor, cycloheximide, and
assessed the levels of full-length PS1 and PS1 fragments over time. In
contrast to the very short (<1.5 h) half-life of full-length PS1 in line
ANPP.8 (Fig. 2C,
lanes 1–6), we now find that the half-life of full-length PS1
in line ANP.24 is apparently extended to 6 h
(Fig. 2C, lanes
7–12). We speculate that the degree to which full-length PS1 can be
"stabilized" will be determined by the relative levels of
coexpressed NCT and APH-1 in those cells. Unfortunately, we were not able to
generate multiple independent lines expressing differing levels of PS1, APH-1,
and NCT, and, hence, this proposal remains to be verified. In any event, our
results offer the suggestion that APH-1 serves as a scaffold protein that
stabilizes full-length PS1 and NCT and that PEN-2 is an essential component of
the apparatus that is responsible for PS1 endoproteolysis.
Analysis of APP Processing in Stable Cell Lines—The finding
that PS1 fragments hyperaccumulate in ANPP lines leads to the prediction that
PS1-mediated -secretase processing of APP-CTFs should be elevated in
these cells. To test this hypothesis, we performed metabolic labeling and
immunoprecipitation studies to evaluate APP processing in the stable cell
lines.
Because the transgenes were introduced into clonal N2a or 293 lines that
harbor integrated copies of APPswe, these "lines" now consist of
populations of cells that express the human APPswe polypeptide at differing
levels. As expected, the synthetic rate of human APPswe (by 10 min
pulse-labeling) and steady-state levels (after 4 h labeling) are variable
between the N2a lines (Fig.
3A, panels I and II).
Immunoprecipitation analysis of radiolabeled cell lysates using the CT15
antibody, revealed a significant reduction of APP-CTFmyc and
APP-CTFmyc in lysates of ANPP.1 and ANPP.7 lines
(Fig. 3A, panel
III, lanes 2 and 4; quantified in
Fig. 3B, panels
I and II). Interestingly, we observed a striking elevation in
the levels of 7 kDa APP-CTFmyc"", or AICD in these
cells (Fig. 3A,
panel III, lanes 2 and 4; quantified in
Fig. 3B, panel
III). Despite the differences in levels of intracellular AICD
polypeptides, however, the levels of secreted A peptides in the
conditioned medium of ANPP lines 1 and 7, when normalized to APP synthetic
levels, were not significantly different from the parental Sw.10 line, or
other lines that express PS1 and varying combinations of other components
(Fig. 3A, panel
V; quantified in Fig.
3B, panel IV).
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We also observed some variation in levels of newly synthesized APPswe (10
min pulse) between the 293 cell lines (Fig.
3C, panel I). However, in lysates of 293ANPP.8
line, that expresses the highest levels of PS1 fragments, we observed high
levels of AICD and marked diminution in levels of APP-CTF-myc and
APP-CTFmyc (Fig.
3C, panel II, lane 2; quantified in
Fig. 3D, panels
I–III). These data are entirely consistent with the data obtained
in the N2aANPP.1 and ANPP.7 lines (Fig.
3A). However, and despite the marked differences in
accumulation of AICD between these cell lines, we were unable to detect any
quantifiable increase in the level of secreted A species in the
conditioned medium from these cell lines
(Fig. 3C, panel
IV; quantified in Fig.
3D, panel IV).
It has been reported that transient expression of APP-CTF (or C99)
along with PEN-2 in a Drosophila S2 cell line that stably expresses
PS1, APH-1, and NCT (S2 ANP cells) leads to an elevation of secreted A
peptides compared with S2 ANP cells that transiently express C99, alone
(41). To validate this finding
in mammalian cells, we transiently transfected 293ANP.24 cells with APPswe
cDNA in combination with either cDNA encoding PEN-2, or a control cDNA
encoding influenza hemagglutinin (HA). Compared with 293ANP.24 cells that
express HA, we observed a small increase in the levels of PS1 fragments and a
corresponding decrease in levels of full-length PS1 in 293ANP.24 cells
transiently expressing PEN-2 (Fig.
4A, lanes 1 and 2). In parallel, we
observed a marked increase in the levels of AICD that is accompanied by a
marked reduction in levels of APP-CTF and CTF in these cells
(Fig. 4B, lanes
1' and 2). Despite this, the levels of A peptides in
the medium are not elevated in any significant way
(Fig. 4C, lanes
1' and 2'; quantified in
Fig. 4D). Thus, in
stable 293 ANPP cells that express all four components of the complex, or 293
ANP cells that transiently coexpress PEN-2, we fail to demonstrate an
elevation in levels of secreted A peptides.
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APP-CTF Is a Substrate for
-Secretase-mediated Generation of A in ANPP
Cells—Having failed to observe elevations in secreted A in
ANPP cells, we considered the possibility that the population of
membrane-tethered APP-CTF, the substrates for -secretase-mediated
production of A, is limiting. To examine this issue, we transiently
transfected 293ANPP.8, or 293PP.22 cell lines with cDNA encoding HA as a
control, or APPswe, and examined the levels of AICD and secreted A. As
we have shown above (Fig.
3C), Western blot analysis with CT15 antibody revealed
that the levels of AICD are elevated and those of CTFmyc and
CTFmyc are reduced in ANPP.8 cells compared with PP.22 cells
(Fig. 5A, lanes
1 and 2). After transient overexpression of APPswe, the level of
AICD is elevated in both cell lines but still much higher in ANPP.8 cells
compared with PP.22 cells (Fig.
5A, lanes 3 and 4). Parallel Western
blot analysis of conditioned medium using the 26D6 antibody revealed that
steady-state levels of secreted A peptides are considerably elevated in
medium of both cell lines transfected with APPswe cDNA
(Fig. 5C, lanes
3 and 4), compared with the same cell lines transfected with
cDNA encoding HA (Fig.
5C, lanes 1, 2, 1', and
2'). Thus, in ANPP cells, membrane-tethered APP-CTFs are fully
competent to serve as substrates to generate more A peptides. However,
the relative level of secreted A in medium of ANPP.8 versus
PP.22 lines following transient expression of APPswe is no different than that
observed in medium of HA transfected cells
(Fig. 5C, compare
lanes 3 and 4 with 1' and 2',
respectively; quantified in Fig.
5D). Hence, under conditions of substrate (i.e.
APP-CTF) excess, the -secretase activity in ANPP.8 cells is
capable of generating additional A peptides, but despite the marked
elevation in steady-state PS1 levels in these cells, we did not observe a
commensurate increase in levels of secreted A peptides.
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Modest Enhancement of -Secretase Processing of Notch in
ANPP Lines—In view of our demonstration that AICD is elevated in
lysates of cells that hyperaccumulate PS1 fragments and components of the PS1
complex, and the similarity in the relative positioning of this processing
event to the scissile sites responsible for -secretase processing of
Notch
(46–48),
we evaluated intramembranous processing of Notch in these cell lines. HEK293
or N2a cell lines were transiently transfected with cDNA encoding an myc
epitope-tagged version of NE, a truncated Notch1 derivative that lacks
the ectodomain (59);
-secretase processing of this Notch derivative occurs in a
ligand-independent, constitutive manner. Selected 293 or N2a cell lines
transiently expressing NE were pulse-labeled with
[35S]methionine for 20 min, then chased for 2 h, and NE
derivatives were immunoprecipitated from detergent-solubilized lysates with
myc-specific antibody, myc15. We show that the level of NICD generated in the
ANPP.8 line after 2 h of chase was 2-fold higher than seen in the
parental Sw.3 or PP.22 line (Fig.
6A, lanes 2, 4, and 6; quantified in
Fig. 6B). Similarly,
the accumulation of NICD in N2a ANPP.1 and ANPP.7 cells was modestly
(40%) elevated compared with P.2, NP.8 or the parental Sw.10 cells
(Fig. 6C, lanes
6–10; quantified in Fig.
6D). Thus, the extent of conversion of NE to NICD
in ANPP lines is not as robust as the conversion of APP-CTFs to AICD. In view
of earlier reports showing that NICD is susceptible to proteasomal degradation
(60,
61), we also analyzed
NE levels in the presence of the proteasomal inhibitor, lactacystin.
The relative increase in accumulated NICD in the presence of lactacystin was
not appreciably different between the lines
(Fig. 6E; quantified
in Fig. 6F), arguing
against differences in the stability of NICD between cell lines examined.
These findings would argue that, although AICD production is elevated in ANPP
cells that hyperaccumulate PS1 fragments, APH-1, NCT, and PEN-2,
processing of Notch is only modestly elevated.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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peptides and S3/NICD, respectively. Three
additional components of the -secretase complex, NCT, APH-1, and PEN-2,
have been identified
(28–30).
Genetic, or RNAi-mediated reduction in expression of these components reduces
the levels of other factors in the complex and results in compromised
production of A peptides and S3/NICD
(24,
29,
39,
40,
51). In addition, all three
components have been shown to coexist with PS1 in high molecular weight
assemblies (24,
41,
42,
45). In the present report, we
offer several important insights into the regulated assembly of the PS1
complex and the role of this assembly on -secretase processing of
APP-CTFs and Notch S2/NEXT. First, and in view of the demonstration that PS1 is subject to endoproteolytic processing and that the accumulation of resulting fragments is highly regulated and saturable, we tested the prediction that the steady-state levels of PS1 derivatives are governed by the association with molecules expressed in limiting levels (15, 18–20). To this end, we assessed the extent to which coexpression of PS1 with APH-1, NCT, and PEN-2 singly, or in combination, modulates the levels of PS1 derivatives. We report that coexpression of PS1 with APH-1 and NCT or APH-1 and PEN2, leads to elevated levels of PS1 derivatives. Furthermore, coexpression of APH-1 increases the steady-state level of NCT, especially the mature, fully glycosylated form, and PEN-2, suggesting that APH-1 is a critical limiting factor to stabilize the PS1 complex. Notably, when PS1 is coexpressed with APH-1 and NCT, full-length PS1 also accumulates to a higher level, and these polypeptides exhibit stability not much different from PS1 fragments. Interestingly, when PEN-2 is additionally expressed, the levels of full-length PS1 are significantly reduced, commensurate with a marked elevation in levels of PS1 fragments. These results suggest that APH-1 and NCT are involved in the stabilization of full-length PS1 and that PEN-2 plays a critical role in the endoproteolytic cleavage of stabilized PS1 protein, a conclusion that was independently reached by Luo et al. (51) and Takasugi et al. (41) using RNAi approaches while this report was in preparation. The one or more mechanisms by which PEN-2 facilitates endoproteolytic cleavage of PS1 are not clear at present. Because PEN-2 does not contain domains homologous to known proteases, it is likely to play an accessory role in the endoproteolysis of PS1. For example, PEN-2 could serve as a critical cofactor for the "presenilinase" enzyme or modify the conformation of the PS1 complex to render the scissile bond more accessible to presenilinase. Our findings confirm our earlier prediction that the regulated, saturable accumulation of PS1-NTF and CTF is mediated by the association with cellular factors that are expressed at limiting levels. In addition, although it remains possible that additional components of the complex are yet to be identified, a tentative conclusion from these studies is that APH-1, NCT, and PEN-2 are components of the minimal complex necessary for establishing the steady-state levels of PS1 fragments.
Second, we demonstrate that the levels of PEN-2 are regulated in a very
similar manner as that described for PS1, mature NCT, and APH-1
(15,
33,
45). In this case, stable
overexpression of an epitope-tagged human PEN-2 results in reduced levels of
accumulated endogenous PEN-2. This replacement phenomenon for PEN-2 further
supports the notion that levels of these proteins are coregulated by
association with other limiting components of the complex. Interestingly, in
ANPP cells that coexpress PS1, APH-1, NCT, and epitope-tagged PEN-2, we did
not observe replacement of endogenous PEN-2. We interpret these findings to
suggest that, in ANPP cells that overexpress all components of the PS1
complex, the factors are now not limiting and, hence, able to stabilize both
endogenous and exogenous PEN-2. Consistent with this interpretation, in ANP
cells that overexpress PS1, APH-1, and NCT, we observed that endogenous
PEN-2 accumulates to higher levels than cells expressing endogenous levels of
the complex.
Third, we have tested the hypothesis that elevated levels of the protease,
be it PS1 alone or in a complex, enhance production of A peptides, AICD,
and S3/NICD. In stable ANPP cell lines that coexpress APH-1, NCT, PEN-2, and
PS1, wherein exogenous PS1 fragment levels are elevated by up to 10-fold over
endogenous levels, there is a consistent and dramatic increase in the
production of AICD accompanied by marked reduction of APP-CTF and
CTF. Furthermore, -secretase-mediated proteolysis of S2/NEXT to
S3/NICD was also elevated, although the extent of increase is not as robust as
that of AICD observed in these cells. These results strongly suggest that
hyperaccumulated PS1 complex does indeed reach the compartments where
membrane-tethered substrates (APP-CTFs and S2/NEXT) reside and that the
complex plays an essential role in catalyzing proteolysis of these substrates
to generate AICD and NICD, respectively. Therefore, it appears that APH-1,
NCT, PEN-2, and PS1 constitute the minimal core of the -secretase
enzyme complex to generate AICD and NICD. Surprisingly, we have failed to
observe a corresponding increase in levels of secreted A peptides in the
conditioned medium of these cell lines. These results are in striking contrast
to three very recent reports
(27,
41,
62) showing the increased
generation of A peptides upon overexpression of four components of the
PS1 complex. It should be noted that two of these studies employed an in
vitro assays consisting of exogenous substrates added to
detergent-solubilized membrane preparations from stably transfected 293 cells,
or a yeast strain that coexpresses all four components
(27,
62). However, neither study
reported on the levels of secreted A from living cells. On the other
hand, Takasugi and colleagues
(41) revealed that transient
coexpression of PEN-2 and C99 in Drosophila S2 cells that stably
overexpress PS1, APH-1, and NCT leads to an increase of 2-fold in
secretion of A peptides. Because the levels of APP-CTFs, the penultimate
substrates of A, are markedly reduced in our ANPP cells, we explored the
possibility that these substrates are limiting because of excessive
proteolysis by hyperaccumulated PS1/-secretase that generates AICD. To
examine this issue, we provided additional levels of APP substrates by
transiently transfecting APPswe. However, whereas the levels of secreted
A peptides are markedly increased in APP-transfected ANPP cells compared
with untransfected cells, there is no difference in the extent to which
A peptides are elevated over cells that express PS1 at endogenous
levels. Finally, and to ask whether we could reproduce the findings of
Takasugi et al. (41)
in mammalian cells, we transiently transfected PEN-2 cDNA into 293ANP.24
cells. In this setting, we show that the levels of PS1 fragments are elevated
and that the levels of AICD are markedly elevated, findings very similar to
those seen in stable ANPP cells. However, we failed to detect an elevation in
the levels of secreted A peptides in the medium of these transiently
transfected cells. At present, we can neither offer a satisfying explanation
for the apparently discordant data on A production obtained from the
in vitro solubilized membrane preparations
(27,
62) and the in vivo
assay system (this report), nor can we find an appealing scenario to
accommodate our failure to replicate, in mammalian cells, the data in
Drosophila S2 ANP cells showing an elevation in A peptide
production by transient overexpression of PEN-2
(41).
Collectively, our findings support the model that overexpression of PS1
fragments and associated components of the complex have differential effects
on production of A, NICD, and AICD. The significance of this observation
in relation to the potential physiological function(s) of APP and AICD, or the
pathogenesis of Alzheimer's disease, is not known. However, our present
demonstration that the generation of AICD can be dissociated from the
production of A peptides strongly argues that A and AICD
production are not temporally linked events but rather two separately
regulated, proteolytic events. These findings also suggest that, although
molecular components constituting the minimal core of the -secretase
complex have been identified, we consider it likely that additional cofactors
are required that modulate -secretase activity in a substrate-selective
manner. It is conceivable that these cofactors could alter -secretase
activity at the level of post-translational modifications, conformational
alterations of individual components of the complex, or subunit
stoichiometries. In this regard, recent studies by Phiel and colleagues
(63) have demonstrated that in
both in vitro and in vivo settings, inhibition of glycogen
synthase kinase 3 activity leads to dramatic reductions in secretion of
A peptides, without any apparent alteration in -secretase
processing of Notch S2/NEXT to generate S3/NICD. In any event, our proposal
offers the attractive hypothesis that therapeutic modalities designed to
selectively inhibit -secretase-mediated production of A peptides
might be identified that do not incur potentially detrimental consequences of
inhibiting intramembranous processing of other substrates that are essential
for modulating important physiological functions during development and in
aging.
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FOOTNOTES |
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Both authors contributed equally to this work.
A research fellow of the Levi Foundation.
¶ A recipient of research fellowships from the Uehara Memorial
Foundation.
|| To whom correspondence should be addressed: Dept. of Neurobiology, Pharmacology, and Physiology, University of Chicago, 947 East 58th St., MC0926, Chicago, IL 60637. Tel.: 773-834-9186; Fax: 773-702-3774; E-mail: ssisodia{at}drugs.bsd.uchicago.edu.
1 The abbreviations used are: PS1/2, presenilins 1 and 2; APP, -amyloid
precursor protein; CTF, C-terminal fragment; NTF, N-terminal fragment;
A, amyloid -peptide; NCT, nicastrin; RNAi, RNA interference; AICD,
APP intracellular domain; RT, reverse transcription; GST, glutathione
S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal
bovine serum; HEK, human embryonic kidney; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; APPswe, Swedish
variant of APP695; HA, hemagglutinin.
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ACKNOWLEDGMENTS |
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