From the Departments of Mammalian Cell Molecular
Biology and § Neuroscience, Amgen Inc., Thousand Oaks,
California 91320-1789
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ABSTRACT |
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Amyloid peptides of 39-43 amino acids (A) are
the major constituents of amyloid plaques present in the brains of
Alzheimer's (AD) patients. Proteolytic processing of the amyloid
precursor protein (APP) by the yet unidentified
- and
-secretases
leads to the generation of the amyloidogenic A
peptides. Recent data suggest that all of the known mutations leading to early onset familial
AD alter the processing of APP such that increased amounts of the
42-amino acid form of A
are generated by a
-secretase activity.
Identification of the
- and/or
-secretases is a major goal of
current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the
long sought
-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and
by characterizing APP processing in mutant Chinese hamster ovary cells
that are deficient in S2P activity and expression. The data demonstrate
that S2P does not play an essential role in the generation or secretion
of A
peptides from cells, thus it is unlikely to be a
-secretase.
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INTRODUCTION |
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A characteristic neuropathological feature of Alzheimer's disease
(AD)1 are amyloid plaques,
containing A, a 39-43-amino acid protein proteolytically derived
from a large type 1 transmembrane protein, the amyloid precursor
protein (APP) (for review see Ref. 1). APP undergoes a series of
proteolytic processing steps in the biosynthetic and/or endocytic
pathway.
- and
-Secretase cleavages generate the
non-amyloidogenic peptide, p3, whereas
- and
-secretase cleavages
lead to the production of the amyloidogenic A
peptide (Fig.
1). A number of different mutations in
APP have been identified that lead to rare forms of early onset
familial Alzheimer's disease (FAD). All of these mutations appear to
alter the proteolytic processing of the precursor, increasing the total
production of the 42-amino acid form of A
, A
42 (for
review see Ref. 2).
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Despite intensive study of APP processing, none of the secretase
activities has been definitively identified, purified, or cloned,
although many enzymes have been proposed as potential secretases (for
review see Ref. 3). Secretase candidates have been difficult to
identify because specific inhibitors are not generally available and it
is unclear to which protease classes - and
-secretases belong. In
addition, it is not known whether
- and
-secretases are each just
one enzyme or are a collection of different enzymes. For example, it
has been suggested, but not proven, that
-secretase cleavages at
positions 40 and 42 are carried out by different proteases (4, 5).
Finally, more than 10 years after the cloning of APP it is still
puzzling how
-secretase cleaves APP within the transmembrane domain
(6).
Recently, Brown and Goldstein (7) noted numerous similarities between
the sterol regulatory element-binding protein (SREBP) site 2 protease
(S2P) and the -secretase(s). Based on these similarities, they
hypothesized that either the S2P may be the
-secretase or that the
two proteases may be closely related (7). The substrate of the S2P,
SREBP, is initially synthesized as an endoplasmic reticulum
membrane-bound precursor. Following a two-step cleavage, the cytosolic
domain of SREBP is released and targeted to the nucleus where it
transcriptionally regulates genes containing the sterol regulatory
element (SRE), including numerous genes involved in cholesterol and
fatty acid metabolism (8).
The similarities between the S2P and -secretase are provocative.
First, the substrates (SREBP and APP) of both enzymes are cleaved
uniquely within the transmembrane domain (9, 10). Second,
both S2P and
-secretase enzymes catalyze the second cleavage of an
obligatory two-step cleavage reaction. Third, both enzymes appear to be
ubiquitously expressed (as are the substrates). Finally, processing of
SREBP is regulated by a six to eight transmembrane domain protein, SCAP
(SREBP cleavage activating protein) (11), which is located in the
endoplasmic reticulum (12). Dominant missense mutations in SCAP have
been shown to alter SREBP processing (11, 13). Although there is no
direct sequence homology, the presenilins (PS1 and PS2 (14, 15)) also
contain six to eight transmembrane domains and are localized to the
endoplasmic reticulum where they are thought to regulate
-secretase
activity. Dominant missense mutations in both PS1 and PS2 account for
the most common forms of FAD, and both in vitro and in
vivo evidence suggest that these mutations in PS1/PS2 somehow
alter
-secretase activity and lead to increased production of
A
42 (for review see Ref. 16). Furthermore, neurons from
PS1 knock-out mice appear to have dramatically reduced
-secretase
activity, suggesting that PS1 may be an activator of
-secretase
(17), much as SCAP activates SREBP processing (11-13, 18).
Recent data have allowed us to directly test whether the -secretase
and the S2P are identical enzymes. Sakai et al. (9) demonstrated that a mutant Chinese hamster ovary (CHO) cell line, CHO
M19 (19, 20), is deficient in the S2P activity. Second, the S2P
cDNA was cloned by complementation of the M19 defect (21, 22);
furthermore, Rawson et al. (22) show that CHO M19 cells do
not express the S2P mRNA due to a large deletion encompassing most,
it not all, of the S2P gene (22).
In this communication we show that over-expression of human (hu) S2P
does not enhance secretion of A from 293 cells, suggesting that
either the S2P is not the
-secretase or that the activity is not
rate-limiting for processing APP to A
. Furthermore, we show that CHO
M19 clones process transfected hu APPsw to A
; both A
40 and A
42 are generated in proportions
indistinguishable from those produced by the wild-type CHO clones.
Finally, we confirm that our CHO M19 clones are auxotrophs and that
they do not express detectable S2P mRNA. These results definitively
show that the S2P is not required for
-secretase processing of APP
to A
40 and A
42.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions--
The sense primer (5'-CGG AGG AGA TCT
CTG AAG TGA ATC TGG ATG CAG AAT TCC GAC ATG ACT C-3')
containing the Swedish mutation and BglII restriction site
and the antisense primer (5'-TAT AGC AGA AGC AGC AAT CTG TAC AG-3')
were used with wild-type APP695 cDNA as template. The
PCR-amplified, BglII-digested DNA fragment was subcloned
into the wild-type APP695 cDNA to generate pGEM3/APPsw695 (Promega). After confirming the DNA sequence, the APPsw fragment (HindIII-XmnI) was subcloned to generate
pBCB/APPsw695 (23). The APPsw695 cDNA
(HindIII-NotI) was subcloned into pCMVi (Cell & Molecular Technologies) to generate pCMVi/APPsw695. pRc/Neo was derived
from pRc/CMV (Invitrogen). It contains the pRc/CMV 1524-bp neomycin
resistance expression cassette (KpnI-SalI) fused to the pRc/CMV 2178-bp vector backbone (SalI-XhoI
fragment). PCR primers for the amplification of codons 1-519 of the
human S2P (22) (GenBankTM accession number AFO 19612) were
synthesized: 5'-GTC TCT AGA GCT GCT ACT ATG ATT CCG GTG TCG-3' and
5'-CAT TAC CGT GCT GTA ACC ATC CAG-3'. PCR amplification was performed
with random primed human fetal brain cDNA as template. The product
was digested with XbaI and cloned into pcDNA3.1()
(Invitrogen) between the XbaI and BamHI (blunt)
sites to generate S2P/pcDNA3.1.
Stable Cell Lines-- A derivative of 293 cells (Cell & Molecular Technologies Inc.) was co-transfected with pCMVi-APPsw695 and pRSV-Puro by the calcium phosphate method (24). Individual puromycin-resistant clones were expanded and analyzed for high APP production by Western dot-blot with monoclonal antibody 22C11 (Boehringer). APPsw 293 clone 101 was used in all experiments reported here; similar results were obtained with clone 117. Wild-type CHO K1 (CHO WT) and CHO M19 cells (19, 20) were co-transfected with pBCB/APPsw695 and pRc/Neo or with pRc/Neo alone using the polyamine LT-1 (Mirus). Individual G418 resistant colonies were expanded and assayed for high APP production by Western blot with antibody 22C11.
Transient Transfections and Cell Culture--
293 cells
over-expressing APPsw (clone 101) were transiently transfected with
S2P/pcDNA3.1, pcDNA3.1, or a -galactosidase-containing vector using DMRIE-C (Life Technologies, Inc.). 293 clone 101 cells
were grown in Dulbecco's modified Eagle's medium with 10% FBS and
1% pen/strep/gln containing 5 µg/ml puromycin. CHO clones were grown
in F12 with 10% fetal bovine serum, 10 µg/ml gentamycin, and 0.5 mg/ml G418.
Electrophoretic Separation of A40 and
A
42, Immunoprecipitations, and Westerns--
Tris-Bicine urea gels were used to resolve A
40 and
A
42 as described (25) using synthetic peptides from
Quality Controlled Biochemicals, Inc. as standards. Proteins were
transferred to polyvinylidene difluoride membranes as described
(26).
Quantitative Analysis of S2P mRNA-- S2P mRNA expressed during transient transfection experiments was quantified using solution hybridization to [35S]UTP riboprobes (27). Riboprobes corresponding to hu S2P amino acids 437-519 and 250-321 (22) in both sense and antisense orientations were used to quantify the S2P mRNA.
RT-PCR--
cDNA template generated with random primers and
total cellular RNA from CHO M19 and WT clones was used to amplify S2P
or -tubulin by PCR. Codons 254-519 of S2P (22) were amplified with
sense, 5'-GTT GGG GTG CTC ATC ACT GAA-3', and antisense, 5'-CAT TAC CGT GCT GTA ACC ATC CAG-3', primers yielding a product of 790 bases.
-Tubulin was amplified with sense, 5'-AAG AAG TCC AAG CTG GAG TTC
TC-3', and antisense, 5'-GTG GTG TTG CTC AGC ATG CAC AC-3', primers
yielding a product of 700 base pairs.
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RESULTS |
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Over-expression of the SREBP S2P Does Not Enhance Production of
A40 or A
42 from APPsw--
To test the
hypothesis that the S2P may be identical to the APP
-secretase, we
generated 293 cells stably expressing human APP695 with the Swedish
mutation (APPsw) (28). If S2P is the same enzyme as the
-secretase,
transient over-expression in cells containing substrate
(e.g. APP-over-expressing cell lines) could lead to an
increase in the production of either A
40,
A
42, or both.
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CHO Cells Lacking S2P Expression Process Hu APPsw to
A40 and A
42--
Recent data indicate
that the cholesterol auxotroph CHO M19 contains a deletion spanning
most, if not all, of the S2P gene and thus represents a cellular S2P
gene knock-out (22). If the activity of S2P is responsible for the
-secretase generation of A
40 and/or
A
42 from APP, then the CHO M19 cells should be unable to
generate A
40 and/or A
42.
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DISCUSSION |
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S2P stands out among the many proteases suggested to be
-secretase because it has several properties that make it an ideal candidate. Only S2P and
-secretase have been postulated to cleave substrates within the membrane in vivo (6, 9, 10). S2P is
widely expressed as is
-secretase. S2P activity is (indirectly) enhanced by a transmembrane protein, SCAP, which is located in the
endoplasmic reticulum (12), and
-secretase activity appears to
require another endoplasmic reticulum transmembrane protein, PS1 (17).
We therefore directly tested the hypothesis that the
-secretase is
the same enzyme as the SREBP S2P (7). First we show that
over-expression of the recently cloned S2P in cells expressing APPsw
does not lead to any enhancement of the production of
A
40 or A
42 (Fig. 2) suggesting that
either the S2P is not the
-secretase or that
-secretase activity
is not limiting. The immediate substrates of
-secretase are the
~10- and ~12-kDa C-terminal fragments (CTFs) generated by
- and
-secretase cleavage of APP, respectively (see Fig. 1 and Ref. 30).
Using antibodies that recognize these CTFs, we can detect both of these
substrates in the 293 APPsw-expressing cells (data not shown), thus it
would appear that the generation of additional A
(and p3) by
over-expression of the authentic
-secretase could occur in these
cells. Although we did not directly show that the S2P protein was
enzymatically active in our transfected 293 cells, using a similar
protocol, Rawson et al. (22) documented that S2P cleavage
was restored and cholesterol auxotrophy of M19 cells was corrected in
CHO cells expressing S2P. Because over-expression of the S2P mRNA
in 293 cells did not lead to generation of additional
A
40 or A
42 we conclude that it is
unlikely to be the same enzyme as the
-secretase.
Second, using S2P-deficient CHO M19 (9, 19, 20, 22) we show here that
these cells process transfected hu APPsw to both A40 and
A
42. Neither the amount nor the ratio of
A
40 or A
42 produced by the mutant CHO M19
clones is different from that produced by the CHO WT clones. Based on
these experimental results we conclude that the SREBP S2P is not
identical to the
-secretase enzyme(s) responsible for the generation
of the C termini of A
40 and A
42.
Although the similarities between -secretase and the S2P are
intriguing, there are a few differences. First, the substrates have
different membrane insertion topology; APP is a type 1, single transmembrane protein (C terminus is cytosolic, N terminus is lumenal)
whereas SREBP has two transmembrane domains (both N and C termini are
cytosolic). Second, although it is not known how PS1 and/or PS2
regulate
-secretase activity (direct versus indirect), it
is clear that SCAP directly interacts with and regulates the SREBP site
1 protease (18); thus the S2P cleavage is only indirectly regulated by
SCAP.
Perhaps, as originally suggested by Brown and Goldstein (7), the S2P
and -secretase(s) are members of the same protease family. Rawson
et al. (22) recently cloned the S2P, and although they found
numerous related genes from different organisms (human, hamster,
Sulfolobus sp., Drosophila sp., Caenorhabditis elegans, and Schistosoma mansoni), the S2P appears to be a novel
member of a new family of metalloproteases. The cDNA encoding the
S2P contains the signature sequence (HEXXH) of zinc
metalloproteases; however it does not belong to any of the well
recognized protease families (31, 32). Analysis of the sequences
suggests that the S2P is a polytopic membrane protein with four or five
transmembrane domains; even the hydrophilic active site,
HEXXH, appears to be buried in a very hydrophobic region of
the protein. These features, along with a serine repeat of variable
length (in different species) and a cysteine-rich domain, may be
hallmarks of this new family. Finding S2P related proteases based on
these motifs or by direct homology screening may yet provide a fruitful
end to the search for the elusive
-secretase(s).
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ACKNOWLEDGEMENTS |
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We thank our colleagues at Amgen for their contributions, especially Jean-Claude Louis and Frank Collins for their support of this research, Roland Lüthy for computational sequence analysis, Paul Denis for CTF assays, Cybil Sundgren and Michael Thomas of Amgen's DNA sequencing group, Joan Bennett for assistance in preparing the manuscript, and Vicki Gottmer for preparing the figures. Finally, we thank T. Y. Chang (Dartmouth) for generously providing the CHO M19 and WT cell lines and Rachael Neve (Harvard Medical School, McLean Hospital) for providing the APP695 cDNA.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Mammalian Cell Molecular Biology, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320-1789. Tel.: 805-447-2493; Fax: 805-499-7464; E-mail: tburgess{at}amgen.com.
1 The abbreviations used are: AD, Alzheimer's disease; FAD, familial AD; APP, amyloid precursor protein; APPsw, Swedish mutation of APP695; CTF, C-terminal fragment; SRE, sterol regulatory element; SREBP, SRE-binding protein; S2P, SREBP site 2 protease; SCAP, SREBP cleavage activating protein; CHO, Chinese hamster ovary cells; PS1/PS2, presenilins 1 and 2, respectively; hu, human; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); Bicine, N,N-bis(2-hydroxyethyl)glycine.
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REFERENCES |
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