From the
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486 and ¶Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5G-1X5, Canada
Received for publication, January 29, 2003 , and in revised form, April 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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APP is processed by two proteases, -secretase and
-secretase, that generate the N and C termini of the A
-peptides, respectively (6). The A
-peptides are generally 40 or 42 amino acids in length (A
40 and A
42, respectively). A
42 is more prone to aggregation and is believed to be a pivotal player in the etiology of AD. There is considerable evidence showing that the presenilins are closely associated with
-secretase-mediated cleavage. Firstly, familial early onset Alzheimer's disease mutations in PS1 and PS2 cause increased production of A
42 in transfected cells or transgenic mice. Secondly, isolated cultured neurons derived from PS1-deficient mice accumulate APP fragments that fail to be processed by
-secretase (7, 8). Thirdly,
-secretase activity is abolished in cultured cells derived from mice that are deficient in both PS1 and PS2 (9, 10). Fourthly, mutagenesis of two conserved aspartates in the transmembrane regions of PS1 significantly reduces
-secretase activity (11). Thus, it was proposed that PS1 is either a unique diaspartyl cofactor for
-secretase or is itself
-secretase, an intramembranous aspartyl protease. Finally, biochemical evidence that PS1 and PS2 contain the active sites of
-secretase was obtained from studies in which presenilins were covalently modified by active site-directed
-secretase inhibitors (12, 13)
Although both PS1 and/or PS2 are essential for the -secretase-mediated cleavage, the dramatically different phenotypes of the PS1-deficient and PS2-deficient mice suggest that these proteins play disparate roles. The PS1-deficient mice exhibit severe developmental defects and perinatal lethality (14, 15), whereas the PS2-deficient mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with increasing age (16). PS1 and PS2 transcripts are present in a wide variety of mammalian tissues; however, the levels of mRNA vary significantly and independently among tissues and during brain development (17). The marked sequence divergence of the putative cytosolic domains in PS1 and PS2 probably enable them to interact with different perhaps overlapping intracellular binding partners. Consistent with this notion, PS1 and PS2 appear to engage in independent high molecular weight protein complexes (18). A functional redundancy between PS1 and PS2 is apparent from the ability of either protein to rescue the egg-laying defect of C. elegans hermaphrodites lacking sel-12 (5). However, the differences/similarities of the PS1 and PS2-mediated
-secretase activities have yet to be fully elucidated.
In this investigation, we aimed to compare the -secretase activities mediated by PS1 and PS2. Blastocyst-derived (BD) cells from wild-type, PS1/PS2+/+, PS1+/PS2/, and PS1/PS2/ mice (19) were used to prepare cellular membranes that were evaluated with an in vitro
-secretase assay (20). In addition, these BD cell lines were transiently transfected with an expression construct encoding the C-terminal fragment of APP (C-100) and the levels of secreted A
were measured. Importantly, the
-secretase activities associated with the PS1+/PS2/ BD cells and PS1/PS2+/+ BD cells are mediated exclusively by the PS1
-secretase complex and the PS2
-secretase complex, respectively. This assertion is supported by the absence of
-secretase activity associated with the PS1 and PS2 doubly deficient BD cells.
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EXPERIMENTAL PROCEDURES |
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Blastocyst-derived Cultures and Cell MembranesBlastocyst-derived cells from PS1+/+PS2+/+, PS1+/PS2/, PS1/PS2+/+, and PS1/PS2/ blastocyst cultures were obtained as described previously (10). Cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and murine leukemia inhibitory factor. BD cell membranes were prepared as described previously (20) with the exception that the cells were broken with a Dounce homogenizer. Protein concentration was determined using a Bio-Rad DC protein assay kit with bovine serum albumin as a protein standard.
Immunoblotting Analysis of the BD Cell MembranesCellular membranes containing PS1 and PS2 were fractionated by 12% SDS-PAGE and electrotransferred to PVDF membranes. The PVDF membranes were probed for the presence of PS1 and PS2 with rabbit anti-PS1-NTF and rabbit anti-PS2-CTF antibodies, respectively. Western blotting signals were detected using the Enhanced Chemifluorescent kit for the PhosphorImager as described above. Methods for immunoprecipitation, in vitro -secretase assay, A
assay, and photoaffinity labeling of membrane proteins were carried out as described elsewhere (12, 20).
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RESULTS |
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Correlation of Anti-PS1 and Anti-PS2 Antibody Signals with Relative Protein AbundanceTo more fully compare PS1- and PS2-mediated -secretase activities, we sought to determine the relative amounts of PS1 and PS2 in the different BD cell lines. Radiolabeled PS1 and PS2 standards were produced by in vitro transcription/translation (TNT) of the corresponding murine presenilin cDNAs in the presence of [35S]Met. The radiolabeled TNT products were fractionated by SDS-PAGE and transferred to PVDF membranes. Fig. 2A, upper panel, shows the phosphorimages of PVDF membranes containing increasing amounts of the radiolabeled TNT products generated with the murine PS1 and PS2 constructs. Murine PS1 and PS2 contain 13 and 17 methionines, respectively. Equal intensity of phosphorimaging signals (after normalization for the different number of methionines in PS1 and PS2) is indicative of equal amounts of protein. The very same blots containing the TNT products were also probed with the anti-PS1-NTF or anti-PS2-CTF antibodies (Fig. 2A, lower panel). The exposure time for the immunoblot signal (5 min) was much shorter than the exposure time for the [35S]Met signal (16 h, no signal was observed after3hof exposure); hence, the immunoblot signal is not corrupted by the [35S]Met signal. The intensities of the anti-PS1-NTF and anti-PS2-CTF immunoblot signals as a function of the amounts of PS1 and PS2, respectively, are depicted in Fig. 2B. We conclude that the immunoblot protocol for detection of PS2-CTF is
1.8-fold more sensitive than the corresponding immunoblot protocol for detection of PS1-NTF.
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Protein Levels of PS1 and PS2 in the BD CellsPresenilin expression in the various BD cells was characterized by immunoblotting using anti-PS1-NTF and anti-PS2-CTF antibodies (Fig. 3A). Both PS1 and PS2 were detected in PS1+/+PS2+/+cell membranes (lanes 1 and 5, respectively). The relative amounts of PS1 and PS2 in the PS1+/+ PS2+/+ membranes were deduced by comparing each immunoreactive signal with the corresponding PS1 or PS2 immunoreactivity standard curves (Fig. 2C). Accordingly, the amount of PS2 in PS1+/+ PS2+/+ cell membranes appears to be 72% of the amount of PS1.
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PS1 but not PS2 was detected in PS1+/PS2/ membranes (lanes 2 and 6, respectively). Conversely, PS2 but not PS1 was detected in PS1/PS2+/+ cell membranes (lanes 3 and 7, respectively). Membranes from PS1/PS2/ BD cells do not possess protein species that cross-react with either the anti-PS1-NTF or anti-PS2-CTF antibodies (lanes 4 and 8, respectively). The PS1 level in PS1+/PS2/ cell membranes (lane 2) was only 6% of that seen with wild-type cell membranes (lane 1) (note the 10-fold higher protein load in lane 2 compared with lane 1). The dramatically lower levels of PS1 in the PS1+/PS2/ cell membranes as compared with the PS1+/+PS2+/+ cell membranes was confirmed by immunoblotting with the anti-PS1-CTF antibody (Fig. 3B). Comparative analysis of PS2 in PS1+/+PS2+/+ and PS1/ PS2+/+ cell membranes (lanes 5 and 7, respectively) reveals a 2.6-fold greater abundance of PS2 in the PS1-deficient cells.
In Vitro -Secretase Activity Catalyzed by BD Cell MembranesThe
-secretase activities catalyzed by the various BD cell membranes were evaluated with the in vitro
-secretase assay using the recombinant substrate, C100/FLAG (20). PS1/PS2/ cell membranes fail to catalyze the formation of the 40- or 42-related cleavage products (data not shown). Importantly, this result signifies that all activity displayed by BD cell membranes using the in vitro
-secretase assay is attributable to PS1 or PS2. Despite the limited amount of PS1 present in the PS1+/PS2/ cell membranes (6% of the level in PS1+/+PS2+/+ cell membranes), the amount of total A
generated by these membranes was 56% of that produced by PS1+/+PS2+/+ cell membranes (Table I). Likewise, membranes prepared from the PS1/PS2+/+ BD cells, which serve as a source of PS2
-secretase activity, catalyze the formation of the A
-peptides from C100/FLAG. However, total A
generated by PS1/PS2+/+ cell membranes is only 11% of that produced by PS1+/+PS2+/+ cell membranes.
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Assessment of the in vitro -secretase activities of the PS1+/PS2/ and PS1/PS2+/+ membranes coupled with the knowledge of the relative abundance of PS1 and PS2 in these membrane preparations (Fig. 3) allow estimates for the comparative enzymatic activities of the PS1
-secretase complex and the PS2
-secretase complex toward the C100/FLAG substrate (Table I). We conclude that the relative activity of the PS1
-secretase complex (residing in the PS1+/PS2/ cell membranes) is 169-fold greater than that of the PS2
-secretase complex (residing in the PS1/PS2+/+ cell membranes) when assayed with C100/FLAG. This conclusion rests on the assumption that PS1 and PS2 are fully engaged in the putative catalytically competent
-secretase macromolecular complex in these membranes.
The amount of A42 generated with PS1+/+PS2+/+ cell membranes was
14% total A
(Table I). PS1+/PS2/ and PS1/PS2+/+ cell membranes also catalyzed the formation of the A
40- and A
42-related species from C100/FLAG. The amount of A
42 (relative to total A
) produced by the PS1- and PS2-containing membranes is
18 and 12%, respectively. Hence, the PS1
-secretase complex may be slightly more proficient than the PS2
-secretase at catalyzing the A
42 cleavage event.
The previously mentioned comparison of the activities of PS1 and PS2 strongly suggests that the PS1 -secretase complex is responsible for the bulk of the in vitro
-secretase activity catalyzed by PS1+/+PS2+/+ cell membranes. However, it is surprising that PS1+/+PS2+/+ and PS1+/-PS2/ cell membranes generate comparable amounts of total A
(Table I) despite 16-fold more PS1 in the former cells compared with the latter cells. The apparent specific activity of the PS1
-secretase complex in the PS1+/PS2/ cell membranes thus appears to exceed the apparent specific activity of the PS1
-secretase complex in the PS1+/+PS2+/+ cell membranes. Varying the amount of the PS1+/+PS2+/+ cell membranes in the assay resulted in a proportional change in the amount of product formation (data not shown). Hence, the level of PS1 in the PS1+/+PS2+/+ cell membranes (when assayed at 2.5 µg) does not appear to simply overwhelm the capacity of the in vitro assay to detect PS1 activity.
-Secretase Activity Displayed by BD Cells Expressing the C-terminal Fragment of APPThe various BD cells were transiently transfected with an expression vector encoding the APP fragment (C100) that corresponds to the membrane-bound C-terminal stub produced by
-secretase cleavage. The BD cells were exposed to the C100 expression vector for 24 h, fresh medium was placed on the cells for an additional 24 h, and the conditioned medium was removed and assayed for A
40 and A
42. The BD cells were cotransfected with a vector encoding green fluorescent protein to control for differences with respect to transfection efficiency and posttransfection cell proliferation rate.
The amount of the A-peptides that accumulated in conditioned media of the various transfected BD cells (after normalizing for transfection efficiency based on the green fluorescent protein levels) is shown in Table I. The PS1/PS2/
BD cells fail to secrete A40 or A
42 (data not shown), which is an observation that is consistent with previous reports (9, 10). Even though the level of PS1 protein in PS1+/PS2/ cells is only 6% of that found in PS1+/+PS2+/+ BD cells (Fig. 3A), the amounts of total A
secreted by the former cells is 38% of that secreted by the latter cells. The level of PS2 protein in PS1/PS2+/+ BD cells is 2.6-fold higher than that in PS1+/+PS2+/+ BD cells; however, the former cells secrete only 30% A
produced by the latter cells.
The relative cellular activity of the PS1 -secretase complex (residing in PS1+/PS2/ BD cells) appears to be 38-fold greater than that of the PS2
-secretase complex (residing in PS1/PS2+/+ BD cells). These results are consistent with the studies carried out with the BD cell membranes, indicating that PS1 exhibits higher relative activity in processing the C100/FLAG substrate. Furthermore, it suggests that secreted A
from PS1+/+PS2+/+ cells is largely produced by PS1-mediated
-secretase activity.
The amount of A42 (relative to total A
) secreted by PS1+/+PS2+/+ BD cells was
13%. The corresponding percentages of A
42 secreted by PS1+/PS2/ BD cells (exclusively PS1 cellular activity) and PS1/PS2+/+ BD cells (exclusively PS2 cellular activity) were
9 and 14%, respectively. Hence, in direct opposition to studies with the cell membranes, it appears that the PS2
-secretase complex in the cellular system may have a slightly greater propensity than the PS1
-secretase complex to catalyze the A
42-cleavage event.
PS1-mediated -Secretase Activity Is Correlated with Abundance of the Photolabeled PS1 SpeciesThe apparent low activity of PS1 in the PS1+/+PS2+/+ BD cells could result from either low specific activity of the PS1-associated
-secretase complex or from PS1 not being fully engaged in the catalytically active
-secretase complex. The active site-directed photoactivatable
-secretase inhibitor, Merck A (Fig. 4), which binds to the active form of enzyme (12), was used as a probe to assess the catalytic competency of PS1 in the BD cell membranes. Merck A displays similar inhibitory potency toward
-secretase activity in membranes prepared from PS1+/+-PS2+/+ and PS1+/PS2/ BD cells (data not shown). Equal amounts of protein from the different BD cell membranes were incubated with Merck A, and the samples were irradiated, leading to photolabeling of active
-secretase. The biotinylated proteins were isolated with streptavidin-beads and analyzed by Western blotting using anti-PS1 and anti-PS2 antibodies (Fig. 4). No labeled species were detected when the PS1/PS2/ cell membranes were treated similarly (data not shown). Moreover, the appearance of the labeled PS1 and PS2 species were blocked when the photolysis was performed in the presence of excess Merck B (data not shown). An analysis of the signals by densitometric scanning revealed 2.2 ± 0.2-fold more photolabeled PS1 in PS1+/+PS2+/+ cell membranes than in PS1+/PS2/ cell membranes. Moreover, there is 2.4 ± 0.3-fold less photolabeled PS2 in PS1+/+PS2+/+ cell membranes than in PS1/PS2+/+ cell membranes. These data indicate that
-secretase activity better correlates with the amount of photolabeled PS1 rather than total amount of PS1 in PS1+/+-PS2+/+ and PS1+/ PS2/ BD cell membranes.
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PS1- and PS2-mediated -Secretase Activity Display Differential Inhibitor SusceptibilityThe potent
-secretase inhibitor, Merck B (21), blocked cleavage of C100/FLAG by PS1+/+ PS2+/+ BD cell membranes (Fig. 5). The IC50 value, 0.26 nM, was indistinguishable from the value that was observed with the detergent extract prepared from HeLa cell membranes (20). The PS1-mediated
-secretase activity catalyzed by PS1+/PS2/ BD cell membranes and the PS2-mediated
-secretase activity catalyzed by PS1/PS2+/+ BD cell membranes were both inhibited by Merck B (IC50 values = 0.15 and 0.81 nM, respectively). Hence, the PS1
-secretase complex appears to be 5.4-fold more susceptible to inhibition by Merck B than is the PS2
-secretase complex. This apparent ability of Merck B to discriminate between PS1 and PS2 was also observed with membranes prepared from mouse embryonic fibroblasts derived from PS1+/+PS2+/+ and PS1+/+PS2/ mice (data not shown). On the other hand, another
-secretase inhibitor, Merck C, displayed more similar inhibitory potency toward
-secretase activities catalyzed by PS1+/PS2/ and PS1/PS2+/+ BD cell membranes (Fig. 5). The apparent IC50 values of Merck C for the PS1- and PS2-mediated
-secretase activities were 9.8 and 18.9 nM, respectively.
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It is noteworthy that the Ki values of the inhibitors determined with the PS1+/+PS2+/+ cell membranes are more similar to the values determined with PS1+/PS2/ BD membranes than to the values determined with PS1/PS2+/+ cell membranes (Fig. 5). These results provide additional evidence that the PS1-containing complex is the major catalyst of -secretase activity in PS1+/+PS2+/+ BD cells and their corresponding cellular membranes.
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DISCUSSION |
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We employed a novel strategy based on standardization of immunoblot signals to quantify the relative levels of PS1 and PS2 proteins in the different BD cell lines. The ratio of PS1 and PS2 proteins in PS1+/+PS2+/+ BD cells is 1:0.7. The amount of PS1 protein in PS1+/PS2/ BD cells is only 6% of that seen in PS1+/+PS2+/+ cells, which is far less than the expected 50% value. The less amount of PS1 protein in PS1+/PS2/ BD cells could reflect more rapid turnover or decreased synthesis of the PS1-associated macromolecular complexes. On the other hand, the amount of PS2 in PS1/PS2+/+ cells is 2.6-fold greater that the level of the PS2 in PS1+/+PS2+/+ cells. The higher level of PS2 in PS1-deficient cells might reflect enhanced stability because of increased availability of critical cellular factors that are typically sequestered by PS1 in wild-type cells.
Our investigation shows that the PS1 -secretase complex displays markedly greater activity than the PS2
-secretase complex both in the in vitro assay using the C100/FLAG substrate and the cellular assay using the C100 substrate. We infer that PS1 is more active than PS2 toward the APP C100 fragments that are generated by
-secretase-mediated processing of APP. These results provide biochemical evidence that PS1 plays a predominant role in the cellular processing of APP, a belief largely based on studies with neuronal cells derived from PS1 or PS2-deficient mouse embryos (7, 16). The fact that PS1- and PS2-mediated activities display different sensitivity to
-secretase inhibitors further indicates that PS1 and PS2 have unique active sites that engender disparate substrate preferences.
Both PS1+/PS2/ and PS1/PS2+/+ BD cells and the corresponding cell membranes displayed -secretase activities that generated both A
40 and A
42. The ratio of A
42 over total A
appeared to be relatively low (<20%) and only slightly different for the PS1- and PS2-mediated
-secretase activities. PS1 actually generated slightly more A
42-related product than PS2 in the in vitro assay system, whereas the situation was reversed in the cellular system. The reason for the ratio difference of A
40 and A
42 when assayed with the in vitro and cellular assays is unclear but it may reflect the contribution of intracellular trafficking or putative/accessory factors in the cell. Nevertheless, our studies thus show that A
42 formation is not necessarily mediated by only one of the two presenilins. Both PS1 and PS2 are seemingly capable of catalyzing this proteolytic cleavage event.
The different PS1 levels in the PS1+/+PS2+/+ and PS1+/ PS2/ cells (and membranes) were not matched by a corresponding disparity in their -secretase activities. The apparent discrepancy between PS1 levels and
-secretase activity is not because of a kinetic effect in our assay system in which the rate of product formation is maximal in the presence of a relatively low amount of PS1. We "titrated" active
-secretase using a photoreactive transition state analog inhibitor to shed light on this enigma. Because the concentration of the compound used in the experiment is much higher than its Ki, the extent of labeling should reveal the relative amount of the active enzyme in the membranes. The amount of photolabeled PS1 in PS1+/+ PS2+/+ cell membranes is only 2-fold greater than that in PS1+/PS2/ cell membranes, even though PS1 in wild type cell membranes is 16-fold more abundant than that in PS1+/ PS2/ cell membranes. Interestingly, the amount of the photolabeled species is more consistent with the relative
-secretase activity displayed by the respective cell membranes. Assuming that PS1 in PS1+/PS2/ cell membranes is fully engaged in the active
-secretase complex, only
14% total PS1 in the PS1+/+PS2+/+ BD cells is engaged in the active
-secretase complex. The components of the PS-1
-secretase complex that are critical for activity remain to be elucidated. Our results indicate that the PS1 heterodimer may be essential but not sufficient for
-secretase activity. This observation is consistent with previous cellular studies in which overexpression of full-length PS1 or coexpression of its NTF and CTF fragments did not increase
-secretase activity (22). Nicastrin (23), APH-1 (24, 25), and Pen-2 (25) are proteins that are purportedly engaged in the complex and thus may be obligatory for
-secretase activity. These putative accessory proteins may be lacking in the PS-associated species that fails to display
-secretase activity. The "non-
-secretase" PS1 complex may mediate other functions such as trafficking and regulation of
-catenin signaling.
-Secretase inhibitors will serve as powerful tools to selectively neutralize the protease-associated functions of the presenilins while leaving the other putative functions intact.
Our observation that only a small portion of the PS1 in PS1+/+PS2+/+ membranes is involved in an active -secretase complex may help to resolve some perplexing issues surrounding
-secretase biology. The assignment of
-secretase activity to the presenilins has been challenged because the presenilins and the
-secretase substrates do not appear to be colocalized to the same cellular site (26). Presenilins predominantly reside in the early compartments of the secretory apparatus, whereas the
-secretase mediated Notch processing occurs at or near the plasma membrane (27). Our discovery that only a small portion of PS1 is engaged in the active
-secretase complex in concert with evidence for a small amount of presenilins on the cell surface (28) may have solved this "spatial paradox."
Presenilins are also involved in the final step of Notch cleavage that generates the Notch intracellular domain, which is involved in transcriptional coactivation of CSL (CBF-1, Su(H), Lag-1) transcription factors (29). Recent studies (30, 31) indicate that complete inhibition of -secretase activity by
-secretase-specific inhibitors blocks the Notch activity and arrests T cell development. It will be important to carry out similar studies aimed at characterizing PS1 and PS2-mediated
-secretase activities toward Notch substrates. Understanding the specificity of PS1 and PS2 toward different substrates and inhibitors will help to develop selective
-secretase inhibitors for AD therapy. Moreover, it is important to determine the applicability of our findings to presenilins expressed by neurons and neural progenitor cells (32). Presenilin-mediated
-secretase activity is believed to participate in at least two critical processes in the brain, neuronal differentiation during neurogenesis (33) and A
production. Abnormal
-secretase activity in neurons and neural progenitor cells may contribute to AD and has implications in other neurodegenerative diseases.
In conclusion, our studies highlight fascinating twists in the link between the presenilins and -secretase activity. Some findings were anticipated such as the dissimilar substrate/ligand preferences of the PS1
-secretase complex and the PS2
-secretase complex. However, other elements in the link between PS1 and
-secretase activity were unexpected such as the apparent heterogeneity with respect to the activity state of PS1 in wild-type BD cells. The striking diversity that is exhibited by the presenilins portends their involvement in a panoply of disparate biological processes that still largely remain to be elucidated.
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FOOTNOTES |
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|| Present address: Lexicon Genetics, Inc., 8800 Technology Forest Place, The Woodlands, TX 77381.
** Present address: Bayer Corporation, B24-2, 400 Morgan Lane, West Haven, CT 06516.
Present address: RRL-617A, Box 459, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.
To whom correspondence may be addressed. E-mail: mingtain_lai{at}merck.com (to M.-T. L.) or liy2{at}mskcc.org (to Y.-M. L.).
1 The abbreviations used are: PS1, presenilin-1; PS2, presenilin-2; BD, blastocyst-derived; AD, Alzheimer's disease; APP, amyloid precursor protein; A,
-amyloid; TNT, transcription/translation; PVDF, polyvinylidene difluoride; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; NTF, N-terminal fragment; CTF, C-terminal fragment.
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ACKNOWLEDGMENTS |
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REFERENCES |
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