Endoproteolytic Cleavage and Proteasomal Degradation of Presenilin 2 in Transfected Cells*

(Received for publication, January 21, 1997, and in revised form, February 24, 1997)

Tae-Wan Kim Dagger , Warren H. Pettingell , Olivia G. Hallmark , Robert D. Moir , Wilma Wasco and Rudolph E. Tanzi §

From the Genetics and Aging Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimer's disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. In this study, we set out to examine the processing and degradation pathways of PS2. For regulated expression of PS2, we have established inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator. Western blot analysis revealed that PS2 was detected as an ~53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, we have found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an ~20-kDa C-terminal fragment and an ~34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. Our studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.


INTRODUCTION

A significant portion of Alzheimer's disease (AD)1 is attributed to specific gene defects leading to familial Alzheimer's disease (FAD) (1-5). Two homologous genes, presenilin 1 (PS1) and presenilin 2 (PS2), are responsible for at least 50% of early onset (>60 years old) FAD (2, 3). PS1 and PS2 are serpentine proteins consisting of six to nine predicted transmembrane domains interspersed with one large and multiple smaller hydrophilic loops (4, 5). At the amino acid level, the two proteins are 67% identical and exhibit significant homology to two Caenorhabditis elegans gene products, sel-12 (approximately 50% identity) which has been predicted to facilitate Notch receptor function (6), and spe-4 (approximately 26% identity) which is involved in cytoplasmic trafficking of proteins during spermatogenesis (7).

PS1 and PS2 are ubiquitously expressed (4, 5) and in brain are expressed primarily in neurons, with similar regional distributions (8-10). The presenilins are localized to the endoplasmic reticulum (ER) and the Golgi apparatus but not the plasma membrane suggesting a potential role in protein processing (8, 11, 43). To date, the PS1 and PS2 genes have been shown to contain 35 different mutations which are inherited in an autosomal dominant fashion in over 60 kindreds with early onset FAD (4, 5, 12; for summary, see Ref. 3). Recent studies suggest that the presenilins may directly or indirectly affect the processing of APP leading to increased production of Abeta 42 (13-16). These results help to explain the relatively high degree of amyloid burden in the brains of FAD patients carrying PS1 and PS2 mutations. The pathogenic mechanism by which presenilin mutations lead to increased beta -amyloid deposition and other neuropathological features of AD remains unclear. To begin understanding the role(s) of PS2 in normal cellular metabolism and AD pathogenesis, we investigated the processing and degradation pathways of PS2.


EXPERIMENTAL PROCEDURES

Materials

Tetracycline and ALLN (N-acetyl-L-leucinal-L-norleucinal) were purchased from Sigma. Lactacystin was supplied by Dr. E. J. Corey (Harvard University, Cambridge, MA). Other protease inhibitors and high quality Triton X-100 were purchased from Boehringer Mannheim. Brefeldin A (BFA) was purchased from Calbiochem.

Fusion Proteins and Antibodies

The glutathione S-transferase fusion protein encoding the large hydrophilic loop domain of PS2 was generated as described (17). Anti-PS2Loop is a polyclonal antiserum generated by immunizing rabbits with gel-purified human PS2 loop fusion protein. Specificity and characterization of the antiserum will be described elsewhere. Monoclonal (M2) and polyclonal (D-8) antibodies raised against FLAG peptide (DYKDDDDK) were purchased from IBI and Santa Cruz Biotechnology, respectively. Mouse monoclonal anti-ubiquitin antibody (Ubi-1) was purchased from Zymed Laboratories, Inc.. Rabbit polyclonal anti-ubiquitin antibodies were purchased from Sigma.

Generation of Founder Cell Line for an Inducible Expression System

Founder cell lines were generated by co-transfecting H4 neuroglioma cells in a 100-mm dish with 10 µg of pUHD15-1, a plasmid encoding a tetracycline-repressible transactivator (18) and 1 µg of pCMVneo. Individual G418-resistant colonies were isolated and characterized by transient transfection with the luciferase reporter plasmid pUHC13-3, whose promoter is induced by the transactivator. Luciferase induction in the presence and absence of tetracycline was measured by Western blot analysis using anti-luciferase antibody (Promega) to identify cells with maximal inducibility and tight regulation (data not shown).

Preparation of cDNA Constructs

The cDNAs for wild-type PS2 were subcloned from the pcDNA3 construct (8) into the tetracycline-inducible expression plasmid pUHD10-3 vector using polymerase chain reaction by Pfu polymerase (Stratagene). FLAG epitopes were added to either 5' or 3' ends of PS2 using polymerase chain reaction with the coding sequence for fusion to the sequence, DYKDDDDK. Resulting constructs, PS2s with either 3'FLAG or 5'FLAG peptides, were verified by DNA sequencing.

Generation of Stably Transformed Cell Lines with Inducible PS2 Constructs

The H4 founder cells were transfected with 10 µg of each construct and 1 µg of pCNH2hygro, conferring resistance to hygromycin. Hygromycin-resistant colonies were isolated in the presence of tetracycline and screened for PS2 expression by Western blot analysis using antibodies against the FLAG epitope-tag upon removal of tetracycline. For each PS2 construct, five clones demonstrating various induction levels with tight regulation by tetracycline were selected and used for further study. For induction of PS2, cells were washed five times with prewarmed phosphate-buffered saline to remove residual tetracycline and then incubated with complete media without tetracycline for the indicated hours.

Cell Fractionation

Cells were fractionated into detergent-soluble and -resistant fractions with CSK buffer (10 mM PIPES (pH 6.8), 100 mM NaCl, 2.5 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml chymostatin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A) (19). Cells were washed with ice-cold phosphate-buffered saline and incubated in CSK buffer for 5 min on ice with gentle rocking. The supernatant (detergent-soluble fraction) was collected, and the insoluble structure that remained on the dish was collected, washed once with CSK buffer, and used as detergent-insoluble fraction. The resulting detergent-resistant pellet which consists primarily of cytoskeletal proteins was further incubated with DNase (300 µg/ml) (19-21).

Nickel Affinity Chromatography

PS2 cells were transiently transfected with ubiquitin constructs either pCW7 (H6M-Ub) or pCW8 (H6M-UbK48R) using LipofectAMINETM according to the manufacturer's instruction (Life Technologies, Inc.). The detergent lysate was prepared in ice-cold buffer (10 mM Tris-HCl (pH 7.4), 1% Triton X-100) containing protease inhibitors (2 mM Pefabloc SC, 5 µg/ml ALLN, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg/ml Nalpha -p-tosyl-L-lysine chloromethyl ketone, and 50 µg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone) from the confluent 100-mm dish. To detect 6 × His-tagged PS2, the detergent-soluble fraction was incubated with nickel-nitrilotriacetic acid spin column (Qiagen, Hilden, Germany) overnight and washed extensively, and bound materials were eluted by passing elution buffer (1 M imidazole, 50 mM phosphate buffer (pH 6.0), 300 mM NaCl, 0.5% Triton X-100) twice through the spin column. Samples were concentrated by freeze-drying, and PS2 immunoreactivity was detected by Western blotting using anti-PS2Loop antibodies.

Western Blotting and Immunoprecipitation

Protein samples were quantitated by the BCA protein assay kit (Pierce). SDS-PAGE was carried out using 4-20% gradient Tris/glycine gels under reducing conditions. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) using a semi-dry electrotransfer system (Hoefer). The blots were blocked with 5% non-fat dry milk in TBST (25 mM Tris (pH 7.6), 137 mM NaCl, 0.15% Tween 20) for 1.5 h, incubated primary antibodies (M2, 3 µg/ml; D-8, 1 to 1000; polyclonal anti-ubiquitin, 1 to 1000; Ubi-1, 1 to 1500) for 1.5 h, and secondary antibodies (horseradish peroxidase-conjugated anti-mouse or rabbit antibodies, 1 to 5000) in TBST. Between steps, the blots were washed with TBST for 30 min. For Western blotting with M2 antibodies, 5% (w/v) non-fat dry milk was included in the incubation steps for primary and secondary antibodies. The blot was visualized using the ECL Western blot detection system (Amersham). For immunoprecipitation, cells were lysed using IP buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% Nonidet P-40, 2 mM EDTA) plus protease inhibitors, and solubilized proteins were subjected to immunoprecipitation. The samples were precleared with protein A conjugated with magnetic beads (Perceptive Diagnostics) for 1 h in the cold room, incubated with either control (rabbit anti-mouse IgG) or anti-FLAG (D-8 at 10 µg/ml) antibodies overnight, further incubated with protein A-magnetic beads (30 µl/sample) for 2 h in the cold room, and washed three times with IP buffer. Immunoprecipitates were collected using a magnetic bead collector, boiled in sample buffer, and subjected to SDS-PAGE and Western blotting using monoclonal anti-ubiquitin antibodies.


RESULTS AND DISCUSSION

Endoproteolytic Processing of PS2

To investigate the processing pathway of PS2, we established a regulated system for the expression of PS2. For this purpose, we developed inducible H4 cell lines expressing epitope-tagged versions of wild-type PS2 using a tetracycline-repressible transactivator (18). In this system, the presence of tetracycline in the culture medium suppresses PS2 expression, while its withdrawal results in induction of PS2 expression. PS2 production following induction was monitored by Western blot analysis using antibodies against either N-terminal or C-terminal FLAG epitope tags or using a polyclonal antisera specific for the large hydrophilic loop (HL-6) of PS2 (alpha PS2Loop). Fig. 1 shows the results of Western blot analysis of a cellular lysate from representative, stably transformed cell lines expressing wild-type PS2 grown in the presence or absence of tetracycline for 48 h. No PS2 was observed for cell lines incubated in tetracycline-containing media, indicating the tight regulation of PS2 expression in this system. The SDS-extracted proteins detected by the monoclonal anti-FLAG antibody, M2, included full-length PS2 with an apparent molecular mass of 54 kDa and high molecular mass species of PS2 (HMW-PS2; Fig. 1) for both N- and C-terminal FLAG-tagged PS2. In addition, a C-terminal 20-kDa fragment (PS2-CTF) and an N-terminal 34-kDa fragment (PS2-NTF) were observed (Fig. 1), indicating that PS2 undergoes endoproteolytic cleavage like its homologue, PS1 (17). Both the N-terminal and C-terminal fragments appeared to be stable cellular products as opposed to degradation products since their presence was not affected by the absence of protease inhibitors in the lysis buffer, and they were not affected by prolonged incubation for up to 12 h at 37 °C (data not shown). Our results reveal that a 54-kDa PS2 protein that undergoes endoproteolytic cleavage to generate 34-kDa N-terminal and 20-kDa C-terminal fragments. The generation of the PS2 endoproteolytic fragments could either be a step in the metabolic pathway of PS2 and/or a processing event necessary for the normal function of PS2.


Fig. 1. Inducible expression and endoproteolytic cleavage of PS2. Detection of C- and N-terminal PS2-derived cellular fragments. Western blot analysis of SDS lysates prepared from stably transformed, PS2-inducible H4 human neuroglioma cells grown in the presence (1 µg/ml) or absence of tetracycline for 48 h. PS2-inducible cell lines were established using the tetracycline-repressible transactivator (18). The presence of tetracycline in the culture medium suppresses PS2 expression, while its withdrawal results in induction of PS2 expression. H4 neuroglioma founder cells were stably transfected with constructs encoding full-length PS2 fused with FLAG epitope peptide in C terminus (clone: WF26; left panel) or in N terminus (clone: 5FW11; right panel). Full-length PS2 with an apparent molecular mass of 53-55 kDa (54K PS2) and a high molecular mass form of PS2 (HMW-PS2) were detected in Western blot analysis using monoclonal anti-FLAG antibody, M2. In addition, 20-kDa C-terminal (PS2-CTF: left panel) and 34-kDa N-terminal (PS2-NTF: right panel) fragments were also detected only in the induced (- tetracycline) lanes.
[View Larger Version of this Image (23K GIF file)]


Detergent Solubility of PS2 Fragments

We next compared the detergent solubility of the N-terminal and C-terminal endoproteolytic fragments. 48 h after induction, a comparison of equal amounts of proteins from the detergent (1% Triton X-100)-soluble and insoluble fractions revealed the PS2-NTF only in the soluble fraction, while the PS2-CTF was enriched in the detergent-resistant fraction (Fig. 2). Loading of equal volumes of the soluble and insoluble fractions, normalized for total cellular proteins, revealed a small amount of PS2-CTF in the soluble fraction, but the majority of the fragment localized to the insoluble fraction. The enrichment of the PS2-CTF with the detergent-resistant cellular fraction (Fig. 2) prepared by a procedure classically employed to isolate either detergent-resistant cytoskeletal structures (19-21, 30, 31) or caveolae-like microdomains (32-34) suggests that the PS2-CTF may be a component of one of these cellular structures. Alternatively, this association could be due to the formation of detergent-resistant HMW-PS2 complexes which localize to this fraction. The detergent insolubility of the PS2-CTF is unlikely to be due to the presence of the FLAG epitope since the PS2-CTF cleaved from the PS2 containing the N-terminal FLAG was also enriched in the detergent-resistant cellular fraction (data not shown).


Fig. 2. Detergent solubility of C- and N-terminal PS2 fragments. Cells expressing either C-terminal or N-terminal epitope-tagged PS2 were induced for 48 h, extracted as described above. Equal amounts of proteins (top panel) were analyzed by Western blotting using anti-FLAG antibody, and equal volumes (bottom panel) were normalized for total cellular proteins using the alpha PS2Loop. While 34-kDa N-terminal fragments (NTF) were only detectable in detergent-extractable cellular pools, the 20-kDa C-terminal fragment (CTF) was highly concentrated in the detergent-resistant cellular fraction.
[View Larger Version of this Image (21K GIF file)]


Induction and Turnover of PS2

We next examined the time course of PS2 induction. Inducible PS2 cell lines were induced and harvested at time intervals up to 72 h. Equal amounts of protein were then analyzed from the detergent-resistant and detergent-soluble fractions (Fig. 3). PS2 carrying the C-terminal FLAG was first detected in the detergent-soluble fraction at 6 h post-induction as a full-length 54-kDa band. Thereafter, increasing amounts of HMW-PS2 were observed along with the appearance of a doublet owing to the presence of a band just below the 54-kDa band. By 72 h post-induction, the lower band of the doublet was not observed, and both the HMW-PS2 and the 54-kDa PS2 bands were less abundant relative to 48 h post-induction. In the detergent-resistant fraction, no obvious PS2 signal was detected by anti-FLAG antibody until 48 h post-induction at which point the PS2-CTF was observed. However, in "high"-expressing PS2 clonal cell lines (e.g. WF9), the PS2-CTF could be observed as early as 24 h post-induction (data not shown).


Fig. 3. Time course of PS2 induction. Inducible PS2 cells (clone WF2) were grown in the absence of tetracycline and harvested at timed intervals up to 72 h. Equal amounts of proteins (30 µg) from detergent-soluble (top panel) and detergent-resistant fractions (bottom panel) were analyzed by Western blotting to detect the 54K PS2, HMW-PS2, and 20K CTF. The 54K PS2 and HMW-PS2 were extractable by the detergent treatment.
[View Larger Version of this Image (61K GIF file)]


The inducible system was next employed to examine the turnover of PS2 containing the C-terminal FLAG (Fig. 4). PS2 was induced for 36 h, and PS2 turnover was assessed by adding tetracycline back to the media to repress further PS2 production (time point 0) and then testing detergent-soluble and detergent-resistant cellular fractions at timed intervals up to 24 h. Levels of HMW-PS2 and full-length 54 kDa PS2 were observed to progressively decrease over time and were largely undetectable after 24 h and 9 h post-repression, respectively, in the soluble fraction. In the detergent-resistant fraction, the PS2-CTF was observed as a highly stable fragment which remained relatively stable over the degradation time course. These data suggest that following cleavage the PS2-CTF is translocated to the detergent-resistant fraction where it exists as a relatively stable polypeptide.


Fig. 4. Turnover of PS2 in the inducible system. H4 transfectants with C-terminal FLAG PS2 constructs (clone: WF9) were grown in the absence of tetracycline for 36 h. To measure PS2 turnover in this system, tetracycline was added back to the culture medium at 36 h post-induction (indicated as time 0), and detergent-soluble and detergent-resistant fractions were prepared at timed intervals up to 24 h and analyzed by Western blotting. Turnover of wild-type 54K PS2 and HMW-PS2 present in the detergent-soluble fraction is shown in the top panel, and metabolism of 20-kDa C-terminal fragments in the detergent-resistant fraction is shown in the bottom left panel. Equal amounts of proteins were loaded in each lane.
[View Larger Version of this Image (46K GIF file)]


Degradation of PS2 by the Proteasome Pathway

To further investigate the degradation of PS2, we tested the effects of a set of known cell-permeable protease inhibitors on the degradation of PS2, including pepstatin A, Pefabloc SC, E-64, leupeptin, and aprotinin. None of these protease inhibitors affected the turnover of the PS2-CTF (data not shown). Next, we tested whether PS2 is polyubiquitinated and degraded by the ubiquitin-proteasome pathway (22-25). For this purpose, we used the proteasome inhibitors, ALLN and lactacystin (26), which are known to induce the accumulation of polyubiquitinated proteins by inhibiting the 20 S proteasome (the catalytic core of the 26 S complex) (27-29). Treatment with ALLN and lactacystin resulted in dramatically increased levels of HMW-PS2 while only full-length PS2 was observed in the absence of proteasome inhibitors (Fig. 5A).


Fig. 5. Polyubiquitination and proteasomal degradation of PS2. A, inhibition of PS2 degradation by the 20 S proteasome inhibitors, ALLN and lactacystin. Wild-type PS2-expressing cells (clone WF2: C-terminal FLAG) were induced for 12 h and further incubated with ALLN (50 µM) or lactacystin (10 µM) for an additional 12 h. Detergent-soluble (Soluble) and detergent-resistant fractions (Insoluble) were prepared and analyzed by Western blotting. B, immunoprecipitation of ubiquitin-positive high molecular weight forms of PS2 (HMW-PS2) by anti-FLAG antibodies. Stable cells expressing C-terminal epitope-tagged PS2 (clone WF9) were incubated for 24 h in the tetracycline-absent media either without (lane 2) or with (lane 3) 50 µM ALLN and were lysed using IP buffer, and soluble proteins were immunoprecipitated with control (rabbit anti-mouse IgG; lane 1) or polyclonal (D-8) anti-FLAG antibodies (lanes 2 and 3). Immunoprecipitates were separated on 4-20% SDS-PAGE, and polyubiquitinated PS2 ([Ub]n-PS2) was detected by Western blot analysis using monoclonal (Ubi-1) ubiquitin antibody. C, binding of PS2 modified with epitope-tagged ubiquitin (H6M-Ub) to nickel affinity columns. Plasmid pCW7 encoding wild-type (H6M-Ub) or pCW8 encoding dominant-negative ubiquitin (H6M-UbK48R) was transiently transfected into uninduced PS2 cells (clone WF2). Transiently transfected PS2 cells were either uninduced (lanes 1, 3, 5, 7, 9, and 11) or induced for 12 h (lanes 2, 4, 6, 8, 10, and 12) in the absence (lanes 1-6) or presence of (lanes 7-12) ALLN. Lysates were subjected to nickel affinity chromatography as described under "Experimental Procedures," and bound materials were analyzed by Western blotting using anti-PS2Loop antibodies.
[View Larger Version of this Image (26K GIF file)]


To further explore the possibility that the HMW-PS2 contained polyubiquitinated PS2, PS2 containing the C-terminal FLAG was immunoprecipitated using polyclonal anti-FLAG antibodies and subjected to immunoblot analysis with a monoclonal anti-ubiquitin antibody (Fig. 5B). Ubiquitin-positive HMW-PS2 was detected and found to be significantly increased following treatment with 50 µM ALLN. These findings were also confirmed using additional monoclonal anti-FLAG and polyclonal anti-ubiquitin antibodies (data not shown).

To determine whether polyubiquitinated HMW-PS2 serves as a degradation intermediate for the full-length PS2, constructs encoding wild-type (H6M-Ub) and dominant-negative ubiquitin (H6M-UbK48R), tagged with poly((6×)-histidine) were transiently transfected into uninduced cells. Lysates from PS2 cells that were either uninduced or induced for 12 h (when the predominant species is full-length PS2 and not HMW-PS2; Fig. 3) in the presence or absence of ALLN were then subjected to nickel affinity chromatography, and bound products (ubiquitinated proteins) were subjected to immunoblot analysis with the alpha PS2Loop antibody (Fig. 5C). In the cells which were not treated with ALLN, no polyubiquitinated HMW-PS2 was observed with the exception of a small amount in one lane (Fig. 5C, lane 6) where dominant-negative ubiquitin was transfected into PS2-expressing cells. In contrast, induced cells which were treated with ALLN and were transiently transfected with H6M-Ub revealed abundant amounts of polyubiquitinated HMW-PS2 (Fig. 5C, lane 10). Meanwhile, the induced cells which were treated with ALLN and were transiently transfected with dominant-negative mutant H6M-UbK48R revealed a small amount of polyubiquitinated HMW-PS2 (Fig. 5C, lane 12) similar to the amount observed in lane 6. These findings indicate that full-length PS2 can be modified by epitope-tagged ubiquitin in vivo to form ubiquitinated HMW-PS2 and degraded through the ubiquitin-proteasome pathway. Thus, polyubiquitinated HMW-PS2 likely serves as an intermediate for full-length PS2 degradation in this system.

To determine the cellular site for polyubiquitination and proteasomal degradation of PS2, we tested the effects of BFA. BFA is known to induce the disassembly of the Golgi complex and led to rapid degradation of full-length PS2 (Fig. 6). However, treatment with both BFA and ALLN resulted in even greater accumulation of HMW-PS2 than with ALLN alone, while no significant increase was observed for PS2-CTF (Fig. 6). Similar results were obtained using lactacystin (data not shown). These results suggest that polyubiquitination and proteasomal degradation of PS2 occur in a pre-Golgi compartment (e.g. ER). These data also indicate that the generation and accumulation of the PS2-CTF may be regulated by additional proteolytic enzymes located in other subcellular sites.


Fig. 6. Effect of BFA on the ER degradation of PS2. Inducible wild-type PS2 cells (clone WF2) were grown in the absence of tetracycline for 12 h and were further incubated for an additional 12 h with ALLN (50 µM), BFA (5 µg/ml), and ALLN plus BFA.
[View Larger Version of this Image (36K GIF file)]


The ubiquitin-proteasome pathway is known to play a role in the selective turnover of intracellular protein substrates via complete degradation. However, this pathway also participates in the functional alteration of specific proteins by limited proteolysis or endocytosis following polyubiquitination (for review, see Refs. 22-25). The proteasome has been shown to degrade ER proteins (35). Thus, the ubiquitination and subsequent degradation of PS2 by the proteasome pathway may serve as a means for regulating the turnover of PS2 in the ER. The ubiquitin-proteasome pathway may also be utilized in the transfected cells to dispose of excess PS2 thereby regulating the amount of PS2 that is available for endoproteolysis. Since there are extremely low levels of endogenous full-length PS1 (17) and PS2 (44), it is tempting to speculate that the proteasome may also play a role in regulating full-length presenilin degradation endogenously. It is unlikely, however, that the proteasome carries out the endoproteolytic cleavage of PS2 into the NTF and CTF since proteasome inhibitors did not block the generation of these fragments (Figs. 5A and 6; data not shown for the PS2-NTF). Two ER proteins that have been shown to be degraded by an ALLN/lactacystin-sensitive proteasomal pathway are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (36, 37) and the sterol regulatory element-binding protein (38, 39). Interestingly, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a key regulatory enzyme of cholesterol biosynthesis, has been shown to span the membrane eight times (40), similar to the topological model recently proposed for PS1 (41, 42). Thus, PS2 is now the second putative eight-transmembrane domain protein which has been localized to the ER (8, 11, 43) and is degraded by the proteasomal pathway.

These data show that PS2 expressed in transfected H4 cell lines undergoes endoproteolytic cleavage, is ubiquitinated, and degraded via an ALLN/lactacystin-sensitive proteasome pathway. Although it is unclear how aberrations in the metabolism of the presenilins ultimately lead to altered processing of APP and increased production of Abeta 42, conformational changes of presenilins due to FAD mutations could conceivably alter their metabolism and adversely affect the processing of APP.


FOOTNOTES

*   This work was supported by grants from NIA and NINDS, National Institutes of Health, and the Metropolitan Life Foundation.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.
Dagger    Recipient of a National Research Service Award.
§   Pew Scholar. To whom correspondence should be addressed: Genetics and Aging Unit, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-6845; Fax: 617-726-5677; E-mail: tanzi{at}helix.mgh.harvard.edu.
1   The abbreviations used are: AD, Alzheimer's disease; FAD, familial Alzheimer's disease; ER, endoplasmic reticulum; ALLN, N-acetyl-L-leucinal-L-norleucinal; PS1, presenilin 1; PS2, presenilin 2; BFA, brefeldin A; Abeta , amyloid beta -peptide; HMW-PS2, high molecular mass forms of PS2; PS2-CTF, presenilin 2 C-terminal endoproteolytic fragment; PS2-NTF, presenilin 2 N-terminal endoproteolytic fragment; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; APP, amyloid beta -protein precursor.

ACKNOWLEDGEMENTS

We thank Cristina Ward and Ron Kopito for providing wild-type and K48R ubiquitin plasmids and Gopal Thinakaran and Sangram Sisodia for anti-PS2Loop antisera. We also thank Suzanne Guénette, Tae Jin Kim, Sean Bong Lee, and Marian DiFiglia for helpful discussions, and Christoph Englert and Sean Bong Lee for help with the tetracycline-inducible expression system.


REFERENCES

  1. Wasco, W., and Tanzi, R. E. (1995) in Molecular Genetics of Amyloid and Apolipoprotein E in Alzheimer's Disease (Dawbarn, D., and Allen, S. J., eds), pp. 51-76, BIOS Scientific, Oxford, UK
  2. Schellenberg, G. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8552-8559 [Abstract]
  3. Tanzi, R. E., Kovacs, D. M., Kim, T.-W., Moir, R. D., Guenette, S. Y., and Wasco, W. (1996) Neurobiol. Dis. 3, 159-168 [CrossRef][Medline] [Order article via Infotrieve]
  4. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T., Mar, L., Foncin, J.-F., Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L., Nee, L., Chumakov, Y., Pollen, D., Wasco, W., Hainus, J. L., Da Silva, R., Pericak-Vance, M., Tanzi, R. E., Roses, A. D., Fraser, P. E., Rommens, J. M., and St. George-Hyslop, P. H. (1995) Nature 375, 754-760 [CrossRef][Medline] [Order article via Infotrieve]
  5. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J. m., Pettingell, W. H., Yu, C., Jondro, P. D., Schmidt, S. D., Wang, K., Crowley, A. C., Fu, Y.-H., Guenette, S. Y., Galas, D., Nemens, E., Wijsman, E. M., Bird, T. D., Schellenberg, G. D., and Tanzi, R. E. (1995) Science 269, 973-977 [Medline] [Order article via Infotrieve]
  6. Levitan, D., and Greenwald, I. (1995) Nature 377, 351-354 [CrossRef][Medline] [Order article via Infotrieve]
  7. L'Hernault, S. W., and Arduengo, P. M. (1992) J. Cell Biol. 119, 55-68 [Abstract]
  8. Kovacs, D. M., Fausett, H. J., Page, K. J., Kim, T.-W., Mori, R. D., Merriam, D. E., Hoillister, R. D., Hallmark, O. G., Mancini, R., Felsenstein, K. M., Hyman, B. T., Tanzi, R. E., and Wasco, W. (1996) Nat. Med. 2, 224-229 [Medline] [Order article via Infotrieve]
  9. Lee, M. K., Slunt, H. H., Lee, M. J., Thinakaran, G., Kim, G., Gandy, S. E., Seeger, M., Koo, E., Price, D. L., and Sisodia, S. S. (1996) J. Neurosci. 16, 7513-7525 [Abstract/Free Full Text]
  10. Page, K., Hollister, R., Tanzi, R. E., and Hyman, B. T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14020-14024 [Abstract/Free Full Text]
  11. Cook, D. G., Sung, J. C., Golde, T. E., Felsenstein, K. M., Wojczyk, B. S., Tanzi, R. E., Trojanowski, J. Q., Lee, V. M.-Y., and Doms, R. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9223-9228 [Abstract/Free Full Text]
  12. Rogaev, E. I., Sherrington, R., Rogaeva, E. A., Levesque, G., Ikeda, M., Liang, Y., Chi, H., Lin, C., Holman, K., Tsuda, T., Mar, L., Sorbi, S., Nacmias, B., Piacentini, S., Amaducci, L., Chumakov, I., Cohen, D., Lannfelt, L., Fraser, P. E., Rommens, J. M., and St. George-Hyslop, P. H. (1995) Nature 376, 775-778 [CrossRef][Medline] [Order article via Infotrieve]
  13. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T. D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., and Younkin, S. (1996) Nat. Med. 2, 864-870 [Medline] [Order article via Infotrieve]
  14. Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C.-M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D., Morgan, D., Gordon, M. N., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., and Younkin, S. (1996) Nature 383, 710-713 [CrossRef][Medline] [Order article via Infotrieve]
  15. Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., Ratovitsky, T., Prada, C.-M., Kim, G., Seekins, S., Yager, D., Slunt, H. H., Wang, R., Seeger, M., Levey, A. I., Gandy, S. E., Copeland, N. G., Jenkins, N. A., Price, D. L., Younkin, S. G., and Sisodia, S. S. (1996) Neuron 17, 1005-1013 [Medline] [Order article via Infotrieve]
  16. Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque, G., Johnson-Wood, K., Lee, M., Seubert, P., Davis, A., Kholodenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R., Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St. George-Hyslop, P., and Selkoe, D. J. (1996) Nat. Med. 3, 67-72
  17. Thinakaran, G., Borchelt, D., Lee, M., Slunt, H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190 [Medline] [Order article via Infotrieve]
  18. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci U. S. A. 89, 5547-5551 [Abstract]
  19. Papadopoulos, V., and Hall, P. F. (1989) J. Cell Biol. 108, 553-567 [Abstract]
  20. Hamaguchi, M., and Hanafusa, H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2312-2316 [Abstract]
  21. Osborn, M., and Weber, K. (1977) Exp. Cell Res. 106, 339-349 [Medline] [Order article via Infotrieve]
  22. Hochstrasser, M. (1995) Curr. Opin. Cell Biol. 7, 215-223 [CrossRef][Medline] [Order article via Infotrieve]
  23. Hochstrasser, M. (1996) Cell 84, 813-815 [Medline] [Order article via Infotrieve]
  24. Ciechanover, A. (1994) Cell 79, 13-21 [Medline] [Order article via Infotrieve]
  25. Finley, D., and Chau, V. (1991) Annu. Rev. Cell Biol. 7, 25-69 [CrossRef]
  26. Fenteany, G., Standaert, R. F., Lane, W. S., Choi, S., Corey, E. J., and Schreiber, S. L. (1995) Science 268, 726-731 [Medline] [Order article via Infotrieve]
  27. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L. (1994) Cell 78, 761-771 [Medline] [Order article via Infotrieve]
  28. Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121-127 [Medline] [Order article via Infotrieve]
  29. Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135 [Medline] [Order article via Infotrieve]
  30. Refolo, L. M., Wittenberg, I. S., Friedrich, V. L., and Robakis, N. K. (1991) J. Neurosci. 11, 3888-3897 [Abstract]
  31. Allinquant, B., Moya, K. L., Bouillot, C., and Prochiantz, A. (1994) J. Neurosci. 14, 6842-6854 [Abstract]
  32. Sargiacomo, M., Sudol, M., Tang, Z., and Lisanti, M. P. (1993) J. Cell Biol. 122, 789-807 [Abstract]
  33. Gorodinsk, A., and Harris, D. A. (1995) J. Cell Biol. 129, 619-627 [Abstract]
  34. Li, S., Okamoto, T., Chun, M., Sargiacomo, M., Casanova, J. E., Hansen, S. H., Nishimoto, I., and Lisanti, M. P. (1995) J. Biol. Chem 270, 15693-15701 [Abstract/Free Full Text]
  35. Wiertz, E. J. H. J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones, T. R., Rapoport, T. A., and Ploegh, H. L. (1996) Nature 384, 432-438 [CrossRef][Medline] [Order article via Infotrieve]
  36. Inoue, S., Bar-Nun, S., Roitelman, J., and Simoni, R. D. (1991) J. Biol. Chem. 266, 13311-13317 [Abstract/Free Full Text]
  37. McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638 [Abstract/Free Full Text]
  38. Sakai, J., Duncan, E. A., Rawson, R. B., Hua, X., Brown, M. S., and Goldstein, J. L. (1996) Cell 85, 1037-1046 [Medline] [Order article via Infotrieve]
  39. Wang, W., Sato, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994) Cell 77, 53-62 [Medline] [Order article via Infotrieve]
  40. Roitelman, J., Olender, E. H., Bar-Nun, S., Dunn, W. A., Jr., and Simoni, R. D. (1992) J. Cell Biol. 117, 959-973 [Abstract]
  41. Li, X., and Greenwald, I. (1996) Neuron 17, 1015-1021 [Medline] [Order article via Infotrieve]
  42. Doan, A., Thinakaran, G., Borchelt, D. R., Slunt, H. H., Ratovitsky, T., Podlisny, M., Selkoe, D. J., Seeger, M., Gandy, S. E., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 1023-1030 [Medline] [Order article via Infotrieve]
  43. Walter, J., Capell, A., Grünberg, J., Pesold, B., Schindzielorz, A., Prior, R., Podlisny, M. B., Fraser, P., St. George Hyslop, P., Selkoe, D. J., and Haass, C. (1996) Mol. Med. 2, 673-691 [Medline] [Order article via Infotrieve]
  44. Kim, T.-W., Pettingell, W. H., Hallmark, O. G., Moir, R. D., Wasco, W., and Tanzi, R. E. (1996) Soc. Neurosci. Abstr.

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.