Intracellular pathways of folded and misfolded amyloid precursor protein degradation

Archives of Biochemistry and Biophysics 451 (2006) 79–90 www.elsevier.com/locate/yabbi

Intracellular pathways of folded and misfolded amyloid precursor protein degradation James F. Hare ¤ Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97219, USA Received 21 February 2006, and in revised form 2 May 2006 Available online 24 May 2006

Abstract A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein -amyloid (A). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when -secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum. © 2006 Elsevier Inc. All rights reserved. Keywords: Protein folding; Amyloid precursor protein; Proteasome; Protease inhibitors; Protein glycosylation; Tunicamycin; Protein traYcking

Amyloid precursor protein (APP1), the precursor to -amyloid (A), has been under intense investigation since its discovery in 1987 [1,2]. The great majority of expressed APP molecules are processed to a shed form in which its N-terminal end is proteolytically released from its transmembranous domain by -secretase, a metalloprotease. A small amount of APP, however, is processed in an alternative pathway employing -, and -secretase activities that *

Fax: +1 503 494 8393. E-mail address: [email protected]. 1 Abbreviations used: A, -amyloid peptide: APP, amyloid precursor protein; APPs, the secreted form of APP; ALLN, N-acetyl-leucyl-leucylnorleucinal; ALLM, N-acetyl-leucyl-leucyl-methioninal; MG-132, carbobenzoxy-leucyl-leucyl-leucinal; E-64, (2S,3S)-transepoxysuccinyl-lleucylamide-3-methylbutane ethyl ester, LC, clasto-lactacystin -lactone; EST, (2S,3S)-trans-epoxysuccinyl-L-leucylamideo-3-methylbutane ethyl ester; SDS, sodium lauryl sulfate, TTE, Tris, Triton, EDTA buVer. 0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.05.002

establish respectively the N- and C-terminal ends of 40–43 amino acid long A. The -secretase cleavage site resides within the A-sequence, making -cleavage competitive with that of  and . A accumulates in the brain of Alzheimer’s patients and contributes to amyloid placques, the pathological manifestations of the disease. The longer forms of A having 42–43 amino acids (A42–43) are more insoluble and are particularly amyloidogenic, most probably due to their propensity to form a soluble, Wbrillar oligomer that is toxic for brain cells [3]. While certain mutations within the APP gene (familial Alzheimer’s disease or FAD mutations) result in increased formation of A, mutations in genes coding for two additional proteins, presenilins I and II (PS1, PS2), also result in early onset Alzheimer’s disease and are coincident with and contribute to increased levels of brain A [4,5]. The cellular location of A generation has proven controversial. A40–43 may

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form in the endocytic pathway [6,7], in the Golgi [8,9], in the endoplasmic reticulum (ER) [10], or on the cell surface [11]. Processing of FAD forms of APP, which generate increased levels of A40, occurs primarily late in the secretory pathway [12], while the site of A42 formation is unaVected by brefeldin and thus occurs earlier in the secretory pathway [13]. DiVerent reported cellular localizations for A formation may result from diVerent processing events, cell type, APP/presenilin mutations, etc. The improper folding and resulting instability of several mutant membrane proteins have been shown to have important disease implications. These include the cystic Wbrosis transport regulator [14] and 1-antitrypsin [15], both of which exhibit mutations that interfere with normal folding events. The misfolded forms of these proteins preclude their escape from the ER, entry into the secretory pathway, and normal function. A number of experiments have indicated that early events in the maturation of APP in the ER may also be critical in its subsequent processing. (1) APP was shown to associate with gp78 (BiP) [16] and with calnexin [17] in the ER. Prolonged interaction with a mutant form of gp78, having reduced ATPase activity, slowed the formation of A42. These experiments suggest that early intermediates in APP maturation interact with ER localized chaperone proteins. (2) The presenilins reside primarily in the ER [18,8] and certain PS mutations increase A formation [19]. As described above there is also evidence for A production within the ER as well [10,13]. (3) PS FAD mutants down-regulate the unfolded protein response (UPR) [20]. The latter is a signaling pathway from the ER to the nucleus that up-regulates the expression of chaperone proteins such as BiP to respond to increased levels of misfolded proteins. UPR results from oxidative stress and extraneous inXuences that interfere with protein folding and expression in the ER. Oxidative stress has also been shown to increase the levels of A [21]. (4) The presence of 10 cysteine residues in the amino terminal folded domain of APP and only three disulWde bonds [22] may cause disulWde bond reshuZing with the resulting potential for misfolding. (5) A screen for proteins that enhance -secretase cleavage of APP identiWed an ER stress protein Herp [23], a previously identiWed homocysteine response protein. (6) Lastly, a proteomics approach has identiWed several ER associated chaperone proteins that are up- or down-regulated in Alzheimer’s patient brains [24]. The above reports underscore a need for more detailed information about APP folding and maturation in the ER. To understand the sequence of events in APP maturation in the ER, I have studied the eVect of protease inhibitors on the degradation and maturation of newly translated APP. Upon Wnding that tunicamycin addition during pulse-labeling causes newly translated APP to be degraded by proteasomes, presumably as a result of protein conformational change, V8 protease sensitivity of immunoprecipitated APP was employed as a probe to detect possible conformational changes that result from lack of N-glycosylation. APP labeled in the presence of tunicamy-

cin was shown to exhibit altered V8 susceptibility that mimicked that seen upon protein denaturation. Materials and methods Cell lines, labeling, and drug treatments SV695, a Chinese hamster ovary (CHO) cell line stably transfected with APP695 (a gift from Dr. S. Sisodia, U. of Chicago), was grown in Delbecco’s modiWed Eagle’s media (DMEM) supplemented with 5% fetal bovine serum, 200–400 g/ml G418, 100 g/ml penicillin, and 100 g/ml streptomycin. An untransfected CHO cell line was grown under the same conditions. A CHO cell line stably transfected with APP750 containing the Swedish mutation was obtained from Dr. N.K. Robakis, Mount Sinai School of Medicine, and grown in McCoy’s media supplemented with G418, penicillin, streptomycin, and 10% fetal bovine serum. For turnover measurements, 30 mm culture dishes were pre-incubated for 30–60 min as indicated in sulfur amino acid depleted DMEM (Sigma), supplemented with 5% dialyzed serum. Cells were then labeled by the addition of 50–100 Ci/ml Easytag EXPRESS protein labeling mix (1175 mCi/mmol, Perkin-Elmer Life Sciences) for the time indicated. Brefeldin A and nocodazole were added 15 min prior to labeling and were also present during the chase. Inhibitors E64, EST, ALLN, ALLM, MG-132, leupeptin, pepstatin, 1,10-phenanthroline, baWlomycin, and clastolactacystin -lactone (LC) were added to chase media in ethanol or water and were present only during the chase unless indicated otherwise. Antiserum and reagents C20 antiserum made to the last 20 amino acids in APP695 was prepared as previously described [25]. EST, ALLN, ALLM, MG-132, and LC were purchased from EMD/Calbiochem. All other reagents were purchased from Sigma or other common sources. Immunoprecipitation and endoproteinase digestion For immunoprecipitation of pulse-labeled and chased protein, 30 mm diameter dishes were placed on ice, washed 3£ with PBS, and extracted with 1.0 ml of 0.02 M Tris, 1% Triton X-100, and 5 mM EDTA (pH 7.4, TTE). After centrifugation as above, mammalian cell protease inhibitor cocktail (1 l, Sigma), 20 l of 5 M NaCl, 50 l of 10% deoxycholate, and 15 packed l of protein A–agarose beads that had been preabsorbed with 3 l of C20 antibody and washed 2£ with TTE were added. After end-over-end rotation for 8–16 h, immunoabsorbed protein was washed 3£ with TTE supplemented with 0.5% deoxycholate and 0.1 M NaCl, and then 60 l of SDS–PAGE sample buVer was added to each washed sample and heated at 100 °C for 3 min. Solubilized proteins were run on 7% or 8–16% SDS or 11% Tris–Tricine–PAGE as indicated, treated with

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Xuoroenhance (RPI), dried, and exposed to Kodak XAR Wlm or phosphorimaging screens (Bio-Rad). Quantitation of radiolabeled bands was performed with Biorad Molecular Analyst software (version 2.1.1). For V8 digestion experiments, 100 mm dishes of cells were preincubated for 30 min with 2.5 ml DMEM lacking methionine and cysteine and labeled for the time indicated (usually 20 min) with 25 Ci [35S]amino acids. After immunoprecipitation and washing as described above, immunoprecipitates were resuspended in 80 l of 0.05 M NaPO4, 1% Triton X-100, 0.1 M NaCl (PTN buVer). 0.025 g of endoproteinase glu-C (V8) or 0.05 g lys-C (EMD/Calbiochem) was added and the suspension incubated at 25 °C for the times indicated. Where indicated, immunoprecipitated APP was Wrst denatured by adding 10 l of 2% SDS and heating at 100 °C for 5 min prior to diluting with 70 l of PTN buVer digesting with protease. Digestions were stopped with the addition of hot 5£ SDS sample buVer and heating at 100 °C for 3–5 min. Samples were run on 11% Tricine gels and processed to Wlm as above.

After washing, cells were lysed, immunoprecipitated, and APP and P83 bands quantitated by phosphorimaging. After plotting the results, the initial rate of APP labeling over the Wrst 10 min as a percent of steady state label (reached in 60 min) was calculated to be 173%/h for transfected cells and 142%/h for untransfected cells while the rate of P83 labeling was calculated to be 8.3%/h. Assuming that all cys and met residues in P83 (5) and APP (28) are labeled to the same extent, the P83 rate of labeling was multiplied by 5.6 (28/5), divided by 1.73 or 1.42 as appropriate, and multiplied by 100 to obtain the percent of APP that is proteolyzed by -secretase. (3) Pulse-chase labeling. Cells were labeled as in (2) in the presence of 50 M MG132 but after a 10 min pulse with [35S]amino acids, dishes were chased for 0–180 min. The peak of label that accumulated in APP after 10 min of chase and that accumulated in P83 over 3 h and divided by 3 were determined by phosphorimaging and the former multiplied by 5.6, divided by the label accumulated in APP after 10 min, and multiplied by 100.

Endoglycosidase H treatment

Statistical analysis of inhibitor eVects

Washed immunoprecipitates were rewashed 2£ in 0.05 M sodium citrate buVer (pH 5.5) and resuspended in 0.04 ml of 0.05 M citrate buVer, 0.04% in SDS and 1% in mercaptoethanol, heated for 5 min at 95 °C, the eluted protein transferred to a new tube and diluted with 1 vol. of 0.05 M citrate buVer. 1 mU of endoglycosidase H (EMD/ Calbiochem) was added and the samples incubated overnight at 37 °C. 5£ sample buVer was added to each prior to heating at 95 °C for 5 min.

The eVect of inhibitors on APP degradation was determined from pulse-chase experiments in the presence of the inhibitor using 8–9 chase points (usually 0–100 min). Aliquots of the lysate were removed prior to immunoprecipitation and counted. Loss of radiolabel from total solubilized protein from Wve independent experiments was determined to average 70 § 8% over the Wrst 80 min of chase. Radiolabel in the chase point as determined from imaging of the immature band and corrected to deviations in the radiolabel recovered in each lysate was Wt to a Wrst order decay curve using regression analysis, and compared to decay from a parallel plot from the same experiment in which no inhibitor was used.

Quantitation of APP proteolysis by -secretase Three methods were employed to evaluate the percent of translated APP that entered the secretory pathway and that was subsequently proteolyzed by -secretase. (1) Blotting with antibody. Cell media was collected after 1, 2, and 3 h, times previously shown to be in the linear range for APPs secretion, and each was concentrated from 4 to 0.2 ml with an Amicon centricon Wlter concentrator. Cell extracts (0.2 ml) and concentrated media were diluted with 5£ sample buVer, run on 8% Tris–glycine gels, and blotted with N-terminal antibody (Sigma). Blots were quantitated by densitometry. APPs recovered in the media was divided by that found in cells to obtain the rate of APPs released per hour as a percent of steady state intracellular APP. This value was divided by the rate of initial APP synthesis as a percent of APP at steady state obtained from continuous labeling experiments (see below). The latter were 173%/h for APP695 expressed in transfected cells and 142%/h for APP750 in untransfected cells. (2) Continuous pulse. Cell cultures in 30 mm diameter dishes were placed in DMEM without methionine/ cysteine in the presence of 50 M MG-132 to inhibit P83 degradation and continuously labeled from 0 to 180 min.

Results Intracellular APP traYcs to diVerent compartments in overexpressing CHO cells Newly translated APP becomes N-glycosylated by ER localized enzymes and a fraction of these molecules leave the ER to become O-glycosylated by Golgi localized enzymes. To assess whether transfected, overexpressing CHO cells represent a viable experimental system to study APP turnover and folding, the eYciency by which newly synthesized APP was able to reach the secretory pathway was measured by the appearance of APPs in the media and the rate of appearance of intracellular P83. Because P83 degradation is inhibited by MG-132 [26], the rate of accumulation of P83 in inhibited cells after either continuous pulse or pulse-chase labeling should represent APP processing by -secretase (Table 1). The results for transfected cells by three methods are in general agreement that the percentage of translated APP695 that is

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Table 1 Processing eYciency of APP in overexpressing versus untransfected cells

Cell line Transfected CHO cells (APP695) Untransfected CHO cells (APP750)

P83/APP £ 100

P83/APP £ 100

APPs/APP £ 100

Continuous labela 27 10

Pulse-labelb 37 —

Immunoblot 24 7.2

a This value represents the radiolabel in APP C-terminal P83 that accumulates in the presence of 50 M MG-132 per hour divided by the radiolabel that accumulates in APP695 per hour with a continuous pulse of [35S]amino acids times 100. See Materials and methods for details. b This value represents the radiolabel in APP C-terminal P83 that accumulates per hour in the presence of 50 M MG-132 divided by the radiolabel that accumulates in APP695 per hour with a 10 min pulse of [35S]amino acids and 1 h chase times 100. See Materials and methods for details.

processed by -secretase is 24–37%. That for APP750 expressed in untransfected cells is 7.2–10% and thus less eYcient. This may be accounted for by the diVerences in processing between APP695 and APP750, the former perhaps being more readily folded. The processing rate for overexpressed APP695 compares favorably with 30% eYciency shown for shedding of endogenously expressed APPs in PC12 cells [27]. N- and O-glycosylated APP appears as a band on SDS–PAGE (N + O) that migrates more slowly than N-glycosylated APP (N, Fig. 1). The immature or N-glycosylated APP is endoglycosidase H sensitive just after labeling as well as after a 70 min chase (see below). Cell associated immature APP is turned over (T1/2 D 35 min) more rapidly than that of cell protein as detected by pulse-chase labeling, immunoprecipitation with C20 antibody, and quantitation of APP695 immature band radiolabel (Fig. 1A). Disappearance of APP may be accounted for by secretion, endocytosis and degradation in the endocytic pathway, or by degradation within the secretory pathway. APP is turned over at about the same rate (T1/2 D 34 min) when precipitated instead with antibody against an N-terminal domain (Fig. 1A), demonstrating that its proteolysis/secretion is not vectorial with respect to exposure to lumenal or cytosolic compartments. Similar values for half-life were also found by treating cells with cycloheximide and following the loss of APP over an 80 min interval by immunoblotting with either C20 or N-terminal polyclonal antibody (results not shown). Moreover, APP immunoprecipitated with N-terminal domain antibody showed no sign of an immunoreactive faster migrating protein on SDS–PAGE that would be expected if its C-terminal tail was being proteolyzed by cytosolic proteases (results not shown). To distinguish between APP turnover due to secretion, endosomal/lysosomal degradation, and secretory pathway degradation, inhibitors of -secretase (1,10-phenanthroline, [28]), endosomal/lysosomal proton pumping (baWlomycin [29]), and a cysteine protease, which acts early in the secretory pathway [25], were employed alone or in combination. Phenanthroline alone or in combination with baWlomycin showed modest inhibition of APP turnover (T1/2 D 55, 60 min). Addition of E64 in combination with the above two classes of inhibitors resulted in complete interruption of APP turnover (Fig. 1). These results indicate that residual APP turnover seen when -secretase and endocytosis inhibitors are both present is accounted for by cysteine protease(s).

Two classes of protease inhibitors aVect APP degradation Since APP turnover was E64 sensitive, a spectrum of protease inhibitors was tested to ascertain their eVects on APP degradation in a pulse-chase experiment. An example of the results obtained is shown after 10 min pulse and 70 min chase (Fig. 2A) and by quantitation over a chase of 120 min from 8-point decay curves Wtted by regression analysis (Table 2). Intracellular loss of APP was found to be most sensitive to the class of cysteine protease inhibitors (E64, EST, and leupeptin [30]), insensitive to proteasome inhibitors MG-132 or ALLN [31], and modest sensitivity to speciWc proteasome inhibitor LC (Table 1, Fig. 2A). The aspartatic protease inhibitor pepstatin did not inhibit (Fig. 2A). The inhibitor proWle changes when cells were pretreated with 30 M tunicamycin for 1 h to prevent N-glycosylation. Although tunicamycin alone had no eVect on APP turnover rate (Table 2), MG-132 inhibited APP turnover in tunicamycin treated cells (Table 2, Fig. 3). Tunicamycin invokes a stress response as well as preventing N-glycosylation [32]. Stress response initiators, however, require longer than 1 h to induce expression of stress related proteins. Thus the channeling of N-glycosylation deWcient APP into a proteasome degradation pathway is more likely the result of incorrect glycosylation followed by misfolding. APP that accumulates in the presence of EST remained sensitive to endoglycosidase H (Fig. 2B). Brefeldin A collapses the Golgi, and simultaneous addition of nocodazole prevents retrograde transport of Golgi proteins to the ER [33]. Addition of brefeldin A and nocodazole to APP expressing cells inhibits APP secretion and prevents terminal glycosylation from occurring since only N-glycosylated APP was seen after labeling and chase (results not shown). APP turnover rate was slowed (T1/2 D 110 min) as would be expected from the interruption of secretion. Loss of APP label became completely sensitive to EST under these conditions (Fig. 2C), thus showing that EST sensitive APP degradation occurs in the ER. Titration curves for EST and MG-132 in tunicamyin treated and untreated cells (Fig. 3A and B) showed that inhibition of control cells with EST and tunicamycin treated cells with MG-132 occurred at <1 M. The concentration required for MG-132 inhibition of cellular proteolysis due to proteasomal degradation is a few micromolar

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Detection of tunicamycin induced conformational alterations by endoproteinase digestion of immunoprecipitated APP

Fig. 1. EVect of inhibitors on the degradation of APP695 in an expressing CHO cell line. (Top) Time course of APP degradation after immunoprecipitation with N or C terminal antibody. After a 10-min. pulse with [35S]amino acids, APP was chased and immunoprecipitated at the indicated chase intervals and proteins separated on 8% SDS–PAGE. (inset) Autoradiogram of immunoprecipitated AP during the chase. N is the Nglycosylated only APP while N + O is APP that has been both N- and Oglycosylated. Turnover of pulse-labeled APP after immunoprecipitation of chased protein with C20 antibody or N-terminal antibody was quantitated by integration of the N-band obtained by phosphorimaging. Bottom. EVect of protease inhibitor combinations on APP turnover after a 10-min pulse with radiolabeled amino acids. Dishes of nearly conXuent SV695 cultures were pulse-labeled with [35S]amino acids for 10 min, washed, and chased in the presence of 5 mM 1,10-phenanthroline (o-phen) to inhibit -secretase, 0.15 M baWlomycin (Baf) to inhibit degradation in the endosome/lysosome pathway, and/or 25 M E64 to inhibit cysteine proteases in the secretory pathway. Cells were extracted, APP precipitated with C20 antibody, and the APP N-glycosylated band seen by phosphorimaging quantitated as described in Materials and methods.

[34] and thus drug inhibition is in the range expected for that shown by cellular cysteine proteases and the proteasome. By contrast, little inhibition of control cells was seen with MG-132 at 2 M (Fig. 3A) while EST inhibited tunicamycin treated cells only by 22% at 5 M (Fig. 3B).

Since tunicamycin pretreatment alters the APP degradation pathway, pathway selection may be determined by conformational changes in the newly translated protein brought about by the absence of N-glycan. Those changes may be detected by altered susceptibility to endoproteinases when APP is immunoprecipitated under relatively mild conditions. A variety of endoproteinases were tested to see if there were diVerences in the proteolytic digest pattern of immunoprecipitated APP seen between control and tunicamycin pretreated cells. Of the endoproteinases tested, (trypsin, chymotrypsin, endoproteinase lys-C, subtilisin, and endproteinase glu-C) only glu-C (V8 protease) and lys-C showed a resulting peptide diVerence between control and tunicamycin treated cells. SpeciWcally, in the case of V8 digestion, two radiolabeled bands at 34 and 29 kDa (arrows in Fig. 4A) of a total of six generated from cells labeled for 20 min were resistant after 60 min V8 digestion in control cells but were eliminated in <20 min in V8 digestion of tunicamycin treated cells. In shorter labeling experiments (see below) the 34 kDa band was absent. The 29 kDa band was found in over 25 experiments with either short or longterm pulse/chase labeling. Pretreatment with A23187, which also elicits the unfolded protein response [35], did not aVect the sensitivity of the two proteolytic fragments (Fig. 4A). Treatment of immunoprecipitates with hot SDS prior to dilution and V8 digestion generated four fragments, all of which were also seen without SDS pretreatment. Lys-C digestion generated three fragments in immunoprecipitates from control cells that were not seen in tunicamycin treated cells (marked by asterisks in Fig. 4B). It was of interest to identify the origin of V8 proteolytic fragments on the linear map of APP to ascertain if the V8 fragments lost as a result of tunicamycin treatment encompass the N-glycosylation site. The sum of apparent molecular weights of the resulting Wve V8 fragments generated from 20 min pulse-labeled cells in the absence of heat and SDS treatments (8, 12, 20, 26, 31, and 34 kDa) totals 131 kDa, This total is a little larger than the apparent size of the holo-protein on SDS–PAGE (110 kDa). The 34 kDa band that appears in longer term experiments could be accounted for by either a partial fragment whose complete fragmentation is impeded by either glycosylation, folding, or other post-translational events. When the splice variant having 770 amino acids was expressed in the same background and labeled and digested with the 695 variant in a parallel experiment, the 31 kDa band was replaced by new band at 37–40 kDa (Fig. 5A). The APP770 splice variant encompasses the KPN protease inhibitor site, which begins at residue V290. Thus, the 29 kDa V8 fragment encompasses residue 289, the site of the KPN insertion. After limited V8 digestion of APP695, the resulting fragments were separated by centrifugation into those that remain bound to the protein A–agarose and those that were released into the supernatant (Fig. 5B). Since only a single band at

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Fig. 2. Inhibitor eVects on APP degradation. (A) EVect of the inhibitors present in a 60 min chase interval after a 10 min pulse (P) with [35S]amino acids. Concentrations are as shown in Table 1 with leupeptin (leu) at 10 M, pepstatin at 25 M, and PMSF at 1 mM. (B) Increased intracellular APP that accumulates in the presence of EST in a pulse-chase experiment remains endoglycosidase H sensitive. APP expressing CHO cultures were pulse-labeled and chased in the absence or presence of 10 M EST and immunoprecipitated from cell lysates after 0, 35, or 70 min of chase. Immunoprecipitates were treated or not treated with endoglycosidase H as described in Materials and methods, run on 8% SDS–PAGE, dried, and exposed to X-ray Wlm. (C) In brefeldin treated cells, APP degradation is completely sensitive to cysteine protease inhibitor EST. Dishes of APP expressing CHO cells were pretreated with 5 g/ml brefeldin A and 20 g/ml nocodazole for 15 min prior to labeling with [35S]amino acids for 10 min. Both drugs were present during the chase interval in which 10 M EST was also present (EST) or absent (Con). Error bars represent the SD, n D 3. Table 2 EVect of drugs on the half-life of intracellular APP in CHO cells Inhibitor

Concentration in media

Half-life (min)

None MG-132 E-64 EST ALLN Lactacystin -lactone BaWlomycin Tunicamycin Tunicamycin/lactacystin -lactone

42 M 25 M 25  M 2M 52 M 20 M 0.1 g/ml 10 g/ml 10 g/ml/20 M

35 30 120 70 25 67 39 26 110

12 kDa remained bound, it likely encompassed the C-terminal amino acids recognized by the protein A bound antibody. APP immunoprecipitated from tunicamycin treated cells ran with a slightly greater mobility than that from untreated cells. When directly compared on 16% Tricine

gels (Fig. 5C) only the 8 kDa V8 fragment from tunicamycin treated cells ran faster than that from control cells, thus showing that this fragment encompassed the N-glycosylation site (N467). All 11 of APP695 cysteine residues are localized to its Nterminal 187 amino acids. Six of these are employed in disulWde bridges that hold together the amino terminal folded growth factor like domain (L28-V123) whose structure has been solved at the 1.8 Å resolution [22]. Labeling of APP695 with [35S]amino acids in the presence of excess methionine to identify this cysteine-rich amino terminal domain resulted in the appearance of only the 26 and 20 kDa V8 fragments (Fig. 6A). Thus the latter two V8 fragments encompass residues from the amino terminal, growth factor like domain. Labeling of APP695 with a short pulse of 5 min (Fig. 6B) resulted in the loss of the 26 kDa fragment in samples pretreated or not pretreated with SDS and heat. Fragments at 34 and 29 kDa, however,

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Fig. 3. Titration of inhibitors EST and MG-132. Cells that had been untreated or pretreated with 10 g/ml tunicamycin for 30 min were pulse labeled for 10 min and chased for 70 min in the presence of diVerent concentrations of MG-132 (A) or EST (B). The amount of APP remaining at 70 min in each case was subtracted from that immediately after the pulse. The value obtained for each concentration of inhibitor was divided by that measured in the absence of inhibitor and multiplied by 100 to obtain percent inhibition. Each point is the average of two determinations.

appeared but converted to a 20 kDa fragment when pretreated with SDS and heat prior to V8 digestion. Thus, the 20 kDa fragment is related to either 29 or 34 kDa fragments or both, while the 26 kDa fragment is absent after a short pulse and thus not protected from V8 digestion under those labeling conditions. This V8 susceptibility of the 26 kDa fragment likely results from delayed folding of the growth factor domain driven by disulWde bond formation, thus leaving the incompletely folded domain accessible to protease. Altered conformation of pulse-labeled and chased APP as detected by V8 proteolysis The V8 proteolytic fragments of pulse-labeled and chased immunoprecipitated APP were compared in Fig. 7A. V8 digestion of the 7 min pulsed and then 45 min chased sample showed the appearance of a larger fragment at 34 kDa (arrow). The new fragment is not eliminated by reduction with dithiothrietol (DTT, Fig. 7B). Since a fraction of the chased APP is O-glycosylated, the new fragment may result from the presence of O-glycans that have been shown to occur on the amino terminal domain of the protein [36]. However, labeling and chasing in the presence of brefeldin A, which eliminates O-glycosylated APP, did not aVect the appearance of the new band (Fig. 7B). These results would indicate the occurrence of a late conformational change in APP structure that takes place even when APP is retained in the ER and prevented from terminal glycosylation in the Golgi.

Discussion As is the case with most secreted proteins, newly translated APP has several alternative fates. It can proceed on to the secretory pathway by leaving the ER, becoming further chemically modiWed in the Golgi, and localizing to the cell membrane where it is shed into the media by the action of -secretase. Alternatively, cell surface localized protein can internalize and become degraded within the endocytic pathway. The third possibility is that it can be degraded prior to accessing O-glycosylation enzymes in the Golgi. Quantitative pulse-chase labeling studies on PC12 cells showed that »30% of the radiolabel found in newly translated APP is recovered in the media as APPs [27] although primary neuronal cultures secrete only 10% of synthesized APP [37]. Translated APP that fails to exit the cell may be secreted but not recognized by antibody or, more likely, is intracellularly degraded [27]. In overexpressing the CHO cell line used in this study, 24–37% of APP695 is processed by the -secretase pathway. Endogenously expressed APP750 is less eYciently processed by -secretase. The processing eYciency for APP695 is similar to that found for endogenously expressed APP695 in PC12 cells (30%, [27]). Remaining protein is intracellularly degraded. Although it is not clear how much APP is intracellularly degraded in the brain without becoming terminally glycosylated, the fate of that protein may be pathologically relevant. The small inhibitory eVect of 1,10-phenanthroline, an inhibitor of -secretase, on APP turnover suggests that inhibition of secretion shifts more of cell surface destined APP into the endocytic pathway. In a similar manner,

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Fig. 4. V8 and Lys C digestion of immunoprecipitated APP from cells labeled in the absence or presence of tunicamycin or A23187. (A) 10 cm dishes of SV695 cells were pretreated with 30 M tunicamycin for 30 min or 5 M A23187 for 60 min and then labeled with [35S]amino acids for 20 min in the presence of the drug. Dishes were extracted and APP immunoprecipitated, washed, and digested with 0.025 g V8 protease for 0, 20, 40, or 60 min as indicated (Materials and methods) prior to separation of proteolytic fragments on 11% Tricine gels. In lanes marked SDS, immunoprecipitates were treated with 10 l of 2% SDS, heated at 100 °C for 5 min before dilution with digest buVer and 60 min V8 treatment. Arrows indicate 34 and 29 kDa bands that were absent in tunicamycin treated cells as well as after SDS and heat treatment prior to V8 digestion. Asterisks mark the position of six fragments seen in digests of control cells. Position of prestained marker proteins (50–10 kDa, Invitrogen) are as indicated. (B) Cells were pretreated with tunicamycin, labeled, and APP immunopreciptated as in (A) but digested with 0.05 g endoproteinase lys-C for 0, 20 or 60 min or for 60 min with prior treatments with sds and heat (sds). Marker proteins are as indicated. Asterisks identify proteolytic fragments found in control but not tunicamycin pretreated cells.

prevention of endocytic degradation brought about by baWlomycin, had little eVect on turnover of O- and N-glycosylated APP, but increased the secretion of APPs into the media ([40]; Hare, unpublished results). Predictably, simultaneous addition of endocytic and -secretase inhibitors in addition to a cysteine protease inhibitor eVectively eliminated APP turnover in intact cells (Fig. 1). Thus all APP695 turnover can be accounted for by the combination of secretion, endocytic removal and subsequent proteolyisis, or degradation on the secretory pathway by a cysteine protease. As shown in present study, cysteine protease inhibitors signiWcantly inhibit intracellular APP turnover, but aVect neither -secretase activity nor secretion since these inhibitors stimulate APPs appearance in the media (Hare, unpublished). In human neuronal cultures E64 also stimulated the secretion of APPs, but had no apparent eVect on turnover of mature or immature intracellular APP [41]. ER cysteine proteases previously identiWed include ERp72 and ER-60 [42] as well as a proteasome inhibitor insensitive pathway implicated in the degradation of an APP transmembrane domain probe [43]. An enzyme that degrades the drug bleomycin was shown to have cysteine protease activity that when over-expressed, increased secretion of A in cells also over-expressing APP. In those cells, however, the

over-expressed bleomycin hydrolase had no eVect on intracellular APP turnover, despite increasing the secretion of APPs [44]. When metabolic stress is imposed, APP is degraded by a system sensitive to classical proteasome inhibitors ALLN, MG-132, and LC. Imposed stress conditions that prevent N-glycosylation (e.g. tunicamycin) aVect APP degradation within an hour, thus making it unlikely that such eVects are dependent on transcriptional events. Longer treatment with tunicamycin (>12 h) is required to activate stress response enzymes [45]. Moreover, another stress inducer, the calcium ionophore A23187, had no eVect on APP turnover (Hare, unpublished) or on the V8 protease digest pattern of immunoprecipitated APP (Fig. 4A). There is a small eVect of proteasome inhibitors on APP turnover in the absence of stress conditions. This may represent proteasomal degradation of APP under non-stress conditions or some non-speciWcity on the part of the inhibitory drugs. Proteasome inhibitors have been shown to have an eVect on the -secretase pathway of APP metabolism [46,47] as well as on presenilin 1 metabolism [48]. APP has been reported to interact with a cytosolic heat shock protein in the presence of proteasome inhibitors but not other protease inhibitors [49]. Proteasome inhibitors were shown to block substantially

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Fig. 5. IdentiWcation of V8 endoproteinase fragments. (A) Twenty-nine kilodalton V8 fragment includes the KPI inset site. CHO cells expressing APP 695 and or APP770 were labeled for 20 min, immunoprecipitated, and digested for 0 or 60 min with 0.5 g V8 protease and the generated fragments run on 11% Tricine gels. In lanes marked sds, immunoprecipitates were Wrst treated with 10 l of 2% SDS and heat prior to dilution and V8 digestion. Asterisks mark the position of 29 and 37 kDa bands seen respectively with 695 and 770 digests. (B) Twelve kilodalton V8 fragment includes the C-terminal residues. After labeling of cells as above, immunprecipitation, and V8 digestion for 15, 30, or 45 min as indicated, labeled bands were separated into supernatant (F) and pellet (B, protein A-agarose bound) after centrifugation. Resulting fragments were run on 11% Tricine gels. Asterisks mark the 12 kDa fragment that remains bound to the protein A-agarose after digestion. (C) Eight kilodalton V8 fragment includes the N-glycosylation site. Untreated and tunicamycin treated cells were pulse-labeled for 20 min, APP immunoprecipitated, digested with V8, and the fragments run on a 16% Tricine gel. The 8 kDa fragment (denoted by line) from the sample obtained from tunicamycin treated cells runs ahead of that from control cells.

the degradation of C99 and thus compete with -secretase for its substrate [50,51]. Thus, the proteasome appears to have a dual function in APP metabolism, degrading both C99 as well as APP that misfolds as a result of under Nglycosylation or potentially other causes. Expression of an APP695 mutant lacking both potential N-glycosylation sites in COS cells resulted in sluggish secretion of APPs and aberrant intracellular processing [53] although the degradation pathway of intracellular APP was not evaluated in this study. Some loss of proteasome function has been reported in age and sex matched Alzheimer’s disease brains [52], and thus the eVect of stress conditions on proteasome degradation of APP in human brain may require further examination. The absence of secondary structure for the amino terminal domain of APP [38,39] may account for its enhanced susceptibility to intracellular proteolytic systems. Conformational plasticity may even direct the protein into alternative degradative pathways. Protein folding/misfolding is diYcult to study in situ. The likelihood that APP is misfolded in the presence of tunicamycin oVered the opportunity to probe conformational alterations by proteolytic digestion of the immunoprecipitated protein. These experiments necessitated that pulse-labeled APP could be extracted and immunoprecipitated cleanly

without perturbing its folded structure. Pulse-labeled APP extracted and immunoprecipitated in the presence of non-ionic detergent appeared as a single 110 kDa band on SDS–PAGE. V8 or lys-C endoproteolysis of protein A–agarose bound APP was able to diVerentiate between that labeled in the presence and absence of tunicamycin (Fig. 4A). No diVerence was seen when cells were stressed with A23187 (Fig. 4B), showing that induction of stress conditions by itself did not bring about altered proteolytic sensitivity. An alternative possibility to explain a diVerence in protease sensitivity is that tunicamycin exposes a cryptic proteolytic site resulting from the absence of attached high mannose N-linked oligosaccharides. Although the nearest glu or asp is E481, 14 residues removed from the glycosylation site (N467), this explanation cannot be ruled out for V8 digestion since the 32 kDa band that becomes V8 sensitive in cells labeled in the presence of tunicamycin could encompass both the KPN insertion site (V289) and the one N-glycosylation site (N467). This distance includes 21.46 kDa of sequence, less than the 32 kDa estimated from its migration on SDS–PAGE. Based upon mobility diVerences of V8 fragments between control and tunicamycin treated cells (Fig. 5C), however, the 8 kDa fragment was tentatively identiWed as encompassing the single N-glycosylation

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Fig. 6. Correlation of APP V8 fragments with amino acid sequence. (A) Cells were labeled for 15 min with [35S]sulfur amino acids (met/cys) or with [35S]sulfur amino acids in the presence of 5 mM cold methionine (cys) added 30 min prior to labeling. Immunoprecipitated APP was digested with V8 protease for 0, 60 min or for 60 min after pretreatment with 2% SDS and heating. Only 26 and 20 kDa fragments are labeled in the presence of cold methionine. (B) Immunoprecipitated APP was digested with V8 protease for 60 min as indicated after a 5 or 20 min pulse with [35S]amino acids. Arrowheads mark the 34 and 29 kDa fragments.

Fig. 7. Appearance of an additional APP V8 band after long-term chase of pulse-labeled APP expressing cells. (A) Cells were pulse-labeled for 10 min and then chased for 0 (pulse) or 60 (chase) min. APP was immunoprecipitated, and digested with V8 protease for 0 or 60 min or Wrst treated with hot SDS and then digested with V8 for 60 min (sds). An additional V8 band appears in sample from chased cells (arrow). (B) The same experiment as in (A) was repeated except that cells were treated with 5 g brefeldin A for 15 min prior to labeling, during labeling, and during the chase interval. The appearance of a new V8 proteolytic fragment only in chased cell, unaVected by brefeldin, is indicated by the arrow. In lanes 5 and 10 immunoprecipitated samples were treated with 5 mM dithiothreitol during V8 digestion.

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site. More to the point is that lys-C digestion was also able to diVerentiate between tunicamycin treated and control APP even though the nearest lysine residues are K435 and K493, both far removed from the putative N467 glycosylation site. The notion that endoprotease sensitivity of immunoprecipitated APP is aVected by subtle conformational alterations of APP is the most likely of several possible interpretations to explain the results. APP may be associated with other accessory chaperone proteins that protect it from proteolytic degradation. Accessory proteins may be prevented from binding to APP by tunicamycin treatment that eliminates N-glycans essential for this interaction. APP was previously shown to associate intracellularly with calnexin [17], Bip [16], and more recently with F-spondin [53]. Alternatively, tunicamyin was shown in another study to alter the metabolism of APP as a result of an indirect eVect of the drug on another, unidentiWed cellular protein [54]. In this latter study, genetic removal of N-glycosylation sites had no eVect on APP secretion into the media although tunicamycin decreased the rate of APP secretion. In that study, however, the eVect of site directed mutation of the two Nglycosylated amino acid residues on intracellular turnover of APP was not determined. The APP domain from residues 290–583, deWned by limited trypsin digestion, was shown by NMR to encompass three -helical domains, one of which includes the N467 glycosylation site [55]. It is interesting to speculate that loss of glycosylation somehow perturbs the three helix bundle and thus opens up the protein enough to direct it to the proteasome pathway. Two conformational alterations occurring during APP maturation were detected by V8 digestion. Short pulses of APP failed to detect the 25 kDa V8 fragment that encompases the N-terminal, cysteine-rich, growth factorlike domain. This data indicates that this domain takes a few minutes after translation is complete to establish its Wnal V8 resistant conformation. An additional proposed conformational change occurs by the completion of a 45 min chase when a novel V8 peptide fragment appears that is not found after a 7–15 min pulse. This apparent change in V8 sensitivity is not due to O-glycosylation because cells labeled and chased in the presence brefeldin were not O-glycosylated but still exhibited the additional V8 fragment not seen in the absence of chase (Fig. 7B). Whether this change in protease sensitivity reXects an additional conformational alteration necessary for APP to escape the ER and thus avoid degradation in the ER is not clear. Protein conformational changes and protein–protein interactions between APP and other proteins that occur prior to its escape from the ER may signiWcantly control APP processing. External stresses that precipitate late onset Alzheimer’s disease may aVect APP processing in a manner similar to that shown by tunicamycin and even redirect newly translated APP from the -secretase to the -secre-

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