Article |
Address correspondence to David Schubert, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 453-4100, ext. 1528. Fax: (858) 535-9062. E-mail: schubert{at}salk.edu
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Abstract |
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Key Words: amyloid precursor protein; proteasome; beta amyloid; secre-tion; MOCA
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Introduction |
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Results |
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To determine the effect of MOCA expression on Aß peptide production, we transfected the MOCA gene into a B103 cell line, which contains the FAD Swedish form of APP (APPsw) and secretes Aß peptides into the medium (Xu et al., 1999). The levels of APPsw secretion were significantly decreased in cells expressing MOCA (Fig. 3 A). The intracellular levels of APPsw were also decreased by MOCA (Fig. 3 B). Therefore, the effects of MOCA on APPsw secretion were similar to those on wild-type APP (Fig. 1). Aß secretion into the culture medium was then measured by immunoprecipitation and radioautography (Fig. 3 C) and independently by the sandwich ELISA (Fig. 3 D). The level of Aß in the medium derived from B103 (APPsw/MOCA) cells was considerably decreased compared with B103 (APPsw) cells (Fig. 3, C and D). We could not detect the intracellular expression of Aß in any of the B103 (APP695) or B103 (APPsw) cell lines using the techniques employed to assay extracellular Aß. The above data show that MOCA decreases the secretion of both APP and Aß.
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To confirm the above data on protein degradation and identify the mechanism which might be involved in the MOCA regulation of APP degradation, the effects of additional protease inhibitors were tested. Cells were treated with chloroquine (50 µM), NH4Cl (5 mM), phosphoramidon (2 µM), N-acetyl-leucyl-norleucinal (ALLN) (27 µM), N-Ac-Leu-Leu-methioninal (ALLM) (27 µM), lactacystin (20 µM), clasto-lactacystin ß-lactone (20 µM), or epoxomicin (10 µM) for 4 and 16 h, and the secretion and the intracellular levels of APP were measured by Western blotting. Lactacystin, ß-lactone, and epoxomicin, which specifically target the proteasome and do not inhibit lysosomal protein degradation (Craiu et al., 1997; Fenteany and Schreiber, 1998; Meng et al., 1999), effectively restored the level of APP secretion in MOCA-containing cells (Fig. 6 A). Similar effects were also observed in cells treated with the peptide aldehydes, ALLN and ALLM, which inhibit proteasomes but also inhibit lysosomal cysteine proteases and calpains (Sherwood et al., 1993; Zhang et al., 1999). In contrast, two lysosomal protease inhibitors, chloroquine and ammonium chloride (Caporaso et al., 1992), did not reverse the MOCA effects on APP secretion. As another negative control, phosphoramidon, a metalloprotease inhibitor, did not affect APP secretion, consistent with the previous reports (Parvathy et al., 1998). The effects of these inhibitors on the intracellular levels of APP were also tested and were comparable to the corresponding effects on APP secretion (Fig. 6 B). These data were confirmed by a kinetic pulsechase analysis. Cells with and without MOCA were labeled for 10 min with [35S]methionine and chased in complete medium in the presence of 10 µM MG132. Fig. 7 A shows that nascent APP molecules are stabilized by proteasome inhibition and that the rate of loss of intracellular APP in cells expressing MOCA becomes similar to that of MOCA-deficient cells. These data strongly support the involvement of proteasomes in the regulation of APP degradation by MOCA.
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MOCA reduces cell-substratum adhesion
The profound decrease in APP secretion caused by MOCA should have biological consequences. APP has multiple functions, but its ability to mediate cell-substratum adhesion has been well-documented (Schubert et al., 1989; Chen and Yankner, 1991; Schubert and Behl, 1993; Jin et al., 1994; Beher et al., 1996; Coulson et al., 1997). Because the inhibition of APP synthesis by antisense nucleotides blocks cell-substratum adhesion (Coulson et al., 1997), it would be predicted that the expression of MOCA has a similar effect and that this phenotype would be reversed by proteasome inhibitors, which restore APP accumulation and secretion. This is indeed the case when the ability of B103 nerve cells, expressing APP, MOCA, or both, to adhere to the extracellular matrix protein laminin are compared. Fig. 8 A shows that the expression of APP695 in B103 cells, which normally lack APP, increases the rate of cell-substratum adhesion to laminin. The expression of MOCA alone in B103 cells lacking APP695 has no effect on adhesion. In contrast, cells expressing both APP and MOCA adhere to laminin at a rate which is indistinguishable from cells expressing no APP. However, when cells expressing APP and MOCA are exposed to the proteasome inhibitor lactacystin for 5 h before the adhesion assay there is an increase in the rate of adhesion to a level indistinguishable from cells expressing APP but no MOCA (Fig. 8 B). Lactacystin does not alter the rate of adhesion of cells which do not express MOCA or wild-type B103 cells. The adhesion data are in agreement with those which show that MOCA expression increases APP breakdown and decreases its secretion (Figs. 1, 2, and 5) and that APP accumulation and secretion can be restored by proteasome inhibitors (Figs. 5 and 6).
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Discussion |
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APP is a type I membrane-spanning protein whose secretion is regulated by a variety of factors including growth factors, neurotransmitters, phorbol esters, extracellular matrix molecules, and stress (Schubert et al., 1989; Mills and Reiner, 1999; De Strooper and Annaert, 2000). The mechanisms involved in the regulation of APP secretion include alternations in APP phosphorylation (Caporaso et al., 1992), the modification of protein glycosylation (Galbete et al., 2000), alternative in gene splicing (Shepherd et al., 2000) and transcription (Ciallella et al., 1994), and also changes in protein degradation (Checler et al., 2000). Although these mechanisms are diverse, it is likely that they are shared with many type I membrane proteins. APP is probably the most studied molecule of this class because of its medical importance.
The intracellular sites of APP metabolism still remain controversial with -secretase cleavage described at the plasma membrane, in the Golgi, or in the post-Golgi secretory vesicles (Mills and Reiner, 1999). Similarly, ß- and
-secretase activities have been identified in the trans-Golgi network (Xu et al., 1997), the endoplasmic reticulum/intermediate compartment (Cook et al., 1997), and the endosome/lysosome system (Haass et al., 1992). Our data do not distinguish between these alternatives but show that secretion of APP and Aß are both reduced in the presence of MOCA. Because we do not observe an intracellular accumulation of APP or intermediate breakdown products of APP, the effect of MOCA on APP processing most likely occurs before APP reaches the cell surface. The pleotropic effects of MOCA on APP degradation may occur in the ER, which is consistent with the subcellular localization of both PS1 and MOCA (Doan et al., 1996; Kashiwa et al., 2000; Xia et al., 2000.
Proteins destined for membranes or secretion are translocated into the ER, folded, assembled, and transported to a cellular destination or secreted (Hurtley and Helenius, 1989). Incorrectly folded proteins, unassembled subunits of multisubunit complexes, and mutated proteins are rapidly eliminated from the cell; misfolded proteins are translocated to proteasomes and degraded (Suzuki et al., 1998). However, in the case of unfolded proteins the conventional route may not be taken because excessive accumulation of these macromolecules within the ER lumen might lead to their aggregation and precipitation, thereby blocking the secretory pathway (Klausner and Sitia, 1990). Because APP binds to the molecular chaperones Bip/G-RP78 and HSC73 and misfolded proteins bound to Bip/GRP78 are degraded, it has been suggested that APP can be retained in the ER as a nascent polypeptide and degraded (Yang et al., 1998; Kouchi et al., 1999). This idea is consistent with data suggesting that the degradation pathway for APP in the ER participates in APP secretion and is distinct from -secretase cleavage (Bunnell et al., 1998) and that the proteasome is involved in APP processing (Hare, 2001). Because APP secretion is restored by proteasome inhibitors and intracellular accumulation of APP is not observed in the absence of proteasome inhibitors, the secretion pathway is not blocked by MOCA. Consistent with previous data (Bunnell et al., 1998), we were unable to isolate a ubiquitin-conjugated APP complex even after the treatment with proteasome inhibitors, and we did not observe any APP intermediates caused by MOCA expression.
Proteasome inhibitors increase the stability of both total and pulse-labeled APP in cells (Figs. 6 and 7) and restore the secretion rate to near control levels in MOCA-expressing cells (Fig. 5 C and Fig. 6). They also increase the intracellular accumulation of MOCA over long periods of time (Fig. 7). Although it cannot be formally ruled out in any studies that proteasome inhibitors block an unknown function to generate the resultant phenotype, we feel that this is unlikely in the experiments described here for two reasons. First, in an experiment where cells are pulse labeled for 10 min and chased in the presence of a proteasome inhibitor there was a rapid inhibition of APP breakdown, bringing it up to the rate of loss of intracellular APP in non-MOCA cells due to secretion (Fig. 7). The effect occurred well before any significant accumulation of other proteins like MOCA. The accumulation in proteins in the ER in the presence of proteasome inhibitors can sometimes indirectly inhibit the secretory pathway; in our case, APP secretion is enhanced. Second, a variety of structurally diverse proteasome inhibitors reversed the effect of MOCA on APP secretion, whereas -secretase inhibitors, lysosomal protease inhibitors, and a metalloprotease inhibitor have no effect. These results strongly suggest that proteasomes are involved in MOCA-induced APP degradation. These data also suggest that nascent APP may pass through an ER environment in which complexes for both protein degradation and protein assembly coexist. In the absence of MOCA, the precursor protein follows the secretory pathway, whereas the expression of MOCA directs a significant fraction of APP to proteasomes where it is degraded. This novel MOCA-mediated pathway presents yet another way in which the expression of specific proteins may be controlled.
Aß amyloid peptides are also generated from various cellular compartments, including the ER, the Golgi apparatus, the trans-Golgi networks, lysosomes, and endosomes, through either a constitutive secretion pathway or through an endocytotic pathway in which cell surface APP moves to the lysosomes or endosomes where Aß is produced (Mills and Reiner, 1999; De Strooper and Annaert, 2000). Aß secretion is decreased by MOCA in our studies, probably because of the rapid degradation of APP, therefore reducing the APP source for the generation of Aß. Because the overall production of Aß is reduced by MOCA, MOCA expression may help to reduce Aß production in the central nervous system. It follows that the loss of MOCA function could lead to AD.
PS1 controls several aspects of APP metabolism (Sisodia, 2000) and protein breakdown (Niwa et al., 1999; Katayama et al., 2000). PS1 is also required for "-secretase" cleavage of Notch-1. However, the proteolytic cleavage of APP and Notch are differentially facilitated (Capell et al., 2000). The PS1-dependent
-secretase processing of APP appears to be nonselective and occurs at multiple sites within the APP transmembrane domain. This is in contrast to the highly selective PS1-dependent processing of Notch (Yu et al., 2001). Our data show that, unlike PS1, MOCA has a minimal effect on Notch-1 degradation and that of five additional membrane proteins (Fig. 1). We also studied the effects of several selective
-secretase inhibitors on APP secretion modulated by MOCA (Fig. 2 B). Consistent with previous observations, there was little effect of these
-secretase inhibitors on APP secretion in the absence of MOCA. In contrast to the proteasome inhibitors, among the
-secretase inhibitors tested, only
-secretase inhibitors III and IV and Calp III partially revert the effect of MOCA effect on APP secretion.
-Secretase inhibitor II, which has no effect, is thought to be an aspartyl protease inhibitor, which selectively inhibits the
-secretase cleavage of APP and Notch-1 proteolysis (Wolfe et al., 1998; 1999; De Strooper et al., 1999; Berezovska et al., 2000; Esler et al., 2000). The other
-secretase inhibitors including III, IV, V, and the potent calpain inhibitor III are dipeptidyl aldehydes targeting cysteine proteases and may block proteasome enzymes, accounting for the inconsistency of their effects on APP secretion affected by MOCA. Finally, the additive effect of PS1 on MOCA-decreased APP secretion was demonstrated (Fig. 2 C). This observation is consistent with the fact that PS1 lowers the secretion of APP in yeast (Evin et al., 2000). MOCA also functions effectively in the presence of mutant PS1. Together, the above data show that the effect of MOCA is very different from that of the PS1-associated
-secretase activity. MOCA contains an SH3 domain, interacts with the Crk adaptor protein, and shares a 40% homology with DOCK180 (Kashiwa et al., 2000). Because DOCK180 interacts with Rac and other small G-proteins, MOCA may also interact with small G-proteins involved in protein breakdown or mediate phosphorylation events between proteins involved in APP trafficking and metabolism.
APP is a potent cell adhesion molecule which binds to both heparin and other extracellular matrix molecules (Schubert et al., 1989; Beher et al., 1996; Wu et al., 1997). Cells which express APP adhere more rapidly to extracellular matrix proteins than cells which do not (Fig. 8) (Schubert and Behl, 1993). Conversely, the inhibition of APP synthesis by antisense techniques blocks cell-substratum adhesion (Coulson et al., 1997). The expression of MOCA in B103 nerve cells, which do not make APP, has no effect on cellular adhesion to laminin, but reduces the rate of adhesion of cells which express APP (Fig. 8). Therefore, the effect of MOCA on cell-substratum is tightly coupled to APP expression. When the rapid breakdown of APP caused by MOCA is blocked by proteasome inhibitors, there is a return to the normal rate of adhesion for B103 cells expressing APP (Fig. 8 B). The simplest explanation for these results is that in the presence of MOCA newly synthesized APP is degraded at such a rapid rate that very little reaches the cell surface so that it is unable to participate in adhesive interactions. Because one adhesion-dependent biological activity of APP is the regulation of neurite outgrowth (Jin et al., 1994), the expression of MOCA during development could regulate cell migration and axon pathfinding. Therefore, the aberrant expression of MOCA or its loss could have pathological consequences in addition to those caused by altered Aß secretion.
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Materials and methods |
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Cells and transfection
The neuronal cell line B103 (Schubert et al., 1974) was grown in DME supplemented with 10% heat-inactivated FBS. B103 cells were stably transfected with APP695 using G418 selection (Schubert et al., 1989) and with various plasmids by Lipofectamine 2000 (Invitrogen) using puromycin selection for MOCA or hygromycin for PS1. The stably transfected cells were subsequently cloned and screened for protein expression by Western blot analysis.
Western blotting, metabolic labeling, immunoprecipitation, and ELISA
For Western blotting, cells were washed twice with ice-cold PBS and lysed in lysis buffer (1% Triton, 50 mM Hepes, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 10 mM Na4P2O7, plus a mixture of protease inhibitors [Complete Mini; Roche]). For secreted protein analysis, semiconfluent cultures (106 cells in 100-mm tissue culture dishes) were washed twice with serum-free medium and incubated for 20 h in 4 ml serum-free medium. The growth conditioned medium was then desalted by passage through a Sephadex G25 column, and 10% of the material was used to determine protein content. For a given experiment, equal amounts of protein (usually 15 µg) were lyophilized and resuspended in 50 µl of sample buffer. Protein concentrations were determined by Coomassie Plus (Pierce Chemical Co.). The same amount of protein from each sample was separated on Novex precast 10% polyacrylamide gels (Invitrogen) and transferred to Immobulin membranes (Millipore). The membranes were blocked with 5% nonfat milk in Tris-buffered saline for 1 h at room temperature. After overnight incubation at 4°C with primary antibodies, the antigens were detected with HRP-conjugated secondary antibodies (Bio-Rad Laboratories) using an ECL kit (Amersham Pharmacia) and exposed to film. For pulsechase experiments, cells were grown to 80% confluency and incubated in methionine-free DME for 90 min. Cells were labeled with [35S]methionine (400 µCi/ml in methionine-free DME plus 5% dialyzed FBS) for 10 min at 37°C, and the medium was replaced with serum-free DME medium plus N1 supplement (Sigma-Aldrich). The media were collected, and cells were lysed after different time periods. Protein concentrations were determined, and the same amounts of protein were immunoprecipitated with antibodies at 4°C overnight. 25 µl of antimouse IgG agarose (Roche) were then added to each sample and incubated at 4°C for 2 h on a rocker platform. The immunoprecipitates were collected by centrifugation and washed four times with the washing buffer (0.1% Triton, 20 mM Hepes, 150 mM NaCl, 10% glycerol). The agarose beads were resuspended in 30 µl SDS-PAGE sample buffer and boiled for 3 min to release the proteins. After 2 min of centrifugation, the supernatants were separated on 10% Tris-glycine gels. For Aß analysis, 1020% acrylamide Tricine gels and longer incubation times were used. The gels were dried and subjected to autoradiography and quantitated by NIH image. Aß production was also measured by a sensitive fluorescence-based sandwich ELISA assay using a kit from Biosource International according to the manufacturer's instructions.
Northern hybridization and RT-PCR
Total RNA was isolated from the cells and brain tissues using Trizol reagents (Invitrogen), and mRNA was purified using a mRNA purification kit (Amersham Pharmacia Biotech). 2 µg of mRNAs from each sample were separated on 1% agarose gels and transferred onto Zeta membranes (Bio-Rad Laboratories, Inc.). Northern hybridization was performed in a UltraHyb buffer (Ambion) with an APP cDNA fragment probe labeled with 32P-dCTP using a rediprime kit (Amersham Pharmacia Biotech). RT reactions were performed for each RNA sample using 1 µg of total RNA in RT buffer composed of 10 mM DTT, 20 µM each of dATP, dCTP, dGTP, and dTTP, and 1 µM of oligo (dT). The solution was heated to 65°C for 5 min and cooled to 37°C for 10 min, and then incubated in the presence of 25 U of AMV RT at 42°C for 1 h. Master mixes for the PCR reactions were used for each sample. The PCR reaction mixture contained forward and reverse primers (1020 pmol each), dNTPs (200 µM each as final concentration), 1x PCR buffer, Taq DNA polymerase (0.5 U) (Roche), and 1 µl of the RT mixture as the source of cDNA. The primers used for PCR reaction were as follows: 5'-ATGGATGCAGAATTCCGACATGAC-3' (forward) (nt 1,9331,956) and 5'-CTAGTTCTGCATCTGCTCAAAGAA-3' (reverse) (nt 2,2352,212) for the APP gene (sequence data available from GenBank/EMBL/DDBJ under accession no. Y00264). Amplification was performed at 94°C for 40 s, 56°C for 1 min, and 72°C for 1 min, for 35 cycles. The PCR reactions with primers and RNAs but without the RT reaction were conducted as controls. After amplification, each sample was electrophoresed on a 1.5% agarose gel visualized by ethidium bromide staining.
Adhesion assay
The cell adhesion assays were performed as described previously (Schubert et al., 1989). Exponentially B103 (APP695/Vector) and B103 (APP695/MOCA) cells were labeled with [3H]leucine for 15 h. The cells were pipetted from the culture dishes and washed three times by centrifugation with Hepes buffered medium containing 0.4% BSA (Calbiochem). No trypsin or chelating reagents were used. Aliquots of 0.2 ml containing 5 x 104 cells were pipetted into 35-mm plastic Petri dishes coated with 5 µg mouse laminin and 2 ml of the above medium. The cells did not attach to uncoated dishes. At the indicated times, the dishes were swirled 10 times, the medium was aspirated, the remaining attached cells were dissolved in 3% Triton X-100, and their isotope content was determined. The data are plotted as the percent of input cells (radioactivity) that adhered at the indicated time and are presented as the average of triplicate plates. Variation between duplicates was <5%.
Immunostaining and laser confocal imaging
Cultured cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 30 min. The fixed cells were blocked in 2% BSA in PBS for 1 h and incubated with primary antibodies followed by fluorescent-conjugated secondary antibodies (Molecular Probes). The cells were then mounted under glass coverslips with antifading media containing 4% N-propyl gallate (Sigma-Aldrich). The cells were examined with a Carl Zeiss MicroImaging, Inc. LSM 5 PASCAL laser scanning microscope. 0.5-µM-thick serial optical sections of the cells were recorded using the Carl Zeiss MicroImaging, Inc. LSM 5 Image Examiner software to obtain images with pixel intensity within a linear range.
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Footnotes |
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Acknowledgments |
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Submitted: 30 October 2001
Revised: 21 May 2002
Accepted: 31 May 2002
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References |
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Beher, D., L. Hesse, C.L. Masters, and K. Beyreuther. 1996. Regulation of amyloid precursor protein (APP)-binding to collagen and mapping of the binding sites on APP and collagen type I. J. Biol. Chem. 271:16131620.
Borchelt, D.R., M.K. Lee, G. Thinakaran, T. Ratovitsky, C.M. Prada, G. Kim, M. Seeger, A. Levey, S. Gandy, N.G. Copeland, et al. 1996. Familial Alzheimer's disease-linked presenilin 1 variants elevate Aß1-42/1-40 ratio in vitro and in vivo. Neuron. 17:10051013.[Medline]
Bunnell, W.L., H.V. Pham, and C.G. Glabe. 1998. Gamma-secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein. J. Biol. Chem. 273:3194731955.
Caporaso, G.L., S.E. Gandy, J.D. Buxbaum, and P. Greengard. 1992. Chloroquine inhibits intracellular degradation but not secretion of Alzheimer ß/A4 amyloid precursor protein. Proc. Natl. Acad. Sci. USA. 89:22522256.[Abstract]
Chen, M., and B.A. Yankner. 1991. An antibody to ß amyloid and the amyloid precursor protein inhibits cell-substratum adhesion in many mammalian cell types. Neurosci. Lett. 125:223226.[Medline]
Chen, Q., H. Yoshida, D. Schubert, P. Maher, M. Mallory, and E. Masliah. 2001. Presenilin binding protein is associated with neurofibrillary alterations in Alzheimer's disease and stimulates tau phosphorylation. Am. J. Pathol. 159:15971602.
Cook, D.G., M.S. Forman, J.C. Sung, S. Leight, D.L. Kolson, T. Iwatsubo, V.M. Lee, and R.W. Doms. 1997. Alzheimer's Aß(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat. Med. 3:10211023.[Medline]
Craiu, A., M. Gaczynska, T. Akopian, C.F. Gramm, G. Fenteany, A.L. Goldberg, and K.L. Rock. 1997. Lactacystin and clasto-lactacystin ß-lactone modify multiple proteasome ß-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation. J. Biol. Chem. 272:1343713445.
De Strooper, B., and W. Annaert. 2000. Proteolytic processing and cell biological functions of the amyloid precursor protein. J. Cell Sci. 113:18571870.
Doan, A., G. Thinakaran, and D.R. Borchelt. 1996. Protein topology of presenilin 1. Neuron. 17:10231030.[Medline]
Evin, G., D. Le Brocque, J.G. Culvenor, D. Galatis, A. Weidemann, K. Beyreuther, C.L. Masters, and R. Cappai. 2000. Presenilin I expression in yeast lowers secretion of the amyloid precursor protein. Neuroreport. 11:405408.[Medline]
Fenteany, G., and S.L. Schreiber. 1998. Lactacystin, proteasome function, and cell fate. J. Biol. Chem. 273:85458548.
Galbete, J.L., T.R. Martin, E. Peressini, P. Modena, R. Bianchi, and G. Forloni. 2000. Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway. Biochem. J. 348:307313.[CrossRef][Medline]
Greenwald, I. 1998. Lin-12/Notch signaling lessons from worms and flies. Genes Dev. 12:17511762.
Hare, J.F. 2001. Protease inhibitors divert amyloid precursor protein to the secretory pathway. Biochem. Biophys. Res. Commun. 281:12981303.[CrossRef][Medline]
Hurtley, S.M., and A. Helenius. 1989. Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5:277307.[CrossRef]
Jin, L.-W., H. Ninomiya, J.-M. Roch, D. Schubert, E. Masliah, D.A.C. Otero, and T. Saitoh. 1994. Peptides containing RERMS sequence of amyloid ß/A4 protein precursor bind cell surface and promote neurite extension. J. Neurosci. 14:54615470.[Abstract]
Katayama, T., K. Imaizumi, N. Sato, K. Miyoshi, T. Kudo, J. Hitomi, T. Morihara, T. Yoneda, and F. Gomi. 2000. Presenilin-1 mutations down-regulate the signalling pathway of the unfolded protein response. Nat. Cell Biol. 1:479485.[CrossRef]
Kim, K.S., G.Y. Wen, C. Bancher, C.M.J. Chen, V.J. Sapienza, H. Hong, and H.M. Wisniewski. 1990. Detection and quantitation of amyloid ß-peptide with 2 monoclonal antibodies. Neurosci. Res. Comm. 7:113122.
Kouchi, Z., H. Sorimachi, K. Suzukui, and S. Ishiura. 1999. Proteasome inhibitors induce the association of Alzheimer's amyloid precursor with Hsc73. Biochem. Biophys. Res. Commun. 254:804810.[CrossRef][Medline]
Marambaud, P., J. Shioi, A. Georgakopoulos, S. Sarner, V. Nagy, L. Baki, P. Wen, S. Ethimiopoulos, Z. Shao, T. Wishiewski. et al. 2002. A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions. EMBO J. 21:19481956.
Meng, L., R. Mohan, B.H.B. Kwok, M. Elofsson, N. Sin, and C. Crews. 1999. Expoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo anti-inflammatory activity. Proc. Natl. Acad. Sci. 96:1040310408.
Naruse, S., G. Thinakaran, J.J. Luo, J.W. Kusiak, T. Tomita, T. Iwatsubo, X. Qian, D.D. Ginty, D.L. Price, D.R. Borchelt, et al. 1998. Effects of PS1 deficiency on membrane protein trafficking in neurons. Neuron. 21:12131221.[Medline]
Parvathy, S., I. Hussain, E.H. Karran, A.J. Turner, and N.M. Hooper. 1998. Alzheimer's amyloid precursor protein -secretase is inhibited by hydroxamic acid-based zinc metalloprotease inhibitors: Similarities to the angiotensin converting enzyme secretase. J. Biochem. 37:16801685.
Scheuner, D., C. Eckman, M. Jensen, X. Song, M. Citron, N. Suzuki, T.D. Bird, J. Hardy, M. Hutton, W. Kukull, et al. 1996. Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's Disease. Nat. Med. 2:864870.[Medline]
Schubert, D., S. Heinemann, W. Carlisle, H. Tarikas, B. Kimes, J. Patrick, J.H. Steinbach, W. Culp, and B.L. Brandt. 1974. Clonal cell lines from the rat central nervous system. Nature. 249:224227.[Medline]
Schubert, U., L.C. Anton, J. Gibbs, C.C. Norbury, J.W. Yewdell, and J.R. Bennink. 2000. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature. 404:770774.[CrossRef][Medline]
Sherrington, R., E.I. Rogaev, Y. Liang, E.A. Rogaeva, G. Levesque, M. Ikeda, H. Chi, C. Lin, G. Li, and K. Holman. 1995. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature. 375:754760.[CrossRef][Medline]
Sherwood, S.W., A.L. Kung, J. Roitelman, R.D. Simoni, and R.T. Schimke. 1993. In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal. Proc. Natl. Acad. Sci. USA. 90:33533357.[Abstract]
Sinha, S., and I. Lieberburg. 1999. Cellular mechanisms of ß-amyloid production and secretion. Proc. Natl. Acad. Sci. USA. 96:1104911053.
Sisodia, S.S. 2000. An accomplice for -secretase brought into focus. Science. 289:22962297.
Soriano, S., A.S.C. Chyung, X. Chen, G.B. Stokin, V.M.Y. Lee, and E.H. Koo. 1999. Expression of ß-amyloid precursor protein-CD3g chimeras to demonstrate the selective generation of ß1-40 and amyloid ß1-42 peptides within secretory and endocytic compartments. J. Biol. Chem. 274:3229532300.
Suzuki, T., Q. Yan, and W.J. Lennarz. 1998. Complex, two-way traffic of molecules across the membrane of the endoplasmic reticulum. J. Biol. Chem. 273:1008310086.
Van Gassen, G., W. Annaert, and C. Van Broeckhoven. 2000. Binding partners of Alzheimer's disease proteins: are they physiologically relevant? Neurobiol. Dis. 7:135151.[CrossRef][Medline]
Wolfe, M.S., and C. Haass. 2001. The role of presenilins in -secretase activity. J. Biol. Chem. 276:54135416.
Wolfe, M.S., W. Xia, C.L. Moore, D.D. Leatherwood, B.B. Ostaszewski, T.T. Rahmati, I.O. Donkor, and D.J. Selkoe. 1999. Peptidomimetic probes and molecular modeling suggest that Alzheimer's -secretase is an intramembrane-cleaving asparty protease. Biochemistry. 38:47204727.[CrossRef][Medline]
Wu, A., M.N. Pangalos, S. Efthimiopoulos, J. Shioi, and N.K. Robakis. 1997. Appican expression induces morphological changes in C6 glioma cells and promotes adhesion of neural cells to the extracellular matrix. J. Neurosci. 17:49874993.
Xia, W., W.J. Ray, B.L. Ostaszewski, T. Rahmati, W.T. Kimberly, M.S. Wolfe, J. Zhang, A.M. Goate, and D.J. Selkoe. 2000. Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid ß-protein generation. Proc. Natl. Acad. Sci. USA. 97:92999304.
Xu, H., D. Sweeney, R. Wang, G. Thinakaran, A.C. Lo, S.S. Sisodia, P. Greengard, and S. Gandy. 1997. Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation. Proc. Natl. Acad. Sci. USA. 94:37483752.
Xu, X., D. Yang, T. Wyss-Coray, J. Yan, L. Gan, Y. Sun, and L. Mucke. 1999. Wild-type but not Alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis. Proc. Natl. Acad. Sci. USA. 96:75477552.
Yang, Y., R.S. Turner, and J.R. Gaut. 1998. The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases Aß40-42 secretion. J. Biol. Chem. 273:2555225555.
Yu, C., S.H. Kim, T. Ikeuchi, H. Xu, L. Gasparini, R. Wang, and S.S. Sisodia. 2001. Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma-secretase processing of the APP and Notch1 transmembrane domains. J. Biol. Chem. 276:4375643760.
Zhang, L., L. Song, and E.M. Parker. 1999. Calpain inhibitor I increases ß-amyloid peptide production by inhibiting the degradation of the substrate of gamma-secretase: evidence that substrate availability limits ß-amyloid peptide production. J. Biol. Chem. 274:89668972.