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Proteasome Inhibitors Induce the Association of Alzheimer's Amyloid Precursor Protein with Hsc73
Biochemical and Biophysical Research Communications 254, 804 – 810 (1999) Article ID bbrc.1998.9977, available online at http://www.idealibrary.com on
Proteasome Inhibitors Induce the Association of Alzheimer’s Amyloid Precursor Protein with Hsc73 Zen Kouchi,* ,† Hiroyuki Sorimachi,‡ Koichi Suzuki,* and Shoichi Ishiura† *Laboratory of Molecular Structure and Function, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; ‡Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and †Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
Received December 2, 1998
Amyloid precursor protein (APP) is a secretory membrane-bound protein that undergoes restrictive proteolysis and degradation with a short life span in the constitutive secretory pathway or in the endosomal/ lysosomal compartment. The degradation machinery, including cellular trafficking and the restrictive cleavage of APP, is poorly understood. To gain further insight into the intracellular degradation mechanism of APP, we searched for effector proteins that interact with APP. We found that a cytosolic molecular chaperon, Hsc73, effectively interacts with the cytoplasmic domain of APP in the presence of proteasome inhibitors. Hsc73 binds to the cytoplasmic domain near the post-transmembrane region of APP and not to the KFERQ-related sequence, KFFEQ, at the C-terminal tail that is assumed to be the selective targeting signal for lysosomal proteolysis. The amounts of Hsc73 that bind to several APP species such as those found in pathological Familial Alzheimer’s disease (FAD), Swedish, or Dutch type mutation, are almost identical, suggesting that an abnormal conformation around the secretory cleavage site or a pathological imbalance in APP processing are not irrelevant to the efficiency of Hsc73 binding. © 1999 Academic Press
Alzheimer’s amyloid precursor protein (APP) is a large secretory membrane-bound protein. It is proteolytically cleaved for secretion in the constitutive secretory pathway or for degradation in the endosomal/ lysosomal compartment (1, 2). The latter endosomal/ lysosomal pathway is a major proteolytic pathway Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; APP, amyloid precursor protein; APP-BP, amyloid precursor proteinbinding protein; Hsc, heat-shock cognate protein; Hsp, heat-shock protein; HSF1, heat shock transcription factor 1; ALLN, N-acetyl-Lleucyl-L-leucyl-L-norleucinal; E64d, 2S,3S-t-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; LC, lactacystin; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
considered to be neccessary for the amyloidogenic processing that results in Amyloid b (Ab) peptide production (3). On the other hand, ubiquitin conjugates are found to be developed in amyloid deposits during pathological neurodegeneration processes, suggesting a significant role for ubiquitin-mediated proteolysis in the formation of abnormal neurite dense bodies (4). Although very little is known about the molecular mechanism of the relationship between ubiquitin-mediated degradation and APP, several reports suggest the involvement of abnormal ATP and the Ub-mediated degradation of cellular proteins in the APP metabolic pathway (5, 6). Using a fraction of rabbit reticulocyte lysates, it was demonstrated that the extracellular, but not the intracellular form, of APP is degraded via ubiquitin modification (5). Recently, APP-BP1, a novel E1-related ubiquitin activating enzyme, was identified as the effector protein that interacts with the cytoplasmic domain of APP by expression screening (6). However, no physiological evidence has been obtained that ubiquitinmediated proteolysis directly participates in APP metabolism in vivo. Many intracellular proteins are subjected to proteasomal degradation or modification by the proteolytic activities of proteasome. Proteasome inhibitors are known to modulate the secretion of sAPPa, a large, extracellular N-terminal fragment, which is cleaved by a putative a-secretase (7). How these intracellular APP degradative processes are controlled remains obscure. We found that Hsc73, a member of the Hsp70 family located in the cytosol, binds to APP in the presence of proteasome inhibitors. It is generally accepted that molecular chaperones such as Hsc73 are involved in the degradation of proteins with abnormal conformations. Hsc73 is known to bind preferentially to a variety of proteins at peptide regions related to KFERQ, a selective targeting signal for lysosomal proteolysis.
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APP also contains this putative KFERQ-like peptide motif, KFFEQ, at the C-terminal tail. Hsc73 interacts with the N-terminal transmembrane side of the cytoplasmic domain of APP independent of the C-terminal KFFEQ sequence. Here we report a novel APP-Hsc73 relationship with the proteasomal degradation system. EXPERIMENTAL PROCEDURES Plasmid construction. Human cDNA was subcloned into pSRDplasmid and p91023. A series of C-terminal truncated APP were generated by PCR using synthetic oligonucleotide primers with introduced stop codon as follows; DC5 was amplified with sense primer 59-ACCTACAAGTTCTTTTGACTCGAGCAGAACTAGACCCCC-39 and anti-sense primer 59-GGGGGTCTAGTTCTGCTCGAGTCAAAAGAACTTGTAGGT-39; DC10 with 59-TCAAAGTTCTTTGAGCAGATGCAGAAC-39 and 59-TCATGGATT TTCGTAGCCGTTCTGCTG-39; DC15 with 59-AAGATGCAGCAGAACTGACTCGAGAATCCAACCTACAAG-39 and 59-CTTGTAGGTTGGATTCTCGAGTCAGTTCTGCTGCATCTT-39; DC20 with 59-CGCCACCTGTCCTGACTCGAGCAGAACGGCTACGAA-39 and 59-TTCGTAGCCGTTCTGCTCGAGTCAGGACAGGTGGCG-39; DC30 with 59-GAGGTTGACGCCTGACTCGAGCCAGAGGAGCGCCA-39 and 59-TGGCGCTCCTCTGGCTCGAGTCAGGCGTCAACCTC-39; and DC43 with 59-CTGAAGAAGAAACAGTGACTCGAGATTCATCATGGTGTG-39 and 59-CACACCATGATGAATCTCGAGTCACTGTTTCTTCTTCAG-39. The amplified PCR products were incubated with DpnI to digest the template DNA. Competent XL-1 blue cells were transformed with mutagenized DNA and subclones were scleened by means of the XhoI sites in the synthetic oligonucleotide primers. Constructs were confirmed by DNA sequencing. FAD mutants including V717F/I/G, Swedish mutation (K670N/M671L), Dutch type (E693Q) were constructed by the methods of Kunkel (a kind gift from Dr. K. Maruyama, Laboratory of Neurochemistry, National Institute for Physiological Sciences) (8). Transient transfection of COS-7 cells. The SV40-transfected African green monkey kidney cell line COS-7 was used for analyses of Hsc73 binding to APP. For transfection cells collected at a density of 3 3 10 6 cells were transfected with 5 mg of circular plasmid DNA/dish by electroporation at the following settings: 0.22 kV, 960 mF as described by the manufacturer. After transfection, the cells were washed three times with phosphate buffered saline (PBS) and aliquots were stored at 280°C. Antibodies. A monoclonal antibody, 22C11, that specifically recognizes residues 66-81 of APP, and anti-Hsc73 were obtained from Boehringer Mannheim. Anti-PN was raised against the secreted N-terminal fragment of APP695. Anti-C100 against residues 597695 of APP was kindly provided by Dr. K. Maruyama (Laboratory of Neurochemistry, National Institute for Physiological Sciences). Immunoprecipitation. Cell lysates were preabsorbed on Protein A-Sepharose for 1 h, incubated with anti-PN or anti-AC100 at 1:100 dilution, and separated on Protein A-Sepharose at 12,000 rpm for 5 minutes. Immunoprecitates were washed three times with TNE buffer and then resuspended in SDS-polyacrylamide gel electrophoresis sample buffer and boiled for 5 min at 95°C. The immunocomplexes were separated on 9% Tris-glycine gels and immunoblot analyses were performed using anti-22C11, anti-PN, and anti-Hsc73. Drug treatments and cell culture. N-acetyl-leucyl-leucyl-norleucinal (ALLN) and E64d were purchased from Sigma and lactacystin (LC) was kindly provided by Dr. S. Omura (The Kitasato Institute). A human glioblastoma cell line, A172, was grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco. BRL) supplemented with 10% fetal bovine serum (FBS) at 37°C. For differentiation into oligodendroglia, A172 cells were cultured in the absence of FBS for 5 days. The cells were incubated with 50 mM of ALLN, 20 mM of E64d, and 30 mM of lactacystin for the indicated time periods.
Immunoblotting. Immunoblot analysis was performed as described previously (9). Proteins were separated by SDS-polyacrylamide gel electrophoresis (10) and transblotted onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). The membranes were first blocked with 5% skim milk in phosphate-buffered saline containing 0.05% Tween 20 (TPBS), and then the blots were treated with APP or Hsc73 antibodies and peroxidase-conjugated anti-mouse or anti-rabbit antibodies and visualized by enhanced chemiluminescence assay (ECL, Amersham). Quantitative densitometry was performed with an Imagemaster densitometer (Pharmacia).
RESULTS AND DISCUSSION A human glioblastoma A172 cell line that expresses detectable amounts of endogenous APP and differentiates into oligodendroglia upon serum deprivation was used for the following experiments. Cells were subjected to serum withdrawal in DMEM and treated with 50 mM ALLN (proteasome inhibitor and a less specific inhibitor of cysteine proteases including calpain and cathepsin B), 20 mM E64d (cell-penetrating cysteine protease inhibitor), or 30 mM lactacystin for 24 h for the indicated intervals (Fig. 1A). We found that immunoprecipitation of APP with anti-C100 and anti-PN, which specifically recognize the C-terminal 100 amino acids of APP and the N-terminal secreted fragment cleaved by a-secretase (sAPPa), respectively, from ALLN-treated cell lysates resulted in the coprecipitation of Hsc73 (Fig. 1B, C), while immunoprecipitation with control IgG did not (Fig. 1B). This demonstrates that full-length APP interacts with Hsc73 in the presence of ALLN. The expression of APP was not significantly affected by treatment with either ALLN or E64d (Fig. 1C). In contrast, treating cells with ALLN but not E64d caused a rapid intracellular accumulation of Hsc73, while the amount of Hsc73 increased slightly during serum withdrawal (Fig. 1C). The amounts of Hsc73 immunoprecipitated with anti-APP in the presence of ALLN increased drastically and the ratio of immunoprecipitable Hsc73 to that of intracellular Hsc73 after 5 days in the presence of ALLN was 4-fold higher than after 1 day in the presence of ALLN (Fig. 1C). These results indicate that the inhibition of proteasome activity promotes the synthesis of Hsc73 and association with APP. These phenomena may be related to a certain differentiational process, since proteasome inhibitors are known to block normal morphological changes to oligodendrocyte and affect sAPPa secretion during serum withdrawal (7,11). To confirm that the APP-Hsc73 complex is induced by the specific inhibition of the proteolytic activity of proteasome, we examined the effects of lactacystin, an irreversible highly specific inhibitor of proteasome, on complex formation. Treatment of the cells with lactacystin also caused a significant increase in Hsc73 content in the material that coimmunoprecipitated with anti-APP as seen in the case of ALLN treatment (Fig. 1D). However, stimulation was slightly lower than in
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FIG. 1. Proteasome inhibitors induce Hsc73 binding to APP during the differentiation of A172 cells into oligodendrocytes. (A) Scheme for inhibitor treatments during the differentiation of A172 cells. A172 cells were grown in DMEM with 10% fetal bovine serum (1FBS), washed once with PBS, and resuspended in DMEM without FBS (2FBS). Cells were then incubated with 50 mM ALLN or 30 mM lactacystin (LC) or 20 mM E64d for the indicated times. The medium was changed and stored daily. (B) Identification of Hsc73-APP complexes by immunoprecipitation. A172 cells were grown for 4 days in the DMEM (2FBS) and then incubated in the presence (2, 4, 6, 8, 10) or absence (1, 3, 5, 7, 9) of 50 mM ALLN for 24 hours. The cells were lysed and subjected to immunoprecipitation (IP) with anti-PN or anti-AC100 or control IgG. The immunocomplexes were analyzed by Western blotting (WB) with the indicated antibodies. (C) ALLN, but not E64d, stimulates Hsc73 binding to APP. The amounts of APP-bound Hsc73 were analyzed by immunoprecipitation with anti-AC100 after incubation for the indicated times as shown in Fig. 1A. Quantitative densitometry from the data in the middle panel was performed with an Imagemaster densitometer (upper panel). The total amounts of intracellular APP and Hsc73 were analyzed by immunoblotting with anti-22C11 and anti-Hsc73, respectively (lower panel). (D) Cells were grown for 1 day (1 6 FBS) or 5 days (5-FBS) in DMEM with or without FBS. Cells were then incubated without (lanes 1, 4, 7) or with 50 mM ALLN (lanes 2, 5, 8) or 30 mM LC (lane 3, 6, 9) for 24 h. The lysates were immunoblotted (lane 10). Quantitative analysis of the amounts of APP-bound Hsc73 were performed by immunoprecipitation with anti-AC100 and immunoblotting with anti-Hsc73. The results shown are based on three sets of immunoprecipitations.
the case of ALLN. Thus the formation of an APP-Hsc73 complex depends specifically on the inhibition of the proteolytic activity of proteasome.
Next, we examined the binding domain of APP to Hsc73 by site-directed mutagenesis to know whether the cytoplasmic domain of APP is necessary for the
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FIG. 1—Continued
interaction with Hsc73. Immunoprecipitation experiments were performed in various APP cDNA transfected-COS-7 cells. We found that a 24 h ALLN treatment increases the amount of Hsc73 immunoprecipited with anti-APP in both wild-type APP695- and control vector pSRD-transfected cells (data not shown). This observation can be explained by the saturability of the formation of APP-Hsc73 complex for prolonged ALLN exposure. To address this issue, we examined the early phase of the APP-Hsc73 complex formation after ALLN treatment in pSRD-, wild-type APP695-, or the entire cytoplasmic domain truncated form of APP (APPDC43)-expressing COS-7 cells (Fig. 2A) Detectable amounts of Hsc73 were observed after 6 h and the complex formation increased significantly and reached a maximal level after 12 h of ALLN treatment in wildtype APP695 expressing cells. This change in the level of Hsc73-APP complex is comparable to the cellular Hsc73 levels induced by ALLN treatment (Fig. 2A lower panel). In contrast, ALLN caused no significant
increase in pSRD-transfected cells even after 12 h treatment, and notably, when APPDC43 was expressed, the APP-Hsc73 complex showed a slight increase after 12 h treatment. Recently, it has been reported that the exposure of cells to various proteasome inhibitors, including lactacystin and peptide aldehydes such as MG132 and ALLN, causes a heat shock response via HSF1 activation, leading to the expression of stress-inducible Hsp70 and an increase in the amount of constitutively expressed Hsc73 (12). There is a time lag in the stress response of hsp gene expression after treatment of proteasome inhibitor relevant to the potency of ALLN in inhibiting proteolytic activity of proteasome (13). To identify the binding regions of APP with Hsc73, we tested the efficiency of a series of C-terminal deletion or familial Alzheimer’s APP mutants to bind Hsc73 (Fig. 2B and unpublished data). The sequential deletion of the C-terminal 15 amino acids of APP did not affect Hsc73 binding, but the further deletions of
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the cytoplasmic domain caused reductions in the binding efficiency to Hsc73 after 9 h treatment (Fig. 2B). The putative KFERQ-like sequence, KFFEQ, locates within the C-terminal 10 amino acids of APP , but this peptide motif is apparently not involved in the Hsc73 binding. In contrast, FAD mutations of APP, including APP717 mutation (V717F/I/G), Swedish mutation (K670N/M671L), and Dutch type (E693Q), did not affect the interaction of APP with Hsc73 (data not shown). These data suggest that the cytoplasmic near transmembrane domain (C-terminal -15-43 amino acids) is essential for the binding to Hsc73 and the irregular conformation around the secretase cleavage site and the pathological imbalance of APP processing are not related to Hsc73 binding efficiency (Fig. 2C). Recently, several APP binding proteins have been found including X11 proteins, Fe65 protein and its related homologues, Fe65L1 and 2, and oligomeric Go protein (6, 14 –16). In proteolytic systems, APP-BP1, which shows sequence similarity to the product of the Arabidopsis auxin resistance gene AXR1 and the Caenorhabclitis elegans gene on chromosome III, was identified as a protein that interacts with the cytoplasmic domain of APP (6). They are putative candidates for directly precluding the molecular mechanism of APP metabolism, however, their physiological and functional significance in APP binding and the mechanism by which they modulate APP metabolism in vivo remain unknown. b-amyloid deposits are found to coexist with extensive amounts of ubiquitin and an hsp27 (acrystallin) in senile plaques, suggesting a pathological disorder between the conformational irrelevance of APP or APP-surrounding proteins and dysfunction in proteolytic systems (17). Evidence that several heat shock proteins might cooperate with the proteolytic machinery is provided by the finding that Hsp90 and Hsc73 are involved in a proteasome complex and in the reduction of protein degradation in mutant strains of hsp70 genes (ssa1 or ssa2) and DnaJ homologue (ydj) in yeast (18 –22). This chaperonin system may function as the conformational maintenance of proteasome, and also raises the possibility that heat shock proteins may contribute to the conformational recognition of aberrant proteins by proteasome.
Generally, various Hsp70 family members appear to bind nascent polypeptides and stabilize the unfolded state of proteins. In the facilitating of these protein maturation events, Hsp70 is known to bind preferentially to exposed stretches of amino acids in unfolded proteins that are enriched in large hydrophobic and aromatic residues. However, several Hsp70-mediated assistance mechanisms of folding distinct from those described above are demonstrated in the folding processes of CFTR (cystic fibrosis transmembrane conductance regulator) and p53 (23, 24). The binding regions of these proteins to Hsp70 are located within functionally significant domains that affect their own biological functions and are associated with pathological mutations. In addition, the cystic fibrosis mutant form of CFTR (DF508) is rapidly degraded in a pre-Golgi compartment and interacts with Hsc70 in the endoplasmic reticulum (25). It is widely known that multiple proteolytic systems, including the ubiquitin-proteasome pathway, contribute to CFTR processing and degradation (26, 27). Recently, cotranslational ubiquitination of CFTR was demonstrated using a cell-free system, but there seems to be no significant difference in the ubiquitination levels between wild-type and DF508 mutants (28). How Hsc70 binding and ubiquitination influence the partitioning of wild-type and mutant proteins into folding and degradation pathways remains obscure. As for the APP protein, it exhibits a short half-life in vivo and is subjected to proteolytic modulation by proteasome inhibitors. This is the first evidence that APP associates with Hsc73 in the presence of proteasome inhibitors. The association between APP and Hsc73 does not occur immediately after treatment with proteasome inhibitor and parallels the time course of hsp induction, suggesting that the complex formation may be mediated via stress response pathways, including the activation of stress kinases or HSF (29). How this APP-Hsc73 complex formation participates in proteasome-mediated proteolysis and the physiological significance of these phenomena must be determined further to understand better disorders in molecular chaperones and proteasome in APP metabolism.
FIG. 2. (A) Hsc73 binding domain of APP. Wild type APP695 or entire cytoplasmic domain-truncated APP (APPD43) was transiently expressed in COS-7 cells. One day after transfection, the cells were incubated with 50 mM ALLN for the indicated times, and the amounts of APP-bound Hsc73 were measured after immunoprecipitation with anti-PN. Quantitative analysis was carried out with an Imagemaster densitometer. Each value is the mean of the results of three experiments. The levels of cellular Hsc73 and APP were determined by Western blot with anti-Hsc73 and anti-22C11, respectively. (B) Hsc73 binds to the cytoplasmic domain of APP. Mock (lane 8), wild-type APP695 (lane 1), and a series of C-terminal truncated APP mutants including DC5 (lane 2), DC10 (lane 3), DC15 (lane 4), DC20 (lane 5), DC30 (lane 6), and DC43 (lane 7), were transiently expressed in COS-7 cells and incubated with 50mM ALLN for 0, 4, or 9 h. The amounts of APP-bound Hsc73 were analyzed by immunoprecipitation with anti-PN and immunoblotting with anti-Hsc73 (upper panel) and quantitated by densitometry after treatment with ALLN for 9 h (lower panel). The intracellular amounts of Hsc73 and APP were analyzed by Western blotting with anti-22C11 or anti-Hsc73, respectively (middle panel). (C) Schematic drawing of APP structure and Hsc73 binding site. The amino acid sequence of cytoplasmic domain is expanded. The asterisks show the positions of Swedish mutation, Dutch type, and V717 mutation. Solid horizontal lines below the amino acid sequence represent the C-terminal deletion mutants used in this study. The amino acids shown in bold type represent the KFERQ-related sequence. 809
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ACKNOWLEDGMENT This work was supported in part by grant-in-aid from the Ministry of Education, Science, Sport and Culture, Japan.
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Proteasome Inhibitors Induce the Association of Alzheimer’s Amyloid Precursor Protein with Hsc73 Zen Kouchi,* ,† Hiroyuki Sorimachi,‡ Koichi Suzuki,* and Shoichi Ishiura† *Laboratory of Molecular Structure and Function, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; ‡Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and †Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
Received December 2, 1998
Amyloid precursor protein (APP) is a secretory membrane-bound protein that undergoes restrictive proteolysis and degradation with a short life span in the constitutive secretory pathway or in the endosomal/ lysosomal compartment. The degradation machinery, including cellular trafficking and the restrictive cleavage of APP, is poorly understood. To gain further insight into the intracellular degradation mechanism of APP, we searched for effector proteins that interact with APP. We found that a cytosolic molecular chaperon, Hsc73, effectively interacts with the cytoplasmic domain of APP in the presence of proteasome inhibitors. Hsc73 binds to the cytoplasmic domain near the post-transmembrane region of APP and not to the KFERQ-related sequence, KFFEQ, at the C-terminal tail that is assumed to be the selective targeting signal for lysosomal proteolysis. The amounts of Hsc73 that bind to several APP species such as those found in pathological Familial Alzheimer’s disease (FAD), Swedish, or Dutch type mutation, are almost identical, suggesting that an abnormal conformation around the secretory cleavage site or a pathological imbalance in APP processing are not irrelevant to the efficiency of Hsc73 binding. © 1999 Academic Press
Alzheimer’s amyloid precursor protein (APP) is a large secretory membrane-bound protein. It is proteolytically cleaved for secretion in the constitutive secretory pathway or for degradation in the endosomal/ lysosomal compartment (1, 2). The latter endosomal/ lysosomal pathway is a major proteolytic pathway Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; APP, amyloid precursor protein; APP-BP, amyloid precursor proteinbinding protein; Hsc, heat-shock cognate protein; Hsp, heat-shock protein; HSF1, heat shock transcription factor 1; ALLN, N-acetyl-Lleucyl-L-leucyl-L-norleucinal; E64d, 2S,3S-t-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; LC, lactacystin; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
considered to be neccessary for the amyloidogenic processing that results in Amyloid b (Ab) peptide production (3). On the other hand, ubiquitin conjugates are found to be developed in amyloid deposits during pathological neurodegeneration processes, suggesting a significant role for ubiquitin-mediated proteolysis in the formation of abnormal neurite dense bodies (4). Although very little is known about the molecular mechanism of the relationship between ubiquitin-mediated degradation and APP, several reports suggest the involvement of abnormal ATP and the Ub-mediated degradation of cellular proteins in the APP metabolic pathway (5, 6). Using a fraction of rabbit reticulocyte lysates, it was demonstrated that the extracellular, but not the intracellular form, of APP is degraded via ubiquitin modification (5). Recently, APP-BP1, a novel E1-related ubiquitin activating enzyme, was identified as the effector protein that interacts with the cytoplasmic domain of APP by expression screening (6). However, no physiological evidence has been obtained that ubiquitinmediated proteolysis directly participates in APP metabolism in vivo. Many intracellular proteins are subjected to proteasomal degradation or modification by the proteolytic activities of proteasome. Proteasome inhibitors are known to modulate the secretion of sAPPa, a large, extracellular N-terminal fragment, which is cleaved by a putative a-secretase (7). How these intracellular APP degradative processes are controlled remains obscure. We found that Hsc73, a member of the Hsp70 family located in the cytosol, binds to APP in the presence of proteasome inhibitors. It is generally accepted that molecular chaperones such as Hsc73 are involved in the degradation of proteins with abnormal conformations. Hsc73 is known to bind preferentially to a variety of proteins at peptide regions related to KFERQ, a selective targeting signal for lysosomal proteolysis.
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APP also contains this putative KFERQ-like peptide motif, KFFEQ, at the C-terminal tail. Hsc73 interacts with the N-terminal transmembrane side of the cytoplasmic domain of APP independent of the C-terminal KFFEQ sequence. Here we report a novel APP-Hsc73 relationship with the proteasomal degradation system. EXPERIMENTAL PROCEDURES Plasmid construction. Human cDNA was subcloned into pSRDplasmid and p91023. A series of C-terminal truncated APP were generated by PCR using synthetic oligonucleotide primers with introduced stop codon as follows; DC5 was amplified with sense primer 59-ACCTACAAGTTCTTTTGACTCGAGCAGAACTAGACCCCC-39 and anti-sense primer 59-GGGGGTCTAGTTCTGCTCGAGTCAAAAGAACTTGTAGGT-39; DC10 with 59-TCAAAGTTCTTTGAGCAGATGCAGAAC-39 and 59-TCATGGATT TTCGTAGCCGTTCTGCTG-39; DC15 with 59-AAGATGCAGCAGAACTGACTCGAGAATCCAACCTACAAG-39 and 59-CTTGTAGGTTGGATTCTCGAGTCAGTTCTGCTGCATCTT-39; DC20 with 59-CGCCACCTGTCCTGACTCGAGCAGAACGGCTACGAA-39 and 59-TTCGTAGCCGTTCTGCTCGAGTCAGGACAGGTGGCG-39; DC30 with 59-GAGGTTGACGCCTGACTCGAGCCAGAGGAGCGCCA-39 and 59-TGGCGCTCCTCTGGCTCGAGTCAGGCGTCAACCTC-39; and DC43 with 59-CTGAAGAAGAAACAGTGACTCGAGATTCATCATGGTGTG-39 and 59-CACACCATGATGAATCTCGAGTCACTGTTTCTTCTTCAG-39. The amplified PCR products were incubated with DpnI to digest the template DNA. Competent XL-1 blue cells were transformed with mutagenized DNA and subclones were scleened by means of the XhoI sites in the synthetic oligonucleotide primers. Constructs were confirmed by DNA sequencing. FAD mutants including V717F/I/G, Swedish mutation (K670N/M671L), Dutch type (E693Q) were constructed by the methods of Kunkel (a kind gift from Dr. K. Maruyama, Laboratory of Neurochemistry, National Institute for Physiological Sciences) (8). Transient transfection of COS-7 cells. The SV40-transfected African green monkey kidney cell line COS-7 was used for analyses of Hsc73 binding to APP. For transfection cells collected at a density of 3 3 10 6 cells were transfected with 5 mg of circular plasmid DNA/dish by electroporation at the following settings: 0.22 kV, 960 mF as described by the manufacturer. After transfection, the cells were washed three times with phosphate buffered saline (PBS) and aliquots were stored at 280°C. Antibodies. A monoclonal antibody, 22C11, that specifically recognizes residues 66-81 of APP, and anti-Hsc73 were obtained from Boehringer Mannheim. Anti-PN was raised against the secreted N-terminal fragment of APP695. Anti-C100 against residues 597695 of APP was kindly provided by Dr. K. Maruyama (Laboratory of Neurochemistry, National Institute for Physiological Sciences). Immunoprecipitation. Cell lysates were preabsorbed on Protein A-Sepharose for 1 h, incubated with anti-PN or anti-AC100 at 1:100 dilution, and separated on Protein A-Sepharose at 12,000 rpm for 5 minutes. Immunoprecitates were washed three times with TNE buffer and then resuspended in SDS-polyacrylamide gel electrophoresis sample buffer and boiled for 5 min at 95°C. The immunocomplexes were separated on 9% Tris-glycine gels and immunoblot analyses were performed using anti-22C11, anti-PN, and anti-Hsc73. Drug treatments and cell culture. N-acetyl-leucyl-leucyl-norleucinal (ALLN) and E64d were purchased from Sigma and lactacystin (LC) was kindly provided by Dr. S. Omura (The Kitasato Institute). A human glioblastoma cell line, A172, was grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco. BRL) supplemented with 10% fetal bovine serum (FBS) at 37°C. For differentiation into oligodendroglia, A172 cells were cultured in the absence of FBS for 5 days. The cells were incubated with 50 mM of ALLN, 20 mM of E64d, and 30 mM of lactacystin for the indicated time periods.
Immunoblotting. Immunoblot analysis was performed as described previously (9). Proteins were separated by SDS-polyacrylamide gel electrophoresis (10) and transblotted onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). The membranes were first blocked with 5% skim milk in phosphate-buffered saline containing 0.05% Tween 20 (TPBS), and then the blots were treated with APP or Hsc73 antibodies and peroxidase-conjugated anti-mouse or anti-rabbit antibodies and visualized by enhanced chemiluminescence assay (ECL, Amersham). Quantitative densitometry was performed with an Imagemaster densitometer (Pharmacia).
RESULTS AND DISCUSSION A human glioblastoma A172 cell line that expresses detectable amounts of endogenous APP and differentiates into oligodendroglia upon serum deprivation was used for the following experiments. Cells were subjected to serum withdrawal in DMEM and treated with 50 mM ALLN (proteasome inhibitor and a less specific inhibitor of cysteine proteases including calpain and cathepsin B), 20 mM E64d (cell-penetrating cysteine protease inhibitor), or 30 mM lactacystin for 24 h for the indicated intervals (Fig. 1A). We found that immunoprecipitation of APP with anti-C100 and anti-PN, which specifically recognize the C-terminal 100 amino acids of APP and the N-terminal secreted fragment cleaved by a-secretase (sAPPa), respectively, from ALLN-treated cell lysates resulted in the coprecipitation of Hsc73 (Fig. 1B, C), while immunoprecipitation with control IgG did not (Fig. 1B). This demonstrates that full-length APP interacts with Hsc73 in the presence of ALLN. The expression of APP was not significantly affected by treatment with either ALLN or E64d (Fig. 1C). In contrast, treating cells with ALLN but not E64d caused a rapid intracellular accumulation of Hsc73, while the amount of Hsc73 increased slightly during serum withdrawal (Fig. 1C). The amounts of Hsc73 immunoprecipitated with anti-APP in the presence of ALLN increased drastically and the ratio of immunoprecipitable Hsc73 to that of intracellular Hsc73 after 5 days in the presence of ALLN was 4-fold higher than after 1 day in the presence of ALLN (Fig. 1C). These results indicate that the inhibition of proteasome activity promotes the synthesis of Hsc73 and association with APP. These phenomena may be related to a certain differentiational process, since proteasome inhibitors are known to block normal morphological changes to oligodendrocyte and affect sAPPa secretion during serum withdrawal (7,11). To confirm that the APP-Hsc73 complex is induced by the specific inhibition of the proteolytic activity of proteasome, we examined the effects of lactacystin, an irreversible highly specific inhibitor of proteasome, on complex formation. Treatment of the cells with lactacystin also caused a significant increase in Hsc73 content in the material that coimmunoprecipitated with anti-APP as seen in the case of ALLN treatment (Fig. 1D). However, stimulation was slightly lower than in
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FIG. 1. Proteasome inhibitors induce Hsc73 binding to APP during the differentiation of A172 cells into oligodendrocytes. (A) Scheme for inhibitor treatments during the differentiation of A172 cells. A172 cells were grown in DMEM with 10% fetal bovine serum (1FBS), washed once with PBS, and resuspended in DMEM without FBS (2FBS). Cells were then incubated with 50 mM ALLN or 30 mM lactacystin (LC) or 20 mM E64d for the indicated times. The medium was changed and stored daily. (B) Identification of Hsc73-APP complexes by immunoprecipitation. A172 cells were grown for 4 days in the DMEM (2FBS) and then incubated in the presence (2, 4, 6, 8, 10) or absence (1, 3, 5, 7, 9) of 50 mM ALLN for 24 hours. The cells were lysed and subjected to immunoprecipitation (IP) with anti-PN or anti-AC100 or control IgG. The immunocomplexes were analyzed by Western blotting (WB) with the indicated antibodies. (C) ALLN, but not E64d, stimulates Hsc73 binding to APP. The amounts of APP-bound Hsc73 were analyzed by immunoprecipitation with anti-AC100 after incubation for the indicated times as shown in Fig. 1A. Quantitative densitometry from the data in the middle panel was performed with an Imagemaster densitometer (upper panel). The total amounts of intracellular APP and Hsc73 were analyzed by immunoblotting with anti-22C11 and anti-Hsc73, respectively (lower panel). (D) Cells were grown for 1 day (1 6 FBS) or 5 days (5-FBS) in DMEM with or without FBS. Cells were then incubated without (lanes 1, 4, 7) or with 50 mM ALLN (lanes 2, 5, 8) or 30 mM LC (lane 3, 6, 9) for 24 h. The lysates were immunoblotted (lane 10). Quantitative analysis of the amounts of APP-bound Hsc73 were performed by immunoprecipitation with anti-AC100 and immunoblotting with anti-Hsc73. The results shown are based on three sets of immunoprecipitations.
the case of ALLN. Thus the formation of an APP-Hsc73 complex depends specifically on the inhibition of the proteolytic activity of proteasome.
Next, we examined the binding domain of APP to Hsc73 by site-directed mutagenesis to know whether the cytoplasmic domain of APP is necessary for the
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FIG. 1—Continued
interaction with Hsc73. Immunoprecipitation experiments were performed in various APP cDNA transfected-COS-7 cells. We found that a 24 h ALLN treatment increases the amount of Hsc73 immunoprecipited with anti-APP in both wild-type APP695- and control vector pSRD-transfected cells (data not shown). This observation can be explained by the saturability of the formation of APP-Hsc73 complex for prolonged ALLN exposure. To address this issue, we examined the early phase of the APP-Hsc73 complex formation after ALLN treatment in pSRD-, wild-type APP695-, or the entire cytoplasmic domain truncated form of APP (APPDC43)-expressing COS-7 cells (Fig. 2A) Detectable amounts of Hsc73 were observed after 6 h and the complex formation increased significantly and reached a maximal level after 12 h of ALLN treatment in wildtype APP695 expressing cells. This change in the level of Hsc73-APP complex is comparable to the cellular Hsc73 levels induced by ALLN treatment (Fig. 2A lower panel). In contrast, ALLN caused no significant
increase in pSRD-transfected cells even after 12 h treatment, and notably, when APPDC43 was expressed, the APP-Hsc73 complex showed a slight increase after 12 h treatment. Recently, it has been reported that the exposure of cells to various proteasome inhibitors, including lactacystin and peptide aldehydes such as MG132 and ALLN, causes a heat shock response via HSF1 activation, leading to the expression of stress-inducible Hsp70 and an increase in the amount of constitutively expressed Hsc73 (12). There is a time lag in the stress response of hsp gene expression after treatment of proteasome inhibitor relevant to the potency of ALLN in inhibiting proteolytic activity of proteasome (13). To identify the binding regions of APP with Hsc73, we tested the efficiency of a series of C-terminal deletion or familial Alzheimer’s APP mutants to bind Hsc73 (Fig. 2B and unpublished data). The sequential deletion of the C-terminal 15 amino acids of APP did not affect Hsc73 binding, but the further deletions of
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the cytoplasmic domain caused reductions in the binding efficiency to Hsc73 after 9 h treatment (Fig. 2B). The putative KFERQ-like sequence, KFFEQ, locates within the C-terminal 10 amino acids of APP , but this peptide motif is apparently not involved in the Hsc73 binding. In contrast, FAD mutations of APP, including APP717 mutation (V717F/I/G), Swedish mutation (K670N/M671L), and Dutch type (E693Q), did not affect the interaction of APP with Hsc73 (data not shown). These data suggest that the cytoplasmic near transmembrane domain (C-terminal -15-43 amino acids) is essential for the binding to Hsc73 and the irregular conformation around the secretase cleavage site and the pathological imbalance of APP processing are not related to Hsc73 binding efficiency (Fig. 2C). Recently, several APP binding proteins have been found including X11 proteins, Fe65 protein and its related homologues, Fe65L1 and 2, and oligomeric Go protein (6, 14 –16). In proteolytic systems, APP-BP1, which shows sequence similarity to the product of the Arabidopsis auxin resistance gene AXR1 and the Caenorhabclitis elegans gene on chromosome III, was identified as a protein that interacts with the cytoplasmic domain of APP (6). They are putative candidates for directly precluding the molecular mechanism of APP metabolism, however, their physiological and functional significance in APP binding and the mechanism by which they modulate APP metabolism in vivo remain unknown. b-amyloid deposits are found to coexist with extensive amounts of ubiquitin and an hsp27 (acrystallin) in senile plaques, suggesting a pathological disorder between the conformational irrelevance of APP or APP-surrounding proteins and dysfunction in proteolytic systems (17). Evidence that several heat shock proteins might cooperate with the proteolytic machinery is provided by the finding that Hsp90 and Hsc73 are involved in a proteasome complex and in the reduction of protein degradation in mutant strains of hsp70 genes (ssa1 or ssa2) and DnaJ homologue (ydj) in yeast (18 –22). This chaperonin system may function as the conformational maintenance of proteasome, and also raises the possibility that heat shock proteins may contribute to the conformational recognition of aberrant proteins by proteasome.
Generally, various Hsp70 family members appear to bind nascent polypeptides and stabilize the unfolded state of proteins. In the facilitating of these protein maturation events, Hsp70 is known to bind preferentially to exposed stretches of amino acids in unfolded proteins that are enriched in large hydrophobic and aromatic residues. However, several Hsp70-mediated assistance mechanisms of folding distinct from those described above are demonstrated in the folding processes of CFTR (cystic fibrosis transmembrane conductance regulator) and p53 (23, 24). The binding regions of these proteins to Hsp70 are located within functionally significant domains that affect their own biological functions and are associated with pathological mutations. In addition, the cystic fibrosis mutant form of CFTR (DF508) is rapidly degraded in a pre-Golgi compartment and interacts with Hsc70 in the endoplasmic reticulum (25). It is widely known that multiple proteolytic systems, including the ubiquitin-proteasome pathway, contribute to CFTR processing and degradation (26, 27). Recently, cotranslational ubiquitination of CFTR was demonstrated using a cell-free system, but there seems to be no significant difference in the ubiquitination levels between wild-type and DF508 mutants (28). How Hsc70 binding and ubiquitination influence the partitioning of wild-type and mutant proteins into folding and degradation pathways remains obscure. As for the APP protein, it exhibits a short half-life in vivo and is subjected to proteolytic modulation by proteasome inhibitors. This is the first evidence that APP associates with Hsc73 in the presence of proteasome inhibitors. The association between APP and Hsc73 does not occur immediately after treatment with proteasome inhibitor and parallels the time course of hsp induction, suggesting that the complex formation may be mediated via stress response pathways, including the activation of stress kinases or HSF (29). How this APP-Hsc73 complex formation participates in proteasome-mediated proteolysis and the physiological significance of these phenomena must be determined further to understand better disorders in molecular chaperones and proteasome in APP metabolism.
FIG. 2. (A) Hsc73 binding domain of APP. Wild type APP695 or entire cytoplasmic domain-truncated APP (APPD43) was transiently expressed in COS-7 cells. One day after transfection, the cells were incubated with 50 mM ALLN for the indicated times, and the amounts of APP-bound Hsc73 were measured after immunoprecipitation with anti-PN. Quantitative analysis was carried out with an Imagemaster densitometer. Each value is the mean of the results of three experiments. The levels of cellular Hsc73 and APP were determined by Western blot with anti-Hsc73 and anti-22C11, respectively. (B) Hsc73 binds to the cytoplasmic domain of APP. Mock (lane 8), wild-type APP695 (lane 1), and a series of C-terminal truncated APP mutants including DC5 (lane 2), DC10 (lane 3), DC15 (lane 4), DC20 (lane 5), DC30 (lane 6), and DC43 (lane 7), were transiently expressed in COS-7 cells and incubated with 50mM ALLN for 0, 4, or 9 h. The amounts of APP-bound Hsc73 were analyzed by immunoprecipitation with anti-PN and immunoblotting with anti-Hsc73 (upper panel) and quantitated by densitometry after treatment with ALLN for 9 h (lower panel). The intracellular amounts of Hsc73 and APP were analyzed by Western blotting with anti-22C11 or anti-Hsc73, respectively (middle panel). (C) Schematic drawing of APP structure and Hsc73 binding site. The amino acid sequence of cytoplasmic domain is expanded. The asterisks show the positions of Swedish mutation, Dutch type, and V717 mutation. Solid horizontal lines below the amino acid sequence represent the C-terminal deletion mutants used in this study. The amino acids shown in bold type represent the KFERQ-related sequence. 809
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ACKNOWLEDGMENT This work was supported in part by grant-in-aid from the Ministry of Education, Science, Sport and Culture, Japan.
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