Binding of Amyloid beta  Protein to the 20 S Proteasome*

(Received for publication, May 17, 1996, and in revised form, September 23, 1996)

Luisa Gregori Dagger , James F. Hainfeld §, Martha N. Simon § and Dmitry Goldgaber

From the Department of Psychiatry and Behavioral Science, School of Medicine, State University of New York, Stony Brook, New York 11794 and the § Biology Department, Brookhaven National Laboratory, Upton, New York 11973

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Neurodegenerative disorders of aging are characterized by the intraneuronal accumulation of ubiquitin conjugates into tangles and inclusions. Ubiquitin conjugates are degraded by cellular particles known as proteasomes. We have previously shown that amyloid beta  protein (Abeta ) inhibits proteasomal activity and thereby blocks ubiquitin conjugate degradation. In the present studies, we found that Abeta binds the 20 S proteasome and forms a proteasome-Abeta complex. The complex was detected by Western blot with anti-Abeta antibodies. Using a 1.4 nm Nanogold-labeled Abeta , we visualized proteasome-Abeta complexes by scanning transmission electron microscopy (STEM). Analysis of the side-on oriented proteasome-Abeta complexes revealed a single gold particle, corresponding to one gold-labeled Abeta , in the middle portion of the proteasome. On end-on views of proteasome-Abeta complexes, gold was detected within the area delimited by the proteasome circular projection. Both STEM views are consistent with Abeta localization inside the proteasome along the peptide channel. Direct interaction of Abeta with the inner catalytic compartment of the proteasome may explain the generation of ubiquitin-containing lesions in Alzheimer's disease and other neurodegenerative disorders. In addition, detection of Nanogold-labeled peptide inside the 20 S eukaryotic proteasome suggests that conformational constraints for protein degradation in eukaryotic proteasomes are different from those in archaebacteria proteasomes.


INTRODUCTION

The ubiquitin/proteasome protein degradation pathway is one of the two major cellular proteolytic systems. In this pathway, damaged and abnormal proteins are targeted for degradation by the covalent attachment of several ubiquitin moieties (ubiquitination) to form ubiquitin conjugates. Ubiquitinated proteins are rapidly degraded by the 26 S proteasome, the proteolytic component of the ubiquitin degradation system (1, 2, 3). Several findings link the ubiquitin/proteasome pathway to some pathological manifestations of neurodegenerative disorders such as Alzheimer's disease (AD),1 Lewy bodies disease, and to a lesser extent normal aging of the brain, as well as pathologies of non-central nervous system-related diseases such as inclusion body myositis (IBM). In these disorders, high levels of ubiquitin and ubiquitin conjugates are detected in abnormal intracellular inclusions and cytoskeletal-derived fibrils (4, 5, 6). In AD, microtubule associated protein tau  is ubiquitinated but is not degraded. Ubiquitinated tau  is found in paired helical filaments forming neurofibrillary tangles (NFT) (7). Development of NFT has been linked directly to neuronal degeneration in AD (8). Other, yet unidentified, proteins also accumulate into intracellular inclusions. These observations suggest that the ubiquitin/proteasome proteolytic pathway may be involved in the formation of ubiquitinated intracellular lesions.

Proteasomes are abundant nonlysosomal multicatalytic proteinases involved in a variety of cell functions (1, 9, 10, 11, 12). Proteasomes are localized both in the cytoplasm and in the nucleus of cells (10). In addition, proteasomes have been detected associated with plasma and internal membranes (10). The 20 S proteasome is the catalytic core of the 26 S proteasome (9, 10, 11). An increasing body of evidence points to a role of proteasomes in the accumulation of ubiquitinated proteins and ubiquitin-containing lesions in neurodegenerative disorders. Antibodies to proteasomes decorate some Lewy bodies and NFT (13). Cultured cells exposed to proteasome inhibitors accumulate ubiquitin conjugates (14, 15) and in at least one neuronal cell line these ubiquitin conjugates accumulated into intracellular AD-like inclusion bodies.2 In addition, we found that in vitro amyloid beta  protein (Abeta ) blocked the degradation of ubiquitinated proteins by inhibiting the proteasome activity (16). Abeta is a 39-42-amino acid peptide, derived from the proteolytic cleavage of the membrane-bound amyloid beta  protein precursor, which has been implicated in AD (17). Intracellular Abeta has been detected in cultured cells (18, 19) and associated with NFT in AD (20, 21) and with cytoplasmic tubulofilaments of vacuolated muscle fibers in IBM (5). We showed that Abeta selectively inhibits the chymotrypsin-like activity in the proteasome (16). Among the proteasome's different proteolytic activities, the chymotrypsin-like activity has been linked directly to the degradation of ubiquitin conjugates. Defects in the yeast proteasome subunits bearing this catalytic specificity cause accumulation of ubiquitin conjugates and reduced protein degradation rates (22). Selective inhibition of the proteasome chymotrypsin-like activity by Abeta is consistent with a critical function of the proteasome-peptide interaction in the accumulation of ubiquitin conjugates in AD. Furthermore, a recent finding that Abeta , used as the bait in a yeast two-hybrid system, binds two distinct yeast proteasome subunits, supports our results on the ability of Abeta to interact with the proteasome (23).

In this work, we characterized the 20 S proteasome-Abeta complex and established the spatial localization of Abeta in the complex. The results provide evidence for a molecular mechanism of Abeta interaction with the proteasome, which may be involved in the accumulation of ubiquitin conjugates in neurodegenerative disorders and other age-related diseases.


EXPERIMENTAL PROCEDURES

Material

Purified proteasomes were provided by Dr. M. Figueiredo-Pereira (Mount Sinai, CUNY, New York). Abeta 1-39C40 was a gift of Dr. C. Gable (University of California, Irvine). Abeta 1-40 (Abeta ) was purchased from K-Biologicals (Rancho Cucamonga, CA). The peptides were dissolved in water at the final concentration of 200 µM and stored in aliquots at -80 °C. Before use, the peptide solutions were microcentrifuged for 10 min. 6E10 is a monoclonal antibody generated against the sequence 1-16 of Abeta (24). Nanogold, silver enhancement kit, and methylamine vanadate were purchased from Nanoprobes (Stony Brook, NY). All other proteins and reagents were obtained from Sigma. Protein concentration was determined using BCA (Pierce) with bovine serum albumin as the standard.

Electrophoretic Analysis of Proteasome-Abeta Complex

Purified 20 S proteasomes (2.3 × 10-7 M) in Tris-HCl, 20 mM, pH 7.4, were incubated with Abeta at the indicated final peptide concentrations for 30 min at room temperature. Samples were analyzed on 5% nondissociating polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane using Tris/glycine/SDS containing 20% methanol as the transferring buffer. The membrane was probed with 6E10 antibodies, and the membrane-bound antibodies were detected by enhanced chemiluminescence (Amersham). Quantitation of immunostained bands was obtained from a phosphoanalyzer (Bio-Rad Laboratories) image using screen-CH plate. Following immunodetection, proteins on the membrane were stained with 0.1% Amido Black and destained with 30% methanol, 10% acetic acid.

Preparation and Characterization of Gold-labeled Abeta

In order to covalently and selectively attach monomaleimido-Nanogold particle to the peptide, we used a synthetic Abeta 1-40 variant in which the last amino acid was substituted with a cysteine residue (Abeta 1-39C40). Ten-fold molar excess Abeta 1-39C40 over the gold particle was used as the coupling conditions. The samples were incubated for 1 h at room temperature according to the manufacturer's instructions. Samples were chromatographed on Superose 12 (FPLC, Pharmacia) to separate the product Abeta 1-39C40-Nanogold (Abeta Au) from unconjugated Nanogold and Abeta 1-39C40. Abeta Au eluted from gel filtration was analyzed on 14% Tris/Tricine PAGE (25) and proteins were transferred onto PVDF membrane. Membrane-bound Abeta Au was detected by immunostaining with 6E10 antibodies and by silver enhancement of gold. Silver enhancement staining does not react with proteins.

Preparation of Cross-linked Proteasome-Abeta Au Complex

The following procedures were performed at room temperature. Purified 20 S proteasomes (2.3 × 10-7 M) were incubated with Abeta Au (10-5 M) for 30 min to form proteasome-Abeta Au complex. As the control, proteasomes were incubated with unconjugated Nanogold. The complex was cross-linked using 0.1% glutaraldehyde for 25 min, and the reaction was stopped by the addition of 0.2 M glycine, pH 7.3, for 1 h. The sample was concentrated to the original volume by ultrafiltration on Microcon-100 (Amicon) and used for STEM analysis. Cross-linked proteasome-Abeta Au complex was subjected to 5% nondissociating PAGE. The proteasome was detected by Coomassie Blue staining and in a parallel experiment, Abeta Au was detected by the silver enhancement method.

STEM Analysis of Proteasome-Abeta Au Complex

Samples for STEM analysis were prepared by applying 5 µl of cross-linked proteasome-Abeta Au complex to a titanium grid with a holey film and 3 nm carbon film on it (26). After 1-min absorption time, the grids were washed, wicked, quick-frozen in liquid nitrogen slush, and freeze-dried slowly overnight. Samples were negatively stained in a low atomic number stain, methylamine vanadate, which is denser than proteins but less dense than the gold particle.


RESULTS AND DISCUSSION

Abeta Forms a Complex with 20 S Proteasome

Abeta and 20 S eukaryotic proteasomes were incubated together to allow the formation of a proteasome-Abeta complex. The complex was analyzed by nondissociating PAGE (Fig. 1) and characterized by Western blotting using 6E10 antibodies to detect Abeta (Fig. 1A) and by protein staining of the same membrane to detect proteasomes (Fig. 1B). Upon incubation of Abeta with the proteasome, anti-Abeta antibodies recognized a new band (Fig. 1A, lane 2) with a slower electrophoretic mobility than free Abeta (Fig. 1A, lane 1). This immunoreactive band was not detected when Abeta was omitted from the reaction (Fig. 1A, lane 3), indicating that proteasomes alone did not form the new band and that there was no cross-reactivity of anti-Abeta antibodies with proteasomes. Furthermore, the same band was not formed when Abeta was incubated without the proteasome (Fig. 1A, lane 1) excluding the possibility that this complex is due to aggregated Abeta formed during incubation. This new band was detected by Coomassie staining of the membrane (Fig. 1B, lane 2) indicating that it contained the proteasomes, thus confirming it was a proteasome-Abeta complex. Under these experimental conditions, Abeta was not degraded by the proteasome since the intensity of the Abeta band as revealed by 6E10 antibody or by protein staining showed no difference between peptide incubated in the presence (lanes 2, Fig. 1, A and B) or in the absence (lanes 1, Fig. 1, A and B) of proteasomes. This observation confirms our previous finding that, under our experimental conditions, the peptide is not degraded by the proteasome (16). However, our results do not exclude that after a prolonged exposure to proteasomes a certain level of Abeta degradation may occur.


Fig. 1. Proteasome-Abeta complex. A and B, electrophoretic analysis of proteasome-Abeta complex. 3 × 10-5 M Abeta was incubated with 2.3 × 10-7 M proteasomes to form proteasome-Abeta complexes (see "Experimental Procedures"). As the controls, peptide and proteasomes were separately incubated under the same conditions. Proteins were separated on 5% nondissociating PAGE and transferred onto nitrocellulose for 60 min at 150 mA at 4 °C using Tris/glycine/SDS and 20% methanol as the transferring buffer. Western blot analysis was performed using anti-Abeta antibodies and detected by enhanced chemiluminescence (A). Antibody detection of Abeta localized inside the proteasome was possible because transferring of the proteins onto the membrane was performed under denaturing conditions which allowed the exposure of Abeta to 6E10 antibodies. Following immunodetection, the same membrane was stained with Amido Black to detect proteins (B). Lanes 1, 3 µg of Abeta ; lanes 2, proteasome-Abeta complex; lanes 3, 3 µg of proteasome. The arrow indicates the position of the complex. C and D, proteasome-Abeta complex formation as a function of Abeta concentration. Proteasome-Abeta complexes were formed using various concentrations of Abeta and analyzed by Western blotting with anti-Abeta antibodies (C). Abeta concentrations were 35, 10, 5, 1, and 0.5 µM (lanes 1-5). In lane 6, Abeta was omitted. In lane 7, proteasomes were omitted. In parallel experiments, various ranges of Abeta concentration were tested (data not shown). The intensity of the bands corresponding to the proteasome-Abeta complexes was quantitated using a phosphoanalyzer, and the values were normalized and combined in the curve shown in D. The values associated with the bands fell in the linear range of detection by 6E10 antibody, which was established in parallel experiments using the same antibodies to detect known concentrations of peptide (data not shown).
[View Larger Version of this Image (27K GIF file)]


We evaluated the formation of the proteasome-Abeta complex at various concentrations of the peptide (Fig. 1C). The intensity of proteasome-Abeta complex bands was quantitated and plotted against peptide concentration (Fig. 1D). The curve in Fig. 1D shows that even at the highest Abeta concentration tested, no saturation of binding was achieved. This phenomenon could be due to the tendency of Abeta to rapidly aggregate when used at concentrations higher than 200 µM. Furthermore, even when in a soluble state, Abeta is present as a mixture of monomer, dimer, tetramer, and higher multimeric forms (27) which preclude any type of standard kinetic analysis. An approximate extrapolation of the curve in Fig. 1D for saturation conditions indicated that 50% binding of Abeta to the proteasome was obtained at a micromolar concentration of Abeta . This range is consistent with the reported IC50 for Abeta inhibition of the proteasome chymotrypsin-like activity of 3-5 µM (16).

Characterization of Proteasome-Abeta Au Complex

In order to establish the spatial localization of Abeta within the proteasome-Abeta complex, we labeled an Abeta variant containing a cysteine residue as the last amino acid (Abeta 1-39C40) with 1.4 nm Nanogold which could be detected by electron microscopy. Formation of gold-labeled Abeta 1-39C40-Nanogold (Abeta Au) was confirmed by Western blotting using 6E10 antibodies to detect Abeta 1-39C40 (Fig. 2A, left panel) and by silver enhancement staining to visualize the gold particle (Fig. 2A, right panel). The same band was detected by both staining methods indicating that the 17-kDa band was gold-labeled Abeta 1-39C40 (Fig. 2A, lane 2). As the controls, unconjugated Abeta 1-39C40 and free Nanogold were analyzed and their migration patterns are shown in Fig. 2A, lanes 1 and 3, respectively. In both cases, the 17-kDa band was not detected.


Fig. 2. Electrophoretic analysis of Abeta Au. A, covalent and select conjugation of monomaleimido-Nanogold to proteins requires the presence of a cysteine residue on the protein. Because Abeta lacks cysteines, we used a peptide variant in which the last amino acid (Val40) was substituted with a cysteine residue (Abeta 1-39C40). Abeta 1-39C40 was coupled to Nanogold as described under "Experimental Procedures" to form Abeta Au in which each labeled Abeta molecule was linked to a single gold particle. 0.1 µg of the product was analyzed by 14% Tris/Tricine PAGE (25) (lane 2), Abeta 1-29C40 (lane 1), and Nanogold (lane 3) were used as the controls. Proteins were transferred onto PVDF membrane for 30 min at 100 mA at 4 °C, and Abeta Au was immunostained with anti-Abeta antibodies (left panel) or stained with the silver enhancement method (right panel). Both staining methods reacted with the same band indicating that Abeta Au migrates as a complex of 17 kDa. Molecular size markers are shown on the right. Note that because gel electrophoresis analysis was performed under denaturing, but not reducing, conditions to prevent thiol degradation of the gold particle, the control lane with the peptide alone shows the monomer and the dimer forms of Abeta 1-39C40 (lane 1). B and C, electrophoretic characterization of proteasome-Abeta Au complex. For STEM analysis, the complexes were cross-linked as described under "Experimental Procedures." Cross-linked proteasomes (B, lane 2) and cross-linked proteasome-Abeta Au complexes (B, lane 3) migrated slightly faster than non-cross-linked proteasomes (B, lane 1). Abeta Au was incubated with proteasome to form proteasome-Abeta Au complex. The complex was detected by Coomassie Blue (B) and silver enhancement staining (C). Both staining methods identified the same band confirming the formation of proteasome-Abeta Au complex. In B, lane 1, 3 µg of non-cross-linked proteasome; lane 2, 3 µg of cross-linked proteasome; lane 3, cross-linked proteasome-Abeta Au complex. In C, lane 1, cross-linked proteasome-Abeta Au complex; lane 2, 0.1 µg of cross-linked Abeta Au; lane 3, 3 µg of cross-linked proteasome to Nanogold.
[View Larger Version of this Image (54K GIF file)]


In order to confirm that Abeta Au maintained the ability to bind the proteasomes, Abeta Au was incubated with the proteasome to form a proteasome-Abeta Au complex. Formation of the complex was analyzed by nondissociating PAGE. Proteasomes were detected by Coomassie Blue staining (Fig. 2B, lane 3) while gold-labeled Abeta Au in the complex was visualized using silver enhancement (Fig. 2C, lane 1). Specificity of labeling is demonstrated by the absence of the proteasome-Abeta Au complex band as detected by silver enhancement staining when proteasomes were cross-linked to unconjugated Nanogold (Fig. 2C, lane 3). The upper band was also not formed when Abeta Au was cross-linked in the absence of proteasomes (Fig. 2C, lane 2). The results in Figs. 1 and 2 indicate that Abeta , as well as gold-labeled Abeta , forms complexes with proteasomes. Furthermore, Abeta Au was able to inhibit ubiquitin-dependent degradation of lysozyme with only a slight increase of the IC50 when compared with Abeta 1-40 (Fig. 3). As already discussed, no further kinetic analysis could be performed due to the anomalous behavior of Abeta in solution. Our result indicates that Abeta Au, similar to unlabeled Abeta (16), interacted with the 26 S proteasome and thereby inhibited its proteolytic activity. Since the 20 S proteasome is the catalytic component of the larger proteasome and contains the chymotrypsin-like activity previously shown to be inhibited by Abeta , we concluded that at least one of the interactions of Abeta Au with the 26 S proteasome is with its 20 S proteasome component. We analyzed this interaction and Abeta localization within the proteasome-Abeta Au complex by electron microscopy. Our observations suggest that the Abeta conformation required for proteasome-peptide interaction was not changed by the presence of the 1.4 nm Nanogold particle at the carboxyl terminus of Abeta . However, based on these experiments, we cannot exclude that coupling of Nanogold to Abeta may change the peptide binding affinity for the proteasome.


Fig. 3. Abeta Au inhibitory effect on ubiquitin-dependent degradation. Ubiquitin-dependent protein degradation was performed using a fraction (fraction II) of the rabbit reticulocyte lysate depleted of ubiquitin and ATP and iodinated lysozyme as the substrate (16). The indicated concentrations of purified Abeta Au were tested and the degradation was measured as the acid-soluble release after 1 h of incubation. Data are reported as the percentage inhibition of the activity measured without the peptide and are mean ± S.E. (values) of three experiments.
[View Larger Version of this Image (21K GIF file)]


STEM Analysis of Proteasome-Abeta Au Complex

Eukaryotic proteasomes are composed of 15-20 different subunits, assembled to form four staked rings in a barrel-shaped structure (10). The regulatory subunits (alpha -type) are located in the outer rings and the catalytic subunits (beta -type) are in the two inner rings with the active sites oriented toward the proteasome internal cavity (9, 28, 29). Proteasomes on electron microscopy are seen in two orientations, either end-on displaying ring-like projections or side-on producing striated rectangular views (29). STEM analysis of proteasome-Abeta Au complexes is shown in Fig. 4. The arrows in Fig. 4A indicate gold particles inside proteasomes. In end-on views (Fig. 4B), we found that the gold particle was in or close to the center of the proteasome circular projection. In side-on views (Fig. 4C), we observed that Abeta Au was located in the middle portion of the four-ring structure. Since each gold particle is linked to a single peptide molecule, our results indicate a predominant one-to-one ratio of proteasome and Abeta in the complex. More than 80% of the labeled proteasomes had one Abeta Au cross-linked, whereas the remaining carried two Abeta Au molecules (last image in Fig. 4, B and C). No cross-linking of unconjugated Nanogold to proteasomes was observed in our control experiments (data not shown). The results from multiple samples of STEM images are schematically summarized in Fig. 4D. Abeta Au was predominantly detected inside the structure, within the area along the peptide channel which runs through the proteolytic structure and is hypothesized to be the route of substrate access into the proteasome for degradation (9, 28, 29). As Fig. 4D indicates, Abeta did not randomly bind the proteasome, but there appeared to be two well defined regions of preferential binding. One between the second and the third ring and the other between the first and the second ring. Although at present, we cannot determine which specific subunits were involved in the interactions, our findings suggest that at least one interaction occurred with beta -type subunits in the inner rings, presumably the subunit with chymotrypsin-like specificity (16). Abeta -proteasome complex formation was not abolished by the addition of 100-fold excess of the peptide Z-Leu-Leu-Leu-p-nitroanilide (data not shown) which is the substrate used to measure the chymotrypsin-like activity (16). These results suggest that Abeta inhibited the proteasome activity not through a direct binding to the catalytic site(s), but via an indirect modification of the same catalytic site(s).


Fig. 4. STEM analysis of proteasome-Abeta Au complexes. Scanning transmission electron micrograph (STEM) of negatively stained proteasome-Abeta Au complexes. A, proteasomes are viewed in side-on and end-on orientations. Proteasome concentration was approximately 2 × 10-7 M. The arrows indicate the gold particle in proteasome-Abeta Au complexes. There were no gold-labeled peptides in the background of the STEM micrographs, indicating specificity of interaction. B, a gallery of end-on views of complexes. C, a gallery of side-on views of complexes. D, the localization of each Abeta Au binding the proteasome was recorded, and the data were condensed onto a single model. Each dot represents one gold label. In the side-on view, all dots were placed in the upper half of the complex due to the proteasome symmetry. Bar = 10 nm.
[View Larger Version of this Image (115K GIF file)]


The current mechanistic model of proteasome peptide degradation proposes that peptides are directed into proteasomes and degraded in the inner cavity, where the active sites of the catalytic subunits are located (28, 29). Recent work by Wenzel and Baumeister (30) supported this model and suggested that only unfolded peptides are able to slide into the peptide channel which functions by size exclusion. By using the same Nanogold reagent we used in our study, they found that the gold particle attached to oxidized insulin B chain was stuck at the entrance of the peptide channel and could not pass the narrow orifice. Our results show that the same gold particle attached to Abeta can enter the proteasome. This difference could be due to the fact that we used eukaryotic proteasomes whereas in Baumeister's work, archaebacteria proteasomes were used. Although the two proteasome structures are very similar, the dimension of the peptide channels might be different (12). In addition to a structural difference, archaebacteria proteasomes are also functionally different since they possess only chymotrypsin-like activity (10, 28, 29), while the more complex eukaryotic proteasomes comprise several distinct proteolytic specificities (9). Thus, our findings suggest that the substrates' conformational constraints for degradation by eukaryotic proteasome may be less stringent than previously reported for archaebacteria proteasomes.

In the degradation of ubiquitin conjugates, the 20 S proteasome requires the addition of other subunits which assemble into the 26 S proteasome. However, within the 26 S proteasome, the 20 S component remains the site with proteolytic activity. Thus, Abeta interaction with and inhibition of the 20 S proteasome bears direct connection with the inhibitory effect of Abeta on the 26 S proteasome-dependent degradation of ubiquitinated proteins. Consistent with this association are the findings that cultured cells exposed to inhibitors of the 20 S proteasome (14, 15) or mutations of its proteolytic subunits (22) caused an accumulation of ubiquitin conjugates. However, our findings do not exclude the possibility that Abeta may interact with the 26 S proteasome at other sites in addition to those of the 20 S proteasome reported here.

Understanding proteasome-Abeta interaction is critical to establishing its role in the accumulation of ubiquitin conjugates and the formation of NFT as seen in AD and other neurodegenerative disorders. Our work proposes a molecular mechanism of proteasome-Abeta interaction. Moreover, the present findings provide evidence that the conformational requirements in peptide degradation by eukaryotic proteasomes are distinct from those of archaebacteria proteasomes.


FOOTNOTES

*   This work was supported by The Alzheimer's Association through Grant ZEN-91-017 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Psychiatry and Behavioral Science, Health Science Center T-10, School of Medicine, State University of New York, Stony Brook, NY 11794-8101. Tel.: 516-444-3426; Fax: 516-444-7534.
1    The abbreviations used are: AD, Alzheimer's disease; IBM, inclusion body myositis; Abeta , amyloid beta  protein; NFT, neurofibrillary tangles; STEM, scanning transmission electron microscopy; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine.
2    M. Figueiredo-Pereira, personal communication.

Acknowledgments

We are particularly grateful to Dr. Maria Figueiredo-Pereira and Dr. Charles Glabe for their very generous gifts. We would also like to thank Dr. Chana Fuchs for her advice and suggestions during the preparation of this manuscript and Dr. Amit Banerjee and Dr. Roy MacCauley for reviewing this manuscript.


REFERENCES

  1. Ciechanover, A., and Schwartz, A. L. (1994) FASEB J. 8, 182-191 [Abstract/Free Full Text]
  2. Gregori, L., Poosch, M. S., Cousins, G., and Chau, V. (1990) J. Biol. Chem. 265, 8354-8357 [Abstract/Free Full Text]
  3. Rechsteiner, M., Hoffman, L., and Dubiel, W. (1993) J. Biol. Chem. 268, 6065-6068 [Free Full Text]
  4. Lowe, J., and Mayer, R. J. (1990) Neuropathol. Appl. Neurobiol. 16, 281-291 [Medline] [Order article via Infotrieve]
  5. Askanas, V., Alvarez, R. B., and Engel, W. K. (1993) Ann. Neurol. 34, 551-560 [Medline] [Order article via Infotrieve]
  6. Mori, H., Kondo, J., and Ihara, Y. (1987) Science 235, 1641-1644 [Medline] [Order article via Infotrieve]
  7. Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron 10, 1151-1160 [Medline] [Order article via Infotrieve]
  8. Braak, H., and Braak, E. (1991) Acta Neuropathol. 82, 239-259 [Medline] [Order article via Infotrieve]
  9. Orlowski, M., and Michaud, C. (1989) Biochemistry 28, 9270-9278 [Medline] [Order article via Infotrieve]
  10. Rivett, J. A. (1993) Biochem. J. 291, 1-10 [Medline] [Order article via Infotrieve]
  11. Hochstrasser, M. (1995) Curr. Opin. Cell Biol. 7, 215-223 [CrossRef][Medline] [Order article via Infotrieve]
  12. Tanaka, K., Tamura, T., Yoshimura, T., and Ichihara, A. (1992) New Biol. 4, 173-187 [Medline] [Order article via Infotrieve]
  13. Kwak, S., Masaki, T., Ishiura, S., and Sugita, H. (1991) Neurosci. Lett. 128, 21-24 [CrossRef][Medline] [Order article via Infotrieve]
  14. Figueiredo-Pereira, M. E, Berg, K. A., and Wilk, S. (1994) J. Neurochem. 63, 1578-1581 [Medline] [Order article via Infotrieve]
  15. Vinitsky, A., Cardozo, C., Sepp-Lorenzino, L., Michaud, C., and Orlowski, M. (1994) J. Biol. Chem. 269, 29860-29866 [Abstract/Free Full Text]
  16. Gregori, L., Fuchs, C., Figueiredo-Pereira, M. E., Van Nostrand, W. E., and Goldgaber, D. (1995) J. Biol. Chem. 270, 19702-19708 [Abstract/Free Full Text]
  17. Selkoe, D. J. (1994) Annu. Rev. Neurosci. 17, 489-517 [CrossRef][Medline] [Order article via Infotrieve]
  18. Turner, S. R., Suzuki, N., Chyung, A. S. C., Younkin, S. G., and Lee, V. M.-Y. (1996) J. Biol. Chem. 271, 8966-8970 [Abstract/Free Full Text]
  19. Martin, B. L., Schrader-Fischer, G., Busciglio, J., Duke, M., Paganetti, P., and Yankner, B. A. (1995) J. Biol. Chem. 270, 26727-26730 [Abstract/Free Full Text]
  20. Perry, G., Cras, P., Siedlak, S. L., Tabaron, M., and Kawai, M. (1992) Am. J. Pathol. 140, 283-290 [Abstract]
  21. Spillantini, M. G., Goedert, M., Jakes, R., and Kung, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3947-3951 [Abstract]
  22. Heinmeyer, W., Kleinschmidt, J. A., Saidowsky, J., Escher, J., and Wolf, D. H. (1991) EMBO J. 10, 555-562 [Abstract]
  23. June 8-9, Cambridge, MASahasrabudhe, S., and Hughes, S. (1995) Alzheimer's Disease: The Promise of New Therapeutics, June 8-9, Cambridge, MA
  24. Kim, K. S., Miller, D. L., Sapienza, J. V., Chen, C.-M. J., Bai, C., Grundke-Iqbal, I., Currie, J. R., and Wisniewski, H. M. (1988) Neurosci. Res. Commun. 2, 121-130
  25. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  26. Wall, J. S., and Hainfeld, J. F. (1986) Ann. Rev. Biophys. Biophys. Chem. 15, 355-376 [CrossRef][Medline] [Order article via Infotrieve]
  27. Podlisny, M. S., Ostaszewski, B. L., Squazzo, S. L., Koo, E. H., Rydel, R. E., Teplow, D. B., and Selkoe, D. J. (1995) J. Biol. Chem. 270, 9564-9570 [Abstract/Free Full Text]
  28. Zwickl, P., Grziwa, A., Puhler, G., Dahlmann, B., Lottspeich, F., and Baumeister, W. (1992) Biochemistry 31, 964-972 [Medline] [Order article via Infotrieve]
  29. Baumeister, W., Dahlmann, B., Hegerl, R., Kopp, F., Kuehn, L., and Pfeifer, G. (1988) FEBS Lett. 241, 239-245 [CrossRef][Medline] [Order article via Infotrieve]
  30. Wenzel, T., and Baumeister, W. (1995) Nature Struct. Biol. 2, 199-204 [Medline] [Order article via Infotrieve]

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