Chaperonin-mediated de novo generation of prion protein aggregates1

doi:10.1006/jmbi.2001.5085 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 313, 861±872

Chaperonin-mediated de novo Generation of Prion Protein Aggregates Johannes StoÈckel and F. Ulrich Hartl* Department of Cellular Biochemistry, Max-PlanckInstitute of Biochemistry D-82152, Martinsried Germany

The infectious prion protein, PrPSc, a predominantly b-sheet aggregate, is derived from PrPC, the largely a-helical cellular isoform of PrP. Conformational conversion of PrPC into PrPSc has been suggested to involve a chaperone-like factor. Here we report that the bacterial chaperonin GroEL, a close homolog of eukaryotic Hsp60, can catalyze the aggregation of chemically denatured and of folded, recombinant PrP in a model reaction in vitro. Aggregates form upon ATP-dependent release of PrP from chaperonin and have certain properties of PrPSc, including a high bsheet content, the ability to bind the dye Congo red, detergent-insolubility and increased protease-resistance. A conserved sequence segment of PrP (residues 90-121), critical for PrPSc generation in vivo, is also required for chaperonin-mediated aggregate formation in vitro. Initial binding of refolded, a-helical PrP to chaperonin is mediated by the unstructured Nterminal segment of PrP (residues 23-121) and is followed by a rearrangement of the globular PrP core-domain. These results show that chaperonins of the Hsp60 class can, in principle, mediate PrP aggregation de novo, i.e. independently of a pre-existent PrPSc template. # 2001 Academic Press

*Corresponding author

Keywords: prion protein; molecular chaperone; chaperonin; GroEL; protein folding

Introduction The prion protein (PrP) is the causative agent of neurodegenerative diseases such as CreutzfeldJakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) and scrapie in sheep. PrP exists in at least two distinct conformational states.1 ± 3 The pathological, infectious form, PrPSc, is a b-sheet aggregate, whereas the normal cellular isoform, PrPC, consists of a largely a-helical, autonomously folded C-terminal domain (residues 126228) and an N-terminal 100 residue segment that is unstructured in solution (at pH 4.5-5.2)4 ± 6 (Figure 1(a)). The N-terminal segment contains a highly conserved octapeptide repeat region that Present address: J. StoÈckel, Curacyte AG, Gollierstr. 70B, D-80339 MuÈnchen, Germany. Abbreviations used: PrP, prion protein; PrPC, cellular isoform of PrP; PrPSc, infectious, scrapie form of PrP; UPrP, unfolded, chemically denatured PrP; N-PrP, native (refolded) PrP; GdnHCL, guanidinium hydrochloride; CD, circular dichroism; FTIR, fourier transformed infrared spectroscopy; CJD, Creutzfeld-Jakob disease; GFP, green ¯uorescent protein; PK, proteinase k. E-mail address of the corresponding author: [email protected] 0022-2836/01/040861±12 $35.00/0

coordinates Cu(II) via histidine residues.7,8 It is assumed that the conversion of PrPC into PrPSc requires the transient accumulation of a conformational intermediate(s).9 However, the PrP core domain re-folds in vitro with extremely rapid kinetics (half-life of folding at 4  C and pH 7, 170 ms) without populating kinetic intermediates.10 Consistent with these ®ndings, no structural intermediates are detected under equilibrium denaturation conditions at pH 5.5.11 At pH 4, however, a soluble monomer of PrP90-231 can be generated that is almost entirely composed of b-sheet and is a precursor of PrP aggregates.12 In vivo a chaperone-like factor may participate in PrPC to PrPSc conversion by either stabilizing or inducing the formation of a structurally reorganized or largely unfolded state of PrPC.13,14 Molecular chaperones recognize and stabilize unfolded or partially folded protein structures, generally suppressing protein aggregation and facilitating ef®cient folding.15 Additionally, certain types of chaperones that form oligomeric ring structures, such as the members of the Hsp100 and chaperonin families, may mediate protein unfolding or disaggregation.16 ± 19 Indeed, yeast Hsp104 and the Escherichia coli chaperonin, GroEL, have been shown to facilitate in vitro the recruitment of # 2001 Academic Press

862 PrPC into pre-existent PrPSc aggregates.20 However, chaperone-mediated de novo formation of PrP b-sheet aggregates from recombinant PrP has not yet been achieved. The mechanism of chaperonin-mediated protein folding has been studied extensively for GroEL, the chaperonin of E. coli. GroEL is a large cylindrical complex composed of two stacked heptameric rings of 57 kDa subunits. It captures non-native polypeptide on multiple hydrophobic sites that are exposed toward a central cavity. Subsequent binding of the ring-shaped co-factor GroES results in discharge of bound protein into an enclosed cage in which folding can occur. Opening and closing of the cage is regulated by the ATPase activity of GroEL.15,21 An interaction between a PrP fusion protein and the apical polypeptide binding domain of the mitochondrial chaperonin, Hsp60, has been detected in a yeast two-hybrid screen.22 Although it seems unlikely that PrP, a secretory protein, can interact with Hsp60 in vivo, we ®nd that among several classes of chaperones present in mammalian cells only the chaperonin has the capacity to induce the aggregation of recombinant PrP de novo, i.e. in the absence of a template of pre-existent PrP aggregates. This effect is critically dependent on the interaction of the chaperonin with the unstruc-

Chaperonin-mediated Prion Protein Aggregation

tured N-terminal segment of PrP and is most pronounced at a mildly acidic pH of 5.5, present in the endosomal compartment where PrPSc formation is thought to occur.23,24 Our results suggest that conditions may exist in which a molecular chaperone or chaperone-like interaction partner of PrP may facilitate the PrPC to PrPSc conversion in spontaneous and familial prion disease.

Results Chaperonin binding of denatured PrP A proteinase K (PK) protection assay was utilized to investigate the interaction of chemically unfolded, recombinant mouse PrP (U-PrP23-231) (Figure 1(a)) with the Hsp60 homolog of E. coli, GroEL. PrP23-231 is the form of PrP after cleavage of N-terminal and C-terminal signals for import into the endoplasmic reticulum and glycolipid addition, respectively. Upon 100-fold dilution from 6 M GdnHCl into GroEL-containing buffer at pH 7, U-PrP23-231 bound to GroEL with high af®nity and was maintained in a non-aggregated, unfolded state. GroEL-bound PrP was highly sensitive towards proteinase K digestion under mild conditions (2.5 mg/ml PK, 25  C) (Figure 1(b)), in

Figure 1. Binding of U-PrP to GroEL. (a) Schematic representation of the structure of PrP23-231. The ®ve octapeptide repeats, the disul®de bond and the location of a-helices and b-sheets are indicated. Sequences without de®ned secondary structure are shown in white. Residues 1-22 corespond to the cleavable signal sequence of the protein. (b) Time-course of proteinase K (PK) digestion (2.5 mg/ml at 25  C) of U-PrP23-231, U-PrP90-231 and U-PrP121-231 upon 100-fold dilution from 6 M GdnHCl into buffer solution (pH 7) containing GroEL. When indicated, GroES and ADP were added prior to PK digestion. Reactions were analyzed by SDS-PAGE and immunoblotting with anti-PrP antibodies. (c) Model showing the topology of GroEL-bound PrP in the presence of GroES/ADP. About 50 % of GroELbound PrP is enclosed in the so-called cis complex and is inaccessible to PK. PrP bound to the trans ring of GroEL is degraded.

863

Chaperonin-mediated Prion Protein Aggregation

which GroEL itself remains intact.25 When incubated with ATP, GroEL and its single ring co-factor GroES form a transient asymmetric complex that is stable in the presence of ADP (Figure 1(c)).26 Approximately half of the GroEL-bound substrate protein can be encapsulated by GroES in the socalled cis-cavity of GroEL and will be inaccessible to externally added protease (Figure 1(c)).27 ± 29 This effect was also seen with PrP23-231 as the substrate (Figure 1(b)). Upon addition of GroES and ADP, about 50 % of GroEL-bound PrP23-231 was protected from PK digestion. Binding to GroEL and encapsulation by GroES was also observed with the chemically unfolded, N-terminally truncated constructs U-PrP90-231 and U-PrP121-231, lacking most or all of the unstructured segment of PrP, respectively (Figure 1(b)). PrP90-231 corresponds to the portion of PrP that becomes protease-resistant in PrPSc. These results suggest that, like many other unfolded polypeptides, U-PrP is bound by the apical domains of GroEL that expose hydrophobic amino acid residues. Binding of GroES to GroEL causes the displacement of PrP from these sites into the enclosed GroEL cavity.

Chaperonin-induced aggregation of unfolded PrP At pH 7 and above, dilution of PrP (isoelectric point pH 9.5) from denaturant to a ®nal concentration of 1 mM results in aggregation rather than in spontaneous refolding, as shown by light scattering measurements (Figure 2(a), trace 4). As expected, aggregation was largely suppressed by GroEL at a stoichiometry of GroEL oligomers to PrP of 1:1 (Figure 2(a), trace 6). ATP-dependent dissociation of preformed GroEL:PrP complexes resulted in aggregation of PrP (Figure 2(a), traces 3 and 5). Only little aggregation was observed when both GroES and ATP were added to preformed GroEL:PrP complexes (Figure 2(a), trace 2), consistent with refolding of PrP occuring in the GroEL-GroES cage prior to its release into bulk solution.21,27 ± 29 In contrast to its tendency to aggregate at pH 7, U-PrP23-231 refolds rapidly and ef®ciently upon dilution from denaturant at pH 5.5, although conversion of PrPC to PrPSc may occur at a similar, mildly acidic pH in early endosomes.23,24 U-PrP does not measurably aggregate at pH 5.5 (Figure 2(b), trace 2) and reaches a mainly a-helical

Figure 2. Aggregation of U-PrP in the presence and absence of GroEL. PrP constructs (®nal concentration 1 mM) were 100-fold diluted from 6 M GdnHCl (U-PrP) into buffer containing additions as indicated and aggregation analyzed photometrically by monitoring turbidity at 320 nm ((a)-(d)) or ®lter retardation assay (e). (a) and (b) UPrP23-231 at pH 7 and pH 5.5, respectively. GroEL (1 mM), GroES (2 mM) and ATP (1 mM) were present as indicated. Arrow marks the addition of ATP and of GroES/ ATP, respectively. (c) U-PrP23-231 in the presence of ATP and increasing mM concentrations of GroEL at pH 5.5. (d) U-PrP90-231 and UPrP121-231 at pH 5.5 in the presence of ATP and GroEL as indicated. (e) Aggregation of U-PrP23231 in the presence of ATP with or without GroEL monitored by ®lter retardation assay at pH 5.5. GroEL alone was analyzed as a control. Left panel, immunostaining of the ®lter with anti-PrP antibody; right panel, immunostaining with antiGroEL antibody.

864 conformation indistinguishable from that of PrPC by far UV CD and infrared spectroscopy (data not shown and Figure 5(c)).30 ± 32 Surprisingly, GroEL induced the pronounced aggregation of U-PrP at pH 5.5 (Figure 2(b), traces 4-6). The light-scattering signal obtained under these conditions exceeded that of spontaneous aggregation of U-PrP at pH 7, suggesting that more or larger PrP aggregates form in the presence of GroEL and ATP at pH 5.5. In contrast, no GroEL-induced aggregation was detectable with Doppel (Figure 2(b), trace 7), a protein homologous to the folded PrP core region (25 % identity) that is not implicated in prion disease.33 At pH 5.5, U-PrP did not form a stable, isolatable complex with GroEL. Nevertheless, GroEL-induced aggregation was signi®cantly enhanced in the presence of ATP (Figure 2(b), traces 4 and 6). Addition of GroES reduced the aggregation of U-PrP only slightly (Figure 2(b), trace 3), due to inef®cient binding of GroES to GroEL at pH 5.5 (data not shown). Formation of large PrP aggregates measured by light scattering was con®rmed by ®ltration of the reactions through a cellulose acetate membrane34 (Figure 2(e)). Upon incubation with GroEL/ATP for ten minutes at pH 5.5, more than 60 % of total U-PrP in the reaction formed aggregates that were readily retained by the ®lter, consistent with the time-course of aggregation shown in Figure 2(b). These aggregates were not solubilized by washing the membrane with 0.1 % SDS. In contrast, most UPrP refolded in the absence of GroEL was not retained on the membrane. Under all conditions tested GroEL remained soluble and did not form detectable aggregates. The observed effect of GroEL to induce PrP aggregation was speci®c for PrP and was not due to a general loss of function of GroEL at pH 5.5. First, GroEL did not induce the aggregation of the PrP homolog Doppel (Figure 2(b), traces 7 and 8). Second, the ability of GroEL to prevent the aggregation of other unfolded proteins such as rhodanese and citrate synthase was preserved at pH 5.5, both in the presence and absence of ATP (data not shown). Enhancement of PrP aggregation by GroEL was optimal at a 1:1 molar ratio of GroEL:PrP (Figure 2(c), trace 5) but was less ef®cient at substoichiometric GroEL concentrations (Figure 2(c), traces 2 and 4), suggesting that GroEL does not facilitate aggregation by bringing multiple PrP molecules into close proximity. When present in excess over PrP, GroEL slowed the kinetics of aggregation, presumably by favoring PrP re-binding to GroEL, thereby reducing the free concentration of aggregation-competent PrP (Figure 2(c), trace 3). Under these conditions aggregation occurred with a clear lag-phase, consistent with a nucleation-dependent process.35 Thus, at pH 5.5 GroEL transiently stabilizes and releases PrP in an aggregation-sensitive conformational state that is not signi®cantly populated during spontaneous refolding.

Chaperonin-mediated Prion Protein Aggregation

Requirement of PrP residues 90-120 for chaperonin-induced aggregation The length of the unstructured N-terminal segment of PrP affects the formation of PrPSc in vivo. In some cases of inherited CJD in humans additional copies of octapeptide repeats (see Figure 1(a)) are inserted in the N-terminal segment of one PrP allele.2 Mice and neuronal cells expressing PrP constructs lacking residues 23-88 are still able to propagate PrPSc 36,37 but mice that express PrP with longer N-proximal deletions cannot be infected.38 Interestingly, upon dilution from denaturant into buffer containing GroEL and ATP at pH 5.5, recombinant U-PrP90-231 also aggregated in a GroEL-dependent manner (Figure 2(d), trace 4), albeit with signi®cantly slower kinetics and smaller signal intensities than observed with UPrP23-231 (Figure 2(b), trace 4). In contrast, GroEL did not induce the aggregation of U-PrP121-231, lacking the complete unstructured segment (Figure 2(d), trace 3), although GroEL bound both U-PrP90-231 and U-PrP121-231 (see Figure 1). Thus, the requirement of residues 90 to 120 of PrP for GroEL-mediated aggregation in vitro mirrors the essential role of this region for the formation of PrPSc in vivo. Epitopes around residues 90-120 are accessible in PrPC but are largely hidden in PrPSc,39 suggesting that this ¯exible region undergoes a conformational change during PrPSc formation. Properties of chaperonin-induced PrP aggregates A hallmark of authentic PrPSc is its strongly increased protease-resistance relative to PrPC.2 Under stringent conditions of PK digestion (20 mg/ ml PK, one hour at 37  C) the aggregates of PrP23231 formed in the presence of GroEL/ATP at pH 5.5 showed a profoundly increased resistance to proteinase K (Figure 3(a), lanes 7-15) under conditions known to completely degrade N-PrP.40 The greatest amount of PK-resistant material (about 20 % of the PrP present in the reaction) was produced at a 1:1 molar ratio of PrP to GroEL (Figure 3(a), lanes 10-12), which also produced the most total aggregation (Figure 2(c)). Ratios of PrP to GroEL that were suboptimal for aggregation (e.g. 10:1) resulted in little protease-resistant material (Figure 3(a), lanes 4-6). Besides some undigested, full-length PrP, PK digestion of the aggregates also generated a product(s) of 16 kDa (Figure 3(a), lanes 8, 11 and 14). This fragment(s) was detected with antibody 3F4, recognizing an epitope at residues 104-113 of PrP, and thus resembles, but is probably not identical to, the PrP fragment 90-231 that is typically derived from authentic PrPSc by PK digestion.2 Interestingly, although GroEL induced the aggregation of PrP independently of ATP (see Figure 2(b)), the aggregates formed in the absence of ATP were not PKresistant (Figure 3(a), lanes 1-3). Similarly, only PK-sensitive PrP aggregates were generated by a

865

Chaperonin-mediated Prion Protein Aggregation

Figure 3. Properties of PrP aggregates generated in the presence of GroEL and ATP. (a) U-PrP23-231 (1 mM, ®nal concentration) was diluted from 6 M GdnHCl into buffer (pH 5.5) containing GroEL (0.1-2 mM) with or without ATP (1 mM) and incubated for 20 minutes at 25  C. Reactions were then incubated with PK (0-20 mg/ ml, one hour at 37  C) and analyzed by SDS-PAGE and immunoblotting with anti-PrP antibody 3F4. *, Position of a PrP dimer observed upon SDS-PAGE. (b) Congo red binding of U-PrP23-231 in the presence and absence of GroEL. Congo red absorbance spectra were recorded for U-PrP23-231 aggregates generated in the presence of GroEL/ATP at pH 5.5 (U-PrP/GroEL), refolded PrP (NPrP), GroEL and buffer control. (c) Recruitment of UPrP23-231 into preformed aggregates. Aggregates were formed at pH 5.5 in the presence of GroEL and ATP as above and then removed from GroEL by centrifugation. Preformed aggregates were resuspended by sonication. Fresh U-PrP23-231 was added to increasing concentrations of preformed aggregates (reactions 1-3) by dilution from 6 M GdnHCl (®nal concentration, 1 mM) and its aggregation measured photometrically after 20 minutes at 25  C. The maximum estimated amount of preformed aggregates used per reaction is indicated (see Materials and Methods). ATP was added to control for trace amounts of GroEL present in preformed aggregates (reaction 4). In reaction 5, 2 mM of U-PrP23-231 was added in the absence of preformed aggregates, demonstrating that aggregation is not simply induced

GroEL mutant protein (D87 K) defective in ATP binding and hydrolysis (data not shown).41 ATP binding causes a signi®cant movement of the apical domains of GroEL42 which may in turn induce a conformational change in bound PrP prior to or concomitantly with its release from GroEL. Such a conformational change may be critical for the formation of PK-resistant aggregates. The PrP aggregates generated in the presence of GroEL/ATP bound the amyloid-staining dye Congo red, as indicated by a shift of the absorbance maximum of Congo red from 502 nm to 533 nm (Figure 3(b)). No signi®cant shift in Congo red absorption relative to the buffer control was detected with refolded PrP (N-PrP23-231) or with GroEL alone (absorption maxima at 500 nm and 506 nm, respectively). Infrared spectroscopy indicated a high b-sheet content of the PrP aggregates (see below). Negative-stain electron microscopy revealed that the aggregates were amorphous rather than ®brillar (data not shown). In summary, by several criteria the GroEL-induced aggregates of PrP have properties resembling those of PrPSc or so-called PrPres (protease-resistant PrP).43 PrP aggregates produced by incubation with GroEL/ATP were able to recruit new unfolded PrP in the absence of GroEL (Figure 3(c)). Addition of U-PrP to increasing amounts of preformed aggregates, isolated from GroEL by centrifugation, resulted in a twofold increase in total aggregate mass within 20 minutes of incubation (Figure 3(c), reactions 1-3), as con®rmed by ®ltration assay (data not shown). Aggregation was not stimulated by addition of ATP, indicating that it was not caused by trace amounts of GroEL remaining in the reaction (Figure 3(c), reaction 4), but was absolutely dependent on the presence of preformed PrP aggregates (reaction 5). No additional aggregation signal was obtained with preformed aggregates alone over the time-course of the experiment (reaction 6). Thus, the PrP aggregates generated by GroEL can recruit additional PrP protein in a GroEL-independent reaction. Recruitment has been observed for other amyloidogenic aggregation processes including PrPSc conversion.35,44 Chaperonin binding of folded PrP In the experiments described above, chemically unfolded PrP (U-PrP) was used as the substrate for GroEL-mediated aggregation. However, in vivo a chaperone-like factor would presumably act on folded, largely a-helical PrPC. Given the rapid rate of spontaneous refolding of U-PrP in vitro,10 the observation that substoichiometric concentrations of GroEL can catalyze PrP aggregation (Figure 2(c))

by an increased PrP concentration. Reaction 6 shows that the light scattering signal of preformed aggregates alone (set to 0 at the beginning of the experiment) is stable over the time-course of the experiment.

866 suggested that the chaperonin may be able to interact with PrP even in conditions where mainly refolded N-PrP is populated. Binding of N-PrP to GroEL was indeed observed, based on the criteria established for U-PrP (see Figure 1). When ®rst refolded at pH 5.5 and subsequently incubated with GroEL, GroES and ADP at pH 7 (allowing formation of a stable GroEL:GroES complex), a signi®cant fraction of total PrP23-231 was protected from PK degradation inside the GroEL:GroES complex (Figure 4(a)). In contrast, the protein was rapidly degraded when added either to GroEL alone or to GroEL/GroES in the absence of nucleotide (Figure 4(a)). GroEL binding was also detectable for N-PrP90-121 but not for N-PrP121-231 or the PrP homolog Doppel, both proteins lacking the unstructured N-terminal segment of PrP (data not shown). In analyzing the binding of N-PrP to GroEL we took advantage of the fact that PrP contains eight tryptophan (Trp) residues and that GroEL lacks Trp entirely.25 Upon incubation of N-PrP23-231 with increasing concentrations of GroEL, a saturable increase in Trp ¯uorescence re¯ecting the formation of a PrP:GroEL complex was observed (Figure 4(b), and insert traces 1 and 2). Fitting the data to a bimolecular binding model indicated that complex formation occurred with high af®nity (Kd of 50 nM). Since seven of the eight Trp residues of PrP are located between amino acid residues 31 and 98 in the unstructured N-terminal segment of PrP, this segment is likely involved in mediating N-PrP binding to GroEL. The increase in ¯uorescence observed upon binding presumably re¯ects the movement of Trp residues into the hydrophobic environment of the apical GroEL domains. As described previously, copper binding reduced the Trp ¯uorescence of N-PrP23-231 by about 50 %7 (Figure 4(b), insert, traces 2and 3). Interestingly, no ¯uorescence increase was seen upon addition of GroEL to N-PrP23-231 in the presence of 25 mM Cu(II) (at pH 7) (Figure 4(b), insert, trace 4), suggesting that copper binding stabilizes the N-terminal segment of PrP in a conformation that differs from that in GroEL-bound PrP. We note that addition of copper cannot prevent GroEL-dependent PrP aggregation at pH 5.5, because below pH 6 PrP loses its ability to bind copper.7 Stop-¯ow experiments showed that binding of N-PrP to GroEL was very fast, occurring at an apparent rate of 44 sÿ1 at 25  C for a single exponential kinetic model (Figure 4(c)). This rate is estimated to be signi®cantly faster than the rate of spontaneous unfolding of the PrP core domain extrapolated to 25  C.10 Considering that the interaction of GroEL with substrate polypeptide is multivalent, binding of the N-terminal region may be followed by trapping of the PrP core domain in a non-native state as the domain unfolds spontaneously.

Chaperonin-mediated Prion Protein Aggregation

Figure 4. Binding of N-PrP to GroEL. (a) Time-course of PK digestion of N-PrP23-231 in the presence of GroEL or GroEL, GroES and ADP (see Figure 1(b)). Reactions were analyzed by SDS-PAGE and immunoblotting with anti-PrP antibodies. (b), N-PrP23-231 (0.125 mM) was mixed with increasing concentrations of GroEL (pH 7) and Trp ¯uorescence emission was recorded. The continuous line represents a curve ®t of the data to a bimolecular binding model. Inset: Emission spectra of N-PrP23-231 (0.5 mM) with (trace 1) or without (trace 2) 0.5 mM GroEL. Trace 3 shows N-PrP23-231 in the presence of 25 mM CuCl2 and trace 4 the same sample after subsequent addition of 0.5 mM GroEL. Background ¯uorescence of GroEL itself is subtracted. (c) Time-resolved change in Trp ¯uorescence upon PrP (0.5 mM) binding to GroEL (0.5 mM) recorded in stop¯ow experiments upon mixing of wild-type N-PrP23231 or N-PrP23-231F175W with GroEL. Data were ®tted to a single exponential (wt PrP) or double exponential (F175W mutant) ¯oating end point equation. Fluorescence intensities are given in arbitray units (AU).

Chaperonin-mediated Prion Protein Aggregation

A mutated version of PrP with an additional Trp in the folded core region, PrP23-231F175W, served to monitor a possible unfolding of the domain in the presence of GroEL, as the single Trp of the wild-type core domain at position 145 is largely exposed and does not exhibit a change in ¯uorescence upon unfolding.10 A core domain construct with the F175W change has been reported to have the same structural properties as the wild-type domain, except that complete unfolding in 6 M GdnHCl results in a 2.5-fold Trp-¯uorescence increase10 (note, however, that in the full-length protein, PrP23-231F175W, the increase in total ¯uorescence upon unfolding of the mutant core domain is expected to be small [  20 %]). Indeed, an additional 15 % ¯uorescence increase occurring with an apparent rate of 5 sÿ1 was consistently observed upon rapid mixing of folded PrP23231F175W with GroEL (Figure 4(c)), presumably

867 re¯ecting at least partial unfolding of the PrP core region upon GroEL binding. We propose that PrP initially binds to GroEL via its unstructured Nterminal segment. This step is followed by spontaneous unfolding of the a-helical core domain of PrP and its stabilization in an unfolded state as a consequence of the high local concentration of hydrophobic binding sites in the GroEL ring. Chaperonin-induced aggregation of refolded PrP It seemed possible that the non-native state of PrP stabilized by GroEL upon binding of N-PrP may be aggregation-prone. Indeed, incubation of N-PrP23-231 with GroEL/ATP at pH 5.5, where NPrP is soluble, resulted in the formation of PrP aggregates with similar ef®ciency as observed with U-PrP (Figure 5(a); see Figure 2b for comparison).

Figure 5. GroEL-dependent aggregation of N-PrP and of PrP23-119-GFP fusion protein. (a) Aggregation of NPrP23-231 (1 mM) in the presence of GroEL (1 mM) or GroEL/GroES (1 mM/2 mM) and ATP (1 mM) at pH 5.5 measured by light scattering. Lysozyme or ovalbumin at concentrations (w/v) equivalent to GroEL were used as controls. Aggregation (absorbance at 320 nm) was measured after 20 minutes in the presence of GroEL/ATP. (b) GroELmediated aggregation of N-PrP23-231 monitored by ®lter retardation assay. N-PrP alone, GroEL/ATP alone or a mixture of N-PrP and GroEL/ATP were analyzed upon incubation as in (a). Left panel, detection with anti-PrP antibody; right panel, detection with anti-GroEL antibody. (c) Infrared spectra of N-PrP23-231 (4 mM) incubated with or without immobilized GroEL as described in Materials and Methods. GroEL promotes a decrease of the absorption maximum at 1664 cmÿ1 and an increase at 1624 cmÿ1 indicating a conformational shift from predominantly a-helical to high bsheet content. (d) Aggregation of N-PrP(23-119)-GFP (1 mM) and GFP (1 mM) at pH 7 either alone or upon incubation with GroEL/ATP. BSA was used as a control protein at a concentration (w/v) equivalent to that of GroEL. Inset: After 20 minutes incubation of N-PrP(23-119)-GFP with or without GroEL/ATP reactions were separated into pellets and supernatants by centrifugation. Pellets were washed and analyzed together with supernatants by SDS-PAGE and immunoblotting with anti-PrP antibodies.

868 These aggregates were retained on a cellulose acetate ®lter (Figure 5(b)) and exhibited a high b-sheet content as indicated qualitatively by a typical shift in the FTIR absorbance maximum from 1664 cmÿ1 to 1624 cmÿ1 (Figure 5(c)). In contrast, N-PrP (in the absence of GroEL) was not retained on the ®lter (Figure 5(b)) and was in a largely a-helical conformation (Figure 5(c)). However, only a small amount of the aggregated PrP formed from N-PrP in the presence of GroEL was PK-resistant (data not shown). Thus, this material differs conformationally from the aggregates generated upon incubation of U-PrP with GroEL and ATP (Figure 3(a)), suggesting that more extensive unfolding of the PrP core domain or addition of PrPSc template is necessary for GroEL-mediated conversion of N-PrP into protease-resistant material.20 Chaperonin-induced aggregation of PrP23-119-GFP To better understand the mechanistic contribution of the unstructured N-terminal segment of PrP to GroEL-mediated aggregation, we constructed a chimeric protein consisting of PrP residues 23-119 and green ¯uorescent protein (GFP). Puri®ed PrP(23-119)-GFP was soluble and had the same ¯uorescence properties as GFP, indicating that the GFP-moiety of the fusion protein was folded. Strikingly, GroEL induced slow but signi®cant aggregation of PrP(23-119)-GFP in an ATPdependent manner, as measured by light scattering, whereas native GFP alone did not interact with GroEL and remained soluble (Figure 5(d)). Upon centrifugation, aggregated but still ¯uorescent PrP(23-119)-GFP was detected in the pellet fraction (Figure 5(d), insert). These results suggest that GroEL transiently stabilizes the N-terminal segment of PrP in a state prone to aggregation and releases it in the presence of ATP. It is possible that this state contains b-conformation, consistent with the recent demonstration that the apical binding domains of GroEL can stabilize a peptide in a bturn con®guration.45 We note that members of the Hsp70 family of molecular chaperones, which transiently stabilize hydrophobic peptides in an extended conformation,46 did not induce the aggregation of PrP(23-119)-GFP and of PrP(23-231) (data not shown).

Discussion We have demonstrated that GroEL, a chaperonin of the Hsp60 class, can induce the formation of PrP aggregates from recombinant prion protein de novo. In principle, a chaperonin-like activity may thus facilitate the accumulation of abnormally folded PrP in the rare spontaneous and inherited forms of prion disease which occur independently of preexistent PrPSc template. The GroEL-induced PrP aggregates have some of the physical properties generally ascribed to PrPSc or PrPres. These include

Chaperonin-mediated Prion Protein Aggregation

a high degree of protease resistance, binding of Congo red and a high b-sheet content. GroELmediated aggregation requires the presence of PrP residues 90-121, the C-terminal part of the unstructured segment of PrP that is essential for the formation of PrPSc in vivo.38 Whether the PrP aggregates generated in the presence of GroEL contain infectious material remains to be investigated. Optimal catalysis of PrP aggregation by GroEL is ATP-dependent and is observed at a mildly acidic pH at which refolded PrP is soluble. Our data suggest that this ``anti-chaperone'' effect is speci®c for PrP and can be explained in terms of the unusual structural and conformational properties of the protein. A model for the interaction of refolded PrP (N-PrP) with GroEL is presented in Figure 6(a). In a ®rst step the unstructured N-terminal segment of PrP is recognized by GroEL with high af®nity and is held in an aggregation-prone state while the PrP core domain may still be folded (denoted PrP-I1). As spontaneous unfolding of the core domain is faster than ATP-dependent dissociation of the GroEL:PrP-I1 complex, PrP is transiently stabilized by GroEL in a non-native conformation (PrP-I2). This intermediate is then released for aggregation. In contrast, sequential binding steps cannot be resolved for the interaction of chemically denatured PrP (U-PrP) with GroEL (Figure 6(b)). Furthermore, the intermediate stabilized by GroEL in this case (denoted PrP-I2*) differs conformationally from PrP-I2, because ef®cient GroEL-mediated formation of protease-resistant PrP aggregates is only observed with U-PrP. The role of GroEL in both of these reactions is essentially to accumulate a folding intermediate(s) of PrP that is poised to aggregation but is not signi®cantly populated during spontaneous unfolding or refolding in the absence of chaperonin. We propose that this mechanism also underlies the previously observed ability of GroEL to enhance the recruitment of acid-denatured hamster PrPC into preformed PrPSc.20 PrP differs markedly from most other globular proteins in that its native state contains a large segment (residues 23-126) that is unstructured in solution and is readily recognized by chaperonin. Our results indicate that this ¯exible region of the protein, or part thereof, can be stabilized by GroEL in a structurally more organised, aggregation-prone state. This state may be characterized by an increased b-strand content, consistent with recent ®ndings that GroEL binds a model peptide in a bturn conformation.47,48 Moreover, several proteins that interact with GroEL physiologically contain extensive hydrophobic b-sheets that may be partially exposed in their folding intermediates and may mediate GroEL binding.49 The critical role of the N-terminal segment of PrP in GroEL-mediated aggregation is underscored by the demonstration that transplantation of this segment to an unrelated protein, GFP, renders this protein aggregation-sensitive in a GroEL and ATP-dependent manner. Furthermore, the PrP homolog Doppel that lacks an

869

Chaperonin-mediated Prion Protein Aggregation

Figure 6. Model of GroELmediated PrP aggregation for refolded PrP (N-PrP) (a) and chemically denatured PrP (U-PrP) (b). GroEL is shown schematically as a vertical cut through the double-ring cylinder. (a) The unstructured N-terminal segment (residues 23-121) of N-PrP mediates initial binding to apical domains of GroEL and is stabilized in an aggregation-prone conformation, presumably enriched in b-strand content (PrP-I1). In a second step the folded, mainly a-helical PrP core domain associates with GroEL upon spontaneous unfolding and is stabilized in a non-native state (PrP-I2). ATP-mediated release of PrP-I2 from GroEL results in formation of b-sheet-rich aggregates with low protease resistance. (b) In the case of U-PrP the N-terminal segment and the unfolded core domain may bind simultaneously to apical GroEL domains. The PrP core domain is stabilized in a different, presumably more unfolded state (PrP-I2*), than in (a). ATP-dependent release of PrP-I2* results in formation of b-sheet-rich aggregates with high protease-resistance.

unstructured region does not undergo GroELinduced aggregation. It is of interest in this context that insertion of additional octapeptide repeats in the N-terminal sequence of PrP causes inherited Creutzfeld-Jakob disease2 perhaps by increasing the propensity of the N-terminal segment to spontaneously adopt an aggregation-prone conformation and/or by increasing its af®nity for a chaperone-like factor. GroEL-mediated generation of protease-resistant PrP aggregates is strictly ATP-dependent. Although at pH 5.5 the interaction of GroEL with PrP is transient, even in the absence of ATP, addition of ATP may enhance the release of PrP from GroEL, as has been shown for other nonnative proteins.25 Additionally, ATP-dependent domain movements in GroEL42 may cause further unfolding of the core domain of bound PrP prior to release for aggregation. In support of this possibility, protease-resistant aggregates were generated preferentially from chemically unfolded U-PrP but only when both GroEL and ATP were present. Conformational stress, such as increased temperature, may facilitate the GroEL-dependent formation of protease-resistant aggregates from refolded, a-helical N-PrP. GroEL-mediated PrP aggregation is a model reaction. It demonstrates that, in principle, de novo conversion of PrP may be facilitated by an extracellular component that mimics the activity of GroEL. Based on our ®ndings, such a factor would be predicted to have multiple binding sites allowing interactions with both the N-terminal segment and the core domain of PrP. The mammalian GroEL homolog, Hsp60, has been shown to bind PrP in a yeast two-hybrid screen,22 but Hsp60 is

located in mitochondria and thus unlikely to interact with PrP in vivo.

Materials and Methods Recombinant proteins Mouse PrP constructs consisting of residues 23-231 (PrP23-231), residues 90-231 (PrP90-231), residues 121231 (PrP121-231), green ¯uorescent protein (GFP), a chimeric construct consisting of PrP residues 23-119 followed by GFP (PrP(23-119)-GFP), and mouse Doppel residues 27-15533 were expressed in E. coli BL21/DE3 strain. To allow detection of PrP with monoclonal antibody 3F4 (Daco, Germany), recognizing an epitope of hamster PrP, amino acid residues V111 and L108 of mouse PrP were altered to Met. Constructs were cloned into pET11d (PrP23-231, PrP90-231, PrP(23-119)-GFP, GFP, Doppel27-155) or into pProEX HTa (PrP23-231, PrP121-231). pET11d (Novagen) constructs were C-terminally extended by the amino acid sequence GGGGSHHHHHH. pProEX HTa-constructs (Lifetech) were N-terminally extended by six His residues and the TEV-protease recognition sequence (removed upon puri®cation with TEV-His6 protease (Lifetech)). The mutation F175W was introduced into PrP23-231 (pProEx HTa) with the Quik-Change kit (Stratagene).10 PrP was puri®ed under denaturing conditions (6 M GdnHCl) by Ni2‡-NTA chromatography, yielding unfolded PrP (U-PrP). PrP23-119-GFP and GFP were puri®ed under native conditions on a Ni2‡-NTA column. U-PrP ( 300 mM) was refolded to N-PrP by 100-fold dilution into 20 mM Mes (pH 6) and concentrated by ultra®ltration. Doppel was puri®ed by the same protocol. Protein concentrations were determined photometrically in 6 M GdnHCl using calculated extinction coef®cients. GroEL and GroES were puri®ed as previously described.50

870 Protection of GroEL-bound PrP by GroES Denatured PrP (U-PrP) (100-fold diluted from GdnHCl) and refolded PrP (N-PrP) (0.5 mM, ®nal concentrations, respectively) was mixed with GroEL (1 mM) in buffer A (50 mM Na-phosphate, pH 7, 50 mM KOAc, 50 mM NaCl, 5 mM Mg(OAc)2). When indicated, GroES (2 mM) and ADP (1 mM) were added after one minute incubation. Bovine serum albumin (BSA) was present to reach similar total protein concentrations for all reactions. Incubation with proteinase K (PK, 2.5 mg/ml) was at 25  C. Aliquots of the reactions were withdrawn at the times indicated. PK activity was stopped with PMSF (1 mM) and reactions were analyzed by SDS-PAGE and immunoblotting with antibody 3F4 (rabbit anti-PrP antiserum A7 in the case of PrP 121-231) and the ECL chemoluminescence system (Amersham). Aggregation assays U-PrP (100 mM) in 6 M GdnHCl, 100 mM Tris (pH 7.5) was diluted (1:100) into either 50 mM Hepes, pH 7 (buffer B) or 50 mM NaOAc, pH 5.5 (buffer C) containing the same salt additions as buffer A (see above). N-PrP was diluted 1:50 into these buffers (1 mM, ®nal concentration). Reactions contained GroEL and GroES (typically 1 mM and 2 mM, respectively) and ATP (1 mM) as indicated. Aggregation was recorded at 25  C by monitoring turbidity at 320 nm.25 Under the conditions analyzed the aggregation behavior of PrP was independent of the six His-tag typically used for puri®cation (data not shown). For ®lter retardation assays,34 U-PrP23-231 or NPrP23-231 (1 mM) was incubated in buffer C/1 mM ATP alone or buffer C/ATP with GroEL (1 mM). GroEL (1 mM) served as a control. After ten minutes at 25  C reactions were ®ltered through a cellulose acetate membrane (0.2 mm, Schleicher & Schuell) using a slot-blot apparatus and washed twice with 200 ml of 0.1 % (w/v) SDS. Membranes were blocked with 5 % (w/v) BSA in PBS/0.1 % (v/v) Tween 20 and PrP aggregates or GroEL detected by immunostaining. To analyze the recruitment of PrP into preformed PrP aggregates, aggregation reactions were performed in the presence of GroEL/ATP as described above. Aggregates were pelleted (one minute, 10,000 g), washed with buffer C, dispersed in the same buffer by sonication (two minutes in a waterbath sonicator) and added to reactions containing fresh U-PrP but no GroEL. The maximum amount of preformed aggregates was estimated assuming that 60 % of total PrP in the reaction had aggregated in the presence of GroEL, corresponding to the amount of pelletable PrP. Turbidity of preformed aggregates was set to 0 and remained constant during the observation period. Protease resistance of PrP aggregates U-PrP23-231 (1 mM, ®nal concentration) was diluted into buffer C with or without GroEL (0-2 mM) as described above and incubated for 20 minutes at 25  C. BSA was added to compensate for differences in total protein content. After adjusting to pH 7 by addition of 2 M Hepes (pH 7), aliquots containing undigested PrP were withdrawn (0.3 mg PrP). The remaining material (2 mg PrP) was pelleted by centrifugation (one minute, 10,000 g), resuspended in 100 mM Na-phosphate, 150 mM NaCl (pH 7) containing 0.02 % Sarkosyl and

Chaperonin-mediated Prion Protein Aggregation 0-20 mg/ml PK. After incubation for one hour at 37  C digestion was stopped with 1 mM PMSF. Spectroscopy For tryptophan ¯uorescence spectroscopy (excitation at 295 nm, Spex Fluorolog 3 spectro¯uorimeter) N-PrP (0.125 mM) and GroEL were mixed in buffer B. Binding kinetics was recorded with a stop-¯ow spectrometer (Applied Photophysics). Syringes contained N-PrP23-231 (1 mM) or N-PrP23-231F175W (1 mM) and GroEL (1 mM). For emission detection a cutoff ®lter (320 nm) was used and PM voltage was recorded. Control experiments in which PrP or GroEL were diluted into buffer alone did not show any signi®cant change in signal. Data (average of 13 traces) were ®tted using a single or double exponential, ¯oating end point equation. For infrared spectroscopy, refolded N-PrP23-231 (4 mM, ®nal concentration) was incubated for 30 minutes at 25  C in 20 mM NaOAc (pH 5.5), 5 mM KOAc, 2.5 mM Mg(OAc)2, with or without GroEL covalently coupled to Af®-Gel 15 (Biorad) in the presence of 1 mM ATP. Immobilized GroEL induced PrP aggregation with similar ef®ciency as free GroEL. Immobilized GroEL was removed by ®ltration through a microcolumn (Molbiol) and PrP dried as a thin ®lm on a CaF2-window. Spectra were recorded on a Perkin Elmer infrared spectrometer. Congo red binding was measured by incubating PrP aggregates generated from U-PrP23-231 at pH 5.5 in the presence of GroEL/ATP with 1 mM Congo red for 30 minutes.51 GroEL and refolded N-PrP were analyzed as controls. Absorption spectra were measured in the range of 400 to 650 nm. To correct for the absorbance of buffers and proteins, absorbance spectra recorded prior to Congo red addition were subtracted.

Acknowledgments We thank W. Schiebel and R. Schiebel for preparing GroEL and GroES, and W. Houry, M. Hayer-Hartl, J. Tatzelt and J. Young for helpful discussion. J. S. was the recipient of a fellowship of the Stipendienprogramm Infektionsbiologie, German Cancer Research Center, Heidelberg. This work was supported by grants from the European Union (BMH4-CT98-6050) and the Deutsche Forschungsgemeinschaft.

References 1. Jackson, G. S. & Clarke, A. R. (2000). Mammalian prion proteins. Curr. Opin. Biol. 10, 69-74. 2. Prusiner, S. B. (1998). Prions. Proc. Natl Acad. Sci. USA, 95, 13363-13383. 3. Weissmann, C. (1996). Molecular biology of transmissible spongiform encephalopathies. FEBS Letters, 389, 3-11. 4. Donne, D. G., Viles, J. H., Groth, D., Mehlhorn, I., James, T. L., Cohen, F. E. et al. (1997). Structure of the recombinant full-length hamster prion protein PrP(29-231): The N-terminus is is highly ¯exible. Proc. Natl Acad. Sci. USA, 94, 13452-13457. 5. James, T. L., Liu, H., Ulyanov, N. B., Farr-Jones, S., Zhang, H., Donne, D. G. et al. (1997). Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proc. Natl Acad. Sci. USA, 94, 10086-10091.

Chaperonin-mediated Prion Protein Aggregation 6. Riek, R., Hornemann, S., Wider, G., Glockshuber, R. & WuÈthrich, K. (1997). NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231). FEBS Letters, 413, 282-288. 7. StoÈckel, J., Safar, J., Wallace, A. C., Cohen, F. E. & Prusiner, S. B. (1998). Prion protein selectively binds copper(II) ions. Biochemistry, 37, 7185-7193. 8. Viles, J. H., Cohen, F. E., Prusiner, S. B., Goodin, D. B., Wright, P. E. & Dyson, H. J. (1999). Copper binding to the prion protein: structural implications of four identical cooperative binding sites. Proc. Natl Acad. Sci. USA, 96, 2042-2047. 9. Cohen, F. E., Pan, K.-M., Huang, Z., Baldwin, M., Fletterick, R. J. & Prusiner, S. B. (1994). Structural clues to prion replication. Science, 264, 530-531. 10. Wildegger, G., Liemann, S. & Glockshuber, R. (1999). Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates. Nature Struct. Biol. 6, 550-553. 11. Hosszu, L. L., Baxter, N. J., Jackson, G. S., Power, A., Clarkel, A. R., Waltho, J. P. et al. (1999). Structural mobility of the human prion protein probed by backbone hydrogen exchange. Nature Struct. Biol. 6, 740-743. 12. Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H. et al. (1999). Reversible conversion of monomeric human prion protein between native and ®brillogenic conformations. Science, 283, 1935-1937. 13. Telling, G. C., Scott, M., Mastrianni, J., Gabizon, R., Torchia, M., Cohen, F. E. et al. (1995). Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell, 83, 79-90. 14. Zahn, R. (1999). Prion propagation and molecular chaperones. Quart. Rev. Biophys. 32, 309-370. 15. Hartl, F. U. (1996). Molecular chaperones in cellular protein folding. Nature, 381, 571-580. 16. Glover, J. R. & Lindquist, S. (1998). Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell, 94, 73-82. 17. Goloubinoff, P., Mogk, A., Zvi, A., Tomoyasu, T. & Bukau, B. (1999). Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc. Natl Acad. Sci. USA, 96, 13732-13737. 18. Shtilerman, M., Lorimer, G. H. & Englander, S. W. (1999). Chaperonin function: folding by forced unfolding. Science, 284, 822-825. 19. Weber-Ban, E., Reid, B., Miranker, A. & Horwich, A. (1999). Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature, 401, 29-30. 20. DebBurman, S. K., Raymond, G. J., Caughey, B. & Lindquist, S. (1997). Chaperone-supervised conversion of prion protein to its protease- resistant form. Proc. Natl Acad. Sci. USA, 94, 13938-13943. 21. Sigler, P. B., Xu, Z., Rye, H. S., Burston, S. G., Fenton, W. A. & Horwich, A. L. (1998). Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581-608. 22. Edenhofer, F., Rieger, R., Famulok, M., Wendler, W., Weiss, S. & Winnacker, E.-L. (1996). Prion protein PrPC interacts with molecular chaperones of the Hsp60 family. J. Virol. 70, 4724-4728. 23. Borchelt, D. R., Taraboulos, A. & Prusiner, S. B. (1992). Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J. Biol. Chem. 267, 16188-16199.

871 24. Caughey, B., Raymond, G. J., Ernst, D. & Race, R. E. (1991). N-terminal truncation of the scrapieassociated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J. Virol. 65, 6597-6603. 25. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L. & Hartl, F. U. (1991). Chaperoninmediated protein folding at the surface of groEL through a `molten globule'-like intermediate. Nature, 352, 36-42. 26. Langer, T., Pfeifer, G., Martin, J., Baumeister, W. & Hartl, F.-U. (1992). Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 11, 4757-4765. 27. Hayer-Hartl, M. K., Weber, F. & Hartl, F. U. (1996). Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis. EMBO J. 15, 6111-21. 28. Mayhew, M., da Silva, A. C., Martin, J., ErdjumentBromage, H., Tempst, P. & Hartl, F. U. (1996). Protein folding in the central cavity of the GroELGroES chaperonin complex. Nature, 379, 420-426. 29. Weissman, J. S., Rye, H. S., Fenton, W. A., Beechem, J. M. & Horwich, A. L. (1996). Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction. Cell, 84, 481-490. 30. Hornemann, S. & Glockshuber, R. (1996). Autonomous and reversible folding of a soluble aminoterminally truncated segment of the mouse prion protein. J. Mol. Biol. 262, 614-619. 31. Mehlhorn, I., Groth, D., StoÈckel, J., Moffat, B., Reilly, D., Yansura, D. et al. (1996). High-level expression and characterization of a puri®ed 142-residue polypeptide of the prion protein. Biochemistry, 35, 55285537. 32. Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D. et al. (1993). Conversion of ahelices into b-sheets features in the formation of the scrapie prion proteins. Proc. Natl Acad. Sci. USA, 90, 10962-10966. 33. Moore, R. C., Lee, I. Y., Silverman, G. L., Harrison, P. M., Strome, R., Heinrich, C. et al. (1999). Ataxia in prion protein (PrP)-de®cient mice is associated with upregulation of the novel PrP-like protein Doppel. J. Mol. Biol. 292, 797-817. 34. Wanker, E. E., Scherzinger, E., Heiser, V., Sittler, A., Eickhoff, H. & Lehrach, H. (1999). Membrane ®lter assay for detection of amyloid-like polyglutaminecontaining protein aggregates. Methods Enzymol. 309, 375-386. 35. Rochet, J.-C. & Landsbury, P. T. (2000). Amyloid ®brillogenesis: themes and variations. Curr. Op. Struct. Biol. 10, 60-68. 36. Fischer, M., RuÈlicke, T., Raeber, A., Sailer, A., Moser, M., Oesch, B. et al. (1996). Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J. 15, 1255-1264. 37. Rogers, M., Yehiely, F., Scott, M. & Prusiner, S. B. (1993). Conversion of truncated and elongated prion proteins into the scrapie isoform in cultured cells. Proc. Natl Acad. Sci. USA, 90, 3182-3186. 38. Shmerling, D., Hegyi, I., Fischer, M., BlaÈttler, T., Brandner, S., GoÈtz, J. et al. (1998). Expression of amino-terminally truncated PrP in the mouse leading to ataxia and speci®c cerebellar lesions. Cell, 93, 203-214.

872

Chaperonin-mediated Prion Protein Aggregation

39. Peretz, D., Williamson, R. A., Matsunaga, Y., Serban, H., Pinilla, C., Bastidas, R. et al. (1997). A conformational transition at the N-terminus of the prion protein features in formation of the scrapie isoform. J. Mol. Biol. 273, 614-622. 40. Kocisko, D. A., Priola, S. A., Raymond, G. J., Chesebro, B., Lansbury, P. T., Jr & Caughey, B. (1995). Species speci®city in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc. Natl Acad. Sci. USA, 92, 3923-3927. 41. Farr, G. W., Scharl, E. C., Schumacher, R. J., Sondek, S. & Horwich, A. L. (1997). Chaperonin-mediated folding in the eukaryotic cytosol proceeds through rounds of release of native and nonnative forms. Cell, 89, 927-937. 42. Roseman, A. M., Chen, S., White, H., Braig, K. & Saibil, H. R. (1996). The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell, 87, 241-251. 43. Caughey, B. & Chesebro, B. (1997). Prion protein and the transmissible spongiform encephalopathies. Trends Cell Biol. 7, 56-62. 44. Dobson, C. M. (1999). Protein misfolding, evolution and disease. Trends Biochem. Sci. 24, 329-332.

45. Chen, L. S. P. B. (1999). The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity. Cell, 99, 757-768. 46. Zhu, X., Zhao, X., Burkholder, W. F., Gragerov, A., Ogata, C. M., Gottesman, M. E. & Hendrickson, W. A. (1996). Structural analysis of substrate binding by the molecular chaperone DnaK. Science, 272, 1606-1614. 47. Buckle, A. M., Zahn, R. & Fersht, A. R. (1997). A structural model for GroEL-polypeptide recognition. Proc. Natl Acad. Sci. USA, 94, 3571-3575. 48. Chen, L. & Sigler, P. (1999). The crystal structure of a GroEL/peptide complex: plasticity asa basis for substrate diversity. Cell, 99, 757-768. 49. Houry, W. A., Frishman, D., Eckerskorn, C., Lottspeich, F. & Hartl, F. U. (1999). Identi®cation of in vivo substrates of the chaperonin GroEL. Nature, 402, 147-154. 50. Hayer-Hartl, M. K., Ewbank, J. J., Creighton, T. E. & Hartl, F. U. (1994). Conformational speci®city of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin. EMBO J. 13, 3192202. 51. Klunk, W. E., Pettefrew, J. W. & Abraham, D. J. (1989). Quantitative evaluation of Congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J. Histochem. Cytochem. 37, 12731281.

Edited by W. Baumeister (Received 11 June 2001; received in revised form 31 August 2001; accepted 4 September 2001)