Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.     

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.     

Chaperone-mediated 26S Proteasome Remodeling Facilitates Free K63 Ubiquitin Chain Production and Aggresome Clearance

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.     

Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1

Abnormal polypeptides that escape proteasome-dependent degradation and aggregate in cytosol can be transported via microtubules to an aggresome, a recently discovered organelle where aggregated proteins are stored or degraded by autophagy. We used synphilin 1, a protein implicated in Parkinson disease, as a model to study mechanisms of aggresome formation. When expressed in na?ve HEK293 cells, synphilin 1 forms multiple small highly mobile aggregates. However, proteasome or Hsp90 inhibition rapidly triggered their translocation into the aggresome, and surprisingly, this response was independent on the expression level of synphilin 1. Therefore, aggresome formation, but not aggregation of synphilin 1, represents a special cellular response to a failure of the proteasome/chaperone machinery. Importantly, translocation to aggresomes required a special aggresome-targeting signal within the sequence of synphilin 1, an ankyrin-like repeat domain. On the other hand, formation of multiple small aggregates required an entirely different segment within synphilin 1, indicating that aggregation and aggresome formation determinants can be separated genetically. Furthermore, substitution of the ankyrin-like repeat in synphilin 1 with an aggresome-targeting signal from huntingtin was sufficient for aggresome formation upon inhibition of the proteasome. Analogously, attachment of the ankyrin-like repeat to a huntingtin fragment lacking its aggresome-targeting signal promoted its transport to aggresomes. These findings indicate the existence of transferable signals that target aggregation-prone polypeptides to aggresomes.     

Backbone and side-chain 1H, 13C and 15N resonance assignments of LEN, a human immunoglobulin κIV light-chain variable domain

¹H, ¹³C and ¹⁵N resonance assignments are presented for a recombinant 114 amino acid human immunoglobulin (Ig) κIV light-chain variable domain (V_L) LEN, which displays a high degree of sequence identity with another human Ig κIV V_L, SMA. While SMA is highly amyloidogenic in vivo and in vitro and has been linked to the pathogenesis of light-chain amyloidosis, LEN is non-amyloidogenic in vivo and can be converted to the amyloid state only in vitro under destabilizing conditions. Measurements of longitudinal and transverse amide ¹⁵N relaxation rates confirm that, as expected, LEN is a dimer at physiological pH and typical concentrations used for NMR studies, and the analysis of secondary chemical shifts indicates that the protein has a high β-sheet content. These findings are consistent with previously published biophysical data and the high-resolution X-ray structure of LEN.     

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.     

Differential degradation of the three fibrinogen chains by proteasomes: involvement of Sec61p and cytosolic Hsp70

HepG2 cells, which synthesize and secrete fibrinogen, accumulate surplus Aalpha and gamma chains. The nonsecreted fibrinogen chains are degraded both by proteasomes and lysosomes, with unassembled chains primarily degraded by proteasomes and an Aalpha-gamma complex by lysosomes. To further determine the mechanisms by which unassembled fibrinogen chains are degraded, and to explain the pools of Aalpha and gamma chains that occur in HepG2 cells, the association of fibrinogen chains with Sec61beta, a component of the translocon, and with a cytosol chaperone, Hsp70, was studied in both HepG2 cells and COS cells expressing single fibrinogen chains. Retrotranslocation from the lumen of the endoplasmic reticulum was shown by treatment with MG132, a proteasome inhibitor. MG132 caused glycosylated Bbeta to accumulate on Sec61beta in COS cells expressing Bbeta and acted similarly with all three fibrinogen chains in HepG2 cells. In HepG2 cells, Bbeta was associated with Sec61beta ahead of Aalpha and gamma chains, suggesting that pools of Aalpha and gamma chains may be caused by unequal rates of retrotranslocation. In COS cells, retrotranslocation into the cytoplasm was demonstrated by the ATP-sensitive association of ubiquitinylated Aalpha, Bbeta, and gamma chains bound to Hsp70. More Aalpha and gamma than Bbeta accumulated on Hsp70 of HepG2 cells, consistent with more rapid degradation of Bbeta. Overexpression of Hsp70 in HepG2 cells resulted in decreased secretion, but not synthesis, of fibrinogen. Decreased secretion may be due to enhanced degradation of unassembled fibrinogen chains, indicating that proteolysis by proteasomes might regulate both the intracellular pools of fibrinogen chains and fibrinogen secretion.     

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome‐like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin‐1 interacted directly with the RuvbL1 barrel‐like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.     

Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.     

Both the environment and somatic mutations govern the aggregation pathway of pathogenic immunoglobulin light chain.

Deposition of monoclonal immunoglobulin light chain (LC) aggregates in tissues is the hallmark of a class of fatal diseases with no effective treatment. In the most prevalent diseases two different types of LC aggregates are observed: fibrillar deposits in LC amyloidosis (AL) and granular aggregates in LC deposition disease (LCDD). The mechanisms by which a given LC forms either type of aggregate are not understood. Although some LCs are more aggregation-prone than others, this does not appear to be due to specific sequence determinants, but more likely from global properties that can be introduced by multiple somatic mutations. Moreover, a single LC isotype can sometimes form both fibrillar and granular aggregates within the same patient. To better understand how the different aggregation pathways arise, we developed a series of in vitro assays to analyze the formation of distinct aggregate types. The recombinant kappa IV LC (SMA) assembles into fibrils when agitated. We now show that SMA can also form granular aggregates upon exposure to copper, and that this aggregation can occur not only in vitro, but also in cells. A constellation of somatic mutations, consisting of His89/His94/Gln96, is sufficient to confer sensitivity to copper on wild-type kappa IV proteins. The formation of both types of aggregates is inhibited by synthetic peptides derived from the LC variable domain. However, the peptide that inhibits fibrillar aggregation is different from the peptide that inhibits copper-induced aggregation. Thus, distinct molecular surfaces of the LC underly each type of aggregate. We conclude that both the intrinsic properties of the sequence and extrinsic conditions govern the aggregation pathway of a LC.     

Shear flow induces amyloid fibril formation

Shear flow is indirectly implicated in amyloid formation in vitro. Despite the association between amyloid fibrils and disease, and the prevalence of flow in physiological systems, the effect of this parameter is uncharacterized. We designed a novel Couette cell to quantitatively investigate shear exposure during fibrillogenesis. Amyloid formation by beta-lactoglobulin was monitored in situ with real-time fluorescence measurements across a range of shear rates. We demonstrate shear-induced aggregation of spheroidal seed-like species. These seeds enhance fibril formation in native beta-lactoglobulin, thereby demonstrating that shear flow generates an amyloidogenic precursor. Furthermore, preformed fibrils are degraded by exposure to high shear rates. Our results have implications for the mechanism of amyloid formation in physiological flow conditions.     

Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer

Protein conformational changes that result in misfolding, aggregation and amyloid fibril formation are a common feature of many neurodegenerative disorders. Studies with beta-amyloid (Abeta), alpha-synuclein and other amyloid-forming proteins indicate that the assembly of misfolded protein conformers into fibrils is a complex process that may involve the population of metastable spherical and/or annular oligomeric assemblies. Here, we show by atomic force microscopy that a mutant huntingtin fragment with an expanded polyglutamine repeat forms spherical and annular oligomeric structures reminiscent of those formed by Abeta and alpha-synuclein. Notably, the molecular chaperones Hsp70 and Hsp40, which are protective in animal models of neurodegeneration, modulate polyglutamine aggregation reactions by partitioning monomeric conformations and disfavoring the accretion of spherical and annular oligomers.     

Interactions between Hsp70 and the hydrophobic core of alpha-synuclein inhibit fibril assembly

Molecular chaperones of the heat shock protein 70 (Hsp70) family counteract protein misfolding in a variety of neurodegenerative disease models. To determine whether human Hsp70 exerts similar effects on the aggregation of alpha-synuclein (alpha-Syn), the key component of insoluble fibrils present in Parkinson's disease, we investigated alpha-Syn fibril assembly in the presence of Hsp70. We found in vitro assembly was efficiently inhibited by substoichiometric concentrations of purified Hsp70 in the absence of cofactors. Experiments using alpha-Syn deletion mutants indicated that interactions between the Hsp70 substrate binding domain and the alpha-Syn core hydrophobic region underlie assembly inhibition. This assembly process was inhibited prior to the elongation stage as we failed to detect any fibrils by electron microscopy. In addition, fluorescence polarization and binding assays suggest that Hsp70 recognizes soluble alpha-Syn species in a highly dynamic and reversible manner. Together, these results provide novel insights into how Hsp70 suppresses alpha-Syn aggregation. Furthermore, our findings suggest that this critical step in Parkinson's disease pathogenesis may be subject to modulation by a common molecular chaperone.     

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/α‐synuclein complex by Hip

The ATP‐dependent protein chaperone heat‐shock protein 70 (Hsp70) displays broad anti‐aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α‐synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation‐prone polypeptides. We show a nucleotide‐dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co‐aggregation phenomenon can be prevented in vitro by the co‐chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.     

Hsc70 protein interaction with soluble and fibrillar alpha-synuclein

The aggregation of α-synuclein (α-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native α-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stress-induced heat shock protein 70 (Hsp70) on α-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with α-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and α-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble α-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble α-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing α-Syn assembly inhibition by Hsc70. We show that Hsc70 binds α-Syn fibrils with a 5-fold tighter affinity compared with soluble α-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight α-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of α-Syn assemblies. We show a reduced cellular toxicity of α-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of α-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.     

Structural transformations of oligomeric intermediates in the fibrillation of the immunoglobulin light chain LEN

LEN is a kappaIV immunoglobulin light chain variable domain from a patient suffering from multiple myeloma but with no evidence of amyloid fibrils. However, fibrils are formed when LEN solutions are agitated under mildly destabilizing conditions. Surprisingly, an inverse concentration dependence was observed on the kinetics of fibril formation because of the formation of off-pathway soluble oligomers at high protein concentration. Despite the fact that most of the protein is present in the off-pathway intermediates at relatively early times of aggregation, eventually all the protein forms fibrils. Thus, a structural rearrangement from the non fibril-prone off-pathway oligomers to a more fibril-prone species must occur. A variety of techniques were used to monitor changes in the size, secondary structure, solvent accessibility, and intrinsic stability of the oligomers, as a function of incubation time. The structural rearrangement was accompanied by a significant increase of disordered secondary structure, an increase in solvent accessibility, and a decrease in intrinsic stability of the soluble oligomeric species. We conclude that fibrils arise from the oligomers containing a less stable conformation of LEN, either directly or via dissociation. This is the first fibrillating system in which soluble off-pathway oligomeric intermediates have been shown to be the major transient species and in which fibrillation occurs from a relatively unfolded conformation present in these intermediates.     

Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid beta (Abeta) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Abeta aggregation. These observations led to the hypothesis that chaperones might interact with Abeta and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Abeta has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Abeta aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Abeta self-assembly and that these chaperones are effective at substoichiometric concentrations (approximately 1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Abeta is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.     

The critical role of the constant region in thermal stability and aggregation of amyloidogenic immunoglobulin light chain

Light chain (LC) amyloidosis (AL) is a fatal disease in which immunoglobulin LC deposit as fibrils. Although the LC amyloid-forming propensity is attributed primarily to the variable region, fibrils also contain full-length LC comprised of variable-joining (V(L)) and constant (C(L)) regions. To assess the role of C(L) in fibrillogenesis, we compared the thermal stability of full-length LC and corresponding V(L) and C(L) fragments. Protein unfolding and aggregation were monitored by circular dichroism and light scattering. A full-length λ6 LC purified from urine of a patient with AL amyloidosis showed irreversible unfolding coupled to aggregation. The transition temperature decreased at slower heating rates, indicating kinetic effects. Next, we studied five recombinant λ6 proteins: full-length amyloidogenic LC, its V(L), germline LC, germline V(L), and C(L). Amyloidogenic and germline proteins showed similar rank order of stability, V(L) < LC < C(L); hence, in the full-length LC, V(L) destabilizes C(L). Amyloidogenic proteins were less stable than their germline counterparts, suggesting that reduction in V(L) stability destabilizes the full-length LC. Thermal unfolding of the full-length amyloidogenic and germline LC required high activation energy and involved irreversible aggregation, yet the unfolding of the isolated V(L) and C(L) fragments was partially reversible. Therefore, compared to their fragments, full-length LCs are more likely to initiate aggregation during unfolding and provide a template for the V(L) deposition. The kinetic barrier for this aggregation is regulated by the stability of the V(L) region. This represents a paradigm shift in AL fibrillogenesis and suggests C(L) region as a potential therapeutic target.     

Effect of methionine oxidation on the structural properties, conformational stability, and aggregation of immunoglobulin light chain LEN

Light chain amyloidoses arise from the overproduction and abnormal deposition of the immunoglobulin light chain in various organs. LEN is the variable domain of an immunoglobulin light chain originally isolated from the urine of a patient suffering from multiple myeloma, with no sign of renal dysfunction or amyloidosis. LEN was shown to form fibrils in vitro under mildly destabilizing conditions. In this work, we investigated the changes induced by methionine oxidation in the structural properties, conformational stability, and aggregation behavior of immunoglobulin light chain domain LEN. We established that LEN was well-protected from oxidation in its native state, but successful oxidation was achieved in the presence of 4 M GuHCl. Oxidation induced noticeable structural changes in LEN and destabilized this protein. The methionine-oxidized LEN preferred to form amorphous aggregates instead of fibrils. The results indicated that the LEN oxidation may play an important role in amorphous deposition of the protein, but not in its fibrillation.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.