Articles pubs.acs.org/acschemicalbiology
Deconstruction of Stable Cross-Beta Fibrillar Structures into Toxic and Nontoxic Products Using a Mutated Archaeal Chaperonin Dmitry Kurouski,† Haibin Luo,‡ Valentin Sereda,† Frank T. Robb,‡ and Igor K. Lednev†,* †
Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222, United States Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, Maryland 21201, United States
‡
S Supporting Information *
ABSTRACT: Our group recently determined that a mutant archaeal chaperonin (Hsp 60) exhibited substantially enhanced protein folding activity at low temperatures and was able to deconstruct refractory protein aggregates. ATP dependent conversion of fibril structures into amorphous aggregates was observed in insulin amyloid preparations (Kurouski et al. Biochem. Biophys. Res. Commun. 2012). In the current study, mechanistic insights into insulin fibril deconstruction were obtained by examination of early stage complexes between Hsp60 and fibrils in the absence of ATP. Activity of the Hsp60 was significantly curtailed without ATP; however, some fibril deconstruction occurred, which is consistent with some models of the folding cycle that predict initial removal of unproductive protein folds. Chaperonin molecules adsorbed on the fibril surface and formed chaperonin clusters with no ATP present. We propose that there are specific locations on the fibril surface where chaperonin can unravel the fibril to release short fragments. Spontaneous coagulation of these fibril fragments resulted in the formation of amorphous aggregates without the release of insulin into solution. The addition of ATP significantly increased the toxicity of the insulin fibril-chaperonin reaction products toward mammalian cells. toxic than mature fibrils.5 Interestingly, a similar mechanism has recently been reported regarding Microcin E46, produced by the bacterium Klebsiella pneumonia. This particular microcin toxin is dispersed from its inert amyloid form in response to environmental change.14 Chaperones and heat-shock proteins are very promising generic tools that help reduce the number of misfolded protein species to favor native protein conformations. Chaperone proteins and protein complexes are highly conserved among species from different kingdoms. Chaperones maintain cellular proteostasis by active and passive folding processes that favor the native conformations of misfolded proteins and nascent polypeptides. For example, the binding of the small heat shock protein αB-crystallin to amyloid β (Aβ) fibrils inhibits fibrillar elongation and reduces the toxicity.15,16 Heat-shock protein 104 from yeast was shown to fragment mature prion fibrils in vitro.17,18 It was shown that a result of fragmentation, the toxicity of fragmented species was increased. Several other heat shock proteins have been reported to have a similar impact on fibrils. For instance, Hsp70 and Hsp90 modulate the assembly of α-synuclein amyloid fibrils.19−22 Human Hsp60 (also known as CCT, chaperonin, or TriC) cooperates with Hsp 70 to prevent the huntingtin protein from fibrillation.23
Specific protein aggregates called amyloid fibrils have been linked to more than 15 severe human maladies, including prionrelated, Alzheimer’s, and Parkinson’s diseases. These important malfunctions are often referred to as “conformational” disorders, which result from the conversion of a normal protein isoform into a specific β-sheet-rich polymeric amyloid form. Recent studies have demonstrated that a broad variety of proteins unrelated to any known conformational disease can adopt β-sheet-rich amyloid forms in vitro and in vivo.1−3 The mechanism of fibril accumulation in amyloid-associated maladies remains elusive. It was previously demonstrated that amyloid fibrils are toxic toward mammalian cells.4,5 They cause a formation of reactive oxygen species and an influx of calcium ions into cells.6 In addition, amyloid fibrils serve as templates for the aggregation of other misfolded and native proteins.7,8 Thus, the degradation or destruction of amyloid fibrils has been the primary focus of potential treatments for protein conformational neuropathy. This approach is fraught with difficulties because amyloid fibrils are the most thermodynamically stable protein forms; only harsh denaturing conditions, such as high pH, pressure, or temperature, or chaotropic solvents at high concentrations are able to disrupt fibrillar structures.9−11 Clearly, these strategies to destabilize amyloid fibrils have practical limitations in therapeutic practice. Substantial evidence suggests that small misfolded protein aggregates are more toxic than larger aggregates.12,13 Significantly, it has been demonstrated that proto-filaments and proto-fibrils, the precursors of mature amyloid fibrils, are more © 2013 American Chemical Society
Received: April 6, 2013 Accepted: July 21, 2013 Published: July 22, 2013 2095
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Archaeal Hsp60 typically forms 1mDa double-ring complexes consisting of eight subunits with a diameter of ∼30 nm and a height of ∼6 Å (Supporting Information (SI) Figure 1). The complexes are structural models for the eukaryotic CCT, except that the number of subunits in archaeal Hsp60 complexes is drastically reduced, compared with eight different subunits in mammalian CCT. The hyperthermophile Hsp60 used in this study is a single ring complex with eight identical subunits. In our previous studies, we constructed a cold-adapted mutant form of Hsp60 from the hyperthermophile Pyrococcus furiosus (Pf) that formed a single-ring, eight-subunit complex that could fragment insulin fibrils in the presence of ATP.24,25,53 We demonstrated that, upon addition of the Hsp60, individual fibril fragments swell and the β-sheet quickly transforms into a mixture of α-helical and unordered protein conformations. Upon further incubation, the fragments coalesced, forming large amorphous aggregates with polydisperse topologies. Questions remain concerning the initial Hsp60 interaction with fibrils and how ATP may modulate these interactions and chaperone activity on macroscopic fibrillar substrates. Hsp60 has a conformational cycle similar to the one of GroEL/ES, despite the fact that this Group I chaperonin differs in several aspects from the archaeal Group II chaperonins in subunit count per ring and the presence of a detachable lid complex, GroES.3,26−28 ATP was previously clearly demonstrated to be essential for the folding activity of GroEL/ES. In the present study, we carried out fibril-chaperonin reactions with the singlering variant of Hsp60 described in our previous publication as MA (Mutant All) with and without ATP.53 ATP hydrolysis is normally considered to be essential for the chaperonins’ activity through the so-called power stroke,28 the repetitive opening and closing of the individual rings through radical, cooperative conformational changes necessary for their folding activity. Previously, we confirmed that ATP is required for the accumulation of helical structure from dissolved fibrils by Hsp60.24 However, transformation of the fibrils by the archaeal single-ring Hsp60 was noted even in the control experiments without ATP in the previous study. In this work, we examine this novel action of Hsp60 in detail.
Figure 1. ThT fluorescence of the fibrillar solution (Fibrils) and the mixture of fibrils and Hsp60 with ATP (Fibrils + Hsp60 + ATP) and without ATP (Fibrils + Hsp60) after 4-h incubation at 37 °C. As controls, intact insulin (Monomeric Insulin) and Hsp60 chaperones (Hsp60) are included.
overall change in fibrillar morphology.24 Fibrils were almost completely deconstructed in the presence of Hsp60 and ATP. We used atomic force microscopy (AFM) imaging to investigate the initial stage of the chaperonin-fibril interaction in the absence of ATP. Reaction mixture aliquots were deposited onto a mica substrate after 5 min of incubation (Figure 2).
Figure 2. Insulin fibrils covered with chaperonins on their surfaces (A−C). Hsp60 were mixed with insulin fibrils without ATP for 5 min. The reaction was terminated by depositing the solution onto the mica surface with quick drying. The scale bar is 500 nm.
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RESULTS AND DISCUSSION Hsp60 Causes Rapid β-Sheet Degradation with and without ATP. Insulin fibrils were selected for this study and prepared as described previously.24,29 Insulin is a very wellstudied peptide hormone that has 51 amino acids organized into two polypeptide chains linked by two interchain and one intrachain disulfide bonds. Upon aggregation into fibrils, insulin molecules undergo a structural change to form a β-sheet-rich conformation.30 The thioflavinT (ThT) fluorescence assay is a universal analytical tool used to detect fibrillar β-sheets. The effects of ATP on the chaperonin-mediated transformation of insulin fibrils by ThT assays showed a decrease in β-sheet content in both the presence (2 mM) and absence of ATP (Figure 1). However, the presence of ATP (Fibril + Hsp60 + ATP) resulted in significantly more ThT signal loss than that measured without ATP (Fibrils + Hsp60), indicating enhanced β-sheet degradation in the presence of ATP. Initial Chaperonin-Fibril Interaction in the Absence of ATP. As previously shown, in the presence of ATP, Hsp60 rapidly deconstruct insulin fibrils to form swollen clumps.24 However, controls in which insulin fibrils were incubated without Hsp60 under the same experimental conditions (20 mM sodium acetate, pH 6.0, 2 mM ATP) at 37 °C exhibited no
Intact Hsp60 single-ring particles are ∼6 Å in height and are clearly visible on the surface of insulin fibrils; particles can be seen on both fibril termini and middle portions (Figure 2, red arrows). Interestingly, the Hsp60 complexes attached to fibrils (marked with red arrows) have larger diameters than the free octameric chaperonin complexes (marked with white arrows). In our previous study, we demonstrated that free Hsp60 complex have around ∼6 Å in height and around 30 nm in width, as evident from the reported AFM images.24 Insulin fibrils were found to have around 8 nm height. At the same time, Hsp60 complexes attached to fibrils have a height around 10−14 nm (SI Figure 2). This might indicate that several chaperonins are adsorbed at the same spot, forming a cluster of chaperonins. More experiments are required for proving that the observed spherical aggregates are Hsp60 clusters. We hypothesize that the fibril surface has hydrophobic hotspots where chaperonins can be adsorbed with high efficiency, forming chaperone clusters. Our results might suggest that there are strategic locations on the fibril surface where Hsp60 can unravel the fibril to form short fragments. When ATP is present, the adsorption of Hsp60 to fibril surfaces leads to the rapid turnover of these initial Hsp60/fibril complexes. As 2096
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evident from our previous microscopic observations, in the presence of ATP, Hsp60 causes substantial fibril fragmentation and deconstruction within minutes.24 A clear understanding of fibril surface organization may shed light on the strategic locations, in future studies. Recently, Tip Enhanced Raman Spectroscopy (TERS) has been demonstrated to be a powerful tool capable of determining the structural organization of the insulin fibril surface with nanometer-scale resolution.31 We plan to use TERS in future studies to characterize the insulin fibril surface and insulin fibril fragments in complexes with chaperonin in greater detail. Hsp60 Has a Noticeable but Less Efficient Activity toward Insulin Fibrils without ATP. To better understand the observed changes in fibrillar structure, we monitored the kinetics of fibril deconstruction by chaperones with and without ATP using a turbidity assay. Not surprisingly, our data indicate that in the presence of ATP, the reaction proceeds more rapidly than in its absence (Figure 3).
Figure 4. SEM images of the amorphous aggregates that formed as a result of insulin fibril degradation by Hsp60 with ATP (a, b) and without ATP (c, d). The scale bar is 200 nm.
ThT assay in Figure 1 indicates that some fibrillar β-sheets remain in the system after prolonged treatment with Hsp60, which could be attributed to the remaining intact fibrils seen with AFM and SEM imaging data. No Soluble Protein Is Released As a Result of Hsp60 Action. We addressed the following two questions in this study: (1) whether soluble proteins are released during fibril transformation and (2) whether individual insulin polypeptides are cleaved during the fibril transformation process. The supernatant, which could potentially contain soluble protein, was analyzed by size exclusion chromatography and SDS-PAGE (SI Figures 3 and 4). This experiment confirmed that free native insulin and protein oligomers were both absent from the supernatant. Thus, we conclude that no soluble protein is released as a result of Hsp60 activity. It is possible, although highly unlikely, that Hsp60 directly cleaved insulin, resulting in oligopeptide fragments within the large protein clumps. To address this possibility, the composition of the amorphous aggregates was analyzed. Both initial insulin fibrils and amorphous aggregates were dissolved in DMSO/TFA and analyzed using ESI-MS. In both cases, a single peak characteristic of monomeric insulin at 5735 m/z was observed, indicating the integrity of the polypeptides (data not shown). Toxicity of Insulin Fibrils and the Products of Their Destruction by Chaperonins. The toxicity of amyloid fibrils, in general, and insulin fibrils, in particular, has been reported previously.4,32,33 Zacko et al. demonstrated that insulin filaments have no cellular toxicity, whereas mature fibrils are toxic to pheochromocytoma (PC 12) cells.5 An MTT assay was used to assess the toxicity of untreated insulin fibrils compared with insulin fibrils after incubation in the presence or absence of chaperonin and ATP using SHSY5Y cells. All samples exhibited dose-dependent cellular toxicity (Figure 5, panel a). Insulin fibrils disaggregated by Hsp60 in the presence of ATP formed highly toxic products (Fibrils + Hsp60 + ATP), which exhibited enhanced cytotoxic effects compared with insulin fibrils alone, at the same dose. However, in the absence of ATP (Fibrils + Hsp60), chaperone’s activity did not result in the formation of toxic products because their toxicity (Fibril + Hsp60) was almost identical to that of insulin fibrils (Fibrils). As evident from this study, Hsp60 destroys fibril architecture and forms amorphous aggregates in the presence and absence
Figure 3. The turbidity assay indicated that ATP enhances the fibril degradation reaction by Hsp60. Reaction conditions: 20 mM.
The decrease in the turbidity of the mixture is associated with the chopping of fibrils into smaller fragments and insoluble aggregates, which precipitate and do not contribute to light scattering, thereby reducing turbidity. Turbidity data indicate that chaperonin exhibits significantly higher fibril deconstruction activity in the presence of 2 mM ATP. The reaction proceeds slowly and reaches a stable plateau after approximately 15 min in the absence of ATP. Morphological Changes of Insulin Fibrils As a Result of Fibril-Hsp60 Interaction. We utilized scanning electron microscopy (SEM) to characterize reaction products that are formed after four hours of fibril exposure to Hsp60 in the presence (2 mM) and absence of ATP. As previously reported, the deconstruction of insulin fibrils by Hsp60 results in the formation of amorphous aggregates.24 Typical images are shown in Figures 4, panels a and b.24 We found that Hsp60fibril coincubation with or without ATP leads to the formation of aggregates with similar morphology (Figures 4, panels c and d). We found that a substantial proportion of the original fibrils and large amorphous aggregates were evident on SEM and AFM images of the final products resulting from chaperonin activity with no ATP present (Figure 4, panel c). This observation supports the hypothesis that Hsp60 is able to deconstruct insulin fibrils to form amorphous aggregates without added ATP, although the efficiency is reduced. The 2097
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hydrophobic surface of oligomers and annular proto-fibrils, which is hidden by sheet stacking interactions in fibrils, is responsible for its toxicity.38 One can imagine that the deconstruction of fibrils could uncover the hydrophobic surface and render products of Hsp60 activity in the presence of ATP more toxic than intact fibrils. At the same time, a similar process of fibril fragmentation by Hsp60 is observed in the absence of ATP; however, these products are substantially less toxic. One possibility is that in the presence of ATP, Hsp60 in addition to large aggregates, form highly toxic oligomeric species. Alternatively, the surfaces of these large aggregates are substantially changed by Hsp60. To investigate these possibilities, we performed SDS-PAGE of fibril solution that was exposed to Hsp60 in the presence of ATP. After sample exposure to Hsp60 at 37 °C the suspension was centrifuged to separate amorphous aggregates, fibrils, and Hsp60 from the soluble phase, which could possibly contain oligomers and/or annular proto-fibrils (SI Figure 4). Our data indicate that neither sample pellet nor supernatants contained prefibrillar oligomers or annular protofibrils within 10−100 kDa range. SDS-PAGE data confirmed the HPLC results discussed, which indicate the absence of soluble protein in the sample supernatants. Therefore, it would be logical to expect that the increase in the fibril-Hsp60 activity in the presence of ATP should be associated with structural alteration of the amorphous aggregates’ surfaces. In our future studies, we plan to use TERS to investigate differences in the secondary structures of amorphous aggregates that are formed from insulin fibrils by Hsp60 in the presence and absence of ATP. In summary, the reaction products (amorphous aggregates and fragmented fibril species) of insulin fibril deconstruction by Hsp60 in the presence of ATP exhibited significantly enhanced cell toxicity compared to untreated insulin fibrils. ATP accelerated the fibril deconstruction of Hsp60 and, concomitantly, increased the toxicity of the product. Recently, striking evidence has accumulated to suggest that the ability to form amyloid fibrils is not unique to a small group of disease-related proteins but may be a generic property of polypeptide chains.1 More than 30 proteins and polypeptides are now known to form macroscopic amyloid fibrils. Because they are implicated in pervasive neurodegenerative diseases, amyloid fibrils have been actively investigated to facilitate therapeutic strategies. Specifically, discovery of efficient bioactive agents that block or reverse protein aggregation to relieve neurodegenerative diseases remains one of the most elusive objectives of pharmaceutical research. One possible approach is to inhibit fibrillation by using a small peptide as a blocker of the fibrillation process. For example, Aitken et al. have demonstrated that various polycyclic compounds may be used therapeutically to blockade amyloid fibril formation.39 The inhibition properties of these compounds are based on aromatic intercalation between the compound and the fibril nuclei.40 Resveratrol, a polyphenolic constituent of red wine, exhibits an antiaggregation activity toward Aβ.41 Resveratrol directly recognizes and modifies soluble oligomers and fibrillar intermediates, and promotes their assembly into highmolecular-weight, nontoxic aggregates.42 An alternative approach based on the utilization of conformation-specific antibodies has been developed.43,44 These antibodies may directly recognize the soluble and insoluble fibrillar intermediates that are believed to have the highest toxicity. In addition, these antibodies do not have affinity toward protein monomers.45−47
Figure 5. Insulin fibrils exhibited a dose-dependent cellular toxicity when treated with chaperonin in the presence (Fibrils + Hsp60 + ATP) and absence (Fibril + Hsp60) of ATP (a). As a control, the cellular toxicity of insulin fibrils was examined (Fibrils). ATP increased the toxicity of the insulin plus fibril and Hsp60 interaction product. Amorphous insulin aggregates (Fibril + Hsp60) resulting from Hsp60 treatment exhibited the same toxicity as the initial fibrils (Fibrils). (b) The cell toxicity (% live cells) of Fibrils (red bar), Fibrils + Hsp60 (green bar), and Fibrils + Hsp60 + ATP (blue bar) at 40 μM. Independently, Hsp60 and ATP are nontoxic to SH-SY5Y cells (gray bar).
of ATP. However, the addition of ATP significantly increases the toxicity of the fibril-Hsp60 reaction product (Figure 5, panel a), confirming that Hsp60 activity is activated in the presence of ATP. Control experiments were done to determine whether cellular toxicity was associated with the presence of chaperonins rather than fibrils and their deconstruction products. Toxicity was determined in the presence of ATP and chaperonin alone (with no fibrils) (Figure 5, panel b). Untreated insulin fibrils (40 μM), under the same conditions, caused significant apoptosis (60 ± 10%). The toxicity of fibrils and Hsp60 (at the same concentration, 40 μM) was almost identical to the toxicity of insulin fibrils alone (Figure 5, panel b, green and red bars, respectively). However, in the presence of ATP, Hsp60 formed highly toxic species from insulin fibrils. We found that the Fibrils + Hsp60 + ATP solution exhibited almost twice the cell toxicity (Figure 5, panel b, blue bar) of the Fibril reaction mix and the Fibril + Hsp60 solution. A number of studies have reported that precursors of amyloid fibrils are more toxic than mature fibrils.12,34 Several types of fibril precursors have been identified recently including prefibrillar oligomers and annular protofibrils.34 Application of antibodies for the detection of fibril precursors revealed that prefibrillar oligomers and annular proto-fibrils have distinct generic structures, dissimilar with those of fibrils.35 This suggests that fibrils, prefibrillar oligomers, and annular protofibrils should have distinctly different mechanisms of toxicity. Several potential mechanisms of oligomers’ and protofibrils’ toxicity have been discussed in the literature. For example, oligomers bind and permeabilize cell membranes while fibrils lack this activity.36,37 Another possibility is that the 2098
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factor. One drop was placed onto freshly cleaved mica and incubated for 2 min, followed by the removal of excess solution. Finally, the mica surface was dried under nitrogen flow. AFM scanning was performed immediately in AC tapping mode using an MFP-3D Bio Asylum Research microscope (Asylum Research, CA, U.S.A.) and Olympus AC160 tips. SEM. Twenty microliters of each analyzed solution was diluted at a 1:400 ratio using distilled water and deposited onto a 200-mesh copper grid. Staining with 1% uranyl acetate was performed 10 min after the deposition. Samples were imaged on a Zeiss Supra SEM in InLense mode with 5 kV EHT. ThT Fluorescence Assays. ThT fluorescence assays were performed on a Fluorolog spectrofluorometer (HORIBA Jobin Yvone, Edison, NJ) at RT. Twenty microliters of the sample aliquot was mixed with 3 mL of ThT (25 μM, Sigma-Aldrich) in 10 mM phosphate buffer (pH 7.4). The ThT emission spectra were recorded between 465 and 550 nm using a 450nm excitation in a 1 cm × 1 cm rectangular cell. The solution was stirred using a magnetic bar for several minutes prior to the fluorescence measurements. A total of three spectra were measured for each sample and then averaged. Toxicity. Neuroblastoma cells (SH-SY5Y) were cultured in a 1:1 (v/v) mixture of Dulbecco’s minimum essential medium (DMEM; Sigma, U.S.A.) and Ham’s F-12 nutrient mixture (Sigma, U.S.A.) supplemented with 10% fetal bovine serum (FBS; Sigma, U.S.A.) and antibiotic antimitotic solution (Sigma, U.S.A.) at 37 °C in a 95% humidified and 5% CO2 incubator. Serum-free medium was used for the cell toxicity assays. Cells were plated onto a flat-bottom 96-well plate (1 × 104 cells per well) and incubated at 37 °C overnight. Once cells attached to the surface, various amounts of mature insulin fibrils (Fibrils), the reaction products of insulin fibrils with Hsp60 (Fibrils + Hsp60 + ATP) or mature fibrils without ATP (Fibrils + Hsp60) were added. The fibril concentration was determined by solvating fibrils in DMSO and measuring the sample absorption at 280 nm (A280). A protein sample (insulin) dissolved in DMSO was used as the reference for the calculation of the protein concentration in the dissolved fibril sample. After a 48-h incubation, the cytotoxicity was assessed using the MTT reduction-inhibition assay. An MTT stock solution (5 mg mL−1) was added to the culture medium (0.5 mg mL−1 final concentration). After 4 h of the incubation at 37 °C, the resultant formazan was extracted with 200 μL of an isopropanol and 0.04 N HCl solution; the absorbance was measured at 570 nm using an automatic plate reader. Control experiments were performed by exposing cells to solutions in an equivalent volume of the same buffer for the same length of time. Cell viability was expressed as a percentage of the absorbance in the wells containing cells treated with fibrils compared to the control wells.
As evident from the present study, archaeal Hsp60 complexes do not require ATP to interact with and adsorb onto the fibril surface. Therefore, we can conclude that the Hsp60 recognizes the surface as a substrate protein. Moreover, in the absence of ATP, Hsp60 retains significant fibril deconstruction activity, which causes amorphous aggregates to form. The HspP60 is a nanomachine with interactive subunits and allosteric interaction is generally considered to be essential for the modification of substrate protein conformations.48 It is tempting to speculate that the binding of substrate proteins results in the allosteric activation of the subunits in the ring in the absence of ATP. However, ATP accelerates Hsp60 activity markedly, favoring fibril degradation, and dramatically increasing the toxicity of the fibril-chaperonin reaction product. The interaction between substrate protein and Hsp60 without ATP suggests that the Hsp60 populates conformational states, which include substrate binding, which supports a mechanism in which such state protein binding sites are available spontaneously.49 This favors the conformational selection model originally proposed by Monod−Wyman−Changeux and recently reviewed and supported by Changeux and Wyman.50 The mutant version of the archaeal chaperonin has a strong and site specific affinity for the fibrils and may be considered to be a valid model system to examine the passive unfolding activity.3 In future studies, HYD mutations in Hsp60 that allow ATP binding but not hydrolysis will be used to distinguish the differential passive fibril dispersion activities of ATP bound vs ATP free conformers of the Hsp60. Our primary motivation for using chaperonins to disintegrate amyloid fibrils is based on the potential to restore native protein conformation and normal biological activity. Increased toxicity of amyloidogenic proteins is typically associated with their dispersion into oligomeric, shorter fibrils.5,13,54 From this perspective, our experiments show the potential to disperse fibrils with no increase in toxicity and may be considered to be an advance toward a future therapeutic strategy for protein conformation diseases.
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METHODS Insulin Fibril Preparation. Bovine insulin (60 mg mL−1, Sigma I5500) was dissolved in water with a final pH adjustment to pH 2.5 by titrating with HCl. The solution was incubated at 70 °C for 2 h, and the fibrillation process was terminated by reducing the temperature to ∼25 °C. To remove nonaggregated proteins, the sample was centrifuged at 14 000 × g for 20 min, and the supernatant was removed. The gelatinous phase, dominated by mature fibrils, was dispersed in an HCl solution at pH 2.5. The centrifugation-dispersion procedure was repeated twice. Chaperonin Preparation. Pf Hsp60 was cloned and expressed in E.coli as previously reported.52,53 The supernatants of the extracts were heated at 70 °C for 30 min and then purified to homogeneity using the following two columns for anion exchange: a HiTrap Q HP cartridge from Bio-Rad (Hercules, CA) and a Bio-Scale macro-prep high Q cartridge from GE Healthcare (Uppsala, Sweden). Chaperonin-Fibril Reaction. Insulin fibrils were mixed with Hsp60 (0.05 mg mL−1) in 20 mM sodium acetate buffer, pH 6.0, with or without ATP (2 mM). Mg ions, essential for Hsp60 activity, were added to the buffer together with sodium chloride at final concentrations of 1 mM and 50 mM, respectively. AFM. An aliquot of the analyzed solution was suspended in sodium acetate buffer, pH 6.0, using a 1:400 (v:v) dilution
% living cells =
(sample absorbance − blank absorbance) (control absorbance − blank absorbance) × 100
The blank absorbance is the absorbance of the medium alone, and the control absorbance is the absorbance of cells and medium alone. At least two independent experiments were performed in triplicate for each concentration (values on X scale on the Figure 5, panel b). Transmittance (Turbidity/Light Obscuration) Measurements. Transmittance readings are based on a measurement of an amount of light that comes through the cuvette cell: 2099
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(9) Brange, J., Andersen, L., Laursen, E. D., Meyn, G., and Rasmussen, E. (1997) Toward understanding insulin fibrillation. J. Pharm. Sci. 86, 517−525. (10) Kim, H. Y., Cho, M. K., Riedel, D., Fernandez, C. O., and Zweckstetter, M. (2008) Dissociation of amyloid fibrils of α-synuclein in supercooled water. Angew. Chem., Int. Ed. 47, 5046−5048. (11) Heldt, C. L., Kurouski, D., Sorci, M., Grafeld, E., Lednev, I. K., and Belfort, G. (2011) Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability. Biophys. J. 100, 2792− 2800. (12) Lindquist, S. L., and Kelly, J. W. (2011) Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: Progress and prognosis. Cold Spring Harb. Perspect. Biol. 3, a004507. (13) Stroud, J. C., Liu, C., Teng, P. K., and Eisenberg, D. (2012) Toxic fibrillar oligomers of amyloid-β have cross-β structure. Proc. Natl. Acad. Sci. U.S.A. 109, 7717−7722. (14) Shahnawaz, M., and Soto, C. (2012) Microcin amyloid fibrils A are reservoir of toxic oligomeric species. J. Biol. Chem. 287, 11665− 11676. (15) Lee, S., Carson, K., Rice-Ficht, A., and Good, T. (2005) Hsp20, a novel α-crystallin, prevents Aβ fibril formation and toxicity. Protein Sci. 14, 593−601. (16) Shammas, S. L., Waudby, C. A., Wang, S., Buell, A. K., Knowles, T. P., Ecroyd, H., Welland, M. E., Carver, J. A., Dobson, C. M., and Meehan, S. (2011) Binding of the molecular chaperone αB-crystallin to αβ amyloid fibrils inhibits fibril elongation. Biophys. J. 101, 1681− 1689. (17) Liu, Y. H., Han, Y. L., Song, J., Wang, Y., Jing, Y. Y., Shi, Q., Tian, C., Wang, Z. Y., Li, C. P., Han, J., and Dong, X. P. (2011) Heat shock protein 104 inhibited the fibrillization of prion peptide 106−126 and disassembled prion peptide 106−126 fibrils in vitro. Intern. J. Biochem. Cell. Biol. 43, 768−774. (18) Arimon, M., Grimminger, V., Sanz, F., and Lashuel, H. A. (2008) Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of αβ fibrils and protofibrils. J. Mol. Biol. 384, 1157−1173. (19) Luk, K. C., Mills, I. P., Trojanowski, J. Q., and Lee, V. M. (2008) Interactions between Hsp70 and the hydrophobic core of α-synuclein inhibit fibril assembly. Biochemistry 47, 12614−12625. (20) Huang, C., Cheng, H., Hao, S., Zhou, H., Zhang, X., Gao, J., Sun, Q. H., Hu, H., and Wang, C. C. (2006) Heat shock protein 70 inhibits α-synuclein fibril formation via interactions with diverse intermediates. J. Mol. Biol. 364, 323−336. (21) Dedmon, M. M., Christodoulou, J., Wilson, M. R., and Dobson, C. M. (2005) Heat shock protein 70 inhibits α-synuclein fibril formation via preferential binding to prefibrillar species. J. Biol. Chem. 280, 14733−14740. (22) Falsone, S. F., Kungl, A. J., Rek, A., Cappai, R., and Zangger, K. (2009) The molecular chaperone Hsp90 modulates intermediate steps of amyloid assembly of the Parkinson-related protein α-synuclein. J. Biol. Chem. 284, 31190−31199. (23) Behrends, C., Langer, C. A., Boteva, R., Bottcher, U. M., Stemp, M. J., Schaffar, G., Rao, B. V., Giese, A., Kretzschmar, H., Siegers, K., and Hartl, F. U. (2006) Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers. Mol. Cell 23, 887− 897. (24) Kurouski, D., Luo, H., Sereda, V., Robb, F. T., and Lednev, I. K. (2012) Rapid degradation kinetics of amyloid fibrils under mild conditions by an archaeal chaperonin. Biochem. Biophys. Res. Commun. 422, 97−102. (25) Luo, H., and Robb, F. T. (2010) Protein folding systems in thermophiles. In The Extremophile Handbook (Horikoshi, K.; Antranikian, G.; Bull, A. T.; Robb, F. T.; Stetter, K. O., Eds.), pp 583−600, Springer, Tokyo. (26) Todd, M. J., Lorimer, G. H., and Thirumalai, D. (1996) Chaperonin-facilitated protein folding: Optimization of rate and yield by an iterative annealing mechanism. Proc. Natl. Acad. Sci. U.S.A. 93, 4030−4035.
the higher the amount of suspended particles present, the less light passes through the cuvette.54 In our experiment, 200-μL samples (20 mM sodium acetate buffer, pH 6.0, 50 mM KCl, 1 mM MgCl2, 0.025 mg mL−1 Hsp60, with or without ATP 0.2 mM) of each reaction solution were loaded into the cuvette cell. The reaction was performed at 42 °C, and light obscuration at 350 nm was recorded for 20 min at 1-min intervals. Size Exclusion Chromatography. FPLC analyses were performed on BioRad BioLogic DualFlow system with Size Exclusion Chromatography (SEC) 125−5 column. For each analysis, 50 μL of the centrifuged (14.000 g for 30 min) supernatant was injected and eluted by 150 mM NaCl, pH 2.3 with a flow rate 1 mL/min. For the protein detection 214, 260, and 280 nm wavelengths were chosen.
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ASSOCIATED CONTENT
* Supporting Information S
Additional figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank I. Baskakov for helpful discussions, A. Shekhtman and J. Washington for help with protein detection, D. Zagorevsky for mass spectroscopy analysis, and M. Shanmugasundaram for helpful discussions on the experimental procedure. This work is supported by the Air Force Office of Scientific Research under Grants AFOSR 03-S-28900 and 9550-10-1-0272 and the National Science Foundation under Grant No. CHE-1152752 (I.K.L.)
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