The effect of small molecules in modulating the chaperone activity of aB-crystallin against ordered and disordered protein aggregation Heath Ecroyd and John A Carver School of Chemistry and Physics, The University of Adelaide, Australia Keywords amyloid fibril; arginine; protein aggregation; small heat-shock protein; aB-crystallin Correspondence H Ecroyd, School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005, Australia Fax: +61 830 34358 Tel: +61 830 35505 E-mail: heath.ecroyd@adelaide.edu.au (Received 12 November 2007, revised 16 December 2007, accepted 20 December 2007) doi:10.1111/j.1742-4658.2008.06257.x Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob diseases Small heat-shock proteins (sHsps), such as aB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of aB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation The effect of these additives is highly dependent upon the target protein undergoing aggregation Importantly, our results show that the chaperone action of aB-crystallin against aggregation of the disease-related amyloid fibril forming protein a-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine) Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some proteinmisfolding diseases Protein aggregation is the result of the mutual association of partially folded intermediate states of a protein, most likely via predominately hydrophobic interactions Protein aggregation can proceed via disordered or ordered mechanisms: which mechanism predominates is thought to be determined by a number of factors, including the rate of unfolding, the amino acid sequence of the protein, the experimental conditions and the nature of the intermediate state(s) that form [1,2] Disordered aggregation results in amorphous aggregates of protein, whilst ordered aggregation produces amyloid fibrils, long threadlike protein structures that are rich in b-sheet and resistant to proteolytic degradation Protein misfolding, and in particular amyloid fibril formation, is associated with a range of diseases, including Alzheimer’s, Parkinson’s and CreutzfeldtJakob diseases, type II diabetes and possibly cataracts [3–5] Protein aggregation is also responsible for inclusion body formation, and therefore the ability to prevent it would be of enormous benefit in recombinant protein production, avoiding the need for resolubilization of the aggregated and precipitated protein Thus, studies aimed at preventing protein aggregation are of interest due to both their biomedical and biotechnological applications In terms of biotechnological applications, small molecules such as guanidine and urea are well-established suppressors of aggregation, and are often used to Abbreviations ANS, 8-anilino-1-naphthalene sulphonate; DTT, 1,4-dithiothreitol; Gdn, guanidine; RCMj-CN, reduced and carboxymethylated j-casein; sHsp, small heat-shock protein; ThT, thioflavin T FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 935 Chaperone activity of aB-crystallin H Ecroyd and J A Carver inhibit aggregation of expressed proteins or to resolubilize proteins that have already aggregated into inclusion bodies [6,7] In suppressing aggregation, these small molecules act by weakening the hydrophobic intermolecular interactions between unfolded or partially folded protein intermediates that are responsible for the aggregation process The amino acid arginine is also often employed as a suppressor of aggregation, and is thought to facilitate correct folding of proteins by destabilizing incorrectly folded structures [8,9] However, high concentrations of guanidine, urea and ⁄ or arginine are usually required for this purpose and must be removed during purification of the recombinant protein In vivo, protein aggregation is prevented through the action of a broad range of highly specialized proteins known as molecular chaperones One such chaperone is a-crystallin, a small heat-shock protein (sHsp) that acts to prevent protein aggregation intracellularly [10] a-Crystallin is present in large concentrations in the eye lens, where it is thought to provide stability and structural support to the other proteins present It is made up of two closely related subunits, aA- and aB-crystallin, which exist at an approximate molar ratio of : in the mammalian lens Moreover, aB-crystallin is found at significant levels in other tissues, such as the heart, kidney, muscle and brain, and its expression is up-regulated in response to stress and pathological conditions [11,12] Recent studies have shown that significant levels of aB-crystallin are found in protein deposits such as those associated with disease [13,14] The molecular chaperone action of aA- and aB-crystallin is manifested by binding to partially unfolded or misfolded target proteins, thus inhibiting their aggregation and precipitation Whilst the chaperone action of aB-crystallin against amorphously aggregating target proteins has been well established, it is only recently that studies have shown that aB-crystallin also acts to prevent ordered amyloid fibril assembly [15–18] Some studies have shown that structural perturbation of a-crystallin and ⁄ or its two subunits (e.g through heating) enhances its chaperone activity against amorphously aggregating target proteins [19– 21], presumably due to increased exposure of its hydrophobic surfaces that facilitate target protein binding [22] In addition to temperature, other treatments (e.g reduction) [23,24] and post-translational modifications (e.g phosphorylation) [18,25,26] that slightly perturb the structure of a-crystallin have been shown to enhance the chaperone activity of the protein against amorphously aggregating target proteins Of particular note, low concentrations of denaturant, such as guanidine hydrochloride (Gdn-HCl) enhance the 936 chaperone activity of a-crystallin against reductioninduced amorphous aggregation of the insulin B-chain [27] Moreover, it was also shown that millimolar concentrations of arginine hydrochloride (Arg-HCl) had a similar effect on the chaperone activity of aB-crystallin [27], which was reported to occur via enhancement of the dynamics of subunit assembly [28] However, to date there have been no reports of the effects of such compounds on the chaperone activity of aB-crystallin against ordered protein aggregation leading to fibril formation In this study, we have explored the potential for small molecules such as Arg-HCl and Gdn-HCl to affect the chaperone activity of aB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation We report that the effect of these additives on the chaperone action of aB-crystallin is dependent on the target protein used, and therefore the results highlight the need to assess the activity of chaperone proteins against a variety of target proteins before drawing conclusions about their generic effects Of particular note, the results from this study show that the chaperone action of aB-crystallin against aggregation of the disease-related amyloid fibril forming protein, a-synucleinA53T, is enhanced in the presence of Arg-HCl and similar positively charged compounds (such as Lys-HCl and Gdn-HCl) Fibril formation by a-synuclein is causally linked to Lewy body formation that occurs in diseases such as Parkinson’s, and the A53T mutant is associated with earlyonset Parkinson’s disease Thus, our results suggest that small molecules that act on sHsps in a similar manner to Arg-HCl may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases Results The effect of Arg-HCl on the chaperone activity of aB-crystallin is target protein-specific In order to investigate the effect of Arg-HCl on the chaperone activity of aB-crystallin, we examined a variety of model target proteins to determine the generic effects of Arg-HCl In particular, we used both ordered (amyloid fibril-forming) and disordered (amorphous) target protein aggregation systems In investigating the effect of Arg-HCl on the chaperone action of aB-crystallin, we also looked at related molecules, to investigate whether any observed effects were specific to Arg-HCl Thus, we also investigated the effects of (a) glycine (Gly), to test whether any effects were attributable to addition of an amino acid to the FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS H Ecroyd and J A Carver Chaperone activity of aB-crystallin solution; (b) lysine hydrochloride (Lys-HCl), to test whether any effects were attributable to adding a basic amino acid; and (c) Gdn-HCl, to test whether any effects of Arg-HCl were attributable to the guanidinium group of the molecule We tested each of these compounds at low (10 mm), intermediate (100 mm) and high (250 mm) concentrations unless otherwise indicated At these concentrations, the additives were found to change the pH of the buffers used in these aggregation assays by < 0.1 units However, at very high concentrations (e.g > 500 mm), some of the compounds had significant effects on the pH of these buffers (i.e increasing the pH by > 0.2 units) In addition, for each assay we used concentrations of aB-crystallin that only partially inhibited aggregation of the target protein in order to enable the effects of the compounds on the chaperone activity to be readily interpreted Disordered (amorphous) aggregation systems Reduction-induced aggregation of a-lactalbumin Upon addition of 1,4-dithiothreitol (DTT), aggregation and precipitation of a-lactalbumin commenced after 25 and reached a plateau after 90 The Fig The effect of additives on the ability of aB-crystallin to prevent the DTT-induced aggregation of a-lactalbumin a-Lactalbumin ( , 0.5 mgỈmL)1) was incubated at 37 °C in 50 mM phosphate buffer, pH 7.2, containing 100 mM NaCl with 20 mM DTT in (A) the absence or (B) the presence of aB-crystallin (0.5 mgỈmL)1), and the change in light scattering at 340 nm was monitored over time For both (A) and (B), the additives were 250 mM of Gly (d), Lys-HCl ()), Arg-HCl ( ) or Gdn-HCl (h) The buffer-only control (r) is also shown in (A) and (B) (C) Percentage protection (mean ± SEM of four independent experiments), calculated 90 after the start of the assay, when a-lactalbumin was incubated with increasing concentrations of the additives, in the absence ( ) or presence ( ) of aB-crystallin The percentage protection that would be expected assuming no influence of the additives on the chaperone activity of aB-crystallin, calculated as described in Experimental procedures, is also shown (j) The asterisks indicate a significant (P < 0.05) decrease in the chaperone ability of aB-crystallin in the presence of that concentration of the additive amount of DTT-induced aggregation of a-lactalbumin was increased in a concentration-dependent manner by the addition of Gly, such that, at 250 mm, light scattering due to its precipitation had increased by 50 ± 7% [mean ± standard error of the mean (SEM)], i.e the calculated percentage protection value was negative because this treatment increased the amount of precipitation compared to that observed when a-lactalbumin was incubated alone (Fig 1A,C) Lys-HCl had a similar concentration-dependent effect However, Arg-HCl had the opposite effect whereby increasing concentrations of Arg-HCl decreased the amount of precipitation, such that, at high concentrations, it had decreased by 60 ± 3% compared to that observed when a-lactalbumin was incubated alone Gdn-HCl had a more complex effect, whereby concentrations up to 100 mm increased the amount of light scattering, but the high concentration (i.e 250 mm) decreased it (Fig 1A,C) Whilst Gly, Lys-HCl and Arg-HCl had no significant effect on the lag phase of precipitation of a-lactalbumin (approximately 25 min), Gdn-HCl decreased it to 15 (Fig 1A) Addition of aB-crystallin at a 1.0 : 1.0 w ⁄ w ratio of a-lactalbumin : aB-crystallin decreased the precipitation of a-lactalbumin by 81 ± 8% The ability of A B C FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 937 Chaperone activity of aB-crystallin H Ecroyd and J A Carver aB-crystallin to protect against this precipitation was significantly decreased in the presence of Gly, LysHCl and Gdn-HCl, such that, when they were present at high concentrations, aB-crystallin had no effect on the amount of light scattering compared to that observed when the additives were present alone (Fig 1C) In contrast, the chaperone action of aBcrystallin against a-lactalbumin was maintained in the presence of intermediate concentrations of Arg-HCl, but was not further enhanced by it (Fig 1C) The significant decrease in the amount of precipitation in the presence of high concentrations of Arg-HCl in the absence of the chaperone precluded analysis of the effect of this concentration on the protective ability of aB-crystallin Reduction-induced aggregation of the insulin B-chain Light scattering due to DTT-induced amorphous aggregation and precipitation of the insulin B-chain commenced after 10 and reached a plateau after 45 (Fig 2A) The amount of precipitation was increased in a concentration-dependent manner by Gly and Lys-HCl compared to that observed when insulin was incubated alone (Fig 2A,C) Addition of Arg-HCl A (up to 250 mm) had a negligible effect on the amount of precipitation Similarly, low and intermediate concentrations of Gdn-HCl had no effect on the precipitation of insulin; however, high concentrations (i.e 250 mm) had a protective effect, decreasing the amount of light scattering by 48 ± 2% (Fig 2A,C) None of the additives used affected the lag phase of the aggregation When incubated in the presence of aB-crystallin alone (at a 1.0 : 1.0 w ⁄ w ratio of insulin : aB-crystallin), the precipitation of insulin was inhibited by 40 ± 4% (Fig 2B,C) Only Arg-HCl significantly (P < 0.05) enhanced this protective activity of aBcrystallin, such that, at 250 mm Arg-HCl, the light scattering due to precipitation of insulin was decreased by 65 ± 8% Low and intermediate concentrations of Gly had no effect on the chaperone activity of aB-crystallin against this target protein, but it was significantly reduced at 250 mm A similar trend was observed for Lys-HCl, with high concentrations significantly inhibiting the ability of aB-crystallin to prevent precipitation (Fig 2C) Gdn-HCl had no effect on the chaperone activity of aB-crystallin against the DTT-induced aggregation and precipitation of insulin B C Fig aB-crystallin protects against the DTT-induced aggregation of insulin, and this activity is enhanced by Arg-HCl Insulin ( , 0.25 mgỈmL)1) was incubated at 37 °C in 50 mM phosphate buffer, pH 7.2, with 10 mM DTT in (A) the absence or (B) the presence of aB-crystallin (0.25 mgỈmL)1) For other details, refer to the legend to Fig In addition, the hash symbol (#) indicates a significant (P < 0.05) increase in the chaperone ability of aB-crystallin in the presence of that concentration of the additive 938 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS H Ecroyd and J A Carver Chaperone activity of aB-crystallin Heat-induced aggregation of catalase We used bovine catalase as the model substrate to test the effect of the small molecules on the chaperone ability of aB-crystallin against a target protein undergoing heat-stressed induced aggregation and precipitation Aggregation of catalase occurs at high temperatures, i.e 55 °C, and these studies aimed to investigate whether these small molecules could further enhance the well-characterized increase in the chaperone activity of aB-crystallin at high temperatures due to changes in its tertiary structure [20,21] The precipitation of catalase commenced after 20 min, and the increase in light scattering due to precipitation of the protein reached a maximum after 90 (Fig 3A) All of the additives tested increased the amount of light scattering due to precipitation of catalase compared to that observed when it was incubated alone Of these, Gdn-HCl had the most dramatic effect, with 250 mm Gdn-HCl increasing the amount of precipitation of catalase by 190 ± 5% (Fig 3A) The presence of aB-crystallin at a 1.0 : 0.5 w ⁄ w ratio of catalase : aB-crystallin inhibited the precipitation of catalase by 71 ± 7% (Fig 3B) This chaperone activity was not affected by increasing concentrations of Gly, but was completely abolished by intermediate and high concentrations of Lys-HCl, and was inhibited by GdnHCl in a concentration-dependent manner (Fig 3B,C) Intermediate concentrations (i.e 100 mm) of Arg-HCl significantly inhibited the ability of aB-crystallin to prevent the precipitation of catalase; however, this effect was not seen at high concentrations of Arg-HCl, i.e the chaperone activity of aB-crystallin was maintained in the presence of 250 mm Arg-HCl Ordered aggregation leading to amyloid fibril formation We employed two models to examine the effect of the small molecules on the ability of aB-crystallin to prevent amyloid fibril formation – a familial mutant of the disease-related protein a-synuclein (i.e a-synucleinA53T) and reduced and carboxymethylated j-casein (RCMj-CN), both of which are natively disordered proteins [29] We employed these systems as they both form fibrils at physiological pH and temperature [30,31], and so can be used to examine the activity of aB-crystallin without confounding factors such as low pH or the presence of other denaturants, which are often required in other amyloid fibril-forming systems A B C Fig Heat-induced amorphous aggregation of catalase is increased by increasing concentrations of the additives Catalase ( , 0.5 mgỈmL)1) was incubated at 55 °C in 50 mM phosphate buffer, pH 7.2, in (A) the absence or (B) the presence of aB-crystallin (0.25 mgỈmL)1) For other details, refer to the legend to Fig FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 939 Chaperone activity of aB-crystallin H Ecroyd and J A Carver In both systems, fibril formation was assessed by an in situ thioflavin T (ThT) fluorescence assay Amyloid fibril formation by RCMj-CN Fibril formation by RCMj-CN, as monitored by an increase in ThT binding, showed a gradual increase over the time course of the assay (Fig 4A) At the end of the assay, electron micrographs of negatively stained RCMj-CN fibrils showed them to be thread-like structures, approximately 100–700 nm in length (Fig 6A,B), similar to those reported previously [30] Addition of Gly slightly increased the degree of ThT binding in a concentration-dependent manner, such that, at 250 mm, there was an increase of 10 ± 1% compared to that observed when RCMj-CN was incubated alone (Fig 4A,C) Lys-HCl, Arg-HCl and Gdn-HCl all decreased the change in ThT fluorescence associated with amyloid fibril formation by RCMj-CN in a concentration-dependent manner, such that, at 250 mm of Arg-HCl and Gdn-HCl, the increase in ThT was almost completely abolished (Fig 4A), precluding analysis of the effect of these concentrations on the chaperone activity of aB-crystallin (Fig 4B,C) None of the compounds had an effect on the morphol- A C 940 B ogy of the amyloid fibrils formed (data not shown) When incubated in the presence of aB-crystallin, the change in ThT fluorescence associated with amyloid fibril formation by RCMj-CN decreased by 30 ± 3% (1.0 : 0.5 w ⁄ w ratio of RCMj-CN : aB-crystallin) (Fig 4B) The amino acids had no significant effect on the chaperone activity of aB-crystallin against this fibril-forming target protein (Fig 4C) At 100 mm, Gdn-HCl had a negative effect on the chaperone activity of aB-crystallin in preventing amyloid fibril formation by RCMj-CN Amyloid fibril formation by a-synucleinA53T At 37 °C, the increase in ThT fluorescence associated with fibril formation by a-synucleinA53T reached a plateau after 140 h (Fig 5A) Electron micrographs of a-synucleinA53T at the end of the assay confirmed the formation of fibrils, which were long (between and nm), straight and unbranched (Fig 6C,D) Addition of Gly and Lys-HCl at 250 mm increased both the rate and magnitude of the change in ThT fluorescence associated with fibril formation by a-synucleinA53T (Fig 5A,C) Overall, Arg-HCl had little effect on fibril formation by a-synucleinA53T, whereas Gdn-HCl at Fig Ordered aggregation of RCMj-CN into amyloid fibrils is inhibited by aB-crystallin but this activity is not affected by ArgHCl The change in ThT fluorescence at 490 nm was used to monitor amyloid fibril formation by RCMj-CN ( , 0.5 mgỈmL)1) in (A) the absence or (B) the presence of aBcrystallin (0.25 mgỈmL)1) For both (A) and (B), RCMj-CN was incubated at 37 °C in 50 mM phosphate buffer, pH 7.2, without shaking for 15 h in the presence of 250 mM of Gly (d), Lys-HCl ()), Arg-HCl ( ) or GdnHCl (h) The buffer-only control (r) is also shown (C) Percentage protection data (mean ± SEM of three independent experiments), calculated 15 h after the start of the assay, for RCMj-CN incubated with increasing concentrations of the additives in the absence ( ) or presence ( ) of aB-crystallin The percentage protection that would result if there was no influence of the additives on the chaperone activity of aB-crystallin, as described in the Experimental procedures, is also shown (j) The asterisk indicates denotes a significant (P < 0.05) decrease in the chaperone ability of aB-crystallin in the presence of 100 mM Gdn-HCl FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS H Ecroyd and J A Carver Fig Amyloid fibril formation by a-synucleinA53T is inhibited by aB-crystallin, and this chaperone activity is enhanced by Lys-HCl, Arg-HCl and Gdn-HCl Fibril formation was induced by incubating a-synucleinA53T ( ; 2.0 mgỈmL)1) with constant shaking at 37 °C in 50 mM phosphate buffer, containing 100 mM NaCl, pH 7.4, for days either in (A) the absence or (B) the presence of aB-crystallin (0.4 mgỈmL)1) and either 250 mM of Gly (d), 250 mM of Lys-HCl ()), 250 mM of Arg-HCl ( ) or 100 mM of GdnHCl (h) The buffer-only control (r) is also shown (C) Percentage protection (mean ± SEM of three independent experiments) for a-synucleinA53T incubated with the additives in the absence ( ) or presence of aBcrystallin ( ) was calculated using data from the 160 h time point The percentage protection that would result if there was no influence of the additives on the chaperone activity of aB-crystallin is also shown (j), the hash symbol (#) denotes a significant (P < 0.05) increase in the chaperone ability of aB-crystallin in the presence of the additive Note that the concentration of Gdn-HCl used in this experiment is 100 mM Chaperone activity of aB-crystallin A B C A Fig Amyloid fibrils formed by the ordered aggregation of RCMj-CN and a-synucleinA53T Electron micrographs of RCMj-CN (0.5 mgỈmL)1, A and B) and a-synculeinA53T (2.0 mgỈmL)1, C and D) 500 lgỈmL)1) following incubation at 37 °C in 50 mM phosphate buffer, pH 7.2, for 15 h and 50 mM phosphate buffer containing 100 mM NaCl, pH 7.4, for days, respectively The scale bars represent lm (A, C) and 0.2 lm (B, D) B C D 250 mm inhibited it by 53 ± 5% This significant decrease in the amount of aggregation in the presence of high concentrations of Gdn-HCl precluded analysis of the effect of this concentration when aB-crystallin was also present Therefore, we also tested Gdn-HCl at 100 mm in these studies (Fig 5), and this concentration was found to inhibit fibril formation by a-synucleinA53T by 21 ± 2% None of the compounds were found to have an effect on the morphology of the fibrils formed by a-synucleinA53T (data not shown), and thus FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 941 Chaperone activity of aB-crystallin H Ecroyd and J A Carver the change in ThT fluorescence is interpreted to be directly attributable to a change in the number of fibrils formed in the presence of these additives In the presence of aB-crystallin (1.0 : 0.2 w ⁄ w ratio of a-synucleinA53T : aB-crystallin), the increase in ThT fluorescence associated with fibril formation by a-synucleinA53T was decreased by 46 ± 3% (Fig 5B,C) Gly had no significant effect on the chaperone activity of aB-crystallin in preventing the increase in ThT fluorescence associated with fibril formation by a-synucleinA53T, but both Lys-HCl and Arg-HCl were found to significantly increase its chaperone activity, such that, at 250 mm, the percentage protection was increased to 27 ± 3% (Lys-HCl) and 99 ± 4% (Arg-HCl) (Fig 5C) Similarly, at 100 mm, Gdn-HCl also significantly increased the chaperone activity of aB-crystallin (84 ± 4%) against this target protein The effect of Arg-HCl on the structure and assembly of aB-crystallin We investigated whether the effects of these additives on the chaperone action of aB-crystallin were attributable to changes in the quaternary structure and oligomerization of the protein We found that, at 250 mm, none of the compounds had a significant effect on the oligomeric size of aB-crystallin as assessed by sizeexclusion chromatography (Fig 7A) (i.e in either the absence or presence of the compounds, aB-crystallin was found to elute with an apparent mass of 580 kDa, which corresponds to the mass of the oligomer reported previously [32]) We also found no significant differences in the accessibility of exposed hydrophobic clusters, as assessed by ANS fluorescence (Fig 7B), or solvent accessibility of the N-terminal tryptophan residues (Trp9 and Trp60), as assessed by intrinsic fluorescence (data not shown), in the presence of these compounds Thus, it appears that the additives may cause subtle changes in the structure of both the target protein and aB-crystallin that lead to changes in the chaperone activity of aB-crystallin for some target proteins but not others Discussion We have investigated the effect of Arg-HCl on the chaperone activity of aB-crystallin against various target proteins undergoing either disordered (amorphous) or ordered (i.e amyloid fibril formation) aggregation We show that the effect of these compounds on the chaperone activity of aB-crystallin is dependent on the target protein undergoing aggregation Thus, our results highlight the need to consider a number of 942 Fig The additives have no effect on the oligomeric size of aBcrystallin (A) or its ability to bind ANS (B) (A) aB-crystallin (1.0 mgỈmL)1), in the absence or presence of 250 mM of the additives, was loaded on to a Superdex 200HR 10 ⁄ 30 column and eluted in 50 mM phosphate buffer, pH 7.2, at a flow rate of 0.4 mLỈmin)1 Calibration of the column was performed using (1) blue dextran, void; (2) thyroglobulin, 670 kDa; (3) c-globulin, 158 kDa; (4) ovalbumin, 44 kDa; (5) myoglobulin, 17 kDa (B) ANS fluorescence of aB-crystallin (0.1 mgỈmL)1) in 50 mM phosphate buffer, pH 7.2, alone ( ) or in the presence of 250 mM of Gly (d), Lys-HCl ()), Arg-HCl ( ) or Gdn-HCl (h), monitored following excitation at 350 nm The samples were maintained at 37 °C for 30 before the fluorescence spectra were obtained aggregation systems in order to assess the effect of various additives and ⁄ or modifications on the overall activity of chaperone proteins Of the target proteins tested, Arg-HCl was found to specifically increase the activity of aB-crystallin against DTT-induced precipitation of insulin at intermediate and high concentrations, and it also increased the activity of aB-crystallin in preventing the aggregation leading to amyloid fibril formation by a-synucleinA53T when used at high concentrations With regard to the latter result, the increase in chaperone activity resulting in the inhibition of fibril formation by a-synucleinA53T was not specific for Arg-HCl as Lys-HCl and Gdn-HCl showed similar effects (Fig 5C) A number of studies have indicated that small molecules, including common metabolites such as pantethine and glutathione [33], can increase the chaperone activity of a-crystallin We confirm here previous results showing that high concentrations of Arg-HCl FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS H Ecroyd and J A Carver (> 100 mm) increase the chaperone activity of aB-crystallin against the DTT-induced precipitation of insulin [27,28] These studies also showed that 100 mm Arg-HCl increases the chaperone activity of a-crystallin against the thermally induced aggregation of f-crystallin at 43 °C [27] Our results indicate that this effect of Arg-HCl is not limited to proteins undergoing disordered (amorphous) aggregation, as Arg-HCl also increases the ability of aB-crystallin to reduce amyloid fibril formation by a-synucleinA53T This result is significant due to the association of this type of protein aggregation with disease Lys-HCl and Gdn-HCl also enhanced the chaperone activity of aB-crystallin against this fibril-forming protein, implying that it is the common positively charged group that plays a role in increasing the activity of aB-crystallin against this target protein To our knowledge, this is the first study that has investigated the effects of small molecules, such as amino acids and Gdn-HCl, on the chaperone function of sHsps against amyloid fibril-forming target proteins Whilst the concentrations used in these studies are high, the results suggest that small molecules such as these may represent important therapeutic leads for increasing the protective ability of chaperone proteins against disease-related amyloid fibril formation Interestingly, none of the compounds tested increased the chaperone activity of aB-crystallin against amyloid fibril formation by RCMj-CN, a milk-derived protein that readily forms fibrils under conditions of physiological pH and temperature The differences in the effect of the small molecules on the chaperone activity of aB-crystallin against the two amyloid fibril-forming target proteins may be attributable to differences in the rate of fibril formation (RCMj-CN forms fibrils much more rapidly than a-synucleinA53T) or the nature of the amyloidogenic intermediate(s) with which aB-crystallin interacts Moreover, we found no generic effect of each compound on the chaperone activity of aB-crystallin We have previously shown that phosphorylation of aB-crystallin, which occurs under conditions of cellular stress [34,35], also has a differential effect on its chaperone activity, increasing the activity against some target proteins, but decreasing it against others [18] Thus, we conclude that aB-crystallin most likely employs various methods of binding (or binding modes) in order to prevent the aggregation of stressed proteins Some of these binding modes (or binding sites) are favoured by phosphorylation or interaction with compounds such as Arg-HCl, whilst others are either not affected or are perturbed Studies using destabilized T4 lysozyme mutants have shown that Chaperone activity of aB-crystallin both aA- and aB-crystallin possess at least two binding modes, and that these are influenced by both external factors (e.g changes in temperature and pH) and intrinsic factors (e.g mutation and phosphorylation) [23,26,36] Various binding modes may facilitate the interaction of aB-crystallin with the various intermediates formed during the aggregation process of diverse targets It may also enable the chaperone protein to better cope with the various types of stresses experienced by cells that cause proteins to unfold Of course, the effect of compounds such Arg-HCl and Gdn-HCl may be also due to changes that they induce in the stability and ⁄ or intermediate states of the target protein itself The denaturant effect of guanidine on proteins is well established; it decreases the stability of the native protein but also suppresses aggregation by weakening the hydrophobic intermolecular interactions between the unfolded states of a protein (i.e increasing the solubility of the unfolded state) In contrast, arginine has been shown to suppress aggregation of some proteins by acting on the unfolded state of the protein and increasing the reversibility of unfolding [37] Arginine had no effect on the stability of the protein’s native state, although it may also interact with it [37] This effect of arginine on protein aggregation has been attributed to the guanidinium group of the compound, which, through electrostatic interactions, prevents the intermolecular interactions leading to aggregation [37–39] However, its effects vary from protein to protein [9] This is clearly evident from our studies in which, even at low concentrations, the aggregation of target proteins examined was affected by the compounds used, and this varied for different target proteins (e.g whilst Arg-HCl at 250 mm had little effect on the aggregation of insulin or a-synucleinA53T alone, it dramatically increased the aggregation of catalase and a-lactalbumin but significantly decreased the ordered aggregation leading to fibril formation by RCMj-CN) As such, consideration not only for the effect of compounds on the activity of the chaperone protein, but also its destabilized target, must be taken into account when examining the effect of an additive on the activity of chaperone proteins We have shown that the mechanism by which the tested molecules influence the activity of aB-crystallin is not through gross quaternary structural changes (as assessed by size-exclusion chromatography; see Fig 6A) or changes in exposure of the tryptophan residues or clustered regions of exposed hydrophobicity (as assessed by intrinsic and ANS fluorescence) of the protein With regard to the effect of Arg-HCl on the mass of aB-crystallin, a previous study [27], using glycerol sedimentation, reported that 300 mm Arg-HCl FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 943 Chaperone activity of aB-crystallin H Ecroyd and J A Carver resulted in a decrease in the size of aB-crystallin, which implies that, at higher concentrations than used in this study, Arg-HCl may have a significant effect on the quaternary structure of aB-crystallin However, at 250 mm, we found that the effect of these additives on the mass of aB-crystallin is negligible, and these data are in agreement with previous work using Gdn-HCl at the same concentration [40,41] Previous studies employing both near and far-UV circular dichroism have also reported that there is little effect of Arg-HCl on the overall secondary or tertiary structure of a-crystallin, but that Arg-HCl mediates an increase in subunit exchange and destabilization of the overall structure of a-crystallin (as assessed by denaturation with urea) [28] Arginine’s side chain, the guanidinium group, is able to interact with a number of functional groups, including the aromatic side chains of some amino acids, through a stacking mechanism [42] The interaction of arginine with aromatic amino acids of aB-crystallin may facilitate its effects Our results suggest that an increase in subunit exchange in the presence of Arg-HCl may only be important in enhancing the chaperone activity of sHsps against certain target proteins Moreover, these are likely to be limited to those situations in which the chaperone forms only a transient complex with the target protein, such as has been described for the amorphous aggregation of a-lactalbumin [43] and amyloid fibril formation by apoC-II [16], as we found no evidence that the overall ability of aB-crystallin to suppress the aggregation of these target proteins was the same after extended time periods In summary, our results show that the effect of small compounds (such as Arg-HCl) on the chaperone activity of aB-crystallin is highly dependent on the aggregating target protein Significantly, we found that Arg-HCl, Lys-HCl and Gdn-HCl increased the ability of aB-crystallin to prevent the ordered aggregation leading to amyloid fibril formation of a mutant form of the Parkinson’s disease-related protein a-synuclein (i.e a-synucleinA53T) These results suggest that, due to their action on molecular chaperone proteins, biologically compatible small molecules, such as Arg-HCl, may be potential candidates as therapeutic agents in the treatment of protein-misfolding diseases Experimental procedures Materials Intrinsic and extrinsic fluorescence Intrinsic tryptophan fluorescence spectra of aB-crystallin (0.1 mgỈmL)1 in 50 mm phosphate buffer, pH 7.2), in the presence or absence of the amino acids or Gdn-HCl, were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian) equipped with temperature control and using a cuvette with a cm path length The excitation wavelength was set at 295 nm, and fluorescence emission was monitored between 300 nm and 400 nm The excitation and emission slit widths were set at nm Samples were maintained at 37 °C for 30 before being assayed For the ANS binding studies, a stock solution of methanolic ANS (100 mm) was diluted 1000-fold into a 0.1 mgỈmL)1 protein solution in 50 mm phosphate buffer, pH 7.2 Emission fluorescence spectra were monitored (400–600 nm) following excitation at 350 nm The excitation and emission slit widths were set at nm Samples were maintained at 37 °C for 30 before being assayed Chaperone activity assays To test the relative chaperone activity of aB-crystallin in the presence or absence of the additives, we monitored the aggregation and ⁄ or precipitation of various target proteins using either ThT fluorescence or turbidity assays (see below) The effect of the additives on aggregation of the target protein (in the absence and presence of aB-crystallin) was assessed at the end of each assay by calculating the percentage protection using the formula: % protection ¼ 100 Â Bovine j-casein was obtained from Sigma Chemical Co (St Louis, MO, USA), and was reduced and carboxymethylated (RCMj-CN) prior to use as described previously [44] 944 Thioflavin T (ThT), 8-anilino-1-napthalene sulfonate (ANS) and b-mercaptoethanol, Arg-HCl, Gdn-HCl, Lys-HCl and Gly were also obtained from Sigma The vector pET24d(+) (Novagen, Madison, WI, USA) containing the gene for expression of human aB-crystallin was a kind gift from W de Jong and W Boelens (University of Nijmegen, Netherlands), and the vector pRSETB (Invitrogen, Carlsbad, CA, USA) containing the human a-synucleinA53T gene was a kind gift from R Cappai (University of Melbourne, Australia) The aB-crystallin and a-synucleinA53T proteins were expressed and purified as described previously [45,46] SDS–PAGE analysis of the purified proteins indicated that they contained < 5% contaminating proteins The concentrations of proteins used in these studies were determined by spectrophotometric methods using a Cary 5000 UV-VisNIR spectrophotometer (Varian, Melbourne, Australia), and calculated extinction coefficients based on amino acid sequences All the buffers in these experiments were passed through a 0.2 lm filter prior to use ðDIc À DIs Þ DIc where DIc and DIs represent the change in absorbance or ThT fluorescence for the target protein in the absence FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS H Ecroyd and J A Carver (control) and presence of the additives (± aB-crystallin), respectively All experiments were independently replicated at least three times, and the results are shown as means ± SEM In each experiment, conclusions regarding the effects of the additives on the chaperone activity of aB-crystallin were drawn based on the measured percentage protection when both the additive and chaperone were present compared to the theoretical percentage protection that would result if there was no influence of the additive on the chaperone activity of aB-crystallin (i.e the effect of the additive on the aggregation of the target protein is independent and different from that of the chaperone) The standard errors associated with this theoretical percentage protection were calculated from the standard errors associated with the means of the measured values (i.e by calculating the square root of the sum of the associated individual errors squared) The maximum percentage protection in these experiments is therefore 100%, i.e complete inhibition of an increase in light scattering or ThT fluorescence ThT assays The formation of amyloid fibrils by RCMj-CN (0.5 mgỈmL)1) and a-synucleinA53T (2.0 mgỈmL)1) was monitored using a ThT binding assay method [47] developed for a 96-well microtitre plate format and adapted as described previously [18] Briefly, fibril formation by RCMj-CN was monitored in real time by incubation at 37 °C in 50 mm phosphate buffer, pH 7.2, without shaking for 15 h Single-point ThT readings were taken for a-synucleinA53T during incubation at 37 °C in 50 mm phosphate buffer containing 100 mm NaCl, pH 7.4, and 0.02% sodium azide for days The microtitre plate containing a-synucleinA53T was subjected to constant shaking between readings All samples were incubated with 10 lm ThT, which did not affect fibril formation for either protein Fluorescence levels were measured with a Fluostar Optima plate reader (BMG Labtechnologies, Melbourne, Australia) with a 440 ⁄ 490 nm excitation ⁄ emission filter set, and the change in ThT fluorescence is reported The change in ThT fluorescence in the absence of the target protein was negligible for each assay The percentage protection for the ThT assays was calculated using the data from the 15-h (RCMjCN) or 160-h (a-synucleinA53T) time points Turbidity assays Light scattering at 340 nm was measured and recorded using a Fluostar Optima plate reader (BMG Labtechnologies) For each assay, the plate was shaken for s after each cycle The change in light scattering at 340 nm for each sample is reported The change in light scattering in the absence of the target protein was negligible for each assay For the heat-induced amorphous aggregation assay, Chaperone activity of aB-crystallin bovine liver catalase (0.5 mgỈmL)1) was incubated at 55 °C in 50 mm phosphate buffer, pH 7.2 For the reductioninduced amorphous aggregation assays, a-lactalbumin (0.5 mgỈmL)1) or bovine insulin (0.25 mgỈmL)1) were incubated at 37 °C in 50 mm phosphate buffer, pH 7.2, containing 100 mm NaCl, and aggregation and precipitation was initiated by addition of DTT to a final concentration of 20 mm (a-lactalbumin) or 10 mm (insulin) The percentage protection for these amorphous aggregation assays was calculated using the data from the 90 time point Size-exclusion FPLC Size-exclusion chromatography of aB-crystallin (100 lL of a 1.0 mgỈmL)1 solution), in the presence or absence of the additives (250 mm), was performed on a Superdex 200HR 10 ⁄ 30 column (Amersham Biosciences, Little Chalfont, UK) Samples were eluted at a flow rate of 0.4 mLỈmin)1 with 50 mm phosphate buffer, pH 7.2, containing the corresponding amino acid or Gdn-HCl at 250 mm The column was calibrated using gel filtration markers (Bio-Rad, Hemel Hampstead, UK) Transmission electron microscopy Samples were prepared for transmission electron microscopy as described 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properties Methods Enzymol 290, 365–383 46 Cappai R, Leck SL, Tew DJ, Williamson NA, Smith DP, Galatis D, Sharples RA, Curtain CC, Ali FE, Cherny RA et al (2005) Dopamine promotes a-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway FASEB J 19, 1377–1379 47 Nielsen L, Frokjaer S, Brange J, Uversky VN & Fink AL (2001) Probing the mechanism of insulin fibril formation with insulin mutants Biochemistry 40, 8397– 8409 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 947 ... effect on the chaperone activity of aB-crystallin against the DTT-induced aggregation and precipitation of insulin B C Fig aB-crystallin protects against the DTT-induced aggregation of insulin,... increased the chaperone activity of aB-crystallin (84 ± 4%) against this target protein The effect of Arg-HCl on the structure and assembly of aB-crystallin We investigated whether the effects of these... on the overall activity of chaperone proteins Of the target proteins tested, Arg-HCl was found to specifically increase the activity of aB-crystallin against DTT-induced precipitation of insulin