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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 αB-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 αB-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 αB-crystallin against aggregation of the disease-related amyloid fibril forming protein α-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 protein-misfolding 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 β-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 Creutzfeldt-Jakob 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 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 α-crystallin, a small heat-shock protein (sHsp) that acts to prevent protein aggregation intracellularly . α-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, αA- and αB-crystallin, which exist at an approximate molar ratio of 3 : 1 in the mammalian lens. Moreover, αB-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 αB-crystallin are found in protein deposits such as those associated with disease [13,14]. The molecular chaperone action of αA- and αB-crystallin is manifested by binding to partially unfolded or misfolded target proteins, thus inhibiting their aggregation and precipitation. Whilst the chaperone action of αB-crystallin against amorphously aggregating target proteins has been well established, it is only recently that studies have shown that αB-crystallin also acts to prevent ordered amyloid fibril assembly [15–18].
Some studies have shown that structural perturbation of α-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 . 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 α-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 chaperone activity of α-crystallin against reduction-induced amorphous aggregation of the insulin B-chain . Moreover, it was also shown that millimolar concentrations of arginine hydrochloride (Arg-HCl) had a similar effect on the chaperone activity of αB-crystallin , which was reported to occur via enhancement of the dynamics of subunit assembly . However, to date there have been no reports of the effects of such compounds on the chaperone activity of αB-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 αB-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 αB-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 αB-crystallin against aggregation of the disease-related amyloid fibril forming protein, α-synucleinA53T, is enhanced in the presence of Arg-HCl and similar positively charged compounds (such as Lys-HCl and Gdn-HCl). Fibril formation by α-synuclein is causally linked to Lewy body formation that occurs in diseases such as Parkinson’s, and the A53T mutant is associated with early-onset 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.
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- Experimental procedures
We have investigated the effect of Arg-HCl on the chaperone activity of αB-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 αB-crystallin is dependent on the target protein undergoing aggregation. Thus, our results highlight the need to consider a number of 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 αB-crystallin against DTT-induced precipitation of insulin at intermediate and high concentrations, and it also increased the activity of αB-crystallin in preventing the aggregation leading to amyloid fibril formation by α-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 α-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 , can increase the chaperone activity of α-crystallin. We confirm here previous results showing that high concentrations of Arg-HCl (> 100 mm) increase the chaperone activity of αB-crystallin against the DTT-induced precipitation of insulin [27,28]. These studies also showed that 100 mm Arg-HCl increases the chaperone activity of α-crystallin against the thermally induced aggregation of ζ-crystallin at 43 °C . 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 αB-crystallin to reduce amyloid fibril formation by α-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 αB-crystallin against this fibril-forming protein, implying that it is the common positively charged group that plays a role in increasing the activity of αB-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 αB-crystallin against amyloid fibril formation by RCMκ-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 αB-crystallin against the two amyloid fibril-forming target proteins may be attributable to differences in the rate of fibril formation (RCMκ-CN forms fibrils much more rapidly than α-synucleinA53T) or the nature of the amyloidogenic intermediate(s) with which αB-crystallin interacts. Moreover, we found no generic effect of each compound on the chaperone activity of αB-crystallin. We have previously shown that phosphorylation of αB-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 . Thus, we conclude that αB-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 both αA- and αB-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 αB-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 . Arginine had no effect on the stability of the protein’s native state, although it may also interact with it . 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 . 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 α-synucleinA53T alone, it dramatically increased the aggregation of catalase and α-lactalbumin but significantly decreased the ordered aggregation leading to fibril formation by RCMκ-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 αB-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 αB-crystallin, a previous study , using glycerol sedimentation, reported that 300 mm Arg-HCl resulted in a decrease in the size of αB-crystallin, which implies that, at higher concentrations than used in this study, Arg-HCl may have a significant effect on the quaternary structure of αB-crystallin. However, at 250 mm, we found that the effect of these additives on the mass of αB-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 α-crystallin, but that Arg-HCl mediates an increase in subunit exchange and destabilization of the overall structure of α-crystallin (as assessed by denaturation with urea) . 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 . The interaction of arginine with aromatic amino acids of αB-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 α-lactalbumin  and amyloid fibril formation by apoC-II , as we found no evidence that the overall ability of αB-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 αB-crystallin is highly dependent on the aggregating target protein. Significantly, we found that Arg-HCl, Lys-HCl and Gdn-HCl increased the ability of αB-crystallin to prevent the ordered aggregation leading to amyloid fibril formation of a mutant form of the Parkinson’s disease-related protein α-synuclein (i.e. α-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.