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Pyrococcus furiosus l-asparaginase (PfA) consists of two distinct α/β domains, a large (1–182) N-terminal domain (NPfA) connected by a linker region (183–200) to a small (201–326) C-terminal domain (CPfA) . Previously we reported active-site mutants of PfA with promising therapeutic and industrial usage. A structure–function relationship was presented to describe the mode of action of this enzyme . With the intention of studying the effect of inter-domain interactions and domain stability in facilitating the function of PfA, we studied individual domains and their folding propensities. In the process we found that NPfA not only acts as an intramolecular chaperone but also functions as a general small molecular chaperone.
Small molecular chaperones are different from intramolecular chaperones by being non-specific to their substrates . Most of these belong to a group of proteins called the small heat shock proteins (sHSPs). sHSPs are ubiquitous proteins having monomer size ranging from 12 to 43 kDa [3, 4]. They form large oligomeric complexes of 9–50 subunits, ranging in molecular weight from 125 kDa to 2 MDa . sHSPs and other small molecular chaperones display significant polydispersity, limiting the availability of structural information [6-8]. To date, a large number of small molecular chaperones/sHSPs have been found with aggregation inhibitory properties against thermally or chemically denatured substrate proteins [9, 10]. For example, bovine α-crystallin, murine HSP25 and human HSP27 have been shown to prevent the thermal aggregation of a variety of model proteins [11-14]. Similarly, chaperones like artemin, tubulin, clusterin etc. have been shown to prevent the refolding-mediated aggregation of a variety of proteins [15-17]. A large number of sHSPs have also been studied from hyperthermophilic archaea, e.g. the sHSP20 from Sulfolobus solfataricus P2, HSP 16.5 from Methanococcus jannaschii and sHSP from Pyrococcus furiosus [18-20]. The involvement of sHSPs in disease and their potential for therapeutic intervention have also been explored recently [21, 22].
The chaperoning efficiency of small molecular chaperones/sHSPs depends upon the type of substrate proteins, the type of chaperone and their mass ratios . Sometimes specific physicochemical conditions are required for chaperones to interact with their substrates [24, 25]. Irrespective of this, all substrate–small molecular chaperone interactions lead to large oligomeric complexes formed primarily by hydrophobic interactions. Protein aggregation is a concentration-dependent phenomenon, where refolding/unfolding intermediates undergo intermolecular interactions through exposed hydrophobic surfaces [26, 27]. Small molecular chaperones prevent aggregation by mimicking these surfaces. Since hydrophobic interactions are non-specific, the interactions of chaperones with substrates are also primarily non-specific. The non-specificity is advantageous as any hydrophobic small molecule may be used to inhibit aggregation of partially denatured proteins, as has been shown earlier .
Here we present data to show that isolated NPfA functions as a small molecular chaperone. During purification of NPfA and CPfA domains (individually cloned and expressed) both appeared as inclusion bodies. Refolding from inclusion bodies yielded only NPfA in the soluble form, whereas CPfA failed to refold and formed insoluble aggregates. Interestingly, when refolded together, both the domains appeared in the soluble fraction suggesting that NPfA probably acts as an intramolecular chaperone for CPfA. Intramolecular chaperones are specific sequences within a polypeptide that are essential for attaining native structure by the polypeptide but may not be necessary for its function. These sequences, also known as propeptide or prosequences, are generally removed autocatalytically or by proteases as soon as the protein attains its native form as has been reported in the case of subtilisin . Intramolecular chaperones are categorized as type I or type II depending on the location of the sequence (at either the N- or the C-terminus) and the assistance they provide in attaining functional form. Regardless of type, the sequences are very specific for their partner proteins . In our case, NPfA does not fall into any of these categories of intramolecular chaperone as it is neither an autocatalytic product, nor the CPfA (which was assisted by NPfA) exists as an independent functional unit. We found that refolded NPfA assembles into polydispersed, large oligomeric complexes. These complexes showed significant surface hydrophobicity, high β-sheet content and remarkable thermal stability. These characteristics are typical of any other small molecular chaperone [31-33]. Small molecular chaperones are independently folded entities that non-specifically assist in the folding of substrate proteins . NPfA when checked for aggregation prevention properties on a variety of proteins qualified well as a typical non-specific molecular chaperone. NPfA prevented thermal aggregation of bovine carbonic anhydrase II (BCA II), α-amylase and malate synthase G (MSG) as well as refolding-induced aggregation of these proteins. The observed chaperoning activity was not due to a bulk solvent effect but rather to direct protein–protein interaction. This is indicated by co-immunoprecipitation of substrate with NPfA and co-elution as a major peak in size exclusion chromatography. Binding of NPfA with substrate proteins was through hydrophobic interactions as observed by 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence. The chaperoning effect was also reflected in NPfA's capacity to dissociate fibrillar aggregates of amyloid-β protein and inhibit polyglutamine (polyQ) conversion to amyloids. Therefore, NPfA appears to be functionally similar to sHSPs. However, NPfA does not show sequence similarity to any known sHSP and hence may be categorized as a separate class of molecular chaperone. To the best of our knowledge, no Pyrococcus furiosus proteins have so far been shown to have domain-specific chaperone activity.
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The studies on stability and interactions between the individual NPfA and CPfA resulted in a novel finding. NPfA not only acts as specific internal chaperone by mediating folding of its own C-domain (CPfA), but also functions as a non-specific molecular chaperone by preventing aggregation of unrelated proteins. In multi-domain proteins, isolated domains often display decreased stability compared with the parent protein and each domain gets stabilized preferentially by inter-domain interactions . For example, in human γ-D-crystallin, folding of the isolated unstable N-terminal domain was found to be nucleated by the relatively stable C-terminal domain . The appearance of CPfA in the supernatant fraction upon refolding in the presence of NPfA indicates that the latter might be working as nucleating partner, assisting the CPfA to fold into soluble form (Fig. 1A).
While studying the molecular organization of isolated domains, we found that NPfA formed large oligomeric complexes of polydispersed nature (Fig. 1B). Owing to the polydispersion, the number of monomers in the oligomeric assembly could not be determined. An ~ 5-fold enhancement in the fluorescence of ANS pointed towards the presence of significant surface hydrophobicity of the folded oligomeric NPfA (Fig. 4A). All these characteristics are typical of small molecular chaperones, which generally form large polydispersed oligomeric complexes surrounding the partially denatured substrate proteins through hydrophobic interactions, keeping the latter in a folding-competent state . Small molecular chaperones are also characterized by the presence of a typical ~ 90 amino acid α-crystallin domain. A blast search revealed that NPfA does not contain any such stretch. Proteins lacking an α-crystallin domain have been reported to show chaperoning activity [37, 38]. The sequence of NPfA was individually aligned with sHSPs from Methanocaldococcus janaschii, Hsp16.9 from wheat, Pyrococcus furiosus sHSP, zebrafish α-A crystallin, human α-B crystallin and other non-HSP chaperones such as human clusterin and human β-tubulin. None of these alignments resulted in any significant sequence similarity with NPfA except β-tubulin, where eight amino acids at its C-terminus were found to be identical with the N-terminus of NPfA (within a stretch of 25 amino acids). Barring lack of sequence similarity, all other properties of NPfA matched well with those of small molecular chaperones. This led us to hypothesize that NPfA also functions as a non-specific molecular chaperone.
To test our hypothesis, we carried out in vitro assays where reduction in aggregation of substrate proteins by NPfA was used as a measure of the extent of its chaperoning efficiency. We studied the aggregation of a variety of proteins that exist as monomers, with size ranging from a small, single-domain, globular protein like BCA II (29 kDa) to a relatively large protein α-amylase (55.5 kDa) and the much larger multi-domain protein MSG (82 kDa). In each case, reduction of light scattering in the presence of NPfA clearly indicated its chaperoning effect (Figs 2 and 3). The chaperoning efficiency varied depending on the size and nature of the target protein. To obtain similar reduction in aggregation, the requirement of NPfA increased with increase in the size of the substrate protein. For the small protein BCA II, the presence of an equimolar concentration of NPfA completely rescued the former from aggregating, whereas higher concentrations of NPfA were required for α-amylase and MSG. Even at such high concentrations of NPfA, complete prevention of aggregation could not be achieved (Fig. 2A–C). This could be because of the dependence of the chaperoning activity of small molecular chaperone/sHSPs on the mass ratios of participating proteins [23, 39]. An alternative explanation could be based on the size of the substrate proteins. With increase in the size of the substrate, the collisional frequency with chaperones vis-à-vis the kinetics of interaction decreases, resulting in reduced chaperoning activity. In a substrate populated environment, the kinetics of interaction amongst partially denatured substrates predominates, causing more aggregation.
The NPfA also facilitated reduction in refolding-mediated aggregation, although at slightly lower efficiency, but significant enough to be noticed (Fig. 3). Recently it has been reported that the chaperoning efficiency of α-crystallin is determined by the differential interaction with aggregation-prone intermediates of different structural stabilities . In our case, the extent of reduction in aggregation was more pronounced in the case of thermal aggregation than refolding-mediated aggregation. This could be because of differential binding affinities of NPfA towards unfolding and refolding intermediates. Such a phenomenon has been observed in the case of α-crystallin . NPfA being thermally stable remains folded at the experimental temperature (Fig. 1D) and binds to thermally denatured aggregation-prone substrates. During the refolding reaction, however, there is a narrow concentration range within which NPfA acts as chaperone (Fig. 3). This is because, at lower NPfA concentrations, there may not be enough NPfA to compete for the available surfaces on partially folded substrate proteins. On the other hand, at higher concentrations, NPfA prefers self-recognition and undergoes homo-oligomerization over hetero-association with substrate proteins.
Efficient prevention of protein aggregation has been achieved by the inclusion of additives like sucrose, glycerol and PEG . These findings were considered to be more of bulk solvent effect rather than direct protein-additive interaction. However, in our case, the co-appearance of both substrate and NPfA bands in the same lane on SDS/PAGE of heat-treated, gel filtration chromatography purified mixtures suggested direct physical interaction between the proteins. This was observed regardless of the type of substrate protein (Fig. 4B). The direct interaction was also evident from studies where heated BCA II co-immunoprecipitated with NPfA-directed antibody (Fig. 4C). The NPfA + CPfA refolded mixture displayed lower ANS fluorescence compared with NPfA alone (Fig. 4A). This suggests that the exposed hydrophobic surface on NPfA gets involved in interaction with CPfA during refolding. This also reflects the general property of small molecular chaperones that are known to bind substrates through hydrophobic interactions [26, 27]. In the case of BCA II aggregation, the presence of NPfA did not lower the ANS fluorescence (Fig. 4A). This may be because of the presence of residual hydrophobic surfaces on both the interacting proteins which bind to ANS.
The predominance of β-sheet structure in folded NPfA (Fig. 1D) fitted well with structural information available for other sHSPs [9, 33]. Moreover, some sHSPs are shown to be associated with protein misfolding diseases, such as Huntington's and Alzheimer's disease [43, 44]. The proteins involved in these diseases, namely polyQ and amyloid-β respectively, form fibrillar amyloidic aggregates. Cross β-sheet structures are the hallmark of such aggregates which propagate by engaging more homologous proteins in the β conformation. Our data showed that NPfA prevents the formation of amyloidic aggregates of polyQ and dissociates the preformed amyloid-β proteins (Fig. 5). In the case of polyQ, this effect is presumably because NPfA mimics the homologous complementary oligomerizing surfaces by extending its own β-sheet scaffold, which engages in heterologous association, thereby preventing amyloid propagation. A possible mechanism by which amyloid-β fibrils are disaggregated by NPfA may be the halting of elongation followed by dissociation, as has been reported in other cases [45, 46]. Alternatively, the intermolecular associative forces between NPfA and amyloid-β are possibly stronger than the homomolecular aggregative forces amongst amyloid-β molecules. Irrespective of whether NfA binds to the fibrils at the ends or sidewise, the dominant interaction of NPfA with amyloid-β releases the latter from their fibrillar structure. Binding of NPfA at the ends will gradually decrease fiber length whereas lateral binding will fragment the preformed fibril at different positions. The latter appears to prevail in our case as we could see smaller oligomers in the incubated samples (Fig. 5D, right-hand panel). The exact mechanism of amyloid dissociation is under investigation. This is a first report where the specific domain of a hyperthermophilic protein has been shown to be endowed with chaperoning activity, while the full-length PfA failed to show this effect. This could be because NPfA is a non-natural product and is an isolated folded polypeptide with its own structural and functional properties very different from its wild-type parent. This finding is of immense physiological relevance as NPfA may be used in heterologous protein expression systems as co-expression partner in improving the solubility of proteins, thereby reducing losses due to inclusion body formation. A thorough investigation of the chaperoning activity of NPfA in a heterologous co-expression system is required. The effect of NPfA on disintegrating amyloid-β proteins is significant as it may be used for therapeutic intervention of protein-aggregation-related diseases.