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Reduction of the amyloidogenicity of a protein by specific binding of ligands to the native conformation

¹ Dipartimento di Scienze Biochimiche, Universitá degli Studi di Firenze, 50134 Firenze, Italy
² Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, Oxford OX1 3QT, UK

Reprint requests to: Giampietro Ramponi, Dipartimento di Scienze Biochimiche, Universitá degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy; e-mail: ramponi{at}scibio.unifi.it; fax: 39-055-4222725; Christopher M. Dobson, e-mail: chris.dobson{at}chem.ox.ac.uk; fax: 44-1865-275921.

(RECEIVED October 4, 2000; FINAL REVISION January 19, 2001; ACCEPTED January 23, 2001)

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
It is known that human muscle acylphosphatase (AcP) is able, under appropriate conditions in vitro, to aggregate and form amyloid fibrils of the type associated with human diseases. A number of compounds were tested for their ability to bind specifically to the native conformation of AcP under conditions favoring denaturation and subsequent aggregation and fibril formation. Compounds displaying different binding affinities for AcP were selected and their ability to inhibit protein fibrillization in vitro was evaluated. We found that compounds displaying a relatively high affinity for AcP are able to significantly delay protein fibrillization, mimicking the effect of stabilizing mutations; in addition, the effectiveness of such outcome correlates positively to both ligand concentration and affinity to the native state of AcP. By contrast, the inhibitory effect of ligands on AcP aggregation disappears in a mutant protein in which such binding affinity is lost. These results indicate that the stabilization of the native conformation of amyloidogenic proteins by specific ligand binding can be a strategy of general interest to inhibit amyloid formation in vivo.

Keywords: Acylphosphatase; aggregation; amyloid fibrils; amyloid formation; ligand binding

Abbreviations: AcP, acylphosphatase • CD, circular dichroism • TFE, 2,2,2-trifluoroethanol • TTR, transthyretin

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
The amyloidoses are a group of diseases where proteins or protein fragments change from their native soluble forms into insoluble aggregates or plaques, and accumulate in a variety of organs and tissues (Kelly 1996; Rochet and Lansbury 2000). This phenomenon has devastating consequences for the function of the organs that lead in many cases to death. To date, nearly 20 different amyloidoses have been described, including Alzheimer's disease (Selkoe 1996), Parkinson's disease (Conway et al. 1998; Spillantini et al. 1998), light chain amyloidosis (Buxbaum and Gallo, 1999), Huntington's disease (Perutz 1999), familial amyloid polyneuropathy (Costa et al. 1978; McCutchen et al. 1995), dialysis-related amyloidosis (Drueke 2000), and the spongiform encephalopathies (Jackson and Clarke 2000).

A recent development in this field has been the discovery that proteins other than those associated with diseases are capable of fibril formation under appropriate conditions in vitro (Guijarro et al. 1998; Litvinovich et al. 1998; Chiti et al. 1999; Gross et al. 1999; Konno et al. 1999; Villegas et al. 2000; Yutani et al. 2000). This finding has two major consequences. First, it has implications for understanding the origin of amyloid formation in vivo because amyloid deposition can no longer be assumed to arise from the unique physical properties of a limited number of protein sequences (Dobson 1999; Rochet and Lansbury 2000). In addition, the ability to study the formation of amyloid fibrils by a wide range of proteins has opened up the opportunity to augment studies of the molecular basis of amyloid fibril formation (Jimenez et al. 1999; Chiti et al. 2000; Ramirez-Alvarado et al. 2000; Villegas et al. 2000). Such studies not only allow fundamental aspects of the process of fibrillization to be investigated in greater depth, but also allow possible strategies to retard or eliminate amyloid deposition to be tested.

In this work we use human muscle acylphosphatase (AcP) to investigate the importance of stabilizing the native state of a globular protein as a possible strategy to contrast amyloid formation in vivo. One of the strategies that can be used to stabilize the native state of a protein relative to unfolded conformations is the addition of ligands that bind specifically to the native state (Sancho et al. 1991; Chiti et al. 1998). In the case of transthyretin (TTR), it was found that binding of specific ligands to the native tetrameric state can inhibit the dissociation of the tetramer into a monomeric conformation that is only partially folded, although it retains a globular structure and a topology similar to that of the native state; such binding was found to inhibit formation of amyloid fibrils because the tetramer is known to be less prone to aggregation than the monomer (Baures et al. 1998; Peterson et al. 1998; Baures et al. 1999; Klabunde et al. 2000). Here we explore the effect of specific ligands on aggregation from a slightly different angle. AcP was shown to be capable of aggregation and amyloid fibril formation in vitro under conditions favoring the conversion of the native state into denatured conformations in which the native topology of the protein is fully disrupted (Chiti et al. 1999). By using AcP as a model system, we assess whether the ligand-mediated stabilization of the monomeric native state of a protein, relative to unfolded or partially unfolded states, can have the potential to retard aggregation and subsequent amyloid formation.

AcP is a 98-residue protein with a structure, in its native state, containing two parallel -helices packed against a five-stranded antiparallel ß-sheet (Saudek et al. 1989; Pastore et al. 1992). Kinetic experiments have indicated that AcP folds and unfolds in a two-state manner (Chiti et al. 1998; van Nuland et al. 1998). A simplified mechanism of fibril formation for AcP is the following (Chiti et al. 1999):

where N is the native and globular state of AcP, and D is an ensemble of denatured conformations. Because aggregation occurs from denatured or partially denatured conformations (D), this process is very slow under physiological conditions that favor the conformational stability of the native state N. Denaturants such as trifluoroethanol (TFE) cause the equilibrium between N and D to be shifted toward the latter, thus favoring aggregation and subsequent fibril formation (Chiti et al. 1999). Consistent with this concept, the analysis of a group of variants of AcP having single amino acid substitutions from the wild-type sequence has shown that the process of aggregation becomes more favorable as the destabilization of the native state caused by mutation becomes larger (Chiti et al. 2000). The most destabilized AcP variants can be readily converted into amyloid fibrils even in the absence of any denaturant, under solution conditions substantially more similar to those found in living systems (Chiti et al. 2000).

The negative correlation between the conformational stability of the native state and the propensity to form amyloid fibrils has encouraged us to assess whether simple compounds with a high affinity for the native conformation of AcP are effective as inhibitors of aggregation and amyloid formation. AcP is an enzyme that binds to and hydrolyzes acylphosphates (Stefani et al. 1997). This natural property of AcP has been exploited by studying the effects on aggregation of substrate analogs that bind to the active site of AcP and thereby stabilize the native state.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Various compounds were tested to examine their binding to native AcP, particularly compounds having a phosphate group or a negative charge for which some indication of high affinity for this protein already exists (Stefani et al. 1997). These compounds include phosphate, sulfate, 2,3-bisphosphoglycerate, ß-glycerophosphate, phenylphosphate, chloride, and adenosine triphosphate (ATP). Because aggregation can be readily induced by addition of TFE, it is important to determine the affinity of the various ligands for native AcP under such conditions. This objective is not straightforward to accomplish with conventional techniques such as optical spectroscopies or equilibrium dialysis, because the native conformation of the protein in the absence of the ligand is unstable under such denaturing conditions. Nevertheless, the affinity of a ligand for the native state of a protein can be determined, under denaturing conditions, by measuring the decrease of the unfolding rate in the presence of increasing concentrations of ligand, a procedure described in detail by Sancho et al. (1991) and Chiti et al. (1998).

Figure 1a shows the change of fluorescence of AcP in the presence of 30% (v/v) TFE. The fluorescence trace fits very well to a single exponential function. No further changes of fluorescence have been observed on the long timescale, indicating that unfolding is a monophasic process under these conditions. The change of the unfolding rate constant on addition of progressive concentrations of the phosphate ligand is reported in Figure 1b. The dependence of the unfolding rate on ligand concentration is given by Chiti et al. (1998):

(1)

where k_u,obs is the observed unfolding rate constant, k_u,E and k_u,EL are the actual rate constants for the free and ligand-bound protein, respectively, K_D is the dissociation constant, and [L] is the ligand concentration. Equation 1 is valid only when the concentration of protein used in the experiments is much lower than that of ligand, which is the case for all of the measurements made here. Furthermore, k_u,EL was found to be several orders of magnitude smaller than k_u,E for AcP (Chiti et al. 1998), causing the equation to be converted into the simpler form

(2)

The K_D value can be considered as the concentration of ligand at which the unfolding rate decreases to one-half with respect to that in the absence of ligand. The fitting of the experimental data reported in Figure 1b to equation 2 provides an estimate of the K_D value for the complex formed by native AcP and phosphate. The analysis was repeated for other putative ligands, and the resulting K_D values are reported in Table 1. The various ligands have extremely diverse K_D values, ranging from a value of 0.083 ± 0.008 mM obtained for the sulfate ion to the value of 21 ± 4 mM obtained for the chloride ion.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Dependence of the unfolding rate constant of AcP (ku,obs) on phosphate concentration in the presence of 30% (v/v) TFE. The decrease of ku,obs is caused by the stabilization of the native conformation of AcP through ligand binding (Chiti et al. 1998). The solid line through the symbols is the best fit of the experimental data to equation 5. The fitting procedure allows the dissociation constant of the complex between native AcP and the ligand to be determined.

(3)

where G is the free energy change of unfolding, whereas k_u,E and k_f,E are the unfolding and refolding rate constants, respectively. The folding rate of AcP does not change with the addition of ligand, as a consequence of the fact that the active site is not yet formed in either the unfolded or the transition state (Chiti et al. 1998). From equation 3 one can therefore derive the change of G of unfolding when the ligand is added, that is, the free energy of stabilization on addition of ligand (G):

(4)

where k_u,obs is the unfolding rate constant in the presence of ligand. Substituting equation 2 in equation 4 we obtain:

(5)

Equation 5 shows that the free energy of stabilization on addition of a ligand depends on both the affinity and concentration of the ligand. The calculated G values on addition of 10 mM concentration of various ligands and of phosphate at various concentrations are reported in Tables 1 and 2, respectively.

The ranges of TFE concentrations at which protein aggregates were observed in each case are shown in Figure 2a and Table 2. The boundaries of these intervals were defined by the TFE concentrations at which the protein produces 50% of the maximum observed increase in CD ellipticity at 215–220 nm and in thioflavine T fluorescence. The results obtained by using CD are in close agreement with those obtained with thioflavine T. Although the upper limit of the range of TFE concentration at which aggregation occurs is similar, within experimental error, at the various ligand concentrations, the lowest TFE concentration that is required for formation of protein aggregates increases with ligand concentration. Figure 2b shows that there is a remarkable correlation between the minimum concentration of TFE that causes protein aggregation after 5 h and the free energy of ligand-induced stabilization of native AcP at different concentrations of phosphate. A value of 0.99 is calculated for the linear correlation coefficient (p < 0.0001), indicating that the correlation is highly significant. Because the aggregation process of AcP becomes faster as the TFE concentration increases (Chiti et al. 2000), the observed shift in the minimal concentration of TFE required for the aggregates to be present after 5 h on addition of phosphate indicates that this compound retards aggregation and amyloid formation.

View larger version (25K): [in this window] [in a new window]   Fig. 2. (a) Ranges of TFE concentration at which protein aggregates are present for wild-type AcP and for the R23Q mutant after incubation of 5 h at the indicated concentrations of phosphate. The samples were incubated at 25°C in 50 mM acetate buffer (pH 5.5) and at protein concentrations of 0.375 mg/mL. Solid and hatched bars indicate results from far-UV CD spectroscopy and thioflavine T fluorescence, respectively. Experimental errors for both the lower and upper TFE concentrations at which aggregates occur are 1. 1% (v/v). (b) Correlation between the minimal TFE concentration required for producing aggregates after 5 h (taken as a measure of the amyloidogenic potential) and the change of G of unfolding on ligand binding (taken as an index of the conformational stability of the native protein). The high r value (0.99) and the low p value (<0.0001) indicate that the correlation is highly significant. The equation of best fit has the form %TFE = (19±1) + (0.48±0.06) * G. The values of TFE concentration on the Y-axis are an average of those determined with the thioflavine T and the CD data. The experimental errors are those indicated in Table 2 for both G and %TFE values.

The analysis was extended to all of the ligands listed in Table 1 to assess whether the inhibiting effect observed for phosphate on aggregation is specific to this compound or, rather, is a general characteristic of molecules binding to AcP. In this case a concentration of 10 mM was used for each ligand to facilitate comparison between the various substances. The free energies of stabilization obtained with a 10 mM concentration of the various ligands are shown in Table 1. The variation in the minimal TFE concentration that is necessary for aggregation after 5 h in the presence of these compounds is shown in Table 1 and in the two panels of Figure 3. The data points appear to be more dispersed compared with the analysis performed with the different concentrations of phosphate, probably because of additional non-specific effects exerted by these compounds on the conversion of soluble AcP into aggregates. Despite this, the statistical analysis clearly shows that the correlation between the minimal TFE concentration and free energy of stabilization is highly significant (r = 0.89, p = 0.003). The results show that only ligands with a relatively high binding affinity for AcP (K_D < 1 mM) are effective at this concentration in inhibiting the aggregation process.

View larger version (25K): [in this window] [in a new window]   Fig. 3. (a) Ranges of TFE concentrations at which protein aggregates are present for wild-type AcP after incubation of 5 h in the presence of 10 mM of the indicated ligands. Experimental conditions and experimental errors are those described in Figure 2. Solid and hatched bars indicate results from far-UV CD spectroscopy and thioflavine T fluorescence, respectively. Adenosine triphosphate and phenylphosphate were not analyzed with CD because of their high absorbance in the far-UV region. (b) Correlation between the minimal TFE concentration required for producing aggregates after 5 h and the change of G of unfolding on ligand binding. An r value of 0.89 and a p value of 0.003 indicate that the correlation is highly significant. The equation of best fit has the form %TFE = (18 ± 1) + (0.40 ± 0.08) * G. The experimental errors are those indicated in Table 1 for both G and %TFE values.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

In our previous study we have shown that single-point mutations destabilizing the native conformation of AcP facilitate aggregation at a denaturant concentration lower than those required for the wild-type protein (Chiti et al. 2000). The minimal TFE concentration required for aggregation was found to correlate closely with the free energy of destabilization of the protein (G) (Chiti et al. 2000). The present investigation is complementary to this previous study. Instead of destabilizing the native conformation of AcP by mutation we have used specific ligands to achieve a global stabilization of the protein. The data obtained with different phosphate concentrations (Fig. 2b) and with various ligands at the same concentration (Fig. 3b), produce best-fitted linear equations similar to each other and to that reported previously from the mutational study (see Figs. 2b, 3b, and 4 legends). This indicates that the dependence of the propensity of AcP to aggregate on conformational stability is unique regardless of whether mutations or ligands are used to modify the stability of the native state. Figure 4 reports a comprehensive analysis in which the two sets of data obtained with ligands and protein mutants are combined to show the relationship between propensity to aggregate and conformational stability over a wide range of G of unfolding, spanning from –22 to 12 kJ mol^-1. The correlation is highly significant (r = 0.97, p < 0.0001); the parameters of the linear expression that fits best to these data are given in the Figure 4 legend.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Correlation between the minimal TFE concentration required for producing aggregates and the change of G of unfolding on either mutation (empty circles) or ligand binding (filled circles). The cross represents the data point for wild-type AcP in the absence of ligands. The r and p values indicate that the correlation is highly significant. The equation of best fit has the form %TFE = (18.25±0.5) + (0.43±0.04) * G, which is very similar to those obtained by separate fits of the data obtained at different concentrations of phosphate (see Fig. 2b legend), at a 10 mM concentration of various ligands (see Fig. 3b legend), or with the mutants (in the latter case, the equation reported previously [Chiti et al. 2000] has the form %TFE = (18.5±1) + (0.46±0.08) * G).

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Materials
AcP was purified from an Escherichia coli-based expression system, as described previously (Taddei et al. 1996). The protein used in this study has cysteine at position 21 replaced by a serine residue to avoid complexities arising from a free cysteine residue (van Nuland et al. 1998). The R23Q mutant of AcP was produced by using an oligonucleotide-directed mutagenesis kit from Pharmacia Amersham (Taddei et al. 1996). It also presents the C21S replacement. Protein concentration was measured by UV absorption by using an ₂₈₀ value of 1.49 mL mg^–1 cm^–1. All compounds used as specific ligands of AcP were purchased from Sigma-Aldrich.

Stopped-flow kinetics
A Bio-Logic SFM-3 stopped-flow device was used to measure the rate of TFE-induced unfolding of AcP in the presence of various concentrations of ligands. In each experiment, 1 volume of a solution containing native AcP was mixed with 10 volumes of a denaturing solution containing TFE and the ligand at the desired concentration. In all experiments, the final conditions after mixing were 0.02 mg mL^–1 AcP, 30% (v/v) TFE, 50 mM acetate buffer (pH 5.5) at 25°C. For each ligand, the unfolding rate of AcP was measured at eight different concentrations. The various kinetic traces obtained in the presence of ligand were fitted to single exponential functions to determine the unfolding rate constant by using the Kaleidagraph software package.

Circular dichroism
Far-UV CD spectra were acquired at 25°C by using a Jasco J-720 spectropolarimeter and cuvettes of 1-mm path length. In a typical experiment, 20–25 samples of AcP at a concentration of 0.375 mg/mL (34 µM) were prepared at different TFE concentrations ranging from 0% to 45% (v/v) in 50 mM acetate buffer (pH 5.5) at 25°C. The far-UV CD spectrum of each sample was acquired after 5 h of incubation. This analysis was repeated in the presence of each of the seven ligands considered here at a concentration of 10 mM. For phosphate, the analysis was also performed at five different concentrations ranging from 0.5 to 50 mM. All mother solutions containing AcP were centrifuged and their protein concentrations measured immediately before preparation of the samples.

Thioflavine T dye binding
Aliquots of the samples prepared as described in the previous paragraph for the CD measurements were also used to test for thioflavine T binding (LeVine 1995). A sample of 133 µL was mixed with 867 µL of 25 µM thioflavine T in 25 mM phosphate buffer (pH 6.0). The fluorescence was measured immediately after mixing by using excitation and emission wavelengths of 440 and 485 nm, respectively. A Shimadzu RF-5000 spectrofluorometer was used for fluorescence measurements.

	Acknowledgments

 
We are grateful to Fabiana Baroni for assistance with this work. We are very grateful for support from the Fondazione Telethon-Italia (F.C.), the Howard Hughes Medical Institute (C.M.D.), and the Wellcome Trust (C.M.D.). The Oxford Centre for Molecular Sciences is supported by the Biotechnology and Biological Sciences Research Council (BBSRC), the Engineering and Physical Sciences Research Council (EPSRC), and the Medical Research Council (MRC). The Dipartimento di Scienze Biochimiche in Florence is supported by the Consiglio Nazionale delle Ricerche (CNR, contributo n. 99.02609.04), the Ministero dell'Universitá e della Ricerca Scientifica e Tecnologica (MURST, fondi PRIN "Folding e misfolding di proteine"), and the Fondazione Telethon-Italia. The stopped-flow apparatus was a generous gift from the Cassa di Risparmio di Firenze.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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