Self-assembly of human latexin into amyloid-like oligomers
© Pallarés et al; licensee BioMed Central Ltd. 2007
Received: 27 April 2007
Accepted: 08 November 2007
Published: 08 November 2007
In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations.
Here, we show that human latexin, a protein that shares the same fold with cystatin C, assembles into stable spherical amyloid-like oligomers that bind thioflavin-T and congo red similarly to common amyloid structures but do not evolve into fibrils. Latexin self-assembly correlates with the formation of a mostly denaturated state rather than with the population of partially structured intermediates during the unfolding process. The results suggest that unfolding of α-helix 3 might be involved in the transition of latexin toward amyloidotic species, supporting the notion of the protective role of the native protein structure against polymerization.
Overall the data herein indicate that latexin could be a good model for the study of the structural and sequential determinants of oligomeric assemblies in protein aggregation processes.
Amyloidoses are a group of protein misfolding diseases characterized by the polymerization of normally innocuous and soluble proteins or peptides into insoluble proteinaceous deposits. No sequence or structural similarities are apparent between any of the proteins that display the ability to form amyloid aggregates in diseases like Alzheimer's disease, type II diabetes, systemic amyloidosis or the transmissible spongiform encephalopaties [1, 2].
There is a striking difference between the amounts of amyloid depositions in various types of amyloid disorders. In systemic lysozyme amyloidosis, for example, the deposits can grow to kilogram quantities in the liver [3, 4], whereas in neurodegenerative diseases, where the quantity of aggregates can be almost undetectable in some cases , there is no clear correlation between the amount of amyloid deposition and the clinical severity of the disease. In Alzheimer's patients, a significant cognitive impairment was observed in the absence of noticeable amyloid deposits in the brain, although the levels of soluble amyloid oligomeric assemblies was found to be greatly elevated [6, 7]. Evidence is accumulating that prefibrillar aggregates are cytotoxic both in vivo and in vitro [7–9], although this question is still open to debate. It still is not evident, however, which particular amyloid structures induce cell death by specific molecular mechanisms and which play the role of "inert" material and cause disease due to their quantity or mechanical properties. In spite of extensive studies of the fibrillar state, mainly by solid state NMR and X-ray diffraction, scarce structural information is available, and there is an increasing interest in the understanding of the biochemical and biophysical properties of metastable polypeptide associations preceding the aggregated states of proteins. Here, we characterize the conformational stability of latexin, an endogenous vertebrate carboxypeptidase inhibitor, and show that it associates into metastable oligomers with amyloidotic properties. Human latexin consists of two topologically equivalent subdomains, each one with a cystatin-like topology, consisting of an α-helix enveloped by a curved β-sheet. These subdomains are packed against each other through the helices and linked by a connecting segment encompassing a third α-helix [10, 11]. Latexin adds up to other proteins unrelated to any known human disease able to form assemblies with the characteristics of amyloid . Latexin oligomerization does not depend on the population of partially structured conformations but probably on the transition of a native helical structured region to an unfolded conformation under conditions that are selectively unfavorable for the folding and nativeness of this polypeptide. In this particular case, conditions promoting self-assembly into highly stable oligomers do not promote further evolution into fibrillar structures suggesting that, in protein deposition, the intermolecular contacts responsible for the final, usually highly ordered, structure of protein aggregates could not necessarily coincide with those promoting the formation of the initial oligomeric assemblies, which may have important consequences for the therapeutics of amyloid diseases.
Conformational stability of latexin
Amyloid-like features of latexin oligomers
Congo red is another amyloid dye, with an absorbance maximum at 490 nm, that increases and shifts to red upon binding to amyloid material. We assayed congo red binding to latexin after protein incubation for one day at different chaotropic agent concentrations. In a fashion very similar to what is observed with Th-T, maximum binding occurs at 6 M urea whereas little binding is detected in protein solutions below 2 M urea and the binding decreases in solutions above 6 M urea (Figure 4A). Latexin incubated at 6 M urea promoted a maximum change in the congo red spectra at 533 nm, similar to that observed for aged solutions of Aβ amyloid peptide . The binding of latexin solutions to both amyloidotic dyes closely correlates with the amount of oligomeric forms they contain, as analyzed by SDS-PAGE. In agreement with the CD data, this indicates that latexin oligomers display amyloid-like conformations. Importantly, the formation of these species correlates with the loss of secondary structure, but not with the formation of an intermediate or the population of native latexin. This observation is evident from the comparison of the traces in Figures 4A and 1B.
A "hot spot" of aggregation in α-helix 3 of latexin
It has been proposed that almost every protein, when incubated under the right conditions, can aggregate . Since globular proteins rarely aggregate from their native states, their destabilization and subsequent increased population of unfolded molecules is well established as a triggering factor in disorders associated with the deposition of proteins that are globular in their normal functional states [23, 24], like β2-microglobulin, lysozyme, transthyretin and the prion protein. Accordingly, many of the proteins involved in depositional disorders are mostly unstructured within the cell . These include amylin, amyloid-β-protein, and α-synuclein, among others. In these cases, protein polymerization and deposition does not require unfolding and can occur by direct self-assembly of the unstructured polypeptide chains. In the last few years, proteins unrelated to any known human disease have been found to convert in vitro into higher order structures that also present a cross-β conformation and fulfill all characteristics of amyloid fibrils [25–27]. Latexin is a vertebrate carboxypeptidase inhibitor with an α/β fold, closely resembling that of cystatins. Interestingly enough, several members of the cystatin family have been shown to form stable oligomeric assemblies and amyloid fibrils [13, 28, 29].
The latexin fold is destabilized at low urea concentrations and the protein becomes mostly unfolded above 6 M urea. A stable equilibrium intermediate populates during the unfolding reaction of latexin. The intermediate most likely corresponds to an open form in which the two domains of latexin have lost their contacts but still conserve most of their secondary structure and probably domain architecture. Nevertheless, this partially folded form of latexin does not appear to be competent for polypeptide self-assembly, since its formation does not overlap with conditions that promote maximum oligomerization. As previously described for myoglobin , the oligomerization of latexin correlates with conditions in which the protein is mostly unstructured, rather than with environments that generate partially folded intermediates. In the case of the structurally related cystatin C, the effect of temperature, pH and denaturant in self-assembly has been assayed . In contrast to latexin, cystatin C self-assembles through the formation of a partially unfolded intermediate, under conditions quite far from those leading to the unfolding of the protein. The process of polymerization involves the initial formation of a dimer by domain-swapping  and subsequent propagation of this effect to form oligomeric assemblies . Consistently, stabilization of the monomer by different means avoid swapping and further cystatin polymerization . Although, in the case of latexin dimers are relevant forms in the oligomerization process, it is unlikely that they are domain-swapped forms, since these species essentially keep the same 3D protein structure as the monomer, while the assemblies of latexin have lost most of the native organization. Also, according to our data, oligomer formation by a mechanism in which dimers act as building blocks is more likely than a propagated (or run-away) mechanism. Overall, latexin differs from protein models in which polymerization depends on the population of partially structured conformations, usually enriched in β-sheet structure [27, 35]. Instead, our data indicate that, like in the case of intrinsically unstructured proteins, the structural precursors in the first stages of latexin polymerization correspond to protein regions containing little, if any, secondary structure.
Provided that self-assembly takes place in conditions where the polypeptide chain is mainly unfolded, the use of prediction algorithms that forecast aggregation-prone regions merely from the protein sequence should allow the detection of the latexin region(s) involved in the formation and maintenance of intermolecular contacts. According to such programs, the latexin region with the highest probability to be involved in amyloid-like intermolecular contacts comprises residues T134-W149. This is supported by the observation that the central sequence of this predicted region, corresponding to the peptide VLHLAWVA, forms well-ordered microcrystals with amyloid properties as analyzed by EM, Th-T binding, CD and FTIR. The formation of peptide microcrystals in physiological conditions is usually associated with rapid formation of strong and specific intermolecular interactions. In the protein context the same strong contacts might become relevant for the oligomerization process of latexin. These microcrystals are of interest since their properties closely resemble those formed by peptides related to fibril-forming proteins like Alzheimer's amyloid-β, tau and prion proteins from which the high resolution atomic structure of a common amyloid cross-beta spine involving the formation of steric zippers has been determined [36, 37]. Experiments are underway in our lab to solve the structure of this peptide assembly.
Overall, the data herein indicate that, in the case of latexin, the formation of oligomeric species occurs maximally under conditions that are specifically adverse to the folding of the protein. This can be easily rationalized, since aggregation-prone regions have their side chains usually hidden in the inner hydrophobic core of the native globular protein or already involved in the network of contacts that stabilizes the secondary and tertiary structure of polypeptides. Specifically, the VLHLAWVA region of latexin is located in α-helix 3, where the establishment of anomalous intermolecular interactions is blocked not only because it belongs to a secondary structure element, but also because its side chains participate in the interface contacts between the two domains of latexin. From the data shown, it appears that opening of the interface is not sufficient to promote the loss of the α-helical structure and oligomerization, but is probably a prerequisite for this event to occur. Once in a non-structured context and exposed to solvent by denaturation, the VLHLAWVA region is ready for the establishment of intermolecular contacts as proven by the analysis of the eight-residue peptide. Thus, latexin constitutes yet another example that illustrates the protective role played by the protein native structure against aggregation.
Protein aggregation generates a very broad range of structures including oligomers, protofibrils, and fibrils. Determining the role of these so-called "prefibrillar species" in assembly is of paramount importance, since such complexes have been proposed for many proteins to be responsible for the cellular toxicity associated with amyloid disease [38, 39]. Dimeric, tetrameric and higher order latexin oligomers were visualized and characterized. From this study, it appears that the latexin system could be an excellent model for the isolation and deep structural and biochemical characterization of highly stable low-molecular weight oligomeric species that appear to be the final association state of latexin under strongly denaturing conditions, despite exhibiting tinctorial and conformational amyloid-like properties. The drastic conditions in which latexin self-assemblies only allow for strong intermolecular contacts to be established and probably prevent the formation of the specific network of non-covalent interactions needed to evolve into the quasi-crystalline structure characteristic of amyloid fibrils. Accordingly, urea has been used to reduce the driving force for Aβ-peptide aggregation, in an effort to isolate stable oligomeric assemblies . Increasing urea concentrations reduced the average size of the aggregates, and the morphology of the aggregates changed from linear fibrils to globular oligomeric structures This behavior indicates that the interactions involved in the formation of the primary, low molecular weight oligomers during protein aggregation do not necessarily overlap with those driving the formation and stabilization of higher order fibrillar assemblies. This is relevant for the therapies of amyloid disease, since it suggests that drugs designed to interfere with the interactions occurring in amyloid fibrils would not necessarily have a significant effect in the initial assembly of pre-fibrillar aggregates, which have been implicated as the toxic species in conformational disorders.
Recombinant latexin was produced in E. coli and purified as previously described . The peptide VLHLAWVA was synthesized by the American Peptide Company, Sunnyvale, CA. Previously a peptide comprising T134-W149 was tried to be synthesized by the same company but its high aggregation tendency made it impossible to obtain it in a soluble form for further purification. Urea, thioflavin-T and congo red were purchased from Sigma. Unless otherwise mentioned, all solutions used for latexin analysis were made in 50 mM phosphate buffer pH 7.5 (buffer N), and 5 mM phosphate buffer pH 7.5 was used for peptide preparations (buffer P). All the experiments were performed at 25°C.
Unless stated otherwise, oligomeric samples were obtained by incubating protein at 1 mg/ml in buffer N in the presence of different concentrations of urea from several hours to several days. The peptide VLHLAWVA was incubated at 500 μM concentration in buffer P for 24 h.
Prediction of sensitive regions for aggregation
To determine which regions of latexin sequence could be involved in aggregation the in-house developed AGGRESCAN software was used  and the predictions cross-checked against TANGO  outputs. Both algorithms detected the sequence stretch around residues 130–150 as the main region which could be potentially involved in the aggregation of latexin from a mainly unfolded state [19, 22]. A peptide covering residues V136-A143 (VLHLAWVA) was synthesized in order to check experimentally its relevance for latexin aggregation.
Circular dichroism and fluorescence spectroscopies
Circular dichroism (CD) spectra in the far-UV region were obtained by using a Jasco-810 spectropolarimeter at 25°C. Spectra were recorded for native proteins and for proteins after incubation at different urea concentrations (ranging from 0 to 9 M) at 100 μg/ml protein concentration. Twenty accumulations were averaged to obtain each spectrum. The mean residue ellipticity at 220 nm was plotted versus urea concentration to obtain denaturation curves. The fitting of the experimental data was performed using the non-linear, least-squares algorithm provided with the software KaleidaGraph (Abelbeck Software). The peptide CD spectra was analyzed immediately upon dilution and after incubation at 500 μM concentration in buffer P for 24 h. Tryptophan fluorescence was excited at 280 nm and recorded at 340 nm. For calculation of the relative population of folded and unfolded species, the linear-dependence of the fluorescence of both the folded and unfolded latexin states on the urea concentrations was included in the fitting of the experimental data.
Dye binding assays
Thioflavin-T binding assays were carried out using aliquots of 100 μl drawn from 1 mg/ml protein samples in buffer N after 24 h incubation. These aliquots were diluted into buffer (10 mM sodium phosphate, 150 mM NaCl) containing 65 μM thioflavin-T, and adjusted to a final volume of 1 ml. Fluorescence data were collected after five minutes to ensure that thermal equilibrium had been achieved. Fluorescence emission spectra were recorded using an excitation wavelength fixed at 440 nm. The Th-T stained samples were also analyzed under UV light using a Leica DMBR microscope. Samples were tested for amyloid-specific congo red binding by the spectroscopic band-shift assay as described by Klunk . Briefly, aliquots of 1 mg/ml protein were diluted in reaction solution (5 mM sodium phosphate/150 mM NaCl, pH 7.0) containing 5 μM congo red and absorption spectra were collected together with negative control solutions of dye in absence of protein and of protein samples in the absence of dye, subtracting the absorption of the dye and the scattering contribution from the samples spectra.
Transmission electron microscopy
Samples containing 1 mg/ml of latexin or 500 μM of VLHLAWVA peptide were incubated as above. A 5 μl aliquot was then placed on carbon-coated copper grids, and allowed to stand for two minutes. The grids were then washed with distilled water and stained with 2% (w/v) uranyl acetate and allowed to stay for two minutes prior to analysis using a Hitachi H-7000 transmission electron microscope operating at an accelerating voltage of 75 kV.
The VLHLAWVA peptide was incubated in buffer P for 24 h at room temperature and prepared essentially as previously described . Briefly, the microcrystals were lyophilized before analysis to reduce water interference in the infrared spectra and the sample applied on top of the germanium crystal in the attenuated total reflection module of a Bruker Tensor FT-IR spectrometer. The structure of the peptide aggregates was then analyzed. For each spectrum, 20 interferograms were collected and averaged. All processing procedures were carried out so as to optimize the quality of the spectrum in the amide I region, between 1550 cm-1and 1700 cm-1.
List of Abbreviations
Attenuated Total Reflectance Fourier Transform Infrared
This work was supported by grants BIO2004-05879 (Ministerio de Ciencia y Tecnología, Spain) and 2005SGR-00037 (AGAUR, Catalonia).
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