Erica Frare

Partly folded states of members of the lysozyme/lactalbumin superfamily: A comparative study by circular dichroism spectroscopy and limited proteolysis

The partly folded states of protein members of the lysozyme (LYS)/α‐lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123‐ to 129‐amino‐acid residues with similar three‐dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium‐binding properties and capability to adopt partly folded states or molten globules in acid solution (A‐state) or on depletion of calcium at neutral pH (apo‐state). Far‐ and near‐UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg‐white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo‐form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the β‐subdomain (approximately chain region 34–57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured α‐domain and a largely disrupted β‐subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.

The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

The aggregation process of wild-type human lysozyme at pH3.0 and 60 degrees C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the beta-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat, trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis.

The calcium‐depleted form of α‐lactalbumin (α‐LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo‐α‐LA at neutral pH. CD spectra revealed that the molten globule of apo‐α‐LA can be obtained upon mild heating at 45°C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far‐ and near‐UV CD spectra of apo‐α‐LA are essentially identical to those of the most studied molten globule of α‐LA at pH 2.0 (A‐state). Proteolysis of the 123‐residue chain of apo‐α‐LA by proteinase K at 4°C occurs slowly as an all‐or‐none process leading to small peptides only. At 37°C, proteinase K preferentially cleaves apo‐α‐LA at peptide bonds Ser34‐Gly35, Gln39‐Ala40, Gln43‐Asn44, Phe53‐Gln54, and Asn56‐Asn57. All these peptide bonds are located at level of the β‐subdomain of the protein (chain region 34–57). Similar sites of preferential cleavage have been observed with the TFE‐ and oleic acid‐induced molten globule of apo‐α‐LA. A protein species given by the N‐terminal fragment 1–34 linked via the four disulfide bridges to the C‐terminal fragment 54–123 or 57–123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo‐α‐LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of α‐LA maintains a native‐like tertiary fold characterized by a rather well‐structured α‐domain and a disordered chain region encompassing the β‐subdomain 34–57 of the protein. Proteins 2002;49:385–397.

The oleic acid complexes of proteolytic fragments of α-lactalbumin display apoptotic activity

The complexes formed by partially folded human and bovine α‐lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha‐lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine α‐lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53–103 and the two‐chain fragment species 1–40/53–123 and 1–40/104–123, these last being the N‐terminal fragment 1–40 covalently linked via disulfide bridges to the C‐terminal fragment 53–123 or 104–123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10‐fold and 15‐fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA‐conditioned anion exchange column and salt‐induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of α‐helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein–OA complex does not imply specific binding of the protein.

Structural and Morphological Characterization of Aggregated Species of α-Synuclein Induced by Docosahexaenoic Acid

The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.

Protein dissection enhances the amyloidogenic properties of α‐lactalbumin

α‐lactalbumin (LA) in its molten globule (MG) state at low pH forms amyloid fibrils. Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1–40/41–123, named Th1‐LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the β‐subdomain of the native protein (1–40/53–123, named desβ‐LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. Th1‐LA and desβ‐LA aggregate at pH 2.0 much faster than the intact protein and form long and well‐ordered fibrils. Furthermore, in contrast to intact LA, the LA derivatives form regular fibrils also at neutral pH, even if at much reduced rate. In acidic solution, Th1‐LA and desβ‐LA adopt a MG state which appears to be similar to that of intact LA, as given by spectroscopic criteria. At neutral pH, both Th1‐LA and desβ‐LA are able to bind the hydrophobic dye 1‐anilinonaphtalene‐8‐sulfonate, thus indicating the presence of exposed hydrophobic patches. It is concluded that nicked Th1‐LA and gapped desβ‐LA are more relaxed and expanded than intact LA and, consequently, that they are more suitable protein species to allow the large conformational transitions required for the polypeptide chain to form the amyloid cross‐β structure. As a matter of fact, the MG of LA attains an even more flexible conformational state during the early phases of the aggregation process at acidic pH, as deduced from the enhancement of its susceptibility to proteolysis by pepsin. Our data indicate that deletion of the β‐subdomain in LA does not alter the ability of the protein to assemble into well‐ordered fibrils, implying that this chain region is not essential for the amyloid formation. It is proposed that a proteolytic hydrolysis of a protein molecule at the cellular level can trigger an easier formation of amyloid precipitates and therefore that limited proteolysis of proteins can be a causative mechanism of protein aggregation and fibrillogenesis. Indeed, a vast majority of protein deposits in amyloid diseases are given by protein fragments derived from larger protein precursors.

Molecular Insights into the Interaction between α-Synuclein and Docosahexaenoic Acid

alpha-Synuclein (alpha-syn) is a 140-residue protein of unknown function, involved in several neurodegenerative disorders, such as Parkinsons disease. Recently, the possible interaction between alpha-syn and polyunsaturated fatty acids has attracted a strong interest. Indeed, lipids are able to trigger the multimerization of the protein in vitro and in cultured cells. Docosahexaenoic acid (DHA) is one of the main fatty acids (FAs) in cerebral gray matter and is dynamically released following phospholipid hydrolysis. Moreover, it has been found in high levels in brain areas containing alpha-syn inclusions in patients affected by Parkinsons disease. Debated and unsolved questions regard the nature of the molecular interaction between alpha-syn and DHA and the effect exerted by the protein on the aggregated state of the FA. Here, we show that alpha-syn is able to strongly interact with DHA and that a mutual effect on the structure of the protein and on the physical state of the lipid derives from this interaction. alpha-Syn acquires an alpha-helical conformation in a simple two-state transition. The binding of the protein to the FA leads to a reduction of the size of the spontaneously formed aggregated species of DHA as well as of the critical aggregate concentration of the lipid. Specifically, biophysical methods and electron microscopy observations indicated that the FA forms oil droplets in the presence of alpha-syn. Limited proteolysis experiments showed that, when the protein is bound to the FA oil droplets, it is initially cleaved in the 89-102 region, suggesting that this chain segment is sufficiently flexible or unfolded to be protease-sensitive. Subsequent proteolytic events produce fragments corresponding to the first 70-80 residues that remain structured and show high affinity for the lipid. The fact that a region of the polypeptide chain remains accessible to proteases, when interacting with the lipid, suggests that this region could be involved in other interactions, justifying the ambivalent propensity of alpha-syn towards folding or aggregation in the presence of FAs.

Covalent α-synuclein dimers: chemico-physical and aggregation properties.

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinsons disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.

Trifluoroethanol-assisted protein folding: fragment 53–103 of bovine α-lactalbumin

Fragment 53--103 of bovine alpha-lactalbumin, prepared by limited peptic digestion of the protein at low pH, is a 51-residue polypeptide chain crosslinked by two disulfide bonds encompassing helix C (residues 86--98) of the native protein. Refolding of the fully reduced fragment (four--SH groups) is expected to lead to three fully oxidized isomers, the native (61--77, 73--91) and the two misfolded species named ribbon (61--91, 73--77) and beads (61--73, 77--91) isomers. The fragment with correct disulfide bonds was formed in approx. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized glutathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was almost quantitative. To provide an explanation of the beneficial effect of TFE in promoting the correct oxidative folding, the conformational features of the various fragment species were analyzed by far-UV circular dichroism measurements. The fully reduced fragment is largely unfolded in water, but it becomes helical in aqueous TFE. Correctly refolded fragment is produced most when the helical contents of the reduced and oxidized fragment in aqueous TFE are roughly equal. It is proposed that 30% TFE promotes a native-like format of the fragment and thus an efficient and correct pairing of disulfides. Higher concentrations of TFE, instead, promote some non-native helical secondary structure in the fragment species, thus hampering correct folding.