Báo cáo khoa học: Protein dissection enhances the amyloidogenic properties of a-lactalbumin potx

Protein dissection enhances the amyloidogenic properties of a-lactalbumin Patrizia Polverino de Laureto 1 , Erica Frare 1 , Francesca Battaglia 1 , Maria F. Mossuto 1 , Vladimir N. Uversky 2,3,4 and Angelo Fontana 1 1 CRIBI Biotechnology Centre, University of Padua, Italy 2 Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Russia 3 Department of Biochemistry and Molecular Biology, Medical School, Indiana University, Indianapolis, IN, USA 4 Molecular Kinetics Inc., Indianapolis, IN, USA Keywords amyloid; a-lactalbumin; circular dichroism; infrared spectroscopy; limited proteolysis; molten globule; protein aggregation Correspondence A. Fontana, CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy Fax: +39 49 827 6159 Tel: +39 49 827 6156 E-mail: angelo.fontana@unipd.it (Received 21 December 2004, revised 23 February 2005, accepted 2 March 2005) doi:10.1111/j.1742-4658.2005.04638.x a-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 resi- dues located at the level of the b-subdomain of the native protein (1– 40 ⁄ 53–123, named desb-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. Th1-LA and desb-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 desb-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 desb-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 desb-LA are more relaxed and expanded than intact LA and, consequently, that they are more suitable protein spe- cies to allow the large conformational transitions required for the poly- peptide chain to form the amyloid cross-b structure. As a matter of fact, the MG of LA attains an even more flexible conformational state dur- ing 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 b-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 lim- ited 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. Abbreviations [h], mean residue ellipticity; ANS, 1-anilinonaphtalene-8-sulfonate; apo-LA, calcium-depleted form of LA; desb-LA, protein species with excision of the 41–52 chain segment; E ⁄ S, enzyme to substrate ratio; LA, a-lactalbumin; MG, molten globule; RP, reverse-phase; TFA, trifluoroacetic acid; Th1-LA, disulfide-crosslinked, nicked protein species of LA with the peptide bond 40–41 cleaved; ThT, thioflavin T. 2176 FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS The propensity to form amyloid fibrils appears to be a general property of polypeptide chains, as under speci- fic experimental conditions a variety of proteins can be induced to aggregate into highly ordered protein aggre- gates [1–10]. Considering that all protein fibrils, inde- pendently of the native structure of the given amyloidogenic protein, adopt the common cross-b structure motif, a large conformational rearrangement has to occur prior to fibrillation [10]. Such changes cannot develop within a tightly packed native protein and thus destabilization of the protein structure and formation of a partly folded state is required. In fact, experimental conditions that favor the partial unfold- ing of the protein are known to trigger the fibrillation process [5–7,10]. It has been proposed that specific interactions between amino acid residues or chain regions of a polypeptide with a high propensity to acquire a b-sheet secondary structure lead to the kernel that determines aggregation of other molecules into the final, well-ordered fibrils [1–3]. However, as even small unfolded peptides are able to form amyloid fibrils, protein conformational parameters are likely to be insufficient to explain the molecular mechanism(s) of fibrillation [11,12]. In a number of cases, a proteo- lytic event of a precursor protein appears to be required for amyloidogenesis [13–16]. Lysozyme and a-lactalbumin (LA) belong to the same protein superfamily, being homologous proteins displaying similarity in their amino acid sequence and overall 3D structure ([17] and references cited therein). Human lysozyme has been shown to form amyloid fibrils in individuals having point mutations in the lyso- zyme gene [18]. Recently, in order to understand better the mechanism of lysozyme fibrillation, the aggregation processes of lysozymes from different sources have been investigated in detail (see [19] for references]. Overall, it seems appropriate to study the aggregation phenomena of proteins belonging to the same protein superfamily. Probably, by studying similar proteins that form fibrils in vitro it will be possible to highlight some peculiar structural features that dictate the overall process of protein fibrillogenesis. Indeed, bovine LA is also able to form amyloid fibrils if the protein is induced to adopt the molten globule (MG) state at low pH [20]. However, fibril formation at pH 2.0 is much more rapid if the protein, upon partial reduction oif its four disulfide bridges, adopts a more open conformation than that of the classical MG in acid solution [21,22]. This finding correlates with the observation that the conformational features of apomyoglobin leading to amyloid precipitates are more expanded than those of a well defined and compact partly folded state of the pro- tein [23]. Similarly, the aggregation process of a SH3 domain at low pH implies significant structural rear- rangements, requiring a more flexible protein species that subsequently forms well-ordered fibrillar structures [24]. Also a partly folded state has been shown to be involved in the fibrillogenesis of a-synuclein, a ‘natively unfolded’ protein [25–27] known to be involved in the pathogenesis of several neurodegenerative disorders [28]. It has been found that a-synuclein undergoes fibrillation under experimental conditions that stabilize a partly folded state, named premolten globule [29]. These various observations can be interpreted as indi- cating that partly folded, but substantially open and dynamic states of proteins are those required for trig- gering the process of fibrillogenesis [10,20]. In this work, we analyse the effects of the dissection of the LA molecule on its conformational features and aggregation processes. Two protein species are herewith examinated, those given by the N-terminal fragment 1–40 covalently linked by the disulfide bridges of the protein to the C-terminal fragment 41–123 (Th1-LA) or fragment 53–123 (desb-LA) (Fig. 1). These species have been prepared by limited proteolysis of LA by thermo- lysin (E.C. 3.4.24.27) in 50% trifluoroethanol at neutral pH or by pepsin (E.C. 3.4.23.1) in acid solution [30–33]. LA is not associated to any specific disease, but several observations have suggested that LA can evoke a variety of physiological effects [34]. Besides its role in modulating the activity of the lactose synthase, new intriguing properties have been evidenced, such as the apoptotic activity in tumor cells of a partly folded variant of LA bound to oleic acid [35,36], the ability of the protein to bind histones [37,38] and the bactericidal activity of some of its chymotryptic peptides [39]. Here, we show that nicking of the 123-residue chain of LA at a single peptide bond (Th1-LA) or removing a 12-resi- due segment (desb-LA) leads to protein species that easily form amyloid fibrils at pH 2.0. It is concluded that the inherent flexibility of these LA derivatives allows the large conformational changes required to form the cross-b-structure of the amyloid fibrils. Of interest, it is also shown that the initial stages of fibril- lation of intact LA at low pH involve protein inter- mediate(s) characterized by enhanced chain flexibility. This study emphasizes that the precursor structures of amyloid fibrils require a more unfolded and ⁄ or flexible state than that of the MG [21,22]. Results Molecular features of Th1-LA and desb-LA Far-UV CD measurements indicate that, in acidic solution at pH 2.0 or at neutral pH in the presence of P. Polverino de Laureto et al. Proteolysis of proteins enhances fibrillogenesis FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS 2177 EDTA, the LA variants Th1-LA and desb-LA adopt a conformation similar to the MG state displayed by intact LA at low pH, retaining a native-like a-helical content (Fig. 2A,B). Furthermore, Th1-LA and desb- LA bind the fluorescent dye 1-anilinonaphtalene-8-sulf- onic acid (ANS), which is considered diagnostic of a protein MG state [40]. While at pH 2.0 the three LA species bind ANS in a similar way, at pH 7.4 the nicked or gapped LA species bind ANS more signifi- cantly than the intact protein, indicating that a larger hydrophobic surface is exposed to the solvent in the LA derivatives (Fig. 2C,D) (see also [32]). The molecular features of LA, Th1-LA and desb-LA at pH 2.0 have been further analysed by limited proteo- lysis, urea-induced unfolding and hydrogen ⁄ deuterium (H ⁄ D) exchange measurements. Limited proteolysis has been conducted at pH 2.0 using pepsin as protease and LA and Th1-LA as substrates [41]. Desb-LA was not analysed, because this species is a product of proteo- lysis of LA at pH 2.0 [30–32]. The comparative suscep- tibility to proteolytic attack by pepsin of LA and Th1-LA is shown in Fig. 3A. The extent of proteolysis was calculated from the amount of residual protein determined by reverse phase (RP)-HPLC analysis of aliquots of the reaction mixtures at various times of proteolysis. After 10 min’ incubation of the proteins with pepsin at pH 2.0, Th1-LA has been  80% cleaved, while LA only  50%. After 20 min’ reaction, the proteolysis is complete for Th1-LA, whereas some intact LA is still present in the proteolysis mixture even after 50 min’ reaction (Fig. 3A). To evaluate further the molecular differences between LA and its derivatives, the effect of urea on the protein tertiary structure was analysed by near-UV CD spectros- copy (Fig. 3B). Earlier it was shown [32] that these LA derivatives retain some tertiary interactions at pH 2.0, as given by a near-UV CD spectrum characterized by a band centered at about 290 nm due to the contribution of tryptophan residues [42]. As shown in Fig. 3B, the increase in urea concentration at pH 2.0 was accompan- ied by a gradual reduction of the CD signal at 291 nm for the three LA species, implying that the urea-medi- ated denaturation is not a cooperative process. Even if the overall processes are quite similar for the three pro- tein species, it seems that intact LA retains some residual structure in the presence of 4 m urea, whereas desb-LA appears to be completely unfolded under the same conditions. In the case of Th1-LA, a complete disappear- ance of the CD signal at 291 nm occurs in 6 m urea. The flexibility of the polypeptide chain of LA and its gapped and nicked species was also analysed by H ⁄ D measurements, i.e. monitoring by ESI-MS the change in the protein mass that results from the replacement of labile protons by deuterium. The fea- tures of the H ⁄ D exchange of the low-pH MG of LA have been already examined [43–46], showing that only the amides in the a-domain are most protected from H ⁄ D exchange [43]. The protein species herewith inves- tigated are characterized by a nicking or a removal of the b-subdomain (Fig. 1) and therefore a significant N C Ala40-Ile41 Leu52-Phe53 1 123 H1 5-11 h1b 23-34 S1 S2 S3 H2 h2 86-98 105-110 h3c H3 H4 Gln-Ala-Ile-Val-Gln-Asn-Asn-Asp-Ser-Thr-Glu-Tyr-Gly-Leu-Phe-Gln 40 45 50 1 40 53 123 1 40 123 41 Th1-LA desβ-LA Fig. 1. (Top) Schematic representation of the 3D structure of LA. The diagram was drawn using the PDB file 1HFZ using the program WEBLAB VIEWER PRO 4.0 (Molecular Simulations Inc., San Diego, CA). The chain segment 41–52 encompassing the antiparallel b-sheet is colored in red. The four disulfide bonds are represented by yellow sticks and the calcium atom by a solid sphere in green. (Middle) Scheme of the secondary structure of the 123-residue chain of LA [58]. The four a-helices (H1–H4) along the protein chain are indica- ted by major boxes and below them the corresponding chain seg- ments are given. The three b-strands (S1, 41–44; S2, 47–50; S3, 55–56) and the 3 10 helices (h1b, 18–20; h2, 77–80, h3c, 115–118) are indicated by small boxes. The amino acid sequence of the chain region 39–54 of LA is explicitly shown and the sites of proteolysis by pepsin and thermolysin are indicated by arrows. (Bottom) Sche- matic structure of the LA derivatives Th1-LA and desb-LA. These protein species are given by the N-terminal fragment 1–40 cova- lently linked by the four disulfide bridges of the protein to the C-ter- minal fragment 41–123 (Th1-LA) or fragment 53–123 (desb-LA). The connectivities of the four disulfide bridges (thin lines) of LA are 6–120, 28–111, 61–77 and 73–91. Proteolysis of proteins enhances fibrillogenesis P. Polverino de Laureto et al. 2178 FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS difference in their H ⁄ D behavior is not expected. In fact, Fig. 3C shows that the time-courses of H ⁄ D exchange for the three LA species are only slightly dif- ferent. However, the lower and the higher extent of exchanged protons by LA and desb-LA, respectively, are consistent with a more compact and a more flex- ible structure in LA and desb-LA, respectively. Aggregation properties of LA derivatives To induce amyloid formation, Th1-LA and desb-LA were dissolved (5 mgÆmL )1 )in10mm HCl pH 2.0, or in 50 mm Tris ⁄ HCl buffer pH 7.4, containing 0.1 m NaCl, and incubated at 35 °C for up to 20 days. The aggregation process was followed by thioflavin T (ThT) binding assay [47,48] and electron microscopy (EM). CD and FTIR measurements have been used to mon- itor the conformational changes during aggregation. The nicked or gapped LA derivatives aggregate very fast (Fig. 4) and form long and well-defined fibrils (Fig. 5). The sigmoid curve of the ThT fluorescence emission at 485 nm vs. time of aggregation (Fig. 4) observed with intact LA is characterized by a lag time and is consistent with a nucleation-dependent elonga- tion model of fibrillogenesis [20]. On the other hand, the lag time is almost not observed with both Th1-LA and desb-LA incubated at pH 2.0 (Fig. 4). Further- more, with the LA derivatives a larger increase in ThT fluorescence is observed upon prolonged incubation at low pH and the intensity of fluorescence reaches a plat- eau after about 70–80 h. Assuming that the ThT fluor- escence enhancement is proportional to the population of well-ordered protein aggregates [47,48], we can con- clude that the amount of fibrils formed by Th1-LA and desb-LA is decidedly larger than that formed by intact LA under similar experimental conditions. EM reveals that the fibrils formed by Th1-LA and desb-LA after 70 h incubation at pH 2.0 are typical amyloid, with a filamentous aspect, unbranched and with a diameter of  10 nm (Fig. 5), quite similar to those formed by intact LA under the same experimental conditions [20]. Aggregation experiments of the LA derivatives have been conducted also at pH 7.4. While intact LA does not form fibrils at neutral pH [20], both Th1-LA and desb-LA after  230 h of incubation do form ordered aggregates. In fact, EM reveals the presence of spher- ical aggregates with dimensions of 4–8 nm (Fig. 5, right panels) and some of them show the typical mor- phology of protofibrils [49]. In the case of Th1-LA, the initial aggregates are rare, if compared to those 250240230220210200190 [ θ 01 x ] 3- ged( . mc 2 . lomd 1- ) -15 -10 -5 0 5 pH 2.0 pH 7.4 fibrils Wavelength (nm) 250240230220210200190 -15 -10 -5 0 5 pH 2.0 pH 7.4 fibrils 400 450 500 550 600 650 ecnecseroulF e vitaleR 0 200 400 600 800 1000 LA Th1-LA desβ-LA pH 2.0 400 450 500 550 600 650 0 10 20 30 40 50 LA Th1-LA desβ-LA pH 7.4 Th1-LA des β -LA AB CD Fig. 2. Conformational characterization of Th1-LA and desb-LA by CD (A, B) and ANS binding (C, D). Far-UV CD spectra of Th1-LA and desb-LA were recorded in 10 m M Tris ⁄ HCl ⁄ 0.1 M NaCl buffer pH 7.4, contain- ing 1 m M EDTA or in 0.01 M HCl ⁄ 0.1 M NaCl pH 2.0, at a protein concentration of 0.05– 0.1 mgÆmL )1 . The far-UV CD spectra of fibrils of Th1-LA and desb-LA refer to those of samples obtained by aggregation of the protein at 35 °C pH 2.0, for 48 h and 65 h, respectively. Fluorescence emission spectra (C, D) of 20 l M ANS in the presence of 10 l M of Th1-LA or desb-LA. The spectra are recorded at 20–22 °C from 390 to 650 nm after excitation at 370 nm in 10 m M HCl pH 2.0, or in 10 mM Tris ⁄ HCl pH 7.4. P. Polverino de Laureto et al. Proteolysis of proteins enhances fibrillogenesis FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS 2179 observed with desb-LA. Moreover, after a prolonged time of incubation, fibrils with a more defined mor- phology develop in the case of desb-LA, but more slowly in the case of Th1-LA. Analysis of the protein secondary structure has been carried out by far-UV CD measurements [50] on aliqu- ots taken at intervals (40–70 h) from a solution of Th1-LA and desb-LA incubated at 35 °C pH 2.0. An extensive conformational rearrangement takes place during the aggregation process, as given by the disap- pearance of the a-helix bands at 208 and at 220 nm and by the development of the band near 217 nm, typ- ical of b-secondary structure (Fig. 2A,B). On the other hand, a sample of intact LA incubated under similar solvent conditions does not develop significant changes of CD spectra (data not shown), even after prolonged time of incubation and thus even if aggregated (Fig. 4). This could be explained by considering that the helical secondary structure, even if present in mod- erate percentages in respect to other types of secondary structures (random coil, b-sheet), shows a CD signal that prevails on the others [50]. To verify if under the acid conditions used to induce fibril formation the protein samples are chemically modified or hydrolyzed, the fibrillar material produced after 10 days of incubation has been analysed by MS, following essentially procedures previously described Time (min) 0 30 60 90 120 150 xe D/Hch )%( egna 0 20 40 60 80 100 Urea concentration (M) 012345678 ]θ[ [/ ]θ 0 mn192 0.0 0.2 0.4 0.6 0.8 1.0 Time (min) 0 102030405060 idnUg ietorP detse)%( n 0 20 40 60 80 100 A B C Fig. 3. Molecular features at pH 2.0 of LA (d), Th1-LA (s) and desb-LA (.) probed by limited proteolysis (A), urea-induced unfold- ing (B) and H ⁄ D exchange rates (C). (A) Susceptibility to proteolysis by pepsin at pH 2.0 of LA and Th1-LA. The percent of undigested protein was calculated from the area of the protein peaks in the RP-HPLC chromatograms of protein samples analysed at different time intervals. (B) Urea-induced unfolding of the tertiary structure of LA, Th1-LA and desb-LA monitored by CD measurements at 291 nm at 20–22 °C pH 2.0. (C) H ⁄ D exchange profiles of LA, Th1-LA and desb-LA at pH 2.0 as monitored by MS. Time of Incubation (h) 0 50 100 150 200 250 300 aleRT evithTF loure ecsn 4 ta ec85n m 0 20 40 60 80 100 desβ-LA Th1-LA LA Fig. 4. Time course analysis of aggregation at 35 °C pH 2.0, of LA, Th1-LA and desb-LA monitored by thioflavin T (ThT) binding. Aliqu- ots (7 lL) were taken from the protein solution after the indicated time and added to a 25 l M solution (500 lL) of ThT in 25 mM phos- phate buffer pH 6.0. The excitation wavelength was at 440 nm and the emission was measured at 485 nm. The protein concentration of each protein sample was 0.4 m M. Proteolysis of proteins enhances fibrillogenesis P. Polverino de Laureto et al. 2180 FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS [19]. In the case of LA, some protein degradation ( 10%) has been actually observed (data not shown). Instead, the desb-LA and Th1-LA fibrils are composed by intact protein molecules. This could be due to the more rapid aggregation process of the LA derivatives in respect to that of the intact protein, likely rendering the aggregated desb-LA and Th1-LA somewhat pro- tected from degradation. Analysis of fibrillogenesis of intact, nicked and gapped LA by FTIR To evaluate the conformational rearrangements of LA and its derivatives during the aggregation process, FTIR measurements [51–53] were conducted on mono- meric and aggregated proteins. Aggregation was moni- tored by analysing the second derivative of the FTIR spectra of protein samples incubated for varying length of time at 35 °C pH 2.0. The second derivative of the FTIR spectrum of LA at pH 2.0 before aggregation (Fig. 6) shows three major bands, one centered at 1648 cm )1 characteristic of a-helix and ⁄ or random structure and two centered at 1632 and 1675 cm )1 related to the antiparallel b-structure [51–53]. These spectral characteristics are in agreement with those reported for intact LA exposed to low pH [20]. After 6–8 h incubation, the main band at 1648 cm )1 shifts to a lower wavenumber (1643 cm )1 ), indicating a preval- ence of random coil and therefore a further structural unfolding of the protein. After 48 h, the bands due to the b-sheet structure (1632 and 1675 cm )1 ) are evident and, upon prolonged incubation (300 h), a band cen- tered at 1616 cm )1 develops, together with a shift of the band at 1675 cm )1 to higher wavelengths. These last bands have been related to fibril formation, as the association of b-sheets cause a splitting of the antipar- allel b-sheet bands [20]. Figure 6 (middle panel) shows the evolution of the FTIR spectrum of Th1-LA during aggregation at pH 2.0. Initially, the spectrum of Th1-LA is quite similar to that of intact LA under the same solvent conditions. A structural rearrangment of Th1-LA appears to take place before initiation of the aggrega- tion process (Fig. 6, 1h). In fact, the FTIR spectrum recorded after 1 h shows a main band at 1645 cm )1 , which is indicative of an enhanced content of dis- ordered structure. The spectrum remains unmodified up to 8–10 h of incubation. After 24 h of Th1-LA incubation, the band at 1643 cm )1 is no more present and a new band at 1616 cm )1 (aggregation band) appears. Upon prolonged incubation, there is a preval- ence of the bands characteristic of protein fibrils (1616 200 nm 200 nm 200 nm 200 nm 200 nm 200 nm des -LA, pH 7.4, 235 h Th1-LA, pH 7.4, 235 h desbb b-LA, pH 2.0, 70 h Th1-LA, pH 2.0, 70 h des -LA, pH 7.4, 720 h Th1-LA, pH 7.4, 800 h 200 nm 200 nm 200 nm 200 nm 200 nm 200 nm Fig. 5. Electron micrographs of Th1-LA and desb-LA fibrils obtained after 70 h incubation of the LA variants at 35 °C pH 2.0. The morphology of the initial aggregates produced after 235 h of incubation of the protein samples at 35 °C pH 7.4, and of those produced after a much pro- longed incubation time are also reported. P. Polverino de Laureto et al. Proteolysis of proteins enhances fibrillogenesis FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS 2181 and 1680 cm )1 ), whereas those of the regular structure reduce their intensity. Overall, the FTIR data indicate that the aggregation processes of LA and Th1-LA are similar, but that Th1-LA aggregates significantly fas- ter. The aggregation process of desb-LA, followed by FTIR analysis, shows a similar behavior to that dis- played by Th1-LA both in terms of rate of appearance and type of FTIR bands (Fig. 6, right panel). Early stages of LA aggregation followed by limited proteolysis In previous studies we have shown that proteolytic probes can be used to analyse structure and dynamics of proteins in their partly folded or MG states [41] and during their aggregation process [24]. Here, we have applied this technique for analysing the early sta- ges in the aggregation process of intact LA. To this end, aliquots of the protein sample of LA incubated at 35 °C pH 2.0, were withdrawn from the reaction mix- ture at various times (from 0 to 200 h) and treated with pepsin in order to test the susceptibility to proteo- lysis of LA during the aggregation process. Fig. 7 shows the reverse phase–HPLC analyses of the proteo- lysis reactions conducted on LA after 0, 8, 24 and 72 h incubation. The pattern of LA proteolysis with pepsin (panel A) is similar to that reported previously [30–32], with the major products being the fragment species 1–40 ⁄ 53–123 (desb-LA), 1–40 ⁄ 104–123 and 53–103. The protein sample left to aggregate for 8 h at pH 2.0 appears to be more sensitive to the protease action, because more fragments and less intact protein are found in the mixture (Fig. 7B). The analysis of the proteolysis of a sample of LA incubated for a pro- longed time (panels C and D) reveals that the protein becomes more and more resistant to proteolysis. In Fig. 7 (bottom), the percent hydrolysis of LA by pep- sin as a function of aggregation time is reported. It is seen that LA at the initial stages of the aggregation process is more easily digested by proteolysis and thus is more unfolded and ⁄ or flexible, a feature that appears to render the polypeptide chain more prone to fibril formation [24]. Discussion Here, we have studied the aggregation processes of LA species with a nick (Th1-LA) or a chain deletion (desb-LA) at the level of the b-subdomain of the 123- residue chain of the protein (Fig. 1). We have also characterized the early stages of protein aggregation in order to get insights into the molecular mechanisms of fibrillogenesis. At pH 2.0, the overall features of the partly folded or MG states formed by Th1-LA and desb-LA appear to be quite similar to that formed by intact LA at low pH under equilibrium conditions, as judged from CD measurements (see also [32]). How- ever, there are some differences in stability and ⁄ or Absorbance h 0 h 6 h 8 4 Wa mc( rebmun e v 1 - ) 00610261 0 461066108610071 h 003 57 6 1 8 4 61 2361 61 6 1 3 4 61 08 61 5 7 6 1 5 7 61 8 4 61 236 1 Absorbance h 0 h 1 h 42 a W e vnu bm er ( cm 1- ) 006102610461066108610071 h 69 2361 9 4 6 1 57 6 1 54 6 1 94 6 1 2361 6161 0861 5 7 61 57 61 sed β -AL Absorbance h 0 h1 h 42 Wa v emunbre( cm 1- ) 0 0 610261 0 461 0 661 0 86 1 00 7 1 h 69 5 761 8461 236 1 6 161 5461 0861 5 7 6 1 5 7 61 8461 2361 Th1-LA tc at nI Fig. 6. Aggregation process of LA, Th1-LA and desb-LA monitored by FTIR. The panels show the time-evolution of the FTIR spectra of the amide I region (continuous line) of LA (left panel), Th1-LA (middle panel) and desb-LA (right panel) during fibril formation at 35 °C pH 2.0. The second derivative spectra (dashed lines) are also shown. Proteolysis of proteins enhances fibrillogenesis P. Polverino de Laureto et al. 2182 FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS flexibility between the three protein species, as inferred from proteolysis experiments, urea-mediated denatura- tion and H ⁄ D exchange measurements (Fig. 3). The major conclusion of this study is that the conforma- tional features of Th1-LA and desb-LA, which are more relaxed and expanded in comparison to intact LA at low pH, are more suitable for triggering the protein fibrillation process. Importantly, the deletion of the b-subdomain in LA does not alter the ability of the protein to assemble into well-ordered fibrils. In this context, it is interesting to recall that in previous studies it has been proposed that the b-domain is of particular significance in triggering the fibril formation in the case of lysozyme ([54] and references cited therein), a protein belonging to the same structural superfamily of LA. Instead, here it is shown that a LA derivative such as desb-LA, lacking the three b-strands of the protein (Fig. 1), is able to aggregate even more readily than the intact protein, implying that the b-sheet region of LA is not required for fibrillogenesis. It may be proposed that the exposure of the hydro- phobic interior of the protein at low pH is sufficient for promoting the aggregation phenomenon, leading ultimately to well-ordered fibrils. An important aspect of this study is the analysis of the molecular features of LA and its derivatives at the initial stages of protein aggregation at pH 2.0. During the lag time, intact LA undergoes significant conform- ational changes, as evidenced from FTIR (Fig. 6) and limited proteolysis experiments (Fig. 7), leading to an increase in the amount of disordered structure in LA at the early stages of fibrillation (after 6–10 h incuba- tion). In fact, less intact LA remains in the proteolysis mixture with pepsin (Fig. 7), indicating that the pro- teolytic degradation increases after incubation of LA at pH 2.0 for 6–10 h. We may infer that LA, at the initial stages of aggregation, exists in a more expanded and flexible conformational state and, for this reason, is more sensitive to proteolytic attack [41]. Import- antly, also Th1-LA and desb-LA appear to initially reach a more unfolded ⁄ flexible state (from FTIR data, see Fig. 6), but faster and in about 1 h only. This is likely due to the fact that Th1-LA and desb-LA are more open and flexible protein species than intact LA. By analogy, similar arguments can be used also to explain why, at variance from the LA derivatives, the intact protein does not aggregate at neutral pH [20]. Clearly, the intact protein is native and rigid at neutral pH and does not populate the partly folded state required for fibrillation. An interesting question here is the apparent discrep- ancy between FTIR data and far-UV CD measure- ments. FTIR data provide evidence of an initial phase in which the three proteins herewith studied develop an increase in the disordered structure and, at longer incubation times, the band characteristic of the b-sheet structure of the amyloid is clearly observed (Fig. 6). The FTIR data are in agreement with the fact that aggregated Th1-LA and desb-LA show a far-UV CD spectrum which is typical of the b-sheet secondary structure (see Fig. 2A,B). On the other hand, far-UV leRative sbA ocnabre mn 622 ta Retention Time (min) 0 5 10 15 20 25 30 0 h 8 h 24 h 41-52 1-40/104-123 LA 1-40/53-123 1-40/53-123 LA 1-40/104-123 41-52 1-40/53-123 LA 1-40/104-123 41-52 53-103 53-103 0 5 10 15 20 25 30 72 h LA Time of Incubation (h) 0 1020304050607080 Per egavaelC tneC 0 20 40 60 80 100 AB CD Fig. 7. Early stages of aggregation of intact LA at pH 2.0 as monit- ored by limited proteolysis. LA was allowed to aggregate at 35 °C pH 2.0. Aliquots (50 lL) were taken at intervals from the LA solu- tion and mixed with a pepsin solution for 45 min at 20–22 °C. (Top) A sample of the proteolysis mixture was analysed by RP-HPLC using a Vydac C18 column (150 · 4.6 mm), eluted with a linear gra- dient of acetonitrile ⁄ 0.1% TFA from 5% to 34% in 4 min and from 34% to 50% in 18 min (Bottom) Time-course of the susceptibility of LA to proteolysis by pepsin during aggregation at 35 °C pH 2.0. The extent of cleavage was calculated by integration of the area of the peaks in the RP-HPLC chromatograms of protein samples ana- lysed at different time intervals. P. Polverino de Laureto et al. Proteolysis of proteins enhances fibrillogenesis FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS 2183 CD spectra measured for intact LA during the entire aggregation process show minor changes in their shape and characteristics mostly of a-helical structure (data not shown). This can be explained by considering that it is likely that residual a-helical structure is still pre- sent in the aggregated mixture of intact LA and that the intensity of the CD signal of the helical structure is much higher than that of other secondary structures, including b-sheet [50]. The key result of this study is that limited proteo- lysis of LA leads to protein species that are much more prone to fibrillogenesis than the intact protein. Therefore, it is shown here, with the model protein LA, that amyloid fibril formation can require proteoly- sis. This is in line with the fact that a large proportion of protein deposits associated with amyloid diseases is made by protein fragments derived from proteolysis of larger protein precursors [2,11,12]. First of all, we may mention here that the prototypic fibril-forming frag- ment Ab in Alzheimer’s diseases is derived from the amyloid precursor protein by a combination of pro- teolytic cleavages given by b- and c-secretase [55]. Caspase-cleavage of the cytoskeletal tau protein is an early event in Alzheimer’s diseases tangle pathology [16]. Human gelsolin is expressed as an 81-kDa intra- cellular protein and its limited proteolysis at the level of peptide bonds 172–173 and 243–244 leads to a 71-residue fragment that is found in protein deposits in individuals with familial amyloidosis [13]. The 34-residue ABri peptide is derived from a putative transmembrane precursor and is found in plaques of familial British dementia [56]. These and other obser- vations [2,11,12], in line with the results of this study, provide an evidence that proteolysis can be a critical prefibrillogenic event. Protein fragments, originating by proteolysis of pro- tein precursors, are particularly vulnerable to aggrega- tion, because they can usually adopt, at most, partly folded states and cannot establish the long-range inter- actions that stabilize the native intact protein. In par- ticular, protein fragments may contain hydrophobic clusters of residues that can trigger protein aggrega- tion. In the case of the LA derivatives Th1-LA and desb-LA, it may be well that the nicking of the 123- residue chain polypeptide chain causes an untighting of the structural domains of LA (see Fig. 1), determin- ing exposure of hydrophobic patches. Furthermore, the fact that the LA derivatives aggregate much more easily at low pH can be explained by considering that a minimization in acid solution of the negative charges of the carboxylates of the Asp and Glu residues in the calcium-binding region of LA can produce a marked decrease in the charge-to-charge repulsions and a concomitant enhancement of the protein association process by favoring intermolecular hydrophobic inter- actions. In recent years, numerous studies have been conduc- ted on the low pH MG state of LA, nowadays consid- ered a prototype MG in protein folding studies [21,22]. A consensus view is that the MG of LA retains signifi- cant native-like structure in the a-domain, while the b-domain (approximately region 50–90) is disordered. From this and previous [32] study, it seems that the MG state at pH 2.0 retains substantial native-like structure that does not allow the protein to form the amyloid precipitate. On the other hand, the low pH MGs adopted by Th1-LA and desb-LA are similar to the MG of intact LA, but they are more open and flexible and thus more prone to aggregation. There- fore, a limited proteolysis phenomenon of a protein can lead to an enhanced population of a rather unfol- ded and ⁄ or flexible protein species that triggers the fibrillation process. Conclusions Substantial data are available to support a model for the amyloid formation in which intermolecular inter- actions between hydrophobic patches in partly folded or MG states of proteins are responsible for protein aggregation [6–10]. The intermediates are more prone to aggregate than the unfolded state because they retain clusters of hydrophobic side chains, which have a strong propensity for aggregation [6]. However, a conformation that is more expanded than typical MG appears to be required before protein assembly into an ordered amyloid-like structure [10,20]. Our observa- tions are in agreement with a general view that a more flexible conformation or a moderately unfolded struc- ture could be the key species triggering fibril forma- tion. In particular, the results of this study highlight the possible role of limited proteolysis as a causative event of fibrillogenesis. It seems possible to propose that a proteolytic attack of a protein at the cellular level can shift the equilibrium between the different protein conformational states towards a species that is more prone to aggregate. Experimental procedures Materials Bovine a-lactalbumin, porcine pepsin, thermolysin and ThT were from Sigma (St. Louis, MO, USA). All other chemi- cals were of analytical reagent grade and were obtained from Sigma or Fluka (Basel, Switzerland). Proteolysis of proteins enhances fibrillogenesis P. Polverino de Laureto et al. 2184 FEBS Journal 272 (2005) 2176–2188 ª 2005 FEBS Isolation of nicked and gapped LA The LA derivatives were obtained by limited proteolysis of the protein, as described previously [30–33]. Proteolysis of bovine LA with thermolysin was performed in a 1 : 1 (v ⁄ v) mixture of 50 mm Tris ⁄ HCl pH 7.0, containing 5 mm CaCl 2 and trifluoroethanol using an enzyme ⁄ substrate (E ⁄ S) ratio of 1 : 20 (w ⁄ w) [30,32]. The reaction was conducted at 20– 22 °C for 6 h and then proteolysis was quenched by acidifi- cation. The proteolysis of LA with pepsin was performed in 0.01 m HCl ⁄ 0.1 m NaCl pH 2.0, for 45 min at 20–22 °C and at an E ⁄ S ratio of 1 : 750 (w ⁄ w) [32]. All digestions of LA were carried out a protein concentration of  1mgÆmL )1 . Proteolysis was stopped by alkalinization of the solution with aqueous ammonia (pepsin) or by acidifi- cation with 4% (v ⁄ v) trifluoroacetic acid (TFA) in water (thermolysin). The LA derivatives (Th1-LA and desb-LA) were purified by micropreparative RP-HPLC on a Vydac C 18 column (150 · 4.6 mm, 5 lm; The Separations Group, Hesperia, CA), eluted at a flow rate 0.6 mLÆmin )1 with a gradient of acetonitrile containing 0.1% TFA and monitor- ing the effluent by absorbance measurements at 226 nm. The proteolysis experiments by pepsin conducted in parallel on LA and its derivative Th1-LA were performed under the same conditions as above described for LA. Fibril formation and characterization The amyloid fibrils of LA and its fragments were prepared by incubating protein samples (5 mgÆmL )1 )in10mm HCl pH 2.0, and in 10 mm Tris ⁄ HCl pH 7.4, at 35 ° C for up to 30 days. To confirm the presence of protein aggre- gates ⁄ fibrils, aliquots of the samples were examined by the ThT fluorescence assay [47,48] and by EM. The ThT bind- ing assay was performed using a freshly prepared 25 lm ThT solution in 25 mm sodium phosphate pH 6.0. Protein samples from suspensions containing aggregates were dilu- ted into the ThT buffer (final volume 500 lL). Fluorescence emission measurements were conducted at 25 °C, using an excitation wavelength of 440 nm and recording the ThT fluorescence emission at 485 nm. EM pictures were taken on a JEOL model JEM-1010 instrument operating at 80 kV. Samples were diluted 20-fold and a drop of the solution was placed on a Formvar-coated nickel grid (400-square mesh, Agar Scientific, Stansted, UK). A drop of uranyl acetate solution (2%, w ⁄ v) was placed on the grid and after a few seconds the grid was washed with deionized water (MilliQ, Millipore, Billerica, MA, USA). Spectroscopic measurements Protein concentrations were determined by absorption measurements at 280 nm on a double-beam Lamda-20 spec- trophotometer from Perkin Elmer (Norwalk, CT, USA). Extinction coefficients (e mgÆmL )1 ) at 280 nm for LA and its derivatives were evaluated on the basis of their amino acid composition [57] and were 2.01 for LA and Th1-LA (1–40 ⁄ 41–123) and 2.23 for desb-LA (1–40 ⁄ 53–123). CD spectra were recorded on a Jasco J-710 (Tokyo, Japan) spectropolarimeter equipped with a thermostated cell holder. Far-UV CD spectra were recorded using a 1 mm pathlength quartz cell and a protein concentration of 0.05– 0.1 mgÆmL )1 . The mean residue ellipticity [h] (degÆcm 2 Æ dmol )1 ) was calculated from the formula [h] ¼ (h obs ⁄ 10)Æ(MRW lc )1 ), where h obs is the observed ellipticity in deg, MRW is the mean residue molecular weight (molecular weight of the protein divided by the number of amino acids), l the optical pathlength in cm and c the pro- tein concentration in mgÆmL )1 . The urea-mediated unfold- ing at pH 2.0 of LA, Th1-LA and desb-LA was monitored by following the near-UV CD signal at 291 nm at 20– 22 °C. The measurements were made after equilibrating the protein samples for 10 min. A protein concentration of  25 lm and a cuvette of 0.5 cm pathlength were used. Fluorescence measurements were performed using a Perkin-Elmer model LS-50 spectrofluorimeter, utilizing a cuvette with 0.1-cm pathlength. An excitation wavelength of 370 nm was used for ANS binding experiments and the emission spectra scanned from 390 to 650 nm [40]. All spec- tra were recorded at 20–22 °C using a 20 lm solution of ANS and a 10 lm solution of protein. ANS-binding experi- ments were conducted with protein derivatives dissolved in 0.01 m HCl ⁄ 0.1 m NaCl pH 2.0, or in 10 mm TrisÆHCl ⁄ 0.1 m NaCl buffer pH 7.4. The concentration of the ANS stock solution was determined using a molar absorption coefficient of 5 · 10 3 m )1 Æcm )1 at 350 nm. FTIR spectra were recorded at 20–22 °C using a Perkin Elmer 1720X spectrometer, purged with a continuous flow of N 2 gas. Solutions of 0.35 mm LA in D 2 O were acidified to the desired pH using DCl. Protein solutions were placed between a pair of CaF 2 windows separated by a 50 lm Mylar spacer. For each protein sample, 50 interferograms were accumulated at a spectral resolution of 2 cm )1 . Buffer spectra were recorded under identical conditions to those of the protein samples and subtracted from the protein spec- tra. The second derivative of the amide I band was used to identify the different spectral components. H/D exchange measurements H ⁄ D exchange measurements [43–46] were conducted by recording the spectra on a Q-Tof Micro (Micromass, Manchester, UK) at a capillary voltage of 3 KV and a cone voltage of 40 V. To perform the H ⁄ D exchange, lyophilized samples of LA, Th1-LA and desb-LA were dissolved in 10 mm HCl pH 2.0, and diluted 35-fold in D 2 O at the same pH to give a final concentration of 10 lm. The deuterium content was deduced from the increase in molecular mass of P. Polverino de Laureto et al. 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LA and 25 °C for the other species, followed by lyophilization Then, the samples were incubated in D2O for 6 h at 65 °C for LA and 38 °C for both Th1-LA and desb-LA and then lyophilized Acknowledgements We thank Mr Vittorio Moretto for assistance in recording FTIR spectra and Dr Marco Crisma for help in the interpretation of the data The assistance of Mr Giuseppe Tognon in the use of transmission electron... removal of the b-subdomain in a-lactalbumin: The protein remains folded and can form the molten globule in acid solution Eur J Biochem 268, 4324–4333 33 Polverino de Laureto P, Frare E Gottardo R & Fontana A (2002) The molten globule of bovine a-lactalbumin at neutral pH induced by heat, trifluoroethanol and oleic acid: A comparative analysis by circular dichroism spectroscopy and limited proteolysis Proteins... 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(1996) Alternative conformations of amyloidogenic proteins govern their behaviour Curr Opin Struct Biol 6, 11–17 6 Fink AL (1998) Protein aggregation: Folding aggregates, inclusion bodies and amyloid Folding Des 3, R9–R23 7 Dobson CM (1999) Protein misfolding, evolution and disease Trends Biochem Sci 24, 329–332 8 Dobson CM (2003) Protein folding and disease: a view from the first Horizon Symposium Nat . deletion of a chain segment of 12 amino acid resi- dues located at the level of the b-subdomain of the native protein (1– 40 ⁄ 53–123, named desb-LA). We have also compared the early stages of the. at the level of the b-subdomain of the 123- residue chain of the protein (Fig. 1). We have also characterized the early stages of protein aggregation in order to get insights into the molecular. results from the replacement of labile protons by deuterium. The fea- tures of the H ⁄ D exchange of the low-pH MG of LA have been already examined [43–46], showing that only the amides in the a-domain