Monitoring the prevention of amyloid fibril formation by a-crystallin Temperature dependence and the nature of the aggregating species Agata Rekas 1,2 , Lucy Jankova 3 , David C. Thorn 4 , Roberto Cappai 5,6 and John A. Carver 4 1 Department of Chemistry, University of Wollongong, Australia 2 Institute for Environmental Research, Australian Nuclear Science and Technology Organization, Menai, Australia 3 ATA Scientific Pty Ltd ANSTO Woods Centre, Lucas Heights, Australia 4 School of Chemistry and Physics, The University of Adelaide, Australia 5 Department of Pathology, Bio21 Institute, The University of Melbourne, Australia 6 Mental Health Research Institute, Melbourne, Australia a-Crystallin is a molecular chaperone of the small heat-shock protein (sHsp) family. It is known to recog- nize and interact with long-lived partially folded pro- teins on their off-folding pathway to prevent their aggregation [1,2]. Two closely related subunits of a-crystallin exist in high concentrations in mammalian lenses, aA- and aB-crystallin; in humans they are pres- ent in a ratio of 3 : 1. Whereas aA-crystallin is lens specific, aB-crystallin is also found extralenticularly in retina, heart, skeletal muscle, skin, kidney, brain, spinal cord and lungs, as well as in CNS glial cells and neurons in some pathological conditions, e.g. Alzhei- mer’s disease and dementia with Lewy bodies [3–5]. The effectiveness of a-crystallin as a chaperone in preventing amorphous aggregation of destabilized proteins increases with temperature [6–8]. a-Crystallin occurs in large supramolecular assemblies of average mass  800 kDa [9], in dynamic equilibrium with Keywords amyloid; dual polarization interferometry; NMR spectroscopy; small heat shock protein; temperature dependence Correspondence J. A. Carver, School of Chemistry and Physics, The University of Adelaide, Adelaide, South Australia 5005, Australia Fax: +61 8 8303 4380 Tel: +61 8 8303 3110 E-mail: john.carver@adelaide.edu.au (Received 22 May 2007, revised 12 October 2007, accepted 16 October 2007) doi:10.1111/j.1742-4658.2007.06144.x The molecular chaperone, a-crystallin, has the ability to prevent the fibril- lar aggregation of proteins implicated in human diseases, for example, amyloid b peptide and a-synuclein. In this study, we examine, in detail, two aspects of a-crystallin’s fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of a-crystallin to suppress fibril formation in j-casein and a-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of a-crystallin. This is consistent with an increased chaperone ability of a-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem 275, 4565–4570]. Sec- ond, dual polarization interferometry was used to monitor real-time a-syn- uclein aggregation in the presence and absence of aB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that aB-crystallin interrupted a-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggrega- tion into fibrillar structures. Abbreviations ANS, 8-anilinonaphthalene 1-sulfonate; DPI, dual polarization interferometry; sHsp, small heat-shock protein; TEM, transmission electron microscopy; TFT, thioflavin T. 6290 FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS dissociated subunits. The rate of subunit exchange in a-crystallin increases with temperature [8]. The correla- tion between the temperature dependency of chaperone efficiency and subunit exchange suggests that it is pri- marily dissociated forms of sHsps that interact with destabilized target proteins [10]. Specifically, enhanced chaperone activity at higher temperatures has been attributed to an increase in the subunit exchange rate [8], and thus the availability of the dissociated, proba- bly dimeric, active forms of a-crystallin [2,11], along with concomitant structural changes in a-crystallin at higher temperatures [6,12,13]. More recently, a-crystallin was found to prevent the formation of amyloid fibrils by various proteins (e.g. Ab peptide, apolipoprotein CII, a-synuclein) [14–18]. Fibrillar aggregation by a number of proteins, includ- ing the aforementioned, forms the basis of many clini- cal disorders (e.g. Ab peptide in Alzheimer’s disease, a-synuclein in Parkinson’s disease, amylin in type II diabetes, b2-microglobulin in dialysis-related amyloido- sis and prion protein in Creutzfeldt–Jakob disease). In comparison with amorphous aggregation, fibril forma- tion is a slower and more ordered pathway of protein aggregation, however, both processes require proteins to adopt a conformation that is only partially folded, either by unfolding of a structured molecule, e.g. a-lact- albumin [19], or, in the case of intrinsically unstruc- tured (also known as natively disordered) proteins such as a-synuclein or j-casein, by stabilizing a conforma- tion that is already relatively unstructured [20]. Whether a protein aggregates amorphously or forms highly ordered fibrillar structures most likely depends on the structural characteristics of the aggregate pre- cursor, which is influenced by environmental condi- tions. With a-lactalbumin, for example, removal of Ca 2+ or the presence of Zn 2+ induces rapid formation of amorphous aggregates, whereas lowering the pH to 2.0 (to give the so-called A state) or reducing three of its four disulfide bonds (to give 1SS-a-lactalbumin) leads to the formation of amyloid fibrils [21]. The first set of conditions gives rise to a highly unstable molten globule state with a relatively rigid conformation, whereas the A state and 1SS-a-lactalbumin both have considerable conformational flexibility. Furthermore, the efficiency of interaction between sHsps and these partially folded species varies greatly and occurs via different binding modes, depending on the conforma- tional properties of the target protein [19,22,23]. During amyloid fibril formation, a protein will pro- ceed from its initial monomeric state through a series of aggregation states, e.g. the amyloidogenic nucleus and other prefibrillar intermediates, culminating in formation of the mature fibril [24]. The increasing complexity of these structures is paralleled by confor- mational changes, often irreversible, which the protein undergoes along its amyloid pathway. These may include conversion to a partially folded intermediate, partial proteolysis, b-sheet formation, ordered intermo- lecular association and the intertwining of two or more protofilaments [24]. Because pathological significance has been ascribed to the early soluble intermediates rather than mature fibrils [25–28], one approach to the treatment of amyloid diseases involves the develop- ment of inhibitors that not only inhibit amyloidogene- sis in its very early stages by interacting with partially folded or very early oligomeric species, but also result in a product which is nontoxic or biodegradable. The role of sHsps in amyloid fibril diseases is contro- versial. They are upregulated in these disease states and are known to interact with partially folded proteins. However, while inhibiting fibril formation, aB-crystal- lin stabilizes prefibrillar neurotoxic forms of Ab-peptide [14,29]. By contrast, aB-crystallin interacts with a-syn- uclein to form large nonfibrillar aggregates, implying that it can redirect a-synuclein from a fibril-forming pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable fibril in favour of easily degradable amorphous aggregates [16]. There are no data available on the effect of sHsps on the cytotoxicity of prefibrillar a-synuclein aggre- gates, however, the unrelated Hsp70 molecular chaper- one reduces the toxicity of prefibrillar and misfolded detergent-insoluble a-synuclein species [30,31]. In this study, we investigated the kinetics of interac- tion of a-crystallin with amyloid-forming a-synuclein and j-casein. a-Synuclein is a 14.4 kDa presynaptic protein of unknown function, which is a main compo- nent of Lewy bodies, the amyloid-rich proteinaceous deposits in Parkinson’s disease. It is intrinsically unstructured, but adopts a predominantly b-sheet con- formation during the formation of cytoplasmic amyloid fibrils in neurons [32]. j-Casein is one of the principal proteins of bovine milk, which together with others caseins (e.g. a s and b), form a unique micellar complex serving as a calcium phosphate transporter. Upon reduction of its intermolecular disulfide bonds, j-casein readily forms fibrils at physiological pH over a wide range of temperatures [33,34], thus providing an excel- lent model for studying the temperature-dependent interaction of amyloid-forming proteins with sHsps. In particular, we examined the effects of temperature on the fibrillation rate of j-casein and a-synuclein and the efficiency of a-crystallin to suppress this aggregation. In addition, we investigated the interaction of aB-crystal- lin with a-synuclein in real time using dual polarization interferometry (DPI) [35,36], a new analytical method A. Rekas et al. a-Crystallin and amyloid fibril formation FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS 6291 for studying protein interaction under physiological conditions. With regards to amyloid fibril formation, it enables the real time study of both fibril elongation and the initial nucleation processes. By monitoring the thickness, average density and mass of the protein deposition layer, it was possible to record in greater detail the kinetics of a-synuclein aggregation in both the absence and presence of aB-crystallin, thereby revealing the stage at which aB-crystallin interacts with a-synuclein to inhibit its fibril formation. Results Temperature dependence of a-crystallin chaperone activity against fibril-forming target proteins The enhanced ability of a-crystallin at elevated temper- ature, i.e. 30 °C and above, to prevent the aggregation and precipitation of amorphously aggregating target proteins has been well characterized [6–8]. The aim of our study was to determine whether a similar tempera- ture dependency existed in the ability of a-crystallin to prevent amyloid fibril formation by either j-casein or a-synuclein. j-Casein As is apparent from transmission electron microscopy (TEM) images (Fig. 1), disulfide-reduced j-casein formed fibrils both at 37 and 50 °C (13.4 ± 2.2 nm in diameter), although a difference in their supramolecu- lar morphology was evident: at 37 °C fibrils were well separated, whereas at 50 °C there was a tendency to further associate to form large conglomerates of tan- gled fibrils. The length of the fibrils varied greatly, but on average, the fibrils formed at 37 °C were shorter (101.1 ± 49.6 nm) than those formed at 50 °C (148.0 ± 88.3 nm), including a larger number of small fragments (up to 20 nm in length) at the lower temper- ature. At 37 °C, the presence of a-crystallin (up to 1 : 1 molar ratio) had little effect on the extent of fibril formation, with longer fibrils of 94.3 ± 28.7 nm, although the overall polydispersity was reduced. A large number of short prefibrillar j-casein species ( 20 nm) were also present, in addition to the spheri- cal aggregates (14–17 nm in diameter) characteristic of a-crystallin. At 50 °C, a-crystallin caused a noticeable reduction in the number of fibrils, including prefibrillar species, but the average length of mature fibrils remained large (152.2 ± 68.7 nm). The fluorescence of j-casein-bound thioflavin T (TFT) at 37 and 50 °C showed a sigmoidal time course (Fig. 2A) typical of nucleation-dependent fibril forma- tion [37]. The initial lag phase corresponds to the formation and accumulation of oligomeric prefibrillar partially folded intermediates that do not bind TFT [38]. The subsequent increase in fluorescence intensity represents elongation of the fibril [39] with a stacked b-sheet conformation. Under stable environmental conditions (e.g. constant temperature), TFT fluorescence can be reliably used to quantify the amount of stacked b sheet, and thus moni- tor the kinetics of fibrillation. However, during experi- ments performed at higher temperatures, a decrease in TFT fluorescence was observed, which suggests that either binding of TFT by proteins or the efficiency of fluorescence are temperature dependent. For this reason, the time course of TFT fluorescence upon interaction with j-casein may reflect other tempera- ture-dependent processes in addition to the formation of amyloid fibrils. For example, at higher temperatures (45–60 °C), the magnitude of TFT fluorescence (maxi- mum intensity value) in the presence of j-casein alone was much lower than at 30–37 °C (Fig. 2A), despite a comparable number of fibrils shown by electron microscopy. Moreover, at higher temperatures, there was a decrease in TFT fluorescence after reaching a maximum value (Fig. 2A; 50 °C data), which may arise from the aggregation of fibrils into large con- glomerates and the possible obstruction of TFT bind- ing sites (Fig. 1). Thus, fibrillation rates (as depicted in Fig. 2B) were reliably estimated from the TFT binding 500 nm -casein, 37°C -casein, 50°C -casein + - crystallin 37°C -casein + - crystallin, 50°C Fig. 1. TEM images of reduced j-casein species formed at 37 and 50 °C in the absence and presence of an equimolar amount of a-crystallin. Images acquired at ·40 000 magnification show a higher level of suppression of j-casein fibrillation by a-crystallin at 50 °C than at 37 °C. a-Crystallin and amyloid fibril formation A. Rekas et al. 6292 FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS data using only the initial period, when the increase in fluorescence was exponential and concentration depen- dent (the length of this exponential period also varied with temperature and was chosen by careful examina- tion of fitting parameters). j-Casein aggregation kinetics depended on tempera- ture and the presence of the inhibitor, a-crystallin. The rate constant for the increase in j-casein TFT binding with time increased with temperature (Fig. 2A,B). However, as assessed by TFT binding, the presence of a-crystallin significantly suppressed fibril formation by j-casein (Fig. 2A,B). At 30 °C, the fibril formation rate was slowest and a-crystallin significantly reduced the number of fibrils without changing the rate of fibril formation (Fig. 2B). Percentage-wise, a-crystallin was least effective at 30–33.5 °C, where temperature-depen- dent increases in the rate of fibril formation were not compensated by a concomitant increase in the ability of a-crystallin to suppress it. Above 33.5 °C, however, the rate of fibril formation by j-casein increased with temperature, and so did the relative efficiency of a-crystallin to inhibit fibril formation (Fig. 2). Amyloid fibril elongation is known to be a first- order reaction [37,39] (A. Rekas, unpublished data on a-synuclein and j-casein). From the Arrhenius law, we have: ln(k app ) ¼ – E A ⁄ RT + ln(A). The activation energy of fibril formation (E A ) was calculated (Table 1) as the slope of the straight line fitted to a plot of ln(k app ) versus 1 ⁄ T, where T is temperature in K, R is the gas constant and A is the frequency (or pre-exponential) factor, expressed in the same units as the apparent first-order rate constant, k app . For reduced j-casein only, E A was 35.5 ± 1.1 kcalÆmol )1 , showing strong temperature dependence of the rate constants (R 2 ¼ 0.995). a-Crystallin reduced the acti- vation energy for j-casein fibril elongation, e.g. for a TFT binding @ 50oC 0 100 200 300 400 500 600 700 0 5 10 15 time (hours) a.u. TFT binding @ 25 o C 0 200 400 600 800 1000 1200 1400 1600 0510 time (hours) a.u. k-cas preinc @ 25 deg k-cas preinc @ 40 deg k-cas preinc @ 60 deg TFT binding @ 37oC 0 1000 2000 3000 4000 5000 6000 7000 0 5 10 15 a.u. 0.10 1.00 10.00 100.00 30 40 50 60 temperature [ o C] kapp [s -1 ] 0 50 100 0 0.25 0.5 κ-cas κ-cas+0.25xα-cr κ-cas+0.5xα-cr κ-cas+1xα-cr κcasein κcas+0.25xαcrys κcas+0.5xα-crys κ cas+1xα-crys A B C 0 0.4 0.8 500 100 250 50 0 0 0.4 0.8 0 1 1 Fig. 2. Temperature dependence of j-casein fibril formation under reducing conditions. (A) Real-time TFT fluorescence data at 37 and 50 °C. (B) Growth-rate constants with temperature in the presence and absence of a-crystallin at the indicated molar ratios. (C) The effects of pre- incubation of j-casein at 25, 40 and 60 °C on its fibrillation potential. Table 1. Comparison of activation energy (E A ) and frequency factor (A) values for j-casein fibril elongation under reducing and nonreducing conditions. a-Crystallin, especially at higher concentrations (0.5 : 1 and 1 : 1 w ⁄ w ratios to j-casein), reduced the activation energy and fre- quency factor for j-casein fibril formation. j-casein +0.25· a-crystallin +0.5· a-crystallin +1.0· a-crystallin Reduced E A (kcalÆmol )1 ) 35.5 ± 1.1 13.8 ± 4.9 18.3 ± 2.6 14.9 ± 2.0 A (h )1 ) range 1.4 · 10 24 )4.2 · 10 25 9.8 · 10 5 )4.8 · 10 12 4.0 · 10 10 )1.2 · 10 14 3.3 · 10 8 )2.0 · 10 11 Native E A (kcalÆmol )1 ) 25.7 ± 1.9 24.9 ± 1.9 1.7 ± 4.4 )0.4 ± 1.2 A (h )1 ) range 1.9 · 10 16 )9.2 · 10 18 6.4 · 10 15 )3.2 · 10 18 5.3 · 10 )3 )5.9 · 10 3 1.2 · 10 )2 )5.3 · 10 )1 A. Rekas et al. a-Crystallin and amyloid fibril formation FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS 6293 1 : 1 molar ratio of j-casein ⁄ a-crystallin, E A was 14.9 ± 2.0 kcalÆmol )1 (R 2 ¼ 0.902). Likewise, the parameter A which is related to the frequency of inter- actions between the molecules, decreased in the pres- ence of a-crystallin. Exposure of j-casein to higher temperatures for 15 min caused a slight decrease in its subsequent fibrillation level when incubated in the presence of reducing agent at 25 °C (Fig. 2C), although the changes in the rate constants were not significant: (1.85 ± 0.09) · 10 )1 Æs )1 , (1.86 ± 0.15) · 10 )1 Æs )1 and (1.79 ± 0.11) · 10 )1 Æs )1 after preincubation at 25, 40 and 60 °C, respectively. The initial lag times increased from 10 to 13 to 20 min for 25, 40 and 60 °C preincu- bation temperature, respectively. These differences indicate that some small irreversible structural changes occur to j-casein with temperature, but they are not sufficient to explain the reduction in maximum fluores- cence intensity and the increase in fibrillation rate that was observed for fibril formation at higher tempera- tures in the experiments described above. Fibril formation by j-casein under nonreducing con- ditions (hereafter referred to as ‘native’ j-casein) was also examined over the temperature range of 30–55 °C. In the absence of reducing agent, the process of fibril- lation proceeded more slowly, especially at higher tem- peratures (Fig. 3), than under reducing conditions (Fig. 2). At the same time, the overall efficiency of a-crystallin to prevent fibril formation was lower, with only equimolar amounts of a-crystallin showing signifi- cant inhibition below 45 °C (Fig. 3B). As seen with the reduced protein, the ability of a-crystallin to suppress fibril formation by native j-casein increased with temperature, as indicated by a significant reduction in activation energy for fibril elongation (E A ) which at a a-crystallin ⁄ j-casein ratio of 1 : 1 (w ⁄ w) was )0.4 ± 1.2 kcalÆmol )1 , compared with 25.7 ± 1.9 kcalÆmol )1 for j-casein only (Table 1). In effect, at high concentrations of a-crystallin, the temperature dependence of j-casein fibril formation was abrogated by the inhibitory action of a-crystallin. a-Synuclein TFT fluorescence data showed differences in the fibril- lation kinetics of a-synuclein at various temperatures (Fig. 4A). From these data, it is evident that the rela- tive ability of aB-crystallin to inhibit a-synuclein aggregation increased with temperature. Also, the max- imum fluorescence over time was relatively unchanged upon increasing the temperature from 37 to 45 °C, but was significantly lower at 60 °C (Fig. 4A), as observed with j-casein at higher temperature (Figs 2 and 3). The temperature dependence of aB-crystallin’s abil- ity to suppress fibril formation, as shown by TFT binding data, was supported by TEM. At 37 and 60 °C, a-synuclein, by itself, formed fibrils of compa- rable length and morphology, however, in the pres- ence of aB-crystallin (at a 1 : 1 molar ratio) fibril formation at 60 °C was almost completely inhibited, while only partial suppression was achieved at 37 °C (Fig. 4B). The reduction in TFT fluorescence at higher temper- atures was demonstrated for preformed fibrils of j-casein and a-synuclein. A constant number of fibrils showed a 36% reduction in TFT fluorescence over the temperature range 28–60 °C for j-casein, and 39% reduction for a-synuclein between 25 and 52.5 °C (Fig. 4C). B A Fig. 3. Temperature dependence of j-casein fibril formation under nonreducing conditions in the absence and presence of a-crystallin. (A) Plots showing real-time TFT fluorescence data at 37 and 55 °C. (B) Changes in fibril growth-rate constants with temperature at the indicated molar ratios. a-Crystallin and amyloid fibril formation A. Rekas et al. 6294 FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS DPI study of the suppression of a-synuclein aggregation by aB-crystallin DPI was used to monitor real-time a-synuclein aggrega- tion and the effect of aB-crystallin on this, particularly at the very early stages of this process. In a DPI mea- surement, the average layer density decreases during fibrillar-type aggregation because the initial dense pro- tein ‘monolayer’ on the surface remains attached, but the subsequent protein deposition occurs by elongating of (pre)fibrillar species, rather than random adherence of nonfibrillar material. This has been observed in DPI examination of the aggregation of other fibril-forming proteins, i.e. the Alzheimer’s amyloid b peptide and the familial mutants (A30P and A53T) of a-synuclein (http://www.farfield-sensors.com/articles/). The signal responses stabilized  10 min after the injection of a-synuclein alone into channel 3 and the resolved data showed a deposition of a protein layer of thickness 4.114 nm, density 0.652 gÆcm )3 and mass 2.531 ngÆmm )2 . Over the next 4 h, a steady decrease in layer density, and an increase in layer thickness and mass were observed (Fig. 5). During the maturation process, these values gradually changed, showing that aggregation proceeded steadily. Specifically, after 60 min the protein layer thickness increased by 0 000 1 0002 0003 0004 0005 000 6 00 07 0008 TFT fluorescence 0 0 0 0 2 0004 0 0 0 6 0008 0 00 0 1 thioflavin T fluorescence 0 0 001 000 2 000 3 00 0 4 0005 02 10 90 60 3 0 2 )setu ni m (em i t thioflavin T fluorescence α -syn α -syn+0.5xα-crys α -syn+1xα-crys 73 o C 54 o C 06 o C 200 nm AB 0 002 0 0 5 2 000 3 0053 0004 005 4 0005 0 605040302 ( e rutarepmet o )C TFT fluorescence (a.u.) κ -casein α -synuclein C Fig. 4. (A) Temperature dependence of a-synuclein fibril formation in the absence and presence of ab-crystallin. Bar graphs show TFT fluo- rescence data at selected time points and 37, 45 and 60 °C. Molar fractions of aB-crystallin over a-synuclein are indicated. (B) Comparison of electron micrographs of a-synuclein species in the absence and presence of aB-crystallin (1 : 1 molar ratio) incubated for 4 h at 37 or 60 °C. (C) Temperature dependence of TFT fluorescence for 1 mgÆmL )1 j-casein (closed symbols) and 2 mgÆmL )1 a-synuclein (open sym- bols); the decrease in TFT intensity accounts for the lower TFT fluorescence levels at higher temperatures shown in Figs 2A, 3A and 4A. A. Rekas et al. a-Crystallin and amyloid fibril formation FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS 6295 0.0746 nm on channel 3, the mass increased by 0.02 ngÆmm )2 and the density decreased by 0.0072 gÆcm )3 . After 2.5 h, the thickness increased by 0.212 nm, the mass increased by 0.05 ngÆmm )2 and the density decreased by 0.0167 gÆcm )3 , from the start of the experiment. By the end of the measurement, the thickness had increased by 0.342 nm, the mass had increased by 0.10 ngÆmm )2 and the density had decreased by 0.024 gÆcm )3 . By contrast, on channel 1, where aB-crystallin was injected together with a-synuclein, the thickness, density and mass of the protein layer were essentially unchanged during the entire experiment (Fig. 5), i.e. the thickness of the layer decreased by 0.014 nm, the layer density decreased by only 0.0086 gÆcm )3 , and the mass decreased by 0.03 ngÆmm )2 . The process of a-synuclein aggregation at 25 °C without agitation is relatively slow, so the thickness did not increase greatly over time. In addition to demonstrating the ability of DPI to moni- tor the aggregation of a-synuclein, this experiment showed that the interaction of aB-crystallin with a-syn- uclein takes place immediately after combining solutions of both proteins and prevents formation of prefibrillar nuclei by a-synuclein, i.e. aB-crystallin interacts with a-synuclein early along its aggregation pathway. To summarize, initial nucleation took place immedi- ately after the protein was bound to the sensor surface as the thickness and mass of the protein layer started to increase with a simultaneous decrease in density after only 10 min. In a parallel experiment (not shown), no change in TFT binding was observed after 24 h incubation of a-synuclein in the absence or pres- ence of a 0.5 molar amount of aB-crystallin at 25 °C without agitation, i.e. under experimental conditions mimicking those of DPI. Therefore, the DPI data refer to prefibrillar a-synuclein aggregation. Species specificity of aB-crystallin interaction with a-synuclein From the DPI results (Fig. 5), it is apparent that aB-crystallin interacts with a-synuclein early during its aggregation pathway (i.e. at the nucleation or proto- fibril stage). Additional experiments were therefore undertaken to determine whether aB-crystallin was as effective at suppressing further fibril formation by more advanced fibrillar forms of a-synuclein. Time course of thioflavin T binding As expected, in the absence of aB-crystallin, an increase in TFT fluorescence was observed for incu- bated a-synuclein. Fibril formation by a-synuclein, as indicated by this increase in fluorescence, was sup- pressed upon the addition of aB-crystallin [16] (Fig. 6A). Interestingly, this effect was observed not only when both proteins were present in the sample from the beginning of incubation, but also in samples containing a significant number of amyloid fibrils (before addition of aB-crystallin at time points between 25 and 65 h). Under these conditions, aB-crystallin prevented, but did not reverse, further fibril formation (i.e. it had no capacity to disassemble existing fibrils), as visible from the stabilization of the level of TFT fluorescence. 2 5.2 3 5.3 4 5.4 5 4 32 1 0 Layer thickness (nm) 55.0 85.0 16 .0 46.0 76. 0 7 .0 4 3210 Layer density (g/cm 3 ) 3 . 1 55. 1 8.1 50 . 2 3 .2 55. 2 8.2 432 1 0 )s r uoh(emi t Mass (ng/mm 2 ) α +nys- α sy r c-B α n i e lcunys - α +nys- α s yr c -B α nielcu n y s - α +nys- α syrc-B α nielcunys- Fig. 5. The DPI data obtained from channel 3 (a-synuclein only; black) and channel 1 (a-synuclein + aB-crystallin; grey), showing a-synuclein physisorption and aggregation. a-Synuclein was at 3.5 mgÆmL )1 and aB-crystallin at 2.5 mgÆmL )1 . The resolved traces of thickness, density and mass are depicted in individual panels. The data shown are from the time of signal stabilization following injection of protein solutions onto the sensors thermostated at 25 °C. a-Crystallin and amyloid fibril formation A. Rekas et al. 6296 FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS Although these data do not exclude the possibility of an interaction between aB-crystallin and fibrillar a-synuclein, they are consistent with the DPI results showing that aB-crystallin interacts with monomeric or nucleated a-synuclein prior to it being incorporated into the growing a-synuclein fibril, and in this way pre- vents further fibril growth. TEM images of a-synuclein species at different stages of its fibril formation (in the absence of aB-crystallin) are also consistent with this proposal. Small globular a-synuclein species were found throughout the entire time course of fibril for- mation (Fig. 6B), including the micrograph at ‘0 h’ (which was actually about 15 min after dissolution of the protein while being kept on ice); and during the plateau phase (after 1 week of incubation). Consider- ing their size of 13–19 nm in diameter, which matches the diameter of a-synuclein fibrils, it is likely that these species are prefibrillar intermediates. Interaction between j-casein and a-crystallin investigated by size-exclusion HPLC, 8-anilino- naphthalene 1-sulfonate binding and NMR spectroscopic studies The interaction and complex formation of destabilized j-casein with a-crystallin, after mixing both proteins in the presence or absence of dithiothreitol, was investi- gated by size-exclusion HPLC. The interaction of a-crystallin with reduced j-casein was also investigated by 8-anilinonaphthalene 1-sulfonate (ANS) binding and NMR spectroscopy, and compared with an analo- gous interaction with native j-casein. The absence of shaking during incubation resulted in j-casein species that did not bind TFT and were therefore nonfibrillar. Size exclusion HPLC Incubation of equal masses of j-casein and a-crystallin in solution at 37 °C for 4 h led to partial formation of a high molecular mass complex between these two pro- teins of  1300 kDa, as shown by size-exclusion HPLC (Fig. 7A). In its native (nonreduced) state, j-casein exists as a large species which eluted at 5 h 45 min from the column, the same elution time as a-crystallin, corresponding to  830 kDa. However, the elution time of the a-crystallin + j-casein mixture was shifted to 5 h 28 min, implying interaction between the two proteins which led to a complex of larger mass. Reduc- tion of j-casein’s intermolecular disulfide bonds led to the appearance a very large aggregate ( 6800 kDa) at an elution time of 4 h 38 min. In the main, the pres- ence of a-crystallin significantly decreased formation of this large aggregate. As a result of the interaction of a-crystallin with reduced j-casein, a complex of similar mass (1500 kDa) to the one with native j-casein was observed with an elution time of 5 h 26 min. A B 0 50 100 150 200 250 300 0 50 100 150 time (hours) thioflavin T fluorescence a-syn a-syn+aB a-syn+aB 25h a-syn+aB 49h a-syn+aB 65h -syn -syn+ B -syn+ B 25h -syn+ B 49h -syn+ B 65h 72 hrs 200 nm 168 hrs 0 hrs Fig. 6. Time course of amyloid fibril forma- tion by a-synuclein (125 l M) in the absence and presence of aB-crystallin (62.5 l M). (A) TFT binding data. aB-crystallin was added to the incubated a-synuclein samples (black diamond) at the beginning of the experiment (black squares) and at later time points, i.e. 25 h (grey triangle), 49 h (black circle) and 65 h (grey diamond). The increase in TFT fluorescence was monitored as described previously [16]. (B) TEM images of a-synuclein species in the absence of aB-crystallin at the indicated times from the beginning of incubation at 37 °C. Images acquired at ·60 000 magnification reveal that small globular protein aggregates (oligomeric intermediates, indicated by arrows) are present alongside fibrils at all stages of the fibril formation time course. A. Rekas et al. a-Crystallin and amyloid fibril formation FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS 6297 ANS binding The level of ANS fluorescence emission (Fig. 7B) indicated that reduced j-casein exposed much more clustered hydrophobicity to solution than nonreduced j-casein, which is consistent with greater unfolding of the protein following disulfide bond reduction. Both reduced and nonreduced j-casein, and a-crystallin, Exposed hydrophobicity 0 500 1000 1500 2000 2500 3000 20 30 40 50 60 70 ANS fluorescence (a.u.) a-crystallin k-casein non-red k-casein reduced k-casein red. + a-crys a-cryst + -cas red (theor) HPLC elution profile A C B 0 20000 40000 60000 80000 100000 33.544.555.566.577.58 elution time (min) A(280) k-cas a-crystallin cas reduced - - - ca s - - - - - crys cas red crys 14.6 kD67 kD669 kD2000 kD temp (°C) Fig. 7. Interaction of a-crystallin with j-casein. (A) Size-exclusion HPLC profiles of j-casein (native and reduced), a-crystallin and their mix- tures. All proteins at 10 mgÆmL )1 were incubated at 37 °C for 4 h prior to chromatography. a-Crystallin decreases the size of reduced j-casein aggregates and also complexes with nonreduced j-casein. Elution times of blue dextran (2000 kDa), thyroglobulin (669 kDa), BSA (67 kDa) and lysozyme (14.6 kDa) are indicated. (B) Maximum ANS fluorescence when bound to j-casein, a-crystallin (both proteins at 0.3 mgÆmL )1 ) and their mixtures, recorded in the temperature range from 25 to 65 °C. At lower temperatures, the interaction between these two proteins (circles) results in greater exposure of hydrophobic regions than the sum of fluorescence values of both component proteins (stars). (C) Superimposed 2D 1 H NMR TOCSY spectra of the NH to a,b,c region of j-casein (red), a-crystallin (blue) and their mixtures (black) acquired at 37 °C. Each protein was dissolved in 10 m M sodium phosphate pH 7.2, 10% D 2 O, at a concentration of 2 mgÆmL )1 . The upper panel shows spectra with reduced j-casein, the lower with native (nonreduced). After combining native j-casein with a-crystallin, additional cross-peaks were observed, which are circled in green. a-Crystallin had little effect on the reduced target protein (a relatively stable unfolded state), but additional cross-peaks were observed for the mixture under native (nonreducing) conditions. a-Crystallin and amyloid fibril formation A. Rekas et al. 6298 FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS showed a decrease in ANS fluorescence with increas- ing temperature. This implies a decrease in the amount of exposed hydrophobicity (due to self-associ- ation) and ⁄ or a decrease in fluorescence emission efficiency with temperature, as was observed for pro- tein-bound ANS in the absence of conformational changes [40]. Notwithstanding, the interaction between j-casein and a-crystallin led to higher level of surface hydrophobicity, as the mixture of reduced j-casein and a-crystallin had a higher ANS-binding level than the sum of both components (Fig. 7B). This effect was largest at lower temperatures (25 °C) and decreased upon heating to 70 °C, at which point the fluorescence of the mixture was equal to the sum of its components. This difference between theoretical values and those of the nonreduced j-casein + a-crystallin mixture was slightly larger than under reduced conditions (not shown). 1 H-NMR spectroscopy Cross-peaks from the NH to aliphatic proton regions of 1 H 2D NMR TOCSY spectra of j-casein, a-crystal- lin and their mixture are shown in Fig. 7C, for reduced and native j-casein (upper and lower panels, respec- tively). As expected, spectra of the a-crystallin aggre- gate show only a few cross-peaks belonging to the highly flexible C-terminal extension in both subunits of 10–12 amino acids [41,42]. Reduced j-casein showed a significant degree of flexibility compared with the native species, as indicated by a large number of intense cross-peaks. Addition of a-crystallin to j-casein caused some additional cross-peaks to be observed, which was particularly pronounced in the case of the native j-casein and a-crystallin mixture, where signifi- cant conformational flexibility was indicated by the appearance of additional cross-peaks. Discussion The temperature dependence of the kinetics of fibril formation by Ab peptide [33,39] and insulin [37] has been described previously. The time course of fibril formation, as monitored by TFT binding, follows a sigmoidal curve. The prefibrillar nuclei (early oligo- meric species) do not bind TFT. They form during the lag time, which is followed by the fibril elongation phase corresponding to an increase in the dye’s fluores- cence. The subsequent plateau phase is associated with a decrease in the concentration of small species, or the aggregation and precipitation of fibrils [37]. The kinet- ics of these three stages of the fibril formation process are temperature dependent [37–39,43]. In this study, the rate of fibril formation of both j-casein and a-synuclein increased with temperature, as monitored by TFT binding. In the presence of a-crystallin, the initial lag phase was longer, which indicates that a-crystallin slowed the formation of pre- fibrillar intermediates. The TEM data are consistent with this conclusion. a-Crystallin undergoes a struc- tural transition at  45 °C which leads to greater unfolding and enhanced chaperone action against amorphously aggregating target proteins [44,45]. This behaviour may contribute to a-crystallin’s enhanced ability to prevent fibril formation at higher tempera- tures. Our data on j-casein showed an exponential depen- dence of the fibril formation rate on temperature. Thus, the rate constants follow Arrhenius’ law, which is consistent with the temperature dependence of fibril elongation rates of the Ab peptide [39]. In addition to decreasing the rates of fibril formation at all tempera- tures for reduced and native j-casein, a-crystallin decreased both the activation energy and the frequency constant of this process. This suggests that the temper- ature-dependent inhibition of j-casein fibrillation by a-crystallin is a function of both ‘activating’ the chaperone ability of a-crystallin, and of the effects of a-crystallin on j-casein, which have not been, as yet, described. If this mechanism of interaction occurs in vivo, it may have important implications in the design of chaperone-based therapeutics against amy- loid diseases. Fibril formation by j-casein in the presence of an inhibitor protein, a-crystallin, is a complex process. Possible components of this reaction include the disso- ciation of large a-crystallin and j-casein oligomers into smaller species, binding of a-crystallin to j-casein, con- formational alteration of j-casein and ⁄ or a-crystallin upon their interaction, dissociation of the complex and subsequent conformational changes (e.g. refolding) of j-casein. The resultant E A and k values are reflective of the entire process (Table 1). Molecular collision rates increase with temperature, and so does the disso- ciation rate of a-crystallin oligomers. In addition, the conformational flexibility of a-crystallin also increases with temperature [2,6,8,11–13], making it potentially more efficient to interact with j-casein and form a transient complex. This is consistent with the observed enhancement of the inhibitory effect of a-crystallin on the rate of j-casein fibrillation at higher temperatures. However, our NMR and fluorescence data also indi- cate a greater unfolding of j-casein upon its interac- tion with a-crystallin. Such partially unfolded j-casein molecules, when released from the complex with a-crystallin would be susceptible to association with A. Rekas et al. a-Crystallin and amyloid fibril formation FEBS Journal 274 (2007) 6290–6305 ª 2007 The Authors Journal compilation ª 2007 FEBS 6299 [...]... at the very early prefibrillar stage, although we cannot deduce whether it acted by preventing the formation of monomeric partially folded species, or by preventing association of a-synuclein monomers into nuclei, or both a-Crystallin and amyloid fibril formation We have shown aB-crystallin’s ability to inhibit a-synuclein fibril formation both in the earliest stages of its aggregation, and during the. .. fibrillar aggregation in conjunction with changes in conformation and dynamics of a-crystallin molecules, contribute to the increased efficiency of suppression by a-crystallin of fibril formation at higher temperatures At higher temperatures, the maximum TFT fluorescence intensity of j-casein and a-synuclein did not correlate with the increased fibrillation rate, i.e fluorescence intensity was lower at temperatures.. .a-Crystallin and amyloid fibril formation A Rekas et al other j-casein molecules In this way, a-crystallin may inhibit fibril formation by binding to the target protein, but in the process ‘activate’ it to form fibrils This speculation is supported by our observation that a-crystallin both decreases j-casein fibril formation rate and lowers the activation energy of this process sHsps... agglomeration of fibrils, as discussed above) By contrast, DPI is a direct tool for studying various types of protein interaction and aggregation in a quantitative manner, by monitoring the thickness and density of the protein deposition layer Moreover, DPI is sensitive to small changes in protein association parameters and thus allows monitoring of processes preceding formation of dye-binding amyloid species, and. .. indicated by NMR spectra collected at 10 °C on freshly prepared samples (and thus not containing any fibrillar species) and by a decreased chaperone effectiveness of aB-crystallin in the presence of a-synuclein [16] Finally, the interaction of aB-crystallin with a-synuclein fibrils (e.g by capping the ends of fibrils) also can be considered, however, addition of aB-crystallin had no effect on the level of a-synuclein... the absence of fibril conglomerates in the presence of a-crystallin in TEM images (Fig 1), which would inhibit binding of TFT The size-exclusion HPLC data indicate complex formation between prefibrillar j-casein (native or reduced) and a-crystallin at 37 °C The size of this complex is less than that of reduced j-casein aggregates formed in the absence of the chaperone, which is consistent with the data... phase of fibril formation (Fig 6A) At each instance, addition of aB-crystallin stopped further formation of stacked b-sheet-rich species with comparable efficiency Recently, two types of a-synuclein prefibrillar intermediates of different structure and aggregation propensity were characterized [38] The first type, of a larger size, accumulates early in fibril formation and rapidly disappears during fibril. .. accumulation of nonfibrillar, potentially toxic oligomers in systems in which fibrillization was inhibited [14,51] and with the absence of intermediate size oligomers of amyloidogenic apolipoprotein C-II in the presence of a-crystallin [15] aB-Crystallin may also bind to monomeric forms of a-synuclein with partially destabilized conformation if any of these were still present in the later stages of fibrillation... contributing factor to the difference in fibrillation rate between the two forms of j-casein and interaction with a-crystallin An increased rate of protein fibrillation does not always result in a greater efficiency of a-crystallin to prevent this For example, molecular crowding increased the rate of fibril formation by a-synuclein, but also decreased the chaperone efficiency of aB-crystallin [16] Furthermore, molecular... bodies and Ab plaques) may be involved in facilitating protein aggregation by binding to nonfibrillar molecules This can occur in two ways: upon sequestration of molecular chaperones by amyloidogenic proteins, the availability of the former to prevent aberrant aggregation is diminished, but also the possibility of partial unfolding of the protein and stabilization of an amyloidogenic intermediate by the . Monitoring the prevention of amyloid fibril formation by a-crystallin Temperature dependence and the nature of the aggregating species Agata Rekas 1,2 , Lucy Jankova 3 ,. detail, two aspects of a-crystallin s fibril- suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of a-crystallin. decreased by 0.0167 gÆcm )3 , from the start of the experiment. By the end of the measurement, the thickness had increased by 0.342 nm, the mass had increased by 0.10 ngÆmm )2 and the density