Kinetic Insights into Zn2+-Induced Amyloid Î²-Protein Aggregation

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Kinetic Insights into Zn -Induced Amyloid #-Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy Jing-Jing Guo, Lin-Ling Yu, Yan Sun, and Xiaoyan Dong J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b12187 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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The Journal of Physical Chemistry

Kinetic Insights into Zn2+-Induced Amyloid β-Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy Jingjing Guo, Linling Yu, Yan Sun, Xiaoyan Dong*

Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

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ABSTRACT: Zn2+ has remarkable impacts on amyloid β-protein (Aβ) aggregation, which is crucial in the pathology of Alzheimer’s disease (AD). However, Zn2+ concentration in human cerebrospinal fluid is commonly too low to interact with Aβ. Only during neurotransmission is there a transient release of high-concentration Zn2+. It is difficult to explore the details of the interaction between Zn2+ and Aβ within such a short time scale by using ordinary analytical methods. Herein stopped-flow fluorescence spectroscopy was used to study the fast aggregation kinetics of Aβ42 in the presence of Zn2+ in the time scale from one millisecond to seconds. It was found that Zn2+ bound to Aβ42 within 1 ms, caused immediate conformational transition around Tyr10, which led to the enhancement of Aβ42 hydrophobicity and then promoted Aβ42 fast aggregation through enhanced hydrophobic interactions. Among the two Zn2+ binding sites on Aβ42 (KD=107 nM and 5.2 µM), the first one of higher affinity had greater impact on the aggregation of Aβ42. Three kinetic phases were observed in the Zn2+-induced Aβ42 fast aggregation, and the fast phase was extremely accelerated by Zn2+, indicating that the accelerated aggregation was mainly in the fast phase. The reactions happened in this phase were closely related to the association of Zn2+ and Aβ42. Moreover, Zn2+ largely broadened the pH range of Aβ42 fast aggregation, from pH5.2 to 6.2 without Zn2+ to pH5.2 to 7.8 in the presence of Zn2+. Besides, the promoting effect of Zn2+ on Aβ42 fast aggregation peaked at pH6.8-7.8, which just covers the pH values of cerebrospinal fluid (pH7.3) and hippocampus (pH7.15-7.35). The findings demonstrated the significant effect of Zn2+ on Aβ aggregation and provided new insights into its working details.

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1. INTRODUCITON Alzheimer’s disease (AD) is a neurodegenerative disorder, which comprises the most prevalent form of dementia. It is characterized by neurofibrillary tangles and amyloid plaques.1,2 Aggregates of the 39-43 amino acid amyloid β-protein (Aβ) are the main components of amyloid plaques found in AD brains.3,4 Besides, autopsy studies showed that there are also high concentrations of metal ions in the amyloid plaques,5 especially Zn2+, whose concentration in the plaques could be up to 1 mM.6 In vitro studies have shown that metal ions, such as Zn2+, Cu2+ and Fe3+, can influence the aggregation of Aβ,7 while the addition of chelates is able to reverse the Zn2+-induced Aβ aggregation,8-10 indicating that the metal ions could interact with Aβ, and co-deposit with it into amyloid plaques. However, Zn2+ concentration in cerebrospinal fluid is commonly at nanomolar level, about two orders of magnitude lower than the dissociation constant for its binding to Aβ (KD=107 nM).11-13 Hence, it is intriguing how Zn2+ at such low concentrations in brain interacts with Aβ. As revealed in later studies, glutamatergic synapses in cerebral cortex and hippocampus transiently release large amounts of Zn2+ during neurotransmission, forming Zn2+ “pulses” whose instantaneous concentration could reach 300 µM,14,15 which is more than enough to interact with Aβ. The Zn2+ “pulses” would be rapidly re-uptaken into synaptic vesicles by zinc transporter ZnT3.14,16 Thus the interactions between Zn2+ and Aβ are most likely to happen during the instant Zn2+ drifting around in synaptic clefts. However, current studies related to the interactions between Zn2+ and Aβ are mostly limited to the use of ordinary analytical methods, and little effort on studying the fast kinetics to simulate the short time scale when Zn2+ “pulses” exist has been made. Noy et al. first employed a stopped-flow apparatus and demonstrated that Zn2+ promoted the aggregation of Aβ40 in several milliseconds.17 However, the detailed mechanism for the interactions between Zn2+ and Aβ in such a short timescale still remains unclear. Besides, Aβ42 rather than Aβ40 is the major variant of Aβ in the core of senile plaques where Zn2+ is co-deposited.18 Moreover, Aβ42 has been found more susceptible than Aβ40 to the influence of Zn2+.19 Therefore, it is intriguing to study the interactions between Zn2+ and Aβ42 by fast aggregation kinetics. In this work, we employed a stopped-flow apparatus with shorter dead time (1 ms), and studied the fast aggregation kinetics of Aβ42 in the presence of Zn2+ under various conditions. The size and conformational transitions of Aβ42 aggregates, as 3



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well as the variations of rate constants, were investigated to reveal the driving force and influential factors of the promoting effect of Zn2+ on Aβ42 aggregation. The findings have provided insights into the important role of Zn2+ in Aβ42 aggregation, and revealed the details of the fast interactions between Zn2+ and Aβ42.

2. EXPERIMENTAL SECTION 2.1. Materials. Lyophilized powdery Aβ42 (>95%) was obtained from GL Biochem (Shanghai, China). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), thioflavin T (ThT), and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). All other chemicals were the highest purity available from local sources. 2.2. Aβ42 Solution Preparation. The lyophilized Aβ42 was stored at -80ºC. The protein was added to HFIP at 1.0 mg/mL, and kept static at 4ºC for 2 h to make the protein (including pre-existing soluble aggregates) be dissolved in HFIP.20,21 The solution was then sonicated for 2 min, followed by centrifugation at 16,000g for 20 min at 4ºC, in order to remove any remaining aggregates. Top 3/4 of the supernatant was carefully collected and lyophilized by vacuum freeze-drying. The resulted freeze-dried Aβ42 was stored at -20ºC immediately. This pre-treated amyloid protein was regarded as monomers in the unstructured conformations.22,23 2.3. Thioflavin T (ThT) Fluorescence Assay. Immediately prior to use, pretreated Aβ42 was dissolved in 20 mM NaOH to a concentration of 275 µM, sonicated in ice bath, and then centrifuged at 16,000g for 20 min at 4ºC to remove the preformed aggregates. The top 3/4 of the supernatant was diluted into 20 mM HEPES buffer solution (150 mM NaCl, pH7.4) containing different concentrations of ZnCl2, resulting in a final Aβ42 concentration of 25 µM. The protein solution was incubated at 37ºC with constant shaking. At different time points, 200 µL of sample was taken and mixed thoroughly with 2 mL ThT (25 µM in 20 mM HEPES buffer solution, pH7.4). The fluorescence intensity values were measured by using a LS55 fluorescence spectrometer (Perkin Elmer, MA, USA), with excitation and emission wavelengths at 440 nm and 480 nm, respectively. Both the excitation and emission slit widths were 5 nm. Each fluorescence intensity value was the average of three replicates represented with its standard deviation, and the value of solution without Aβ42 was subtracted as background. In order to test if Zn2+ might quench the ThT fluorescence, Aβ42 fibrils 4


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preformed after incubation at 37 ºC for 24 h from 25 µM Aβ42 monomers were first mixed with ThT solution, and then Zn2+ was added to a final concentration of 25 µM to check the fluorescence changes. 2.4. Fast Kinetics Experiment. The SX20 stopped-flow apparatus (Applied Photophysics, Leatherhead, UK) was utilized for the fast kinetics studies. Pretreated Aβ42 was dissolved in DMSO to a concentration of 275 µM. After sonication in ice bath, the Aβ42 solution was drawn into one syringe of the stopped-flow apparatus as Solution A. The other syringe was filled with Solution B (20 mM HEPES buffer solution with predetermined ZnCl2 concentration, NaCl concentrations and pH value). Pure DMSO is an anti-aggregation solvent for Aβ, but its inhibiting effect fades after being diluted with an aqueous solution.21,24 Therefore, Aβ42 in Solution A kept monomeric state till mixing with Solution B (aqueous solution). One volume of Solution A and 10 volumes of Solution B were mixed rapidly in the stopped-flow apparatus, resulting in a final Aβ42 concentration of 25 µM. Instantly after the mixing, the Rayleigh light scattering intensity (R) was monitored with the instrument equipped with a 290 nm optical filter, and the excitation wavelength set at 435 nm. The tyrosine fluorescence intensity (F) was measured by setting the excitation and emission wavelengths at 276 and 303 nm, respectively. All the measurements were performed at 37ºC in triplicate, and the average value was reported. The data were collected and analyzed by using the Pro-Data software. 2.5. Kinetic Analysis. Rayleigh light scattering intensity (R) and tyrosine fluorescence intensity (F) reflect the aggregate size of Aβ42 and the conformational transition around Tyr10 in Aβ42, respectively.17 To examine the influence of a specific factor, X (Zn2+ or Na+) on Aβ42 aggregation, relative Rayleigh light scattering intensity (RX) and tyrosine fluorescence intensity (FX) are defined as, ,

=

(1)

,

,

=

(2)

,

where R∞,X and F∞,X are the Rayleigh light scattering intensity (R) and tyrosine fluorescence intensity (F) at equilibrium, respectively. R∞,X=0 and F∞,X=0 are the R∞,X and F∞,X values when the concentration of X is 0, respectively. Each of the four parameters at equilibrium was obtained from the average value of the last 2000 readings in the corresponding kinetic curve measured by the stopped-flow apparatus. The ratio of the R (or F) value in the presence of 25 µM Zn2+ to that without Zn2+ 5



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(η) was used to illustrate the influence of 25 µM Zn2+ on Aβ42 aggregation. = =

,

(3a)

, ,

(3b)

,

where R∞,25Zn and F∞,25Zn are the R and F values at equilibrium with 25 µM Zn2+, respectively, while R∞,0Zn and F∞,0Zn are the R and F values at equilibrium, respectively, under the same conditions except that Zn2+ concentration is 0. Besides, the kinetic curves determined by the stopped-flow spectroscopy were fitted to the multi-exponential equations that describe aggregation of different kinetic phases,25,26 = + ∑ exp (− )

(4a)

= + ∑" " exp (−" )

(4b)

where Rt and Ft represent the R and F values at time t, respectively, R∞ and F∞ are the R and F values at infinite time, respectively, kRi (or kFj) is the rate constant of phase i (or j), ARi (or AFj) is the amplitude of phase i (or j), and t represents the time after mixing (s). The rate constants fitted from the kinetic traces were averaged and reported with standard deviations.

3. RESULTS AND DISCUSSION 3.1. Zn2+ Distinctly Influences Aβ42 Aggregation in Different Time Scales. ThT is a kind of fluorescent dye that can bind into the hydrophobic grooves on the cross-β-sheet aggregates.27,28 Once binding to Aβ, ThT emits enhanced fluorescence at 480 nm with an excitation at 440 nm. Therefore, the intensity of ThT fluorescence reflects the aggregation state of Aβ. Because Zn2+ did not show a quenching effect on the ThT fluorescence of Aβ42 fibrils (Figure S1), Aβ42 was first co-incubated with different concentrations of Zn2+. The variations of ThT fluorescence intensities over time are exhibited in Figure 1. As shown in Figure 1A, Zn2+ inhibited the fluorescence intensity in a concentration dependent manner. This is because Zn2+ redirected the pathway of Aβ42 aggregation, leading to the formation of non-fibrillar aggregates.29,30 By viewing the aggregation of the first 2 h (Figure 1B), however, it can be seen that Zn2+ enhanced the ThT fluorescence intensity as well as the aggregation velocity of Aβ42, which indicates that Zn2+ accelerated the formation of β-structured Aβ42 aggregates in a short time scale. The enhancement of ThT fluorescence intensity occurred at the very beginning of the test, suggesting that the interaction between Zn2+ 6


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and Aβ42 was so quick that it took place within the dead time of the ThT fluorescence assay. Since the Zn2+ “pulses” released during neurotransmission also exist in seconds,14 the fast aggregation kinetics deserve a detailed examination by using stopped-flow spectroscopy, to study the interaction between Zn2+ and Aβ42 within a short time frame.

Figure 1. Effect of Zn2+ concentration on Aβ42 aggregation kinetics detected by ThT fluorescence. (A) represents the dynamics at the full time range; (B) represents the data of the first 2 h. The final concentration of Aβ42 was 25 µM, while the final concentrations of Zn2+ were 0, 12.5 and 25 µM in the three incubations. Solid lines are drawn to guide the eye. 3.2 Fast Aggregation Behavior Affected by Zn2+. The fast aggregation behavior of Aβ42 was monitored by stopped-flow spectroscopy with the Rayleigh light scattering and tyrosine fluorescence intensities. Figure 2A shows immediate and significant increase of Rayleigh scattering intensity upon the addition of Zn2+, and the 7



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final intensity increased with increasing Zn2+concentration. Since Rayleigh light scattering intensity reflects the aggregate size of Aβ, the results indicate that Zn2+ accelerated Aβ42 aggregation and increased the size of aggregates with increasing Zn2+concentration. It was reported that the binding between Zn2+ and Aβ42 plays a role as bridging between Aβ42 molecules,31,32 so this bridging effect of Zn2+ could be one of the reasons that lead to the enlargement of Aβ42 aggregates. Moreover, the first few seconds of aggregation (Figure 2B) revealed that under the influence of Zn2+, the initial Rayleigh scattering intensity was higher than that without Zn2+. This indicates that the promotion effect of Zn2+ on Aβ42 aggregation started within the dead time of the stopped-flow apparatus (1 ms). Comparing with Noy’s work,17 in which less aggregation-prone Aβ40 was used, the results herein revealed that in the presence of Zn2+, Aβ42 aggregated much faster than Aβ40. Along with previous report,19 this finding further confirms that Zn2+ had greater influence on the aggregation of Aβ42 than of Aβ40.

Figure 2. Kinetic curves of Rayleigh scattering intensity during Aβ42 aggregation with different concentrations of Zn2+. (A) represents the data in the full time range (15 s) and (B) represents the data within the initial 3 s.

In Aβ42 sequence, there is only one tyrosine residue (Tyr10) that is capable of generating intrinsic fluorescence, so the tyrosine fluorescence of Aβ42 reflects the conformational transition around Tyr10. The change of tyrosine fluorescence took place quickly and kept stable afterward (Figure S2), so the results within 10 s are reported in Figure S3 for clearer view. It is seen that Zn2+ altered Aβ42 conformation around Tyr10. This observation is consistent with literatures,33-35 which reported that Zn2+ bound with His6, His13 and His14 in Aβ. Because Tyr10 resides between His6 and His13, the neighboring conformation could be readily affected by the binding of Zn2+. Besides, Figure S3 shows that the variation of tyrosine fluorescence intensity 8


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was almost complete within the dead time, implying that the conformational transition around Tyr10 was very quick, and it might be a cause of the fast aggregation of Aβ42 induced by Zn2+. To exclude the influence of other factors such as the NaCl concentration, and to embody the impact of Zn2+ concentration on Aβ42 aggregation, RX defined by equation (1) was introduced for analysis. As shown in Figure 3A, the RX values were always greater than unity, and became larger as Zn2+ concentration increased. This indicates that Zn2+ enlarged the aggregate size of Aβ42 in a concentration dependent manner. Moreover, the RX value increased faster when Zn2+ concentration was less than 25 µM. In other words, Zn2+ had greater impact in promoting Aβ42 aggregation at Zn2+/Aβ42

Kinetic Insights into Zn2+-Induced Amyloid Î²-Protein Aggregation

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