Structural Insights into the Inhibition of Amyloid Fibril Formation by

Subscriber access provided by Bethel University

Article

Structural Insights into the Inhibition of Amyloid Fibril Formation by Fibrinogen via Interaction with Prefibrillar Intermediates Naoki Yamamoto, Taiki Akai, Rintaro Inoue, Masaaki Sugiyama, Atsuo Tamura, and Eri Chatani Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00439 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Structural Insights into the Inhibition of Amyloid Fibril Formation by Fibrinogen via Interaction with Prefibrillar Intermediates Naoki Yamamoto†§, Taiki Akai†, Rintaro Inoue ‡ , Masaaki Sugiyama ‡ , Atsuo Tamura†, and Eri Chatani†* †Graduate

School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501,

Japan ‡Institute

for Integrated Radiation and Nuclear Science, Kyoto University, 2, Asashiro-Nishi,

Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan

*To

whom correspondence should be addressed: Eri Chatani: Graduate School of Science, Kobe

University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan; [email protected]; Tel. +81-78-803-5673, FAX: +81-78-803-6442.

Table of Contents Graphic

Keywords: protein aggregation, small-angle X-ray scattering (SAXS), amyloid, protein misfolding, chaperone, amyloid intermediates, amyloid fibril inhibitor 1 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Abnormal protein aggregation tends to result in the formation of β-sheet rich amyloid fibrils, which are related to various kinds of amyloidoses and neurodegenerative diseases. The susceptibility to aggregation of protein molecules is dealt with by proteostasis in living systems, in which molecular chaperones play an important role. Recently, several secreted proteins have been focused on as extracellular chaperones with a potency to suppress the formation of amyloid fibrils, although a whole picture including their inhibition mechanisms has not been understood so far. In this study, we investigated the inhibitory effect of fibrinogen (Fg), one of the extracellular proteins referred to as a potential member of chaperones, on fibril formation. Insulin B chain was used as an amyloid-formation model system because its prefibrillar intermediate species in the nucleation phase were well characterized. We revealed that Fg efficiently inhibited the amyloid fibril formation via a direct interaction with the surface of the prefibrillar intermediates. Smallangle X-ray scattering experiments and a stoichiometry analysis suggested a structure model where the surface of the rod-shaped prefibrillar intermediates is surrounded by Fg molecules. From such a specific manner of interactions, we propose that the role of Fg is to disturb fibril growth by confining the nuclei even when the nucleation occurs inside the prefibrillar intermediate. The structural property of the B-chain intermediates complexed with Fg would provide insights into general principles of functions of chaperones and other potential chaperone-like proteins involved in amyloid-related diseases.

2 ACS Paragon Plus Environment

Page 2 of 47

Page 3 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION Protein misfolding often results in accumulation of abnormal aggregates of proteins that are related to amyloidoses and neurodegenerative diseases 1, 2. To prevent the occurrence of such diseases, misfolded proteins have to be quickly repaired or eliminated. The normality of protein functions in proteostasis is maintained by activating stress-responsive signaling pathways where a large number of molecular chaperones play critical roles

3-5.

Various kinds of intracellular

chaperones, many of which are heat shock proteins (Hsps), have been identified to contribute to refolding of misfolded proteins in cytoplasm, or in endoplasmic reticulum of eukaryotes 3, 6-8. While the majority of chaperones play a role in facilitating correct folding of just-translated proteins or in the recovery of misfolded proteins to prevent aggregation, some of them, e.g., Hsp104/ClpB, can act as a protein disaggregation machinery 9, 10. Furthermore, a deep involvement of the chaperone systems in amyloidoses and neurodegenerative diseases has also been proposed by a considerable accumulation of experimental evidence. For example, α-crystallin is proved to interact with fibril forming proteins

11.

Hsp70 and Hsp90 working together with their co-chaperones inhibit fibril

formation and oligomerization that are related to the neurodegenerative diseases, as reported by investigations on model organisms of neurodegenerative diseases 12. Human Hsp70, a member of Hsp70 family, is known to serve as a powerful disaggregase system for Parkinson’s disease-related α-synuclein amyloid fibrils together with DNAJB1 and Apg2 13. Moreover, remarkable capability of endoplasmic reticulum chaperones to prevent the secretion of toxic aggregates has also been found 7. In addition to the above-mentioned intracellular chaperones, an increasing number of secreted proteins, such as clusterin, haptoglobin, and α2-macroglobulin, have recently been proposed as extracellular chaperones, which prevent the formation of amyloid fibrils 14. In an in 3 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 47

vivo model system, it is revealed that a complex of clusterin with misfolded proteins is delivered to intracellular lysosome for degradation, suggesting the anti-amyloidosis action

15.

A strong

association of extracellular chaperones with Alzheimer’s disease is also demonstrated from the analysis of an Alzheimer’s diseases-model mouse crossed to clusterin knockout mice

16.

It is

reported that amyloid fibril formation of amyloid β (Aβ) peptide, calcitonin or lysozyme is inhibited by α2-macroglobulin or haptoglobin 17. Furthermore, a differential proteomics analysis shows that several extracellular chaperones are over-expressed in human plasma of transthyretin amyloidosis patients, implying relationships between these putative extracellular chaperones and amyloidoses or neurodegenerative diseases

18.

Exploring molecular functions of these and other

potential extracellular chaperones is important not only for understanding the molecular mechanism underlying anti-fibrillation activities but also for paving ways for clinical treatments of amyloid-related diseases. The aim of this paper is to scrutinize the inhibition mechanism of the amyloid fibril formation by fibrinogen (Fg), a candidate for the extracellular chaperones 14. Fg is the third most populated protein in mass in plasma, next to albumin and γ-globulin 19, which is typically known to participate in the blood coagulation initiated by external injuries. It possesses a rod-like tertiary structure and it is composed of two α, β, and γ domains, respectively, which are covalently connected by disulfide bonds with each other, resulting in the molecular mass of ~340 kDa 20. It is reported by an in vitro study that human Fg shows inhibitory activities against the thermal-induced aggregation of citrate synthase and the fibril formation of a yeast prion protein Sup35(NM)

21.

Furthermore, proteomic analyses prove that Fg is an interaction partner with transthyretin in plasma and that the glycation of Fg, which occurs prominently in patients of familial amyloidotic polyneuropathy, decreases its chaperone activity 22. Recently, it is shown that fibril formation of


Page 5 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

CsgA is inhibited by Fg

23.

Because many of plasma proteins have been suggested to prevent

amyloid fibril formation 14, it would be interesting to clarify molecular mechanisms underlying the inhibition of the fibril formation by Fg. However, any structural information about interactions requisite for the inhibition has not been provided yet, because of the complexity of fibril-formation pathways in many cases. Here, by using the amyloid fibril formation of insulin B chain, we investigated molecular mechanisms of the inhibition of fibril formation by Fg. We previously identified a pathway of an amyloid fibril formation of insulin B chain via prefibrillar intermediates, in which a monomeric insulin B chain first associates to organize the first prefibrillar intermediate and then a conversion to the second prefibrillar intermediate responsible for nucleation occurs

24.

Because these

intermediate species that precede the nucleation and subsequent elongation of mature amyloid fibrils are well characterized, insulin B chain can be used as a good model system to understand the mechanisms of inhibition, especially for the nucleation phase. In this paper, the insulin B chain fibrillation reaction in the presence of Fg was scrutinized using circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and size exclusion chromatography (SEC) combined with thioflavin T (ThT) assay. Furthermore, the interaction model was constructed based on smallangle X-ray scattering (SAXS) profiles and a titration experiment by proton nuclear magnetic resonance (NMR) spectroscopy. Based on the results, we concluded that the inhibition of the fibril formation by Fg is achieved via a specific interaction with the surface of the B-chain prefibrillar intermediates at a submicromolar-order dissociation constant.

MATERIALS AND METHODS Preparation of B chain and Fg.

B chain was prepared from human insulin (Wako Pure


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 47

Chemical Industries, Ltd., Japan; UniProtKB P01308) as described in a previous study 24. The Bchain solution dissolved in 10 mM NaOH was stored at -80 °C before use. The concentration of B chain was determined by using the absorption coefficient of 0.90 (mg/mL)-1cm-1 at 280 nm

24.

Purity of B chain was confirmed by using 1H-NMR as described 24. Fg from bovine plasma (Wako Pure Chemical Industries, Japan), which consists of α chain (UniProtKB P02672), β chain (UniProtKB P02676), and γ chain (UniProtKB P12799), was used without further purification except for the measurements of SEC, DLS, and SAXS. In the analysis of DLS, SEC, and SAXS, Fg was purified by using SEC as described later in the part of SEC, so that large aggregation components did not interfere with the experimental results. The ability of Fg to inhibit the amyloid fibril formation did not change depending on the presence or absence of purification (data not shown).

Formation of B chain amyloid fibrils / prefibrillar intermediates.

The purified B-chain stock

solution in 10 mM NaOH was diluted using Fg or buffer solution containing 50 mM Tris-HCl whose pH value was 8.7 and 10 mM HCl. These buffer conditions were permanently used in this paper unless otherwise noted and the peptide or protein concentration prior to the mixing were adjusted to appropriate values so that their final concentrations would become desirable values. The final concentration of B chain was set to 1.4 mg/ml (400 μM), and that of Fg varied ranging from 0 to 3.5 mg/ml (0 to 10 μM) depending on analyses. The sample solutions were then incubated at 25 ºC under agitated or quiescent conditions.

In the case of agitated conditions, the samples

were mixed in a circular movement at 1,200 rpm using ThermoMixer C (Eppendorf, Hamburg, Germany). The formation of prefibrillar intermediates or amyloid fibrils was monitored using the fluorescent dye, ThT, as previously reported 24.


Page 7 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Atomic force microscopy (AFM).

5 μL of a sample was loaded to a mica plate, left for one

minute, and then rinsed using 1 ml of Milli-Q. We used this normal sample preparation method unless otherwise indicated. AFM images were obtained using the dynamic force mode with Probestation NanoNavi II/IIe (SII Nanotechnology, Japan). The sweep rate was set to 0.5 or 1.0 Hz with the recording of 256 × 256 points per image.

CD spectroscopy.

CD spectra were recorded using the CD spectrometer, J-720 or J-1100

(JASCO, Japan) from 250 to 190 nm. Liquid samples were placed in a quartz cell with a path length of 0.2 mm. The path length was short enough to prevent the HT (high tension) from exceeding 700 volts even in the presence of the highly-concentrated Fg of 3.5 mg/ml. Each scan was performed at 100 nm/min or 200 nm/min, and several individual scans were integrated and averaged to obtain one spectrum at 25 ºC. The mean residue molar ellipticity, [θ], which is molar ellipticity normalized by the number of amino acid residues, was calculated as follows; [ ] 

100 / deg  cm 2  dmol 1 clN aa

-(1)

where θ, c, l, and Naa represent experimentally-obtained ellipticity (in deg), the molar concentration of a sample (in M), the path length of a cell (in cm), and the number of amino acid residues, respectively. In the measurement of B chain incubated with Fg, the spectrum of Fg was subtracted to observe net changes in the structure of B chain, assuming that the structure of Fg is unchanged (see also RESULTS). For the analysis of time dependence, mean molar ellipticity at 216 nm ([θ216]) was plotted against reaction time and then fit using the following biexponential function;

( ) 𝑡

( ) 𝑡

[𝜃]216(𝑡) = [𝜃]2 ―([𝜃]1 ― [𝜃]0)exp ― 𝜏1 ―([𝜃]2 ― [𝜃]1)exp ― 𝜏2 -(2) where τi and [θ]i represent the apparent time constants of the ith phase and the asymptotic value of


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 47

molar ellipticity after the completion of the ith phase, respectively.

DLS.

In order to estimate particle sizes, DLS experiments were performed using Zetasizer Nano-

S (Malvern Instruments, Worcestershire, UK) or a ALV system consisting of a 22 mW He−Ne laser, an avalanche photodiode mounted on a static/dynamic compact goniometer, ALV/LSE-5003 electronics, and an ALV-5000 correlator (ALV, Langen, Germany). In the measurements with Zetasizer Nano-S, a He-Ne laser at a wavelength of 633 nm was introduced to a sample cuvette and back scattered light was detected using an avalanche photodiode at a scatter angle of 173° at 25 °C. Data collection and processing were performed using the software, Dispersion Technology Software 5.00 (Malvern Instruments Ltd., UK). The measurements with the ALV system were carried out at the scattering angle of 90° at 25 °C where data collection and processing were performed using the software, ALV-Correlator Software Version 3.0. CONTIN analysis was used for obtaining size distribution, and the hydrodynamic diameter, Dh, was calculated by DLS-derived diffusion coefficient of the solutes, DT, based on Stokes-Einstein-Debye equation;

Dh 

k BT 3 0 DT

-(3)

where kB, T, and η0 represent the Boltzmann constant, temperature, and viscosity, respectively. The value of the viscosity, η0, was set to be that of the buffer solution, i.e. 0.94 mPa·s.

SEC.

For purifying Fg and monitoring the interaction between B chain and Fg, SEC was

performed using a column, HiPrep 16/60 Sephacryl S-300 HR (GE Healthcare, Japan) equipped with ÄKTA Start (GE Healthcare, Japan). For the purification of Fg, the Fg powder of 20-30 mg was dissolved in 1ml buffer solution of 100 mM Tris-HCl and 10 mM HCl (pH 8.5). The solution was centrifuged at 21,500 ×g and the supernatant was applied for the SEC. The elution was 8 ACS Paragon Plus Environment

Page 9 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

performed with the same buffer. A main elution peak was collected and the purity was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For the analysis of the interaction between B chain and Fg, a mixture of 1.4 mg/ml B chain and 3.5 mg/ml Fg were incubated for 2 hours under quiescent conditions and 1 ml of the reaction mixture was injected as a sample, which was then eluted with the same methods as that used for the purification of Fg.

Titration experiment monitored by 1H-NMR.

The number of Fg bound to B chain was

monitored by a titration experiment monitored by NMR. 1.4 mg/mL B chain solution mixed with several concentrations of Fg (0-10 mg/mL, i.e., 0-29 μM) was prepared and incubated under quiescent conditions for 2.5 h at 25 °C. Then, the solution was diluted by 4-fold using a same buffer solution, and after 5-min incubation, a 1H-NMR spectrum was measured. The NMR experiments were performed as described in our previous work

24

with minor modifications. Briefly, the

measurements were performed using AVANCE III HD (Bruker, Germany) with the Lamor frequency of 1H of 400.13 MHz. The spectra were recorded using zgesgp pulse program which was pre-installed in the control software, Topspin (Bruker, Germany). The amount of Fg-unbound B chain, or monomeric B chain, is known to be monitored by two peaks originating from two ε protons of histidine residues in B chain 24. However, some of resonant peaks originating from Fg completely overlapped with one of the peaks and the other was also partially eclipsed (see also RESULTS). Therefore, a spectral deconvolution was performed using three Lorentzian functions to obtain the peak area of the former ε proton of a histidine side chain. A curve fit was performed to deconvolute the partially-eclipsed histidine peak using the equation below; 　　　　　　　　　 3 Ak I     　　　　　　　　　　  a  b -(4) 2 k 1       k k


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 47

where I(δ) is peak intensity at the frequency δ, and Ak, δk, and γk represent the center amplitude, center frequency, and damping factor of the NMR peak being focused on. The peak area and full width at half-maximum (FWHM) were described as πAk/γk and 2γk, respectively. The second and the third terms in the right side of the equation represent a baseline. A resultant titration curve was analyzed based on the biomolecular interaction model 25, 26. In this model, one Fg molecule binds to a binding site of the B chain intermediate composed of n monomers. The reaction scheme is represented as; (B chain) + Fg ⇄ (B chain) -Fg n

n

where (B chain)n, Fg and (B chain)n-Fg represent the free binding site in the B chain intermediate, the free Fg, and a complex of the B chain intermediate and Fg, respectively; n denotes the number of B chain constituting the binding site for a single Fg. In this scheme, the apparent Kd is defined as; 𝐾d =

[B chain(free)][Fg(free)] 𝑛[(B chain)𝑛 ― Fg]

-(5)

where [B chain(free)], [Fg(free)], and [(B chain)n-Fg] represent concentrations of the free B chain, the free Fg, and the complex, respectively. A titration curve was fitted using the equation below; 𝐴([Fg]0) = 𝐴0 ―(𝐴0 ―𝐴∞)𝑓

-(6)

where f 

(B chain) n  Fg B chain(free)  (B chain) n  Fg

.

2    n[Fg]0 nK d  1 n[Fg]0 nK d 4n[Fg]0     1    1    2 [B]0 [B]0 [B]0 [B]0  [B]0    

A([Fg]0) represents the NMR peak intensity at an arbitrary Fg concentration, and A0 and A∞ represent peak intensities in the absence of Fg and at infinite Fg concentrations, respectively. f


Page 11 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

represents a fraction of B chain interacting with Fg where [B]0 and [Fg]0 represent total concentrations of the B chain and Fg, respectively. As the titration curve showed, some of B chain remained to be unbound to Fg in the high-concentration limit. Therefore, the total concentration of B chain participating in the interaction was corrected by [B]0{(A0-A∞)/A0} and this value was used instead of [B]0. All fittings were carried out using IGOR Pro 6.37 (WaveMetrics, Inc.).

Small-angle X-ray scattering.

The SAXS profiles were obtained by using NANOPIX (Rigaku

Corporation, Japan). Cu Kα line was used as the beam source and the camera length was set to 1.33 m. 1.4 mg/mL B chain solution was pre-incubated under quiescent conditions for 2 h at 25 °C, and then a concentrated Fg solution was added to the B chain prefibrillar intermediates formed. In this case, the final concentration of Fg and B chain was 3.5 mg/ml and 1.27 mg/ml, respectively. Sample liquid was introduced to a tailor-made sample cell which were composed of a pair of quartz windows with the path length of ~1 mm. The temperature was kept at 25 ºC using circulating water. A scattering pattern was collected for 30 min and then integrated. The scattering vector q (q = 4πsin(θ/2)/λ, where λ and θ indicate X-ray wavelength and the scatter angle, respectively) ranged from 0.006 to 0.23 Å-1. The Porod region of the log-log plots was analyzed using the equation below 27; log 𝐼(𝑞) = log 𝐼(0) + 𝑎 ∙ log (𝑞) -(7) where I(0) and a represent the intensity at q = 0 and the slope of a log-log plot, respectively. The cross-section plots for cylindrical shape were analyzed using the equation below 27, 28; 𝑅2𝑐

log {𝐼(𝑞) ∙ 𝑞} = 𝐴 ― 2 𝑞2 -(8) where A represents the constant at q = 0 limit, and Rc represents the base radius of gyration, or the radius of gyration of cross-section. The maximum q value for the fitting region was determined 11 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 47

with the criterion Rc q < 1.3. The radius of a rod of the base radius of gyration Rc, i.e., Rp, is described as; Rp  2Rc .

-(9)

The length of prefibrillar intermediates, L, is obtained by Broersma’s relationship;

DT 

 L ln 3 0 L  Rp k BT

   

-(10)

where DT is the diffusion coefficient of the solutes obtained by DLS. kB, T, and η0 represent the Boltzmann constant, temperature, and viscosity, respectively.

RESULTS Inhibition of the B-chain amyloid fibril formation in the presence of Fg.

The capability of Fg

to inhibit the fibril formation was monitored using ThT fluorescence. Fig.1A shows time dependencies of ThT fluorescence intensitiy of insulin B chain in the presence or absence of Fg, and four representative results are shown. In this analysis, the formation of the amyloid fibril was accelerated by agitating the samples at 1,200 rpm, and the concentrations of B chain and Fg were set to be 1.4 mg/ml (400 μM of monomer equivalent) and 3.5 mg/ml (10 μM of monomer equivalent), respectively. These concentrations were used hereafter unless otherwise noted. In the absence of Fg, B chain first formed prefibrillar intermediates, as indicated by a slight increase in ThT intensity. A large increase in the ThT intensity was then observed at 30-80 min, indicating amyloid fibril formation in agreement with our previous work

24.

The fibril formation was

confirmed by the AFM image taken at 90 min (Fig. 1B). The uncertain time-delay of the abrupt increase in the intensity is probably due to slight differences in the sample condition, which is usually seen in amyloid fibril formation. However, the large increase was always observed between


Page 13 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. (continued)

30 and 80 min. In contrast to this, when Fg was added at the starting point of the reaction, only a gradual and slight increase of ThT intensity was observed without a large increase in the later time region (Fig. 1A). Even though the intensity in the early period appeared larger than that in the 13 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Effects of Fg on B chain fibrillation. (A) Time courses of ThT fluorescence intensities of B chain amyloid fibrillation. The concentrations of B chain and Fg were set to 1.4 mg/ml (400 μM) and 3.5 mg/ml (10 μM), respectively, and the reaction was performed under agitated conditions. Results of four repeated experiments are shown in the absence or presence of Fg, respectively. The inset shows a magnified view of the fluorescence time course. (B) AFM images for the clarification of morphology of the final products without (left) or with Fg (right). The reaction mixtures at 90 min in panel a were sampled and analyzed. The scale bars repr esent 1 µm. The heights of fibrils and granular particles were in the range of 4.5-15 nm and 2.5-7.0 nm, respectively. (C) Distribution of hydrodynamic diameters monitored by DLS. The results at 10, 20, 30, 60, and 90 min without or with Fg are shown. In the lower panel, the hydrodynamic diameter of Fg is also shown for reference. (D) Time-dependent changes of CD spectra without or with Fg. In the presence of Fg (lower panel), net changes in the structure of B chain are indicated by subtracting the spectrum of Fg from each experimental spectrum. (E) Raw CD spectra of B chain or B chain incubated with Fg after 30-min incubation, respectively. The raw CD spectrum of Fg is also shown. A simulated CD spectrum obtained by summing those of B chain alone and Fg alone is overlayed.

absence of Fg, the magnitude was still much lower than the intensity of the fibril. Consistent with this observation, no fibril-like structures were confirmed by AFM in the presence of Fg; instead, a large number of particles were observed in the image (Fig. 1B), indicating that B-chain prefibrillar intermediates were accumulated in the presence of Fg. It is interesting that some of the particles are not spherical, but more or less rod-like or ellipsoidal. In the SAXS experiment the detail of shapes of prefibrillar intermediates in the presence of Fg is discussed. The larger intensity in the presence of Fg in the early time period compared to that in the absence of Fg is possibly due to the higher affinity of ThT dye to a B chain-Fg complex than to B chain intermediates. The direct interaction between B chain and Fg will be examined later. The inhibition of the amyloid fibril formation by Fg was also confirmed by DLS measurements and CD spectra. Fig. 1C shows the time dependence of size distributions obtained by DLS measurements. In the absence of Fg, B chain first formed prefibrillar intermediates whose size was around 50 nm. Although a minor component was additionally observed at around 104 nm at 10 min, it was ignorable in terms of volume fraction. From 60 min further development of the size was observed, which is consistent with the fibril formation as indicated by ThT fluorescence and AFM results. On the other hand, in the presence of Fg, the size was kept at an average diameter


Page 14 of 47

Page 15 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

of 40 nm up to 90 min, supporting the interruption of amyloid fibril formation from the prefibrillar intermediates. CD spectra also showed similar tendencies. As shown in the upper panel of Fig. 1D, in the absence of Fg, the spectra initially showed a minimum position of ~208 nm up to 60 min and then the minimum was remarkably shifted to ~220 nm at 90 min. On the other hand, the spectra kept similar to that observed in the initial spectral change observed in the presence of Fg within the measurement period of 90 min as shown in the lower panel in Fig. 1D. Here, it should be noted that the spectrum of Fg, which possesses a typical α-helical shape 29, was subtracted. This operation was based on the assumption that the structural change of Fg was negligible upon the interaction with B chain. This presumption seems to be valid because the raw CD spectrum of the mixture of Fg and B chain was almost overlapped with a simulated spectrum obtained by adding a raw spectrum of Fg to that of B chain as shown in Fig. 1E. Consequently, although the measurable range was limited to 208 nm because the protein concentration increased due to the coexistence of Fg, the difference spectrum showed a similar shape with the spectrum of the prefibrillar intermediates formed without Fg during the measurement time (Fig. 1D, dotted black line). From this result, it was supported that the reaction stopped at the stage of the prefibrillar intermediates in the presence of Fg. In the previous study, it was revealed the structure of the prefibrillar intermediates was a mixture of a random-coil and β-sheet structure 24. Similarly, the result of the CD study indicates that the secondary structure of prefibrillar intermediates stabilized by Fg is also composed of a mixture of a random-coil and β-sheet structure.

Evidence of interaction between B-chain prefibrillar intermediates and Fg.

The

accumulation of a significant amount of the prefibrillar intermediates in the presence of Fg as suggested in Fig. 1 raises a possibility that Fg interacts with the prefibrillar intermediates. To clarify


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

this point, SEC was performed. A mixture of B chain plus Fg was prepared, and after 2-hour incubation under quiescent conditions, in which the incubation was performed without any agitation, the solution was applied to a HiPrep 16/60 Sephacryl S-300 HR column. In the previous study, the prefibrillar intermediates were shown to survive as a metastable state for a long time under quiescent conditions

24

and therefore, these conditions were used in the experiments

described hereafter to observe the prefibrillar intermediate states readily. The formation of a multitude of granular particles were confirmed by the AFM image after the incubation (Fig. 2A, left), and it was also confirmed that such granular particles were not observed in the case of Fg alone but only smaller particles were observed (Fig. 2A, right). As a result of elution, two major peaks, termed E1 and E2, were observed at around 38 and 42 ml, respectively, and a minor peak termed E3 was also observed at around 100 ml (red curve in Fig. 2B). E1 is almost identical to the void volume of the column (38 ml). From the comparison of this elution pattern with those of Fg only (black) and B chain only (blue) samples, the E1 peak was identified as a newly emerging peak derived from the complex of Fg and the Bchain prefibrillar intermediates. The result of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) verified that the E1 peak contained both Fg and B chain, and the E2 and E3 peaks, which were assumed to be unbound Fg and B chain, respectively, were also confirmed by SDS-PAGE (Fig. 2C). The 1H-NMR spectrum of Fg mixed with B-chain monomers, which was reproduced by a sum of individual B-chain and Fg spectra, eliminated another possibility that Fg might interact with B chain monomers (Fig. S1A). We also checked whether Fg interacts with the B-chain amyloid fibril by comparing 1H-NMR spectra of Fg with or without Bchain fibrils. The result suggested negligible interaction of Fg with amyloid fibrils (Fig. S1B) and therefore, it was concluded that Fg specifically interacts with the prefibrillar intermediates.


Page 16 of 47

Page 17 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. SEC analysis of the interaction between the prefibrillar intermediates of B chain (1.4 mg/ml) and Fg (3.5 mg/ml). (A) AFM images of the Fg-B chain complex in the quiescent condition (left) and Fg alone as a control (right). (B) SEC profile of the mixture of the B chain prefibrillar intermediates and Fg. The profiles of the prefibrillar intermediates and Fg are also shown as comparisons. The inset shows a magnified view of the low-absorption areas. E1, E2, and E3 indicate eluted fractions applied for SDSPAGE.(C) SDS-PAGE of the eluted fractions shown in panel a. Three bands observed between 75 and 50 kDa (indicated by *) are α, β, and γ chains of Fg. Some of these chains remained disulfide-bonded and provided additional bands at around 140 kDa and 100 kDa (indicated by #), which was especially eminent for the Fg sample mixed with the prefibrillar intermediates possibly due to prevention of the reduction of the disulfide bond by B chain. B chain, which possesses the molecular weight of ~3.4 kDa, exists at around the predicted position (indicated by †). M indicates the molecular mass marker. SDSPAGE was performed by a standard protocol at the acrylamide concentration of 12 % vol/vol.

It should be noted that any peaks corresponding to the prefibrillar intermediates were not detected in the elution pattern of B-chain only sample (blue curve in Fig. 2B); the experimental peak at around 100 ml as well as a satellite small peak at around 80 ml were assigned to monomeric and small oligomeric B chain, respectively, based on the elution volume. This observation is due to dissociation of the prefibrillar intermediates caused by dilution during the SEC analysis. The prefibrillar intermediates are marginally stable and in equilibrium with monomers under the conditions of 1.4 mg/ml B chain 24. Since sample solution was diluted severely during the elution 17 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 47

of SEC, it is thus explained rationally that the B chain concentration became less than the critical monomer concentration of the prefibrillar intermediates, resulting in dissociation. In contrast to this, a detectable amount of the Fg-bound prefibrillar intermediates survived even under the same elution conditions, suggesting that the dissociation of the prefibrillar intermediates was suppressed by the presence of Fg. There remains one question about whether Fg can suppress the fibrillation even after the prefibrillar intermediates are formed. To answer this question, Fg was added to B chain solution after the formation of the prefibrillar intermediates. As a result, no marked increase in ThT fluorescence intensity typical for the fibril formation was observed, only with a slight increase to a similar level to that when Fg was added to the B-chain solution from the beginning (Fig. S2A). Furthermore, the CD spectrum after the addition of Fg was similar to that of the sample in which Fg was co-incubated with B-chain from the beginning (Fig. S2B). These results indicate that the reaction stopped without any significant changes in the prefibrillar intermediate structures.

Effects of Fg on the formation of the prefibrillar intermediates.

In the previous study, a two-

step process involving the first and second prefibrillar intermediates was observed under quiescent conditions

24.

Here, we checked effects of Fg on the formation of these two prefibrillar

intermediates by tracing time-dependent changes in CD spectra (Fig. 3). The mean residue ellipticity of the Fg-B chain complex at 216 nm was monitored, and two time constants 21±3 min and 1,200±200 min, which are the resultant value ± S.D. calculated by curve fitting, were obtained (red plots). When these time constants were compared with those in the absence of Fg (black plots in Fig. 3; τ1 = 14±2 min and τ2 = 390±30 min, which are the resultant value ± S.D. calculated by curve fitting), the time constants, especially the latter one, appeared markedly longer than that


Page 19 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. Effects of Fg on the formation of the B-chain prefibrillar intermediates. The concentrations of B chain and Fg were set to 1.4 mg/ml and 3.5 mg/ml, respectively, and time courses of average molar ellipticity at 216 nm ([θ]216) without or with Fg were monitored under quiescent conditions. For the data with Fg, the [θ]216 value of Fg was subtracted at each time point. The inset shows a magnified view in the early time period. Solid lines represent fitted curves obtained by using eq. 2. In the absence of Fg; τ1 = 14±2 min and τ2 = 390±30 min with [θ]0, [θ]1, and [θ]2 of -5,900±200, -8,300±100, and -11,200±100 deg·cm2·dmol-1, respectively. In the presence of Fg; τ1 = 21±3 min and τ2 = 1,200±200 min with [θ]0, [θ]1, and [θ]2 of -5,700±300, -9,800±200, and -12,700±200 deg·cm2·dmol-1, respectively.

without Fg. These results imply that even in the presence of Fg, B chain forms first and second prefibrillar intermediates. However, the formation of the second prefibrillar intermediate is decelerated by Fg. Time-dependent measurements of DLS also implied that Fg slowed the development of the second prefibrillar intermediates (Fig. S3), qualitatively consistent with the result of the time course of the CD spectra. Furthermore, the values of mean residue ellipticity corresponding to the equilibrium points after the formation of the first and second prefibrillar intermediates in the presence of Fg (i.e., [θ]1, and [θ]2 of -9,800±200 and -12,700±200 deg·cm2·dmol-1, respectively, which are the resultant value±S.D. calculated by curve fitting) were slightly lower than that in the absence of Fg ([θ]1, and [θ]2 of -8,300±100 and -11,200±100 deg·cm2·dmol-1, respectively) (Fig. 3). It was therefore indicated that the fractions of the first and second prefibrillar intermediates were increased by the interaction with Fg.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Stoichiometry in the complex of the B chain prefibrillar intermediate and Fg.

Page 20 of 47

We next

estimated the number of Fg bound to the B chain prefibrillar intermediates by using 1H-NMR. It was impossible to detect NMR signals of the prefibrillar intermediates because of the large molecular weight, and it was thus difficult to monitor interactions between Fg and the prefibrillar intermediates directly. Here, we found that the binding of Fg to the prefibrillar intermediates prevented dissociation of the prefibrillar intermediates upon dilution; signals of histidine ε protons in 1H-NMR spectra fully recovered upon a 4-fold dilution of the 1.4 mg/ml B-chain prefibrillar intermediates (Fig. S4A), indicating the dissociation of the prefibrillar intermediates to monomers, whereas the recovery of the signal was suppressed in the presence of Fg (Fig. S4B). This result indicates that the amount of the B-chain prefibrillar intermediate bound to Fg is able to be estimated by the extent of the suppression of the signal recovery after the sample dilution. By taking advantage of the strong resistance to the dissociation upon the dilution in the presence of Fg, the titration experiment was performed on 1.4 mg/ml prefibrillar intermediates with increasing amount of Fg. No chemical exchange effects due to interactions of Fg with insulin B chain monomers need to be considered, since any spectral changes of B chain were not detected when Fg was added (Fig. S1A). Serious obstruction by some nonspecific aggregation can also be excluded in light of the SAXS result (see later section), in which it was demonstrated that the prefibrillar intermediate structure was kept even after the addition of Fg. Fig. 4A shows the result of the experiment, in which the NMR spectra were recorded after the sample was diluted by 4 times. The preparation of the samples was performed under quiescent conditions. Although two well-isolated peaks originating from ε protons in the histidine residues of the B chain peptide were observed at 7.705 ppm (termed peak 1) and 7.685 ppm (termed peak


Page 21 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4. NMR analysis for the binding of Fg to the prefibrillar intermediates of B chain. (A) A magnified view of signals of histidine ε protons. Dashed lines represent the fitted curves obtained by using eq. 4. (B) A titration curve obtained by tracing the area of peak 1 in panel a. Molar concentration corresponding to weight concentration of Fg is provided as an upper horizontal axis. Dashed line represents a fitted curve obtained by using eq. 6. The values of ThT intensity at 210 min observed in panel c is also plotted to obtain the binding stoichiometry of Fg to the prefibrillar intermediates with the level of inhibition of the fibril formation. (C) Time courses of the fibril formation in the presence of various kinds of concentrations of Fg as monitored by ThT. The concentration of B chain was 1.4 mg/ml (400 μM of monomer equivalent), and the reaction was performed under agitated conditions.

2) before the addition of Fg, some of resonant peaks probably originating from histidine ε protons of Fg (termed peak 3) severely overlapped with the peak 2 and also partly with the peak 1 in the presence of Fg. Then, a spectral deconvolution using three Lorentzian functions was performed to quantify net peak area of the ε proton of histidine side chains of the B chain. The resultant peak area of peak 1, which was successfully isolated from the overlap of the signals of free Fg, is shown in Fig. 4B as a function of the Fg concentration. The area initially decreased as the concentration 21 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of Fg increased and finally reached a plateau point. When the titration plot obtained was analyzed using eq. 6 where a single Fg molecule was assumed to bind to a site of B chain prefibrillar intermediates composed of n molecules of B-chain monomers (see MATERIALS AND METHODS), a good fitting was achieved as shown as a dashed line in Fig. 4B. The value of n obtained was 29±4, which means that ~30 B chain molecules associate with one Fg molecule. Based on the number and molecular mass of Fg and B chain (Fg; ~340 kDa, B chain; ~3.5 kDa), the mass ratio of the complex can also be calculated as Fg:(B chain)n = 340: 3.5×30 = 3.5:1.0. By using stoichiometry along with SAXS results as described later, the binding manner of Fg will be deduced in DISCUSSION. The apparent binding constant (Kd) was 880±660 nM, indicating that the binding is fairly strong. At the plateau point, 25 % of B chain remained as a monomer (Fig. 4B), which was smaller than that in the absence of Fg (40 % in the previous work) 24. This result indicates that the equilibrium shifted to the prefibrillar intermediates upon the interaction of Fg, in good agreement with the values of mean residue ellipticity [θ]1 and [θ]2 in Fig. 3, as well as the resistance to dissociation of the prefibrillar intermediates during the SEC experiment by the binding of Fg (Fig. 2). The titration curve clearly showed that the most amount of the prefibrillar intermediates formed the Fg-complex when the concentration of Fg is 3.5 mg/ml, representing that the concentration of Fg was enough to suppress the fibril formation. Then we next scrutinized if the extent of the Fg binding has any correlation with the level of inhibition of the fibril formation. We monitored the fibril formation by varying the concentration of Fg. As shown in Fig. 4C, final ThT values after the fibrillation reaction decreased as the Fg concentration increased, and furthermore, the timing where the marked increase in ThT fluorescence intensity started, i.e., a starting point of the fibril formation, was also delayed. When the ThT value at 210 min was plotted against the Fg concentration and compared with the amount


Page 22 of 47

Page 23 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

of free B chain as monitored by NMR, the final ThT fluorescence intensity was fairly proportional to the amount of free B chain (Fig. 4B), supporting the idea that the capability of Fg to inhibit the fibril formation is due to the complex formation with the prefibrillar intermediates.

Specification of reaction steps blocked by the interaction of Fg with the B-chain prefibrillar intermediates.

It was previously clarified that the second prefibrillar intermediates of B chain

can serve as a precursor of amyloid nucleation 24. Although they showed metastable features, they could become amyloid nuclei as an on-pathway species upon agitation. Interestingly, it was found that the fibril formation could be enhanced by applying a short pulse of ultrasonic wave. By taking advantage of the availability of the time-controlling of fibril formation using the ultrasonic pulse, specific points of Fg inhibition on the pathway of the B-chain amyloid fibril formation were sought out. When the ultrasonic treatment was performed on the second prefibrillar intermediates in the absence of Fg, the amyloid fibril formation was observed after a subsequent incubation (Fig. 5A), whereas any fibrillation was not observed in the presence of Fg (Fig. 5B). Although the spectra in the presence of Fg were slightly deviated downwards from that before the ultrasonic treatment, this is conceived to be due to the time-dependent increase in the amount of the prefibrillar intermediates, considering that they showed an isoelliptic point slightly below 210 nm; a similar behavior was also previously observed during the formation of the prefibrillar intermediates

24.

This result

indicated a possibility that the interaction of Fg with the prefibrillar intermediates inhibits fibrillation at a step of nucleation. However, a significant inhibition of fibrillation was also observed when Fg was added immediately after the ultrasonic pulse was application, i.e., even after the nucleation (Fig. 5C). It was thus concluded that the conclusive role of Fg is to prevent the participation of the nuclei in subsequent elongation, presumably by confining the nuclei inside the


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Effect of Fg on the amyloid fibril formation of B stimulated by the ultrasonic treatment (UT). B chain (1.4 mg/ml) was pre-incubated for 26 h to form the second prefibrillar intermediates, to which UT and/or Fg were added. In all the experiments, the final concentration of B chain or Fg was 1.24 mg/ml or 3.5 mg/ml, respectively. (A) CD spectra measured before and after UT to the prefibrillar intermediates in the absence of Fg. After the spectrum of the prefibrillar intermediates was measured (before treatment), UT was applied. The spectrum was recorded 67 h after the UT treatment (after UT). (B) CD spectra measured before and after UT to the prefibrillar intermediates in the presence of Fg. After the spectrum of the prefibrillar intermediates was measured (before treatment). Fg was added and then the spectrum was recorded (+Fg, before UT). Immedately after that, UT was applied. The spectrum was recorded 65 h after the UT treatment (+Fg, after UT). (C) CD spectra measured before and after the addition of Fg to the prefibrillar intermediates treated by UT. After the spectrum of the prefibrillar intermediates was measured (before treatment), UT was applied and then the spectrum was recorded (UT, before +Fg). Immediately after that, Fg was added. The spectrum was recorded 63 h after the incubation (UT, after +Fg).

prefibrillar intermediate. As for the question about whether Fg inhibited nucleation, it remained unanswered because of the present situation that a direct observation of nuclei formation has not been accomplished yet due to a too slight amount of nuclei produced by the ultrasonic treatment 24.


Page 24 of 47

Page 25 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

SAXS analysis of the complex of the B chain prefibrillar intermediate and Fg.

SAXS

measurements were performed to obtain structural insights of the complex composed of the B chain prefibrillar intermediate and Fg. For this purpose, the complex was prepared by adding Fg after the formation of the B-chain prefibrillar intermediates. Fig. 6A shows the scattering profiles of the SAXS measurements of the complex of Fg and the prefibrillar intermediates (green) as well as those of a free form of the prefibrillar intermediates (blue) and Fg only (black). As for the shape of the B-chain prefibrillar intermediate, it was indicated to possess a rod-like structure from a slope obtained by fitting the profiles using eq. 7

27,

which was close to -1 (gray lines in Fig. 6A),

suggesting that the prefibrillar intermediates kept a rod-like structure even when complexed with Fg. The slope of Fg was also close to -1, in good agreement with the long shape structure of human Fg, which is homologous to bovine Fg 20. The base radii of inertia of these rod-like structures, Rc, were then obtained by analyzing a cross-section plot of each profile as shown in Fig. 6B. From the Rc values obtained by linear fitting using eq. 8 (gray lines in Fig. 6B and Table 1), the apparent radius of a rod Rp could be calculated by using eq. 9, which is also shown in Table 1. The Rp value of Fg was ~25 Å, which was consistent with the reported value of the radius of human Fg, i.e., ~25 Å 20. The Rp value of the Fg-B chain complex was ~61 Å, which were significantly larger than that of the free form of the B-chain prefibrillar intermediate, ~33 Å. With the Rp values obtained by the above analyses, the length of the rod-like structures, L, were then estimated using Broersma’s relationship where a diffusion coefficient of a rod-like molecule is described by a function of the length and radius (eq. 10) 30, 31. The hydrodynamic diameter, which was obtained by DLS (Table 1, see also Fig. S5), was used for the calculation (see MATERIALS AND METHODS for detailed procedures). The obtained L values are summarized in Table 1. The length of Fg, 600 Å, was consistent with that of


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. SAXS profiles of the B chain-Fg complex as well as Fg and B chain. The concentration of B chain and Fg were 1.4 mg/ml and 3.5 mg/ml, respectively, except for the complex where the final concentration of B chain was 1.27 mg/ml because of the dilution by the Fg addition. (A) Log-log plots of the intensity vs the q value. Gray lines show the result of curve fitting performed using eq. 7. The black slopes of -1, -2, or -4 indicate guides for the eyes. (B) Cross-section plots for cylindrical shape. Gray lines show the result of curve fitting performed using eq. 8. The inset shows a magnified view in a low-q region for B chain + Fg.

the reported structure, i.e. ~500 Å 20, supporting validity of the estimation. Interestingly, the lengths of the free form of the prefibrillar intermediate and the Fg-complex were very similar (i.e., ~2,700 Å and ~2,900 Å, respectively), indicating that Fg binds to the side surface of the prefibrillar intermediates. The SAXS structural analysis was also performed on another Fg-B chain complex that was formed upon their simultaneous incubation, i.e. the same condition as performed in Fig. 1-5. As a result of the measurement 2 hours after the incubation, by which the complex formation almost


Page 26 of 47

Page 27 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table 1. Structural features obtained from SAXS and DLS measurements sample Fg B chain B chain + Fg

Rc /Å (fit range) 17.7±0.1 (0.01 < q < 0.056) 23.5±0.9 (0.01 < q < 0.04) 43.2±3.2 (0.01 < q < 0.016)

Rp /Å

Dh / Å

L /Å

25.0±0.1

190±4

600±20

33.2±1.3 610±40 2,700±200 61.1±4.5 760±10 2,900±500

The base radius of gyration, Rc, and the radius of a rod, Rp, was obtained by using eq. 8 and 9, respectively. The hydrodynamics diameter, Dh, was obtained by the DLS measurement, and the length, L, was calculated by using eq. 10. completed, a similar SAXS profile with a slope close to -1 was obtained as shown in Fig. S6, supporting that the complex possesses a rod-like structure. Interestingly, the length of the complex calculated by eq. 10 was 870 Å, which is about three times shorter than the former case, while the Rp value of the complex was almost similar (Fig. S6 and Table 1). This result suggests that Fg slowed down the elongation of the prefibrillar intermediates, which would correspond to the retardation of the formation of the second prefibrillar intermediates observed in the time-dependent changes in CD spectra (Fig. 3). On the other hand, the radius of the complex was 73.8±3.3 Å, which was similar to that of the former case (61.1±4.5 Å), supporting the idea that Fg interacts with the side surface of the prefibrillar intermediates.

DISCUSSION A chaperone-like activity of Fg to prevent amyloid fibril formation of insulin B chain.

From

the results of ThT fluorescence, CD, AFM, and DLS measurements, we have demonstrated the inhibition of amyloid fibril formation of insulin B chain by Fg. The most noteworthy finding of 27 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

this study is a specific interaction of Fg molecules with the surfaces of prefibrillar intermediates of B chain, which has been clarified by virtue of significant population of the prefibrillar intermediates in early stages of the fibrillation pathway. Similar to our case, a significant number of chaperones have been suggested to prohibit the amyloid fibril formation by interacting with prefibrillar intermediates rather than monomers or fibrils; the interaction of clusterin and several other Hsps with oligomers or protofibrils has been directly revealed by using transmitted electron microscopy 32,

surface plasmon resonance 33, immunoblotting 34 and SEC 35, 36. The fundamental similarity in

the inhibition manner of these chaperones with that of Fg suggests that Fg can serve a chaperonelike function against amyloid formation. Although it has often been difficult to scrutinize structural characteristics of prefibrillar intermediates complexed with chaperone proteins due to their transient and low stability, the prefibrillar intermediates of B chain were metastable and accounted for a major fraction of the total mass of B chain as far as kept quiescent, which allowed the application of SAXS analysis as discussed below. Prefibrillar intermediates often play important roles in early stages of amyloid fibril formation, and therefore, the structural property of the B-chain intermediates complexed with Fg will provide insights into general principles of functions of chaperones and chaperone-like proteins involved in amyloidoses and other neurodegenerative diseases.

Structural prediction of the complex of Fg and B-chain prefibrillar intermediates. Based on the SAXS results in addition to the stoichiometry by NMR, a possible model of Fg binding to the B chain prefibrillar intermediate has been constructed. From the SAXS experiment, it was clarified that the B-chain prefibrillar intermediates possessed a rod-like structure whose radius and length were ~33 Å and 2,700 Å, respectively (Table 1). The prefibrillar intermediate kept a rod-like


Page 28 of 47

Page 29 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

structure even after the binding of Fg. The radius of the rod of the Fg-B chain complex was ~61 Å (Table 1), which was larger than that of the free form of the prefibrillar intermediate. On the other hand, the length, ~2,900 Å, appeared almost similar (Table 1). Given that the radius of Fg was ~25 Å, the increase in the radius by the Fg binding can be explained reasonably when a single layer of Fg molecules surrounds the surface of the prefibrillar intermediates. When it is assumed that Fg molecules are aligned around the circumference of the prefibrillar intermediate with the long axis parallel to that of the prefibrillar intermediate, seven Fg molecules are supposed to surround the surface of the prefibrillar intermediate. Given that the length of the complex was ~2,900 Å, 4 to 5 repeats of the alignment of 7 Fg molecules, i.e., 28-35 Fg molecules, are estimated as the number necessary for covering the lateral surface of the prefibrillar intermediate. According to this model, the volume ratio of Fg to the B-chain prefibrillar intermediate is calculated to be 3.3-4.2. Under the assumption that mass densities of the prefibrillar intermediates and Fg are the same, the value would correspond the mass ratio. Interestingly, it was close to the mass ratio obtained by the NMRtitration experiment, i.e. ~3.3 (Fig. 4B), suggesting validity of this model. In the AFM images of Fg-B chain complex, the number of granular particles are much more frequently observed than rod-like objects (Fig. 1B and 2A). On the other hand, the SAXS experiments suggested that Fg-B chain complex possesses a rod-like structure. The discrepancy between the structural model constructed by SAXS and the result of the AFM is probably due to the washing process in the sample preparation for AFM; it was clarified that the prefibrillar intermediates are fragile and unstable against washing in the previous study

24.

Even if they are

complexed with Fg, the prefibrillar intermediate structure would not be resistant to washing, resulting in a partial disruption of the structure.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Possible scenarios of the inhibition of the B-chain amyloid fibril formation by Fg.

Page 30 of 47

With

schematic cartoons of the B-chain prefibrillar intermediates complexed with Fg molecules, a possible inhibition mechanism is summarized in Fig. 7. The B-chain fibrillation occurs via two prefibrillar intermediates; the first one is formed initially and then the second one appears later. The SAXS experiments have clarified that these prefibrillar intermediates possessed rod-like structures (Fig. 6). In the presence of Fg, the Fg molecules selectively interact with the surface of the prefibrillar intermediates. As for interactions contributing to the specific binding of Fg to the prefibrillar intermediates, higher hydrophobicity of the prefibrillar species than that of monomeric form was suggested from the measurement of fluorescence intensity of 8-anilinonaphthalene-1sulfonic acid, although marked difference in the intensity was not observed for amyloid fibrils (data not shown). As another possibiltiy, the previous analysis of binding motifs of DnaK by screening peptide sequences of its substrate proteins has suggested that chaperones tend to bind with hydrophobic residues in target proteins

37

and a sequence LVEALYL has been suggested a

recognition motif in insulin B chain 38. The same peptide region might be involved in the preferable interaction of Fg with the B-chain prefibrillar intermediates, although further investigation would be necessary to identify it. As for binding sites of Fg, it has been reported that the C-terminus domain of the α chain in human Fg has chaperon activity to Sup35 and transthyretin 21, 39. However, bovine Fg used in this study does not have the C-terminus domain, and it is thus suggested that the site of Fg used for the interaction with the B-chain prefibrillar intermediates would be different from that used for Sup35 and transthyretin. The interaction of Fg with the second prefibrillar intermediate would be responsible for the suppression in light of the previous knowledge that the second prefibrillar intermediate is crucial for nucleation 24. The interaction model where a layer of Fg molecules surrounds the side


Page 31 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 7. Schematic picture of the inhibition of the amyloid fibril formation of B chain by Fg. See details in the main text.

surface of the prefibrillar intermediate suggests that the inhibition of fibril formation would be ascribed to the prevention of monomer B chains from approaching the nuclei. Although it was difficult to verify whether Fg can also inhibit nucleation, there still remains a possibility that this mechanism also occurs to inhibit fibril formation. In addition, the deceleration of the formation of the prefibrillar intermediates, especially the second one, as suggested by the time course of CD spectra (Fig. 3) and DLS (Fig. S3), would also contribute to the inhibition of the fibril formation. As represented by equilibrium arrows, conformational conversions among the monomers and the prefibrillar intermediates of B chain have been proved reversible

24.

Interestingly, this

equilibrium appeared to shift towards the prefibrillar intermediates in the presence of Fg, as indicated by the resistance to dilution in SEC (Fig. 2), the increase in the absolute values of [θ]1 and [θ]2 in CD (Fig. 3), and the decrease in monomer concentration in the NMR measurement (Fig. 4). Also in the ThT fluorescence, the higher intensity in the early time period may be partly due to the increased population of the prefibrillar intermediates (Fig. 1A and S2); however, the increment


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 47

was still much higher than expected, which may be due to changes in ThT affinity and/or binding manners by the interaction of Fg to the prefibrillar intermediates. This observation complies with the holding model, which is one of the inhibition mechanisms as deduced from the study on the inhibition of Aβ1-42 by Hsp 70/40 and 90 40. According to this model, thermodynamic trapping of aggregation-competent species by the binding to the chaperone molecules results in decrease in the concentration of proteins available to the amyloid formation. Alternatively, if the Fg binding actually inhibits amyloid nucleation of B chain, the stabilization of the second prefibrillar intermediates might contribute to raising the nucleation energy barrier and relatively destabilizing the nucleation state.

Implication of the roles of Fg in the proteostasis system.

There are a considerable number of

reports demonstrating that prefibrillar aggregates play key roles in an early phase of amyloid formation

41-46.

In light of deep involvement of the prefibrillar aggregates in amyloid formation,

the current study on the Fg-mediated inhibition is significant in that it has advanced understanding of the inhibitory effect of amyloid formation targeting the prefibrillar aggregates. When we consider physiological significance, blocking undesirable fibrillation from the prefibrillar intermediates in intercellular spaces will be the most crucial role of Fg to prevent amyloid fibrilrelated diseases. However, there remains a concern that the thermodynamically trapped prefibrillar intermediates have an adverse effect on in vivo systems. An answer to this question may be hinted at by some examples of neutralization of toxic oligomers by the binding of molecular chaperones; α-synuclein oligomers were rendered harmless to neuroblastoma cell lines upon forming a complex with Hsp90

36,

and clusterin antagonized the toxicity in Caenorhabditis elegans by the


Page 33 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

preincubation with oligomers of Aβ1-42

33.

In a similar way, Fg may have an effect to diminish

cytotoxicity of prefibrillar aggregates in vivo, although direct evidence has not been obtained yet. It may also be possible that the Fg binding to the prefibrillar intermediates also promotes proteolysis by enhancing delivery to lysosomes or other intracellular degradation systems, as was observed for clusterin 15. The complex of Fg and the prefibrillar intermediates might also serve as a kind of reservoir to rescue aggregation-prone proteins; in the case of α-crystallin, a family of sHsps which possesses dynamic oligomers 47, it adsorbs aggregating target proteins to form a high molecular weight complex and sequesters until they are refolded back to the native state 11. Future investigations will be needed to address more detailed biological relevance. Very recently, studies on the formation of fibrin clots have found that Fg interacting with Aβ1-42 led to the formation of fibrin clots with an abnormal structure that were more resistant to plasmin-induced degradation

48, 49,

which provided an intriguing hypothesis that Fg is a critical

factor in Alzheimer’s diseases 50. When Fg intrudes in the nervous system via a disrupted blood brain barrier, the co-aggregation of Fg and Aβ peptides would realistically occur, which may be responsible for the accumulation of Aβ peptides in brain blood vessels known as cerebral amyloid angiopathy in the Alzheimer’s disease. If Fg interacts with oligomers or protofibrils composed of Aβ peptides, our present study might provide a hint for the molecular basis for the co-aggregation of Fg and Aβ peptides. Further studies across Aβ and other amyloid-prone proteins should be performed in order to examine the generality of the chaperone-like activity of Fg, and also negative effects. Further in vivo as well as in vitro studies have to be conducted to see the whole picture of the role of Fg on proteostasis.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACCESSION CODES human insulin

P01308

Fg from bovine plasma

P02672 (α chain); P02676 (β chain); P12799 (γ chain)

ASSOCIATED CONTENT Supporting Information (SI) The Supporting Information is available free of charge. Figure S1. Negligible interactions of Fg with insulin B chain in a form of monomers or amyloid fibrils. Figure S2. Inhibition of fibril formation of B chain by the addition of Fg after the preformation of prefibrillar intermediates. Figure S3. Time dependence of the hydrodynamic diameter in the formation of the B-chain prefibrillar intermediates of B chain with or without Fg. Figure S4. Prevention of dissociation of the B chain prefibrillar intermediates complexed with Fg upon dilution. Figure S5. Hydrodynamic diameter distributions of the prefibrillar intermediates of B chain complexed with Fg. Figure S6. SAXS analysis of Fg-B chain complex that was formed by a simultaneous incubation of Fg and B chain.

AUTHOR INFORMATION Corresponding Author *Email:

[email protected].


Page 34 of 47

Page 35 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Present address §Division

of Biophysics, Physiology, School of Medicine, Jichi Medical University, 3311-1

Yakushiji, Shimotsuke, 329-0498, Japan Funding This work was funded by JSPS KAKENHI Grant Numbers JP16H04778, JP16H00772, JP17H06352, JP16K17783, JP26288079, JP15K13289, JP18H05229, and JP17K07361. Notes The authors declare no competing financial interest.

ACKNOWLEDMENTS The SAXS measurements were carried out under the Visiting Researchers Program of Institute for Integrated Radiation and Nuclear Science, Kyoto University. The SAXS measurements were supported by the Project for Construction of the Basis for the Advanced Materials Science and Analytical Study by the Innovative Use of Quantum Beam and Nuclear Sciences at Institute for Integrated Radiation and Nuclear Science, Kyoto University. This work was supported by JSPS Core-to-Core Program, A. Advanced Research Networks.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES [1] Dobson, C. M. (2003) Protein folding and misfolding, Nature 426, 884-890. [2] Landreh, M., Sawaya, M. R., Hipp, M. S., Eisenberg, D. S., Wuthrich, K., and Hartl, F. U. (2016) The formation, function and regulation of amyloids: insights from structural biology, J. Intern. Med. 280, 164-176. [3] Wickner, S., Maurizi, M. R., and Gottesman, S. (1999) Posttranslational quality control: folding, refolding, and degrading proteins, Science 286, 1888-1893. [4] Lindquist, S. L., and Kelly, J. W. (2011) Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: progress and prognosis, Cold Spring Harbor Perspectives in Biology 3. [5] Kim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M., and Hartl, F. U. (2013) Molecular chaperone functions in protein folding and proteostasis, Annu. Rev. Biochem. 82, 323-355. [6] Grimminger-Marquardt, V., and Lashuel, H. A. (2010) Structure and function of the molecular chaperone Hsp104 from yeast, Biopolymers 93, 252-276. [7] Vincenz-Donnelly, L., Holthusen, H., Korner, R., Hansen, E. C., Presto, J., Johansson, J., Sawarkar, R., Hartl, F. U., and Hipp, M. S. (2018) High capacity of the endoplasmic reticulum to prevent secretion and aggregation of amyloidogenic proteins, EMBO J. 37, 337-350. [8] Landreh, M., Rising, A., Presto, J., Jornvall, H., and Johansson, J. (2015) Specific chaperones and regulatory domains in control of amyloid formation, J. Biol. Chem. 290, 26430-26436. [9] Winkler, J., Tyedmers, J., Bukau, B., and Mogk, A. (2012) Chaperone networks in protein disaggregation and prion propagation, J. Struct. Biol. 179, 152-160. [10] Nillegoda, N. B., Wentink, A. S., and Bukau, B. (2018) Protein disaggregation in multicellular organisms, Trends Biochem. Sci. 43, 285-300. [11] Ecroyd, H., and Carver, J. A. (2009) Crystallin proteins and amyloid fibrils, Cell. Mol. Life Sci. 66, 62-81. [12] Lackie, R. E., Maciejewski, A., Ostapchenko, V. G., Marques-Lopes, J., Choy, W. Y., Duennwald, M. L., Prado, V. F., and Prado, M. A. M. (2017) The Hsp70/Hsp90 chaperone machinery in neurodegenerative diseases, Front. Neurosci. 11, 254. [13] Gao, X., Carroni, M., Nussbaum-Krammer, C., Mogk, A., Nillegoda, N. B., Szlachcic, A., Guilbride, D. L., Saibil, H. R., Mayer, M. P., and Bukau, B. (2015) Human Hsp70 disaggregase reverses Parkinson's-linked α-synuclein amyloid fibrils, Mol. Cell 59, 781793. [14] Wyatt, A. R., Yerbury, J. J., Dabbs, R. A., and Wilson, M. R. (2012) Roles of extracellular chaperones in amyloidosis, J. Mol. Biol. 421, 499-516. 36 ACS Paragon Plus Environment

Page 36 of 47

Page 37 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[15] Wyatt, A. R., Yerbury, J. J., Berghofer, P., Greguric, I., Katsifis, A., Dobson, C. M., and Wilson, M. R. (2011) Clusterin facilitates in vivo clearance of extracellular misfolded proteins, Cell Mol. Life Sci. 68, 3919-3931. [16] Wojtas, A. M., Kang, S. S., Olley, B. M., Gatherer, M., Shinohara, M., Lozano, P. A., Liu, C. C., Kurti, A., Baker, K. E., Dickson, D. W., Yue, M., Petrucelli, L., Bu, G., Carare, R. O., and Fryer, J. D. (2017) Loss of clusterin shifts amyloid deposition to the cerebrovasculature via disruption of perivascular drainage pathways, Proc. Natl. Acad. Sci. USA 114, E6962E6971. [17] Yerbury, J. J., Kumita, J. R., Meehan, S., Dobson, C. M., and Wilson, M. R. (2009) α2Macroglobulin and haptoglobin suppress amyloid formation by interacting with prefibrillar protein species, J. Biol. Chem. 284, 4246-4254. [18] da Costa, G., Ribeiro-Silva, C., Ribeiro, R., Gilberto, S., Gomes, R. A., Ferreira, A., Mateus, E., Barroso, E., Coelho, A. V., Freire, A. P., and Cordeiro, C. (2015) Transthyretin amyloidosis: chaperone concentration changes and increased proteolysis in the pathway to disease, PLOS One 10, e0125392. [19] Doolittle, R. F. (1984) Fibrinogen and fibrin, Annual Review of Biochemistry 53, 195-229. [20] Kollman, J. M., Pandi, L., Sawaya, M. R., Riley, M., and Doolittle, R. F. (2009) Crystal structure of human fibrinogen, Biochemistry 48, 3877-3886. [21] Tang, H. D., Fu, Y., Cui, Y. J., He, Y. B., Zeng, X., Ploplis, V. A., Castellino, F. J., and Luo, Y. Z. (2009) Fibrinogen has chaperone-like activity, Biochem. Bioph. Res. Commun. 378, 662-667. [22] da Costa, G., Gomes, R. A., Guerreiro, A., Mateus, E., Monteiro, E., Barroso, E., Coelho, A. V., Freire, A. P., and Cordeiro, C. (2011) Beyond genetic factors in familial amyloidotic polyneuropathy: protein glycation and the loss of fibrinogen's chaperone activity, PLOS One 6, e24850. [23] Swasthi, H. M., Bhasne, K., Mahapatra, S., and Mukhopadhyay, S. (2018) Human fibrinogen inhibits amyloid assembly of biofilm-forming CsgA, Biochemistry 57, 6270-6273. [24] Yamamoto, N., Tsuhara, S., Tamura, A., and Chatani, E. (2018) A specific form of prefibrillar aggregates that functions as a precursor of amyloid nucleation, Sci Rep 8, 62. [25] Heymann, J. B., Zakharov, S. D., Zhang, Y. L., and Cramer, W. A. (1996) Characterization of electrostatic and nonelectrostatic components of protein membrane binding interactions, Biochemistry 35, 2717-2725. [26] Hille, J. D. R., Denkelder, G. M. D., Sauve, P., Dehaas, G. H., and Egmond, M. R. (1981) Physicochemical studies on the interaction of pancreatic phospholipase A2 with a micellar substrate-analog, Biochemistry 20, 4068-4073. [27] Glatter, O., and Kratky, O. (1982) Small angle X-ray scattering, Academic Press, London ; 37 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

New York. [28] Holasek, A., Wawra, H., Kratky, O., and Mittelbach, P. (1964) Small-angle scattering of Bence-Jones Protein, Biochim. Biophys. Acta 79, 76-87. [29] Martin, S. C., and Bjork, I. (1990) Conformational changes in human fibrinogen after in vitro phosphorylation and their relation to fibrinogen behaviour, FEBS Lett. 272, 103-105. [30] Broersma, S. (1960) Viscous force constant for a closed cylinder, J. Chem. Phys. 32, 16321635. [31] Newman, J., Swinney, H. L., and Day, L. A. (1977) Hydrodynamic properties and structure of Fd virus, J. Mol. Biol. 116, 593-603. [32] Arimon, M., Grimminger, V., Sanz, F., and Lashuel, H. A. (2008) Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Aβ fibrils and protofibrils, J. Mol. Biol. 384, 1157-1173. [33] Beeg, M., Stravalaci, M., Romeo, M., Carra, A. D., Cagnotto, A., Rossi, A., Diomede, L., Salmona, M., and Gobbi, M. (2016) Clusterin binds to Aβ1-42 oligomers with high affinity and interferes with peptide aggregation by inhibiting primary and secondary nucleation, J. Biol. Chem. 291, 6958-6966. [34] Yerbury, J. J., Poon, S., Meehan, S., Thompson, B., Kumita, J. R., Dobson, C. M., and Wilson, M. R. (2007) The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures, FASEB J. 21, 2312-2322. [35] Huang, C., Cheng, H., Hao, S., Zhou, H., Zhang, X., Gao, J., Sun, Q. H., Hu, H., and Wang, C. C. (2006) Heat shock protein 70 inhibits α-synuclein fibril formation via interactions with diverse intermediates, J. Mol. Biol. 364, 323-336. [36] Daturpalli, S., Waudby, C. A., Meehan, S., and Jackson, S. E. (2013) Hsp90 inhibits αsynuclein aggregation by interacting with soluble oligomers, J. Mol. Biol. 425, 4614-4628. [37] Rudiger, S., Germeroth, L., Schneider-Mergener, J., and Bukau, B. (1997) Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries, EMBO J. 16, 1501-1507. [38] Ballet, T., Brukert, F., Mangiagalli, P., Bureau, C., Boulange, L., Nault, L., Perret, T., and Weidenhaupt, M. (2012) DnaK prevents human insulin amyloid fiber formation on hydrophobic surfaces, Biochemistry 51, 2172-2180. [39] Tang, H. D., Fu, Y., Zhan, S. L., and Luo, Y. Z. (2009) αEC, the C-Terminal extension of fibrinogen, has chaperone-like activity, Biochemistry 48, 3967-3976. [40] Evans, C. G., Wisen, S., and Gestwicki, J. E. (2006) Heat shock proteins 70 and 90 inhibit early stages of amyloid β-(1-42) aggregation in vitro, J. Biol. Chem. 281, 33182-33191. [41] Chimon, S., Shaibat, M. A., Jones, C. R., Calero, D. C., Aizezi, B., and Ishii, Y. (2007) Evidence of fibril-like β-sheet structures in a neurotoxic amyloid intermediate of 38 ACS Paragon Plus Environment

Page 38 of 47

Page 39 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Alzheimer's β-amyloid, Nat. Struct. Mol. Biol. 14, 1157-1164. [42] Bleiholder, C., Dupuis, N. F., Wyttenbach, T., and Bowers, M. T. (2011) Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation, Nat. Chem. 3, 172-177. [43] Nors Perdersen, M., Fodera, V., Horvath, I., van Maarschalkerweerd, A., Norgaard Toft, K., Weise, C., Almqvist, F., Wolf-Watz, M., Wittung-Stafshede, P., and Vestergaard, B. (2015) Direct correlation between ligand-induced α-synuclein oligomers and amyloid-like fibril growth, Sci. Rep. 5, 10422. [44] Potapov, A., Yau, W. M., Ghirlando, R., Thurber, K. R., and Tycko, R. (2015) Successive stages of amyloid-β self-assembly characterized by solid-state nuclear magnetic resonance with dynamic nuclear polarization, J. Am. Chem. Soc. 137, 8294-8307. [45] Pavlova, A., Cheng, C. Y., Kinnebrew, M., Lew, J., Dahlquist, F. W., and Han, S. (2016) Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau, Proc. Natl. Acad. Sci. USA 113, E127-E136. [46] Chatani, E., and Yamamoto, N. (2018) Recent progress on understanding the mechanisms of amyloid nucleation, Biophys. Rev. 10, 527-534. [47] Inoue, R., Takata, T., Fujii, N., Ishii, K., Uchiyama, S., Sato, N., Oba, Y., Wood, K., Kato, K., Fujii, N., and Sugiyama, M. (2016) New insight into the dynamical system of αB-crystallin oligomers, Sci. Rep. 6, 29208. [48] Ahn, H. J., Zamolodchikov, D., Cortes-Canteli, M., Norris, E. H., Glickman, J. F., and Strickland, S. (2010) Alzheimer's disease peptide β-amyloid interacts with fibrinogen and induces its oligomerization, Proc. Natl. Acad. Sci. USA 107, 21812-21817. [49] Cortes-Canteli, M., Paul, J., Norris, E. H., Bronstein, R., Ahn, H. J., Zamolodchikov, D., Bhuvanendran, S., Fenz, K. M., and Strickland, S. (2010) Fibrinogen and β-amyloid association alters thrombosis and fibrinolysis: a possible contributing factor to Alzheimer's disease, Neuron 66, 695-709. [50] Petersen, M. A., Ryu, J. K., and Akassoglou, K. (2018) Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics, Nat. Rev. Neurosci. 19, 283-301.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC

ACS Paragon Plus Environment

Page 40 of 47

Page 41 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. Effects of Fg on B chain fibrillation. (A) Time courses of ThT fluorescence intensities of B chain amyloid fibrillation. The concentrations of B chain and Fg were set to 1.4 mg/ml (400 μM) and 3.5 mg/ml (10 μM), respectively, and the reaction was performed under agitated conditions. Results of four repeated experiments are shown in the absence or presence of Fg, respectively. The inset shows a magnified view of the fluorescence time course. (B) AFM images for the clarification of morphology of the final products without (left) or with Fg (right). The reaction mixtures at 90 min in panel a were sampled and analyzed. The scale bars repr esent 1 µm. The heights of fibrils and granular particles were in the range of 4.5-15 nm and 2.5-7.0 nm, respectively. (C) Distribution of hydrodynamic diameters monitored by DLS. The results at 10, 20, 30, 60, and 90 min without or with Fg are shown. In the lower panel, the hydrodynamic diameter of Fg is also shown for reference. (D) Time-dependent changes of CD spectra without or with Fg. In the presence of Fg (lower panel), net changes in the structure of B chain are indicated by subtracting the spectrum of Fg from each experimental spectrum. (E) Raw CD spectra of B chain or B chain incubated with Fg after 30-min incubation, respectively. The raw CD spectrum of Fg is also shown. A simulated CD spectrum obtained by summing those of B chain alone and Fg alone is overlayed.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 42 of 47

Page 43 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. SEC analysis of the interaction between the prefibrillar intermediates of B chain (1.4 mg/ml) and Fg (3.5 mg/ml). (A) AFM images of the Fg-B chain complex in the quiescent condition (left) and Fg alone as a control (right). (B) SEC profile of the mixture of the B chain prefibrillar intermediates and Fg. The profiles of the prefibrillar intermediates and Fg are also shown as comparisons. The inset shows a magnified view of the low-absorption areas. E1, E2, and E3 indicate eluted fractions applied for SDS-PAGE.(C) SDS-PAGE of the eluted fractions shown in panel a. Three bands observed between 75 and 50 kDa (indicated by *) are α, β, and γ chains of Fg. Some of these chains remained disulfide-bonded and provided additional bands at around 140 kDa and 100 kDa (indicated by #), which was especially eminent for the Fg sample mixed with the prefibrillar intermediates possibly due to prevention of the reduction of the disulfide bond by B chain. B chain, which possesses the molecular weight of ~3.4 kDa, exists at around the predicted position (indicated by †). M indicates the molecular mass marker. SDS-PAGE was performed by a standard protocol at the acrylamide concentration of 12 % vol/vol.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Effects of Fg on the formation of the B-chain prefibrillar intermediates. The concentrations of B chain and Fg were set to 1.4 mg/ml and 3.5 mg/ml, respectively, and time courses of average molar ellipticity at 216 nm ([θ]216) without or with Fg were monitored under quiescent conditions. For the data with Fg, the [θ]216 value of Fg was subtracted at each time point. The inset shows a magnified view in the early time period. Solid lines represent fitted curves obtained by using eq. 2. In the absence of Fg; τ1 = 14±2 min and τ2 = 390±30 min with [θ]0, [θ]1, and [θ]2 of -5,900±200, -8,300±100, and -11,200±100 deg·cm2·dmol-1, respectively. In the presence of Fg; τ1 = 21±3 min and τ2 = 1,200±200 min with [θ]0, [θ]1, and [θ]2 of -5,700±300, -9,800±200, and -12,700±200 deg·cm2·dmol-1, respectively.


Page 44 of 47

Page 45 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4. NMR analysis for the binding of Fg to the prefibrillar intermediates of B chain. (A) A magnified view of signals of histidine ε protons. Dashed lines represent the fitted curves obtained by using eq. 4. (B) A titration curve obtained by tracing the area of peak 1 in panel a. Molar concentration corresponding to weight concentration of Fg is provided as an upper horizontal axis. Dashed line represents a fitted curve obtained by using eq. 6. The values of ThT intensity at 210 min observed in panel c is also plotted to obtain the binding stoichiometry of Fg to the prefibrillar intermediates with the level of inhibition of the fibril formation. (C) Time courses of the fibril formation in the presence of various kinds of concentrations of Fg as monitored by ThT. The concentration of B chain was 1.4 mg/ml (400 μM of monomer equivalent), and the reaction was performed under agitated conditions.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Effect of Fg on the amyloid fibril formation of B stimulated by the ultrasonic treatment (UT). B chain (1.4 mg/ml) was pre-incubated for 26 h to form the second prefibrillar intermediates, to which UT and/or Fg were added. In all the experiments, the final concentration of B chain or Fg was 1.24 mg/ml or 3.5 mg/ml, respectively. (A) CD spectra measured before and after UT to the prefibrillar intermediates in the absence of Fg. After the spectrum of the prefibrillar intermediates was measured (before treatment), UT was applied. The spectrum was recorded 67 h after the UT treatment (after UT). (B) CD spectra measured before and after UT to the prefibrillar intermediates in the presence of Fg. After the spectrum of the prefibrillar intermediates was measured (before treatment). Fg was added and then the spectrum was recorded (+Fg, before UT). Immedately after that, UT was applied. The spectrum was recorded 65 h after the UT treatment (+Fg, after UT). (C) CD spectra measured before and after the addition of Fg to the prefibrillar intermediates treated by UT. After the spectrum of the prefibrillar intermediates was measured (before treatment), UT was applied and then the spectrum was recorded (UT, before +Fg). Immediately after that, Fg was added. The spectrum was recorded 63 h after the incubation (UT, after +Fg).


Page 46 of 47

Page 47 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 6. SAXS profiles of the B chain-Fg complex as well as Fg and B chain. The concentration of B chain and Fg were 1.4 mg/ml and 3.5 mg/ml, respectively, except for the complex where the final concentration of B chain was 1.27 mg/ml because of the dilution by the Fg addition. (A) Log-log plots of the intensity vs the q value. Gray lines show the result of curve fitting performed using eq. 7. The black slopes of -1, -2, or 4 indicate guides for the eyes. (B) Cross-section plots for cylindrical shape. Gray lines show the result of curve fitting performed using eq. 8. The inset shows a magnified view in a low-q region for B chain + Fg.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Schematic picture of the inhibition of the amyloid fibril formation of B chain by Fg. See details in the main text.


Page 48 of 47

Structural Insights into the Inhibition of Amyloid Fibril Formation by

Recommend Documents