BBA - and Proteomics 1867 (2019) 416–425

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BBA - Proteins and Proteomics

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Modulation of aggregation of fibroin by synergistic effect of the complex T of curcumin and β-cyclodextrin ⁎ Priyanka Dubeya, Pramit K. Chowdhuryb, Sourabh Ghosha, a Department of Textile Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. b Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.

ARTICLE INFO ABSTRACT

Keywords: Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has de- Silk fibroin monstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored Curcumin the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized con- β-Cyclodextrin coction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils witha Self-assembly diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk Amyloidogenic aggregation fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non- polar residues, which are the core components for nucleation dependent aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.

1. Introduction intermediate species, or amyloid fibril catalyzed secondary nucleation mechanism [5]. The self-assembly/aggregation of proteins asserts different roles in The silk fibroin protein can be projected as a model protein tostudy many of the regular physiological activities due to its specific structural the inhibition of fibrillation due to a host of similarities between the and functional properties, for example aggregation of explicit proteins amyloid forming and fibroin proteins [4,6,7]. Moreover, studies fo- play an imperative role during blood coagulation and hemostasis [1]. cusing on self-assembly process of silk fibroin protein can not only On the other hand, anomalous protein assembly and aberrant ag- provide a platform to explore the assembly process from nano-fibril to gregation could lead to devastating disorders such as cancers, cystic hierarchical structures in vitro, but also generate novel insights for de- fibrosis, Alzheimer's, Parkinson's and prion diseases [2]. Therefore, signing new silk-based functional biomaterials [8]. Recent reports in- requirement for a therapeutic agent for inhibiting the amyloid assembly dicated that silk fibroin-based biomaterials occasionally promote is of significant interest. A variety of small molecules have beenex- amyloidogenesis in vivo [9]. Another study showed that fibroin could plored to inhibit the protein aggregation [3,4]. However, the mechan- assemble into elongated fibrillar morphology comparable to amyloid isms for inhibiting the protein aggregation using small compounds are depending upon the processing conditions, concentration of fibroin and still poorly understood, which results in slow development of new concentration of ethanol [10]. Furthermore, treatment with less than therapeutics against amyloid pathogenesis. Thus, strategies for mod- 40% (v/v) of ethanol to the silk fibroin represents a nucleation de- ulation of protein assembly would be of considerable interest in order to pendent fibrillation, which was not observed in case of higher con- address the aberrant aggregation. Furthermore, the controlled in- centration of ethanol [10,11]. Therefore, fibroin was used as model vestigation of conformational transitions, fibrillation and inhibition of protein to enhance our understanding of controlling the amyloidogen- aggregation of amyloidogenic proteins could shed light on the patho- esis-like aggregation and to modulate its self assembly process. genesis as well. Such studies are however challenging due to aggrega- Silk fibroin protein mainly consists of two distinct structural con- tion propensity of amyloidogenic proteins, leading to lack of under- formations namely, random coil, silk I (which is metastable, water-so- standing in terms of reproducible kinetics data, limited evidence of luble, helix-like S-shaped zigzag/crankshaft or repeated β-turn type II

⁎ Corresponding author. E-mail address: [email protected] (S. Ghosh). https://doi.org/10.1016/j.bbapap.2019.01.009 Received 28 September 2018; Received in revised form 14 December 2018; Accepted 18 January 2019 Available online 21 January 2019 1570-9639/ © 2019 Elsevier B.V. All rights reserved. P. Dubey et al. BBA - Proteins and Proteomics 1867 (2019) 416–425 conformation) and silk II (which consists of highly ordered crystalline β-cyclodextrin and silk fibroin using PatchDock program (http:// regions) [12,13]. Various factors such as shear, addition of ethanol bioinfo3d.cs.tau.ac.il/PatchDock/index.html)[28]. The N-terminal se- (alcohol), metallic ions and pH induce a structural transition from the quence of silk fibroin protein available in PDB (PDB id: 3UA0) was used random coil structure to silk I to the β–sheet rich silk II structure during for the docking studies. PatchDock requires the receptor and ligand the self assembly process [14–17]. Previously, we have illustrated that molecules in PDB format and calculates the three-dimensional trans- optimized concentration of calcium ions can modulate the self-assembly formations based on the geometry based docking algorithm. It segre- of random coil silk fibroin solution to β–sheet rich silk II conformation gates the molecules into concave, convex and flat patches based on through α-helical intermediates [18]. Moreover, the precise control of Connolly dot surface Representation [28]. It further calculates the self-assembly of fibroin protein can also be utilized for distinct im- complementary patch based on Geometric Hashing algorithm for three munological responses to silk biomaterial [8,18]. dimensional transformations that evaluate the scoring function based In the current study we have focused on controlling the aggregation on both atomic desolvation energy and geometric fit. PatchDock re- kinetics of silk fibroin in the presence of combination of twocom- moves the redundant solutions using RMSD (root-mean-square devia- pounds, curcumin and β-cyclodextrin. Addition of β-cyclodextrin and tion) clustering. curcumin has been reported previously to inhibit the aggregation of protein partially or completely [7,19] and hence disfavouring the self- 2.3. Isolation of regenerated aqueous solution of fibroin from cocoons assembly of protein into highly organized fibrillar structures. A bi- phenolic compound, curcumin [(1,7-bis(4-hydroxy-3-methoxyphenyl)- Fibroin solution from Bombyx mori cocoons was obtained as re- 1,6-heptadiene-3,5-dioene] is extracted from the roots of the Curcuma ported previously [18]. Degumming was carried out by boiling the longa [20]. It is a potent anti-inflammatory agent and also has some cocoons twice, for 20 min each in 0.02 M Na2CO3 to remove the remedial effects on Alzheimer's disease [21] due to its binding capacity protein. Fibers obtained after degumming were washed thoroughly to amyloid fibrils [22]. Though curcumin has been widely accepted as a with ultrapure milli-Q water and kept for drying overnight. Further, chemotherapeutic agent, its limitations such as low bioavailability, 9.3 M LiBr was used to dissolve the fibers at 60 °C for 3–4 h. The re- inadequate tissue absorption, poor solubility in water or physiological sulted protein solution was dialyzed using ultrapure milli-Q water for pH medium [23,24], fast hydrolysis in alkaline medium and poor sta- 48 h at 25 °C. The final concentration of regenerated fibroin solution bility have been a major limitation towards its widespread usage [25]. was 6 wt%. Thus, it is essential to combine the curcumin with a carrier in order to improve above-mentioned properties. 2.4. Congo red (CR) dye preparation and binding assay β-cyclodextrin belongs to another promising class of compounds that have been used for modulating the self-assembly of proteins [19] A 20 mM stock solution of CR was prepared in deionised water to and inhibiting the amyloid fibril formation [26,27]. β-cyclodextrin is a get the final concentration of 20 μM during experiments. The CR solu- non toxic, cyclic oligomer consisting of seven D-glucopyranose units, tion was filtered thrice with 0.2 μm filter. Absorbance was recorded at linked by α-1, 4 linkage and has been known to inhibit amyloid β-oli- 540 nm after 75 min incubation at 25 °C using a spectrophotometer gomerization [19]. β-cyclodextrin has a hydrophilic exterior with a (UV-2450, Shimadzu, Japan). CR was added 15 min prior to spectral hydrophobic cavity, which makes it an appropriate host for aromatic analysis in all the samples. guest molecules in water. Therefore, it may act as a carrier for curcumin For studying the aggregation kinetics of fibroin with ethanol using and thereby help overcome some of the aforementioned limitations of CR dye a 50 mg/ml stock solution of fibroin was diluted with deionised the curcumin. In the current work, we have used this combination of water to obtain a final concentration of 1 mg/ml. Fibroin solution was curcumin and β-cyclodextrin to induce controlled conformational further incubated with different concentrations of ethanol (0, 5, 10, 20, translation and fibrillation of the silk fibroin protein and prevent its 30, 40%) for 75 min at 25 °C. CR absorbance was recorded at 540 nm aggregation, which could potentially elucidate pathogenesis of amy- for all the samples at regular time intervals from 0 to 75 min. loid-forming proteins. This study is also significant because the iden- Aggregation inhibition of fibroin treated with curcumin, β-cyclodextrin tification of secondary structural conformations that shed light onen- and combination of curcumin and β-cyclodextrin was also studied using abling the protein to remain as soluble and functional rather than in CR dye. A stock solution of silk fibroin at a concentration of 50 mg/ml aggregated form, has a noteworthy impact under physiological condi- was diluted to obtain a solution having the concentration of 1 mg/ml tions [8]. This study can further be explored to control the fibroin and was treated with different concentrations of curcumin (0, 10, 20, protein conformations by precisely tuning its self assembly process for 30, 40, 50 & 60 μM), β-cyclodextrin (0, 10, 25, 50, 75, 100, 150 & designing and developing the advanced silk biomaterials for various 200 μM) and combination of both curcumin and β-cyclodextrin at biomedical applications. various concentrations (2.5 μM curcumin with 5 μM β-cyclodextrin, 5 μM curcumin with 10 μM β-cyclodextrin, 10 μM curcumin with 20 μM 2. Materials and methods β-cyclodextrin, 15 μM curcumin with 30 μM β-cyclodextrin and 20 μM curcumin with 40 μM β-cyclodextrin) at 25 °C for 75 min. Silk fibroin 2.1. Materials alone (without adding any compound) at 0 min was used as a control in all three cases. Bombyx mori cocoons were procured from Central Silk Silk fibroin with 20% (v/v) ethanol in the presence of the CRdye Technological Research Institute (Central Silk Board), Bangalore, was observed under polarized light using Leica DM2500 P microsystem,

Ministry of Textiles, Government of India. Sodium carbonate (Na2CO3) (Leica Weitzlar, Germany) as described [29]. and lithium bromide (LiBr) were provided by Merck (Mumbai, India). Congo red dye and curcumin were purchased from Sigma-Aldrich (St. 2.5. Turbidity measurements Louis, MO, USA). β-cyclodextrin was obtained from Hi-media (Mumbai, India). All other used chemicals were of analytical grade. Turbidity of silk fibroin with 20% (v/v) ethanol, silk fibroin with optimized ratio of a combination of curcumin with β-cyclodextrin, and 2.2. Docking of curcumin and β-cyclodextrin into N-terminal domain of silk silk fibroin only (without any compounds) were monitored at 600nm fibroin using a UV–visible spectrophotometer (UV-2450, SHIMADZU, Japan) from 0 to 75 min. All samples were incubated at 25 °C and baseline Firstly we carried out the in silico molecular docking analysis of fi- correction was carried out using the respective solutions without the broin to predict the most stable complex structure between curcumin, protein.

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2.6. Circular dichroism measurements 3.2. Effect of ethanol on silk fibroin fibrillation detected by Congo red(CR)

Far-UV CD spectra were carried out using Jasco J-815 CD spectro- CR dye has the capability to bind to amyloid-like proteins with a β- meter (Jasco Corporation, Tokyo, Japan) in the range of pleated sheet conformation [32]. The CR data of fibroin with ethanol 190 nm–260 nm with a quartz cuvette of path length of 0.1 cm. Fibroin showed a gradual increase in the rate of fibrillation as the ethanol solution at a concentration of 1 mg/ml with 20% (v/v) ethanol and concentration was increased. Fibroin from 20 to 40% ethanol showed fibroin with combination of curcumin and β-cyclodextrin wasin- maximum fibrillation at 75 min whereas fibroin without ethanol (con- cubated at 25 °C for 75 min however spectra of only fibroin solution was trol) showed no fibrillation. A similar observation occurred in case of recorded at 0 min incubation. All spectra were recorded in triplicates. fibroin with 5–10% ethanol wherein no relevant change in fibrillation was observed, as illustrated in Fig. 2a. The conformational change in silk fibroin after addition of ethanol could be attributed to thedehy- 2.7. Attenuated total reflectance-fourier transforms infrared spectroscopy drating effect of ethanol on the silk fibroin polypeptide backbone, which enhances strong intramolecular interactions between the mole- ATR-FTIR was performed using Perkin-Elmer (Spectrum BX Series, cular chains of fibroin, leading to the transformation from a pre- MA, USA) instrument in absorption mode, aided with DTGS dominantly random coil structure to that of an extended β-sheet. Hy- (Deuterated Triglycine Sulfate) IR detector. The amide groups in pro- drogen bonding and electrostatic interactions further stabilize this self- tein shows high intensity in amide I range as compared to amide II. assembly. Ethanol with a concentration of 20% (v/v) was the optimum Therefore, a total of 64 scans per sample were carried out at a resolu- concentration used to induce the aggregation of silk fibroin, since above −1 −1 tion of 4 cm in the amide I region (1700 to 1600 cm ). After ap- this concentration the aggregation kinetics were too fast to monitor, plying a high pass filter, Origin Pro 8.5 (Origin Lab Corporation, while below that concentration the reaction was significantly slower. Northampton, MA, USA) was used for deconvolution of spectra [30] Interestingly, silk fibrils exhibited green birefringence in the presence wherein, spectra were curve-fitted by Gaussian peaks [31]. The number of the CR dye, akin to amyloids as depicted in Fig. 2b. and positions of peaks are defined from the results of the second deri- vatives of the spectra and were area-normalized and baseline corrected. 3.3. Effect of curcumin, β-cyclodextrin and their combination onthe aggregation profile of fibroin detected byCR 2.8. Dynamic light scattering (DLS) measurements A range of curcumin concentrations from 0 to 60 μM was used to monitor the fibrillation/aggregation inhibition of fibroin treated with DLS technique was used for determining the hydrodynamic radius 20% (v/v) ethanol, using CR absorbance at 540 nm. By increasing the of molecules of fibroin only, fibroin with 20% (v/v) ethanol and fibroin curcumin concentration a simultaneous decrease in fibrillation was with combination of curcumin and β-cyclodextrin using a Malvern observed. The CR absorbance followed a cooperative relationship with Zetasizer Nano ZS instrument (Worcestershire, UK). Distilled water was silk fibroin fibrillation as demonstrated by the sigmoidal curvein used for sample preparation. Each sample (2 ml) was injected manually Fig. 3(a). A 40 μM concentration of curcumin was found to be most in 10 mm path length cuvette and the data recorded. effective for inhibition of aggregation of the fibroin solution incubated with 20% (v/v) ethanol. Similarly, different concentrations of 2.9. Atomic force microscopy (0–200 μM) of β-cyclodextrin were also used to investigate their effects on the aggregation inhibition of fibroin incubated with 20% (v/v) AFM (Dimension ICON with Scan Asyst, Bruker) of fibroin with and ethanol. Data showed that at 100 μM and above β-cyclodextrin sup- without 20% (v/v) ethanol and combination of curcumin and β-cyclo- pressed the aggregation significantly; however at concentrations less dextrin mixture were carried out in tapping mode with silicon probe than 100 μM, aggregation was not prevented as illustrated in Fig. 3(b). (Tap 300 Al-G, Budget Probes, Sofia, Bulgaria). All samples (10 μl) were In order to investigate the effect of combination of curcumin andβ- mounted on mica, washed with deionised water and dried overnight. cyclodextrin on aggregation suppression, different concentrations of curcumin with β-cyclodextrin were used in a 1:2 ratio due to the stoi- chiomeric relationship as reported in earlier studies [33]. Data showed 3. Results that 15 μM curcumin with 30 μM β-cyclodextrin suppressed the ag- gregation completely and hence was much more effective when com- 3.1. In-silico molecular docking of curcumin and β-cyclodextrin with N- pared to the 2.5 μM curcumin with 5 μM β-cyclodextrin, 5 μM curcumin terminal domain of silk fibroin using PatchDock with 10 μM β-cyclodextrin and 10 μM curcumin with 20 μM β-cyclo- dextrin as depicted in Fig. 4. The docking model of all compounds was chosen based upon the best scores predicted by the PatchDock program. Fig. 1 represents the 3.4. Turbidity analysis interaction of curcumin, β-cyclodextrin and the complex of these two with fibroin as obtained from molecular docking studies. The Patch- Turbidity measurements were carried out at 600 nm in order to Dock server generated the geometric score of all the docked molecules investigate the difference in fibril formation as a function of time.Silk with top 20 scoring solutions. fibroin with 20% (v/v) ethanol showed maximum absorbance (0.65)at The geometric shape complementarity score of curcumin and β- 75 min. However after addition of the optimized combination of 15 μM cyclodextrin with fibroin was 10,590 (Table S1) and 9306 (Table S2). curcumin and 30 μM β-cyclodextrin the aggregation and subsequent However, fibroin docked with the complex of curcumin and β-cyclo- fibrillation was suppressed as evident by absorbance of 0.12, whichwas dextrin in 1:1 and 1:2 ratios predicted a score of 11,368 and 14,452 close (0.05) to control (i.e. only silk fibroin at 0 min). Hence, data respectively as illustrated in (Table S3, S4). Therefore, it can be de- (Fig. 5) corroborated the CR findings as illustrated in Fig. 4. Based on duced that β-cyclodextrin and curcumin together form a stable complex the findings of CR binding and turbidity data, only fibroin at0min that offered more geometrical complementarity for the silk fibroin N- (control), fibroin with 20% (v/v) ethanol till 75 min and fibroin treated terminal region at 1:2 ratio as compared to only curcumin, only β-cy- with complex till 75 min was considered for further spectroscopic clodextrin or 1:1 complex of both as depicted in Fig. 1. Based on the analysis. The turbidity measurements were carried out till 300 min in above stated in silico analysis all further experimental validations were order to clarify whether the combination inhibits or delays the ag- conducted using similar (1:2) ratio. gregation process. No aggregation was observed till 300 min in the

418 P. Dubey et al. BBA - Proteins and Proteomics 1867 (2019) 416–425

Fig. 1. In silico analysis of N-terminal domain of fi- broin wherein, α-Helix, β- sheet and β- turn are shown in red, yellow and green respectively is docked with (a) curcumin (blue), (b) β-cyclodextrin (magenta), (c) Complex of 1:1 curcumin and β-cy- clodextrin (cyan) and (d)1:2 curcumin and β-cyclo- dextrin (purple) using PatchDock docking server.

Fig. 2. Congo red absorbance of (a) 1 mg/ml fibroin solution incubated with different concentrations of ethanol for 75 min at 25 °C monitored at540nm regular time intervals (b) Silk fibroin with 20% ethanol in the presence ofthe CR dye under plane polarized light. presence of optimized combination of curcumin and β-cyclodextrin Fig. 3. Aggregation inhibition of 1 mg/ml fibroin incubated with 20% ethanol (data not shown). treated with (a) 0–60 μM of curcumin, (b) 0–200 μM of β-cyclodextrin (β-CD) monitored by Congo red at 540 nm. 3.5. Fibroin secondary conformation monitored by circular dichroism solution in random coil conformation even after 75 min incubation, CD spectroscopy of only fibroin at 0 min (control), fibroin incubated similar to that of the control (fibroin at 0 min) as depicted in Fig. 6. with 20% (v/v) ethanol and treated samples (fibroin incubated with 20% ethanol (v/v) and combination of curcumin and β-cyclodextrin) 3.6. ATR-FTIR analysis were performed in order to estimate the secondary conformation of fibroin in the presence of different compounds. Our data showthatfi- ATR-FTIR analysis was carried out in the amide I broin solution at 0 min showed random coil structure, while fibroin (1600–1700 cm−1) region and was further deconvoluted [31] in order incubated in the presence of 20% (v/v) ethanol showed β-sheet con- to validate the results obtained using CD spectroscopy. Peaks in the formation at 75 min. When the fibroin solution with 20% (v/v) ethanol range of 1616 to 1637 cm−1 are characteristic of β-sheet conformation. was incubated with the optimized combination of curcumin and β-cy- The peaks in the range 1611–1630 cm−1 indicates cross β region clodextrin, it prevented the aggregation process and kept the fibroin characteristic of amyloid structure. Peaks in the range of

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in Table 1.

3.7. DLS studies

DLS was performed to investigate the average size and size dis- tribution of fibroin in different conditions. The DLS data of fibroinat 0 min showed the presence of low molecular weight species with an average diameter of ~10 nm. Fibroin treated with 20% (v/v) ethanol at 75 min however showed the presence of higher molecular weight spe- cies as expected with the diameter peaking at ~1000 nm. Incubation Fig. 4. Aggregation inhibition of 1 mg/ml fibroin solution incubated with 20% with combination of curcumin and β-cyclodextrin suppressed the ethanol till 75 min by combination of 15 μM curcumin and 30 μM β-cyclodex- higher molecular weight species formation and led to the low molecular trin at 540 nm detected by Congo red. weight species with an average diameter of ~30 nm species as illu- strated in Fig. 8.

3.8. AFM analysis

Silk fibroin incubated with 20% (v/v) ethanol at 25 °C showedfi- brillation at 75 min with a height of about 3.2 nm, whereas silk fibroin without ethanol at 0 min did not show any fibrillation. After addition of the combination of 15 μM curcumin and 30 μM β-cyclodextrin to silk fibroin with 20% (v/v) ethanol, it did not show fibrillation evenafter 75 min incubation (Fig. 9). Therefore a similar trend in accordance with CR and ATR-FTIR data was observed using AFM.

Fig. 5. Turbidity of 1 mg/ml fibroin with 20% ethanol, fibroin with ethanol 3.9. Effect of optimized combination of curcumin and β-cyclodextrin on treated with combination of 15 μM curcumin and 30 μM β-cyclodextrin, only aggregation inhibition of higher concentration of fibroin (5 mg/ml) detected silk fibroin (control) was monitored at 600 nm using UV–visible spectro- by CR photometer. In order to confirm the efficacy of this optimized combination of 1638–1655 cm−1 are characteristic of the random coil conformation 15 μM curcumin with 30 μM β-cyclodextrin on higher fibroin con- while those for α-helical structure lie in the range of 1656 to centration (5 mg/ml) treated with 20% ethanol, CR analysis was per- 1662 cm−1 [31]. The deconvolution data of only silk fibroin (control) formed at 540 nm. Data showed that at 15 μM curcumin with 30 μM β- at 0 min showed a 91.6% random coil conformation with 6.8% β-sheet cyclodextrin was unable to suppress the aggregation completely. structure at 1647 and 1625 cm−1 respectively. On the contrary, the However, with simultaneous increase in the concentration of combi- content of β-sheet increased to 59% at 1625 cm−1 when fibroin was nation of curcumin and β-cyclodextrin, aggregation was inhibited. The incubated with 20% (v/v) ethanol till 75 min. Interestingly, the intense maximum aggregation inhibition occurred at the 1:2 stoichiometry band at 1625 cm−1 decreased to 15.3% when 1:2 complex of curcumin with a concentration of 75 μM curcumin and 150 μM β-cyclodextrin as and β-cyclodextrin was added to fibroin incubated with 20% (v/v) depicted in Fig. 10. Therefore it can be concluded that this combination ethanol till 75 min and showed predominantly random coil (84.7%) acted in a dose dependent manner. conformation (Fig. 7). The band at 1625 cm−1 is characteristic feature of amyloid fibrils due to presence of inter-molecular β-sheets con- 4. Discussion formation [34,35]. Therefore, a similar trend in accordance to CD spectroscopy was obtained with ATR-FTIR. Aberrant self-assembly of amyloid fibrils is a causative agent of Based on aforesaid data, it can be deduced that combination of more than 20 different diseases such as Alzheimer's disease, Parkinson's curcumin and β-cyclodextrin effectively suppressed the aggregation disease, Prion disease etc. [36] Amyloids are generally produced by process of silk fibroin in the presence of 20% (v/v) ethanol as illustrated soluble or unfolded peptides that aggregate to form insoluble and

Fig. 6. CD spectroscopy of (a) 1 mg/ml fibroin solution at 0 min without ethanol showed random coil structure (b) with 20% ethanol at 75 min showedβ-sheet conformation and (c) fibroin with 20% ethanol treated with combination of curcumin and β-CD at 75 min showed random coil conformation.

420 P. Dubey et al. BBA - Proteins and Proteomics 1867 (2019) 416–425

Fig. 7. ATR-FTIR spectroscopy of (a) 1 mg/ml fibroin solution (control) at 0 min (b) with 20% ethanol at 75 min and (c) fibroin with 20% ethanol treatedwiththe combination of curcumin and β-cyclodextrin at 75 min.

Table 1 inherently stable fibers characterized by β-sheet structure [37]. These ATR-FTIR of Fibroin at 0 min, Fibroin with 20% ethanol at 75 min and Fibroin are formed by either via nucleation dependent or seeded polymerization with 20% ethanol treated with curcumin and β-cyclodextrin in the amide I process [38]. Such self-assembly occurs in vivo due to mutations, dis- −1 region (1600–1700 cm ). parity in protein synthesis and degradation, or impaired molecular Wave-number Deconvulated (%) Band chaperones. Curcumin has been shown to bind to amyloid-β peptide and inhibit the oligomerization of the amyloid-β monomers to amyloid 1) Silk fibroin at 0 min (control) fibrils [39]. Another study has demonstrated the capability of curcumin 1625 6.8 β-sheet 1647 91.6 Random coil in crossing the blood-brain barrier and preventing neurons from dif- 1704 1.6 β-sheet ferent toxic effects which may lead to the formation of amyloid-β [40]. Hence, curcumin plays an effective role for inhibiting the amyloid ag- 2) Silk fibroin incubated with 20% ethanol at 75 min 1625 59 β-sheet gregation in vitro and in vivo of a wide variety of aggregated proteins. 1692 41 β-turn However, an urgent requirement for new formulation is much re- 3) Silk fibroin incubated with 20% ethanol treated with curcumin and β-CD at75min commended in order to overcome inherent limitations of curcumin, 1625 15.3 β-sheet such as poor bioavailability, stability and low water solubility. 1652 84.7 Random Coil A large number of non-mammalian, naturally occurring proteinac- eous materials, such as silkworm silk fibroin, spider silk, curli from Escherichia coli and Sup35 from Saccharomyces cerevisiae demonstrate a β-sheet rich structure comparable to amyloids [41]. Bombyx mori silk

Fig. 8. DLS of (a) 1 mg/ml fibroin solution (control) at 0 min, (b) fibroin solution incubated with 20% ethanol at 75 min and (c) fibroin with 20% ethanoltreatedwith the combination of curcumin and β-cyclodextrin.

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Fig. 9. AFM of (a) Only 1 mg/ml fibroin solution (control) at 0 min (b) incubated with 20% ethanol at 75 min showing fibrillation (c) fibroin with20%ethanol combination of curcumin and β-cyclodextrin at 75 min.

microenvironment [41]. This is of significant clinical interest, as silk scaffold-based tissue engineering strategies may lead to amyloidogen- esis at the site of the injury in inflammatory milieu. Hence strategies to counteract such amyloidogenesis would have crucial relevance in re- generative medicine. Moreover, the different secondary conformations have significant influence on different immune responses when exposed to cells in vivo or in vitro [8]. We have earlier demonstrated that human monocytes, cultured on fibroin with silk I conformation was inept to elicit monocyte responsiveness; while fibroin with silk II conformation showed notable immune response signified by increased gene expres- Fig. 10. Congo red data of 5 mg/ml fibroin solution incubated with 20% sion of IL-6 and IL-1β [8]. In addition, deposition of protein on the ethanol at 75 min treated with different concentrations of the combination of surface of the surgical instruments can also be controlled by tuning the curcumin and β-cyclodextrin. secondary conformation of protein by modulating its self-assembly process [49]. Hence, strategies to modulate secondary conformation fibroin consists of light chain (25 kDa) and heavy chain (391kDa) can also be potentially used to fabricate versatile engineered bioma- polypeptides linked by disulphide bond(s). Fibroin contains the GA- terials by modulating its self-assembly process to tailor-made secondary GAGY, GAGAGVGY, and repetitive hexapeptide GAGAGS sequences, conformations [8,50]. Such biomaterials can be used to prepare stra- which contribute towards its hydrophobicity [42,43]. These repetitive tegically designed scaffolds, for biological surface engineering and drug hexapeptide sequences GAGAGS of fibroin play a decisive role in the delivery purposes. To achieve all the above stated objectives, a generic secondary structure transitions leading to the formation of anti-parallel model polymorphic fibroin protein was chosen, which has the cap- β-sheet structure, similar to that of amyloid-β peptide associated with ability to show transition from a water-soluble random coil form to Alzheimer's disease [43–45]. There are remarkable similarities between aggregated silk I to insoluble β-sheet (silk II) rich structure or vice versa silk fibroin and amyloid-β peptide, which make the former agood thereby providing a significant possibility to achieve various functional choice to serve as a model protein for aggregation related studies. Both outcomes in vivo [51]. Therefore, we selected the Bombyx mori silk fi- fibroin and amyloid-β peptide share a similar nucleation dependent broin for studying its self assembly process in the presence of different mechanism for fibril formation [46]. The amyloid-β peptide and α-sy- compounds such as curcumin and β-cyclodextrin. nuclein consist of VGGVV and VGGAVVAGV hydrophobic sequences, Our molecular docking analysis predicted a significantly high score respectively, which is remarkably analogous to GAGVGAGYG sequence in case of curcumin and β-cyclodextrin complex (1:2) with fibroin of Bombyx mori silk fibroin [47,48]. Also, silk fibroin protein consists of (14452) as compared to fibroin docked with either curcumin (10590) or repetitive sequences of AHGGYSGY similar to PHGGGWGQ octapeptide β-cyclodextrin (9306) alone (Fig. 1) and the 1:1 complex (11368). Tang sequence in prion protein, which is involved in the pathogenesis of et al. reported that, based on the criteria of molecular dimensions, one Prion disease [16]. The X-ray diffraction analysis of amyloid fibrils molecule of curcumin and two molecules of β-cyclodextrin might form indicates the presence of oriented repeating structure, similar to that a coherent complex since it is difficult to restrict curcumin completely observed for Bombyx mori silk fibroin [44]. Interestingly, silk bioma- inside one β-cyclodextrin cavity [52]. Another study has demonstrated terials have been shown to occasionally cause amyloidogenic deposits that curcumin and β-cyclodextrin have a stoichiomeric relationship at a based on secondary conformations, processing conditions [9]. Injection ratio of 1:2 33 wherein each of the benzene rings of curcumin was en- of regenerated silk fibrils to mice caused deposition of mild to moderate closed within a single β-cyclodextrin cavity, stabilized by hydrophobic extent of amyloids in spleens of five out of seven mice [41]. Fibroin interactions and van der Waal forces. Furthermore, curcumin is a mo- fibrils reduced the initial lag phase in amyloid deposition, which lecule with electron donor groups, which is capable of forming hy- highlighted that even non-pathogenic β-sheet rich protein fibrils may drogen bonds within the cavity of β-cyclodextrin and may lead to more lead to amyloidogenesis, based on chronic inflammatory stable complexation at 1:2 ratio [33]. Hence, based on abovementioned

422 P. Dubey et al. BBA - Proteins and Proteomics 1867 (2019) 416–425 understandings, in the present study, it was decided to exploit this decrease in the efficiency of inhibition. Taken together these results particular combination of curcumin and β-cyclodextrin (1:2 ratio), to suggest that both aromatic interactions involving the phenyl rings and modulate the aggregation of silk fibroin protein. the phenolic –OH moieties act in unison in bringing about the in- Few recent studies showed inhibition of aggregation induced by hibitory effects of curcumin. metallic ions in silk fibroin using different polyphenols (curcumin [7], Similar conclusions have also been reached at by other groups for epigallocatechin gallate) [4]. However, it would be interesting to polyphenols in general, including curcumin, wherein both interaction evaluate inhibition strategies for aggregation induced by alcohol in silk with aromatic residues of the inhibitors and amyloidogenic hydro- fibroin, which is much more potent and faster than metallic ionsasan phobic amino acids of the amyloid forming protein, and polar inter- aggregation stimulating agent [18]. Our CR-binding data show that actions of the –OH groups of the polyphenols with polar side when used alone, curcumin at a concentration of 40 μM or β-cyclo- chains leading to weakening of intrastrand hydrogen bonds of the ag- dextrin at 100 μM concentration was needed to effectively block fibroin gregates, play important roles in abrogating aggregation [58–61]. A aggregation induced by 20% (v/v) ethanol in fibroin solution (Fig. 3). simulation study has demonstrated that curcumin preferentially as- However, the combination with 1:2 ratio (15 μM curcumin:30 μM β- sociates with and other aliphatic residues of amyloid-β pep- cyclodextrin) showed aggregation inhibition in even less than half of tides, these residues being abundant in silk fibroin [62]. the concentration required for any of these compounds when used The other compound β-cyclodextrin was not only utilized as a car- alone (Fig. 4). Recently, Yao et al. [7] have demonstrated that ag- rier of curcumin for increased bio-availability be able to prevent the gregation of fibroin was induced by Al (III), whereas addition ofcur- aggregation of fibroin (Fig. 3b). β-cyclodextrin is capable of seques- cumin reduced the aggregation of fibroin solution. However, they did tering the hydrophobic groups, which helps in reducing the amyloid not quantify the percentage of aggregates formed and how much dis- formation of proteins [63,64]. Thus, β-cyclodextrin has been demon- ruption of aggregation was achieved after addition of curcumin. strated to work as a pseudo-chaperone and act as appropriate folding Therefore, the complete inhibition of fibril formation may not have aid for many proteins [65]. Few studies have illustrated that β-cyclo- been accomplished. In the current study, our ATR-FTIR data showed dextrin interacts with the aromatic (phenylalanine, tyrosine, and tryp- that 1 mg/ml concentration of fibroin solution incubated with 20% tophan) and histidine residues of proteins and accommodate these re- ethanol (v/v) showed 59% β-sheet conformation at 75 min. However, sidues in the hydrophobic cavity of β-cyclodextrin [66,67]. Curcumin after addition of the curcumin and β-cyclodextrin combination, it inhibits the formation of intermolecular hydrogen bonding between the showed 84.7% random coil, which is reasonably close to control sample molecular chains of fibroin, while β-cyclodextrin sequesters the (only fibroin at 0 min) sample i.e. 91.6% random coil (Fig. 7). This monomeric units in its hydrophobic cavity thereby forbidding them to finding was in accordance with our CD measurements (Fig. 6). Also, this assemble further. Furthermore, the hydrophobic core of β-cyclodextrin synergistic combination works in a dose dependent manner (Fig. 10). can also interact with the non-polar residues which are core compo- Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6,-heptadiene- nents for nucleation dependent protein aggregation. Indeed fibroin is a 3,5-dione) consists of a two aromatic rings both of which contain an O- predominantly hydrophobic protein and has a majority of glycine, methoxy phenolic group linked by α, β-unsaturated β-diketone moiety. alanine, and valine residues, that are known to interact directly with These aromatic phenolic groups facilitate hydrophobic interactions the nonpolar cavity of β-cyclodextrin [68]. Therefore it is evident from with the side chains of the protein while the linker contributes towards the present study that combination of curcumin and β-cyclodextrin the flexibility of the molecule [53]. The diketo group of curcumin en- could tune the secondary conformational translation and fibrillation of ables keto-enol tautomerism, which leads to either donating or ac- fibroin protein and block it's self-assembly, which could potentially cepting the lone pair for hydrogen bonding. Therefore, it may inhibit elucidate pathogenesis of amyloid-β (and other similar amyloid forming the β-sheet formation by steric hindrance and alternative hydrogen proteins) since both these proteins share a similar fibrillation me- bonding [53]. Curcumin has been known to prevent the conformational chanism. Moreover as mentioned earlier tuning/modulating the self changes followed by fibrillogenesis [3]. Yamada and co-worker de- assembly process of silk fibroin could lead to the synthesis ofbio- monstrated that curcumin might be accountable for explicit binding to technologically relevant engineered biomaterials, for regenerative free available amyloid-β further inhibiting the fibril formation [21]. medicine, drug delivery, immunotherapy and vaccines etc. The insights The aggregation of silk fibroin is mainly caused by the intra- and inter- so obtained regarding the control of the nucleation step of the self-as- molecular interactions between hydrophobic β-sheet forming domains sembly process through curcumin and β-cyclodextrin will help address [54]. The –OH groups on aromatic phenyl rings of curcumin might whether injectable silk fibroin hydrogel or scaffolds may lead to block the inter-molecular hydrogen-bonding of fibroin proteins and synthesis of functionally beneficial amyloids or aggregates akin to pa- inhibit the formation of β-sheets. This would further lead to decrease in thological amyloids during tissue regeneration [69]. We also propose fibril production as a result of aggregation prevention of the fibroin that silkworm fibroin protein may be used as a valuable model system protein. Moreover, curcumin's benzene rings also show hydrophobic to investigate inhibition of fibrillation mechanisms for amyloidogenic interactions with the tyrosine, tryptophan and phenylalanine aromatic systems, specially under controlled inflammatory microenvironment residues present in the significant proportion (~8%) in fibroin solution [70]. [55]. An earlier study has shown that curcumin could bind and mini- mize aggregation of amyloid-β due to its capability of specific binding 5. Conclusion with amyloid structures rather than binding to primary structure [22]. It has further been shown using molecular modelling that polyphenolic The optimized combination of curcumin and β-cyclodextrin blocked compounds like curcumin are capable of acquiring a three-dimensional the conformational transition in secondary structures from random coil pharmacophore conformation that might be crucial for preventing the to β-sheet (silk II)-rich fibrillar structure for silk fibroin in the presence amyloid fibrillation [56]. A recent study has focussed on various deri- of 20% ethanol. We propose that curcumin inhibited intermolecular vatives of curcumin in order to find out the factors that are responsible hydrogen bonding between fibroin chains, and simultaneously β-cy- for curcumin-like molecules being able to act as inhibitors of amyloid clodextrin sequesters the monomeric units in its hydrophobic cavity formation [57]. The authors showed that for compounds wherein one of and blocked further assembly. This strategy of modulation of fibroin the phenyl rings was absent; these were far less effective than curcumin protein self-assembly might be used as a potent methodology to unravel in decreasing the aggregation of amyloid-β. Moreover, the presence of aggregation inhibition. Moreover, the present study can further be ex- an –OH group on the phenyl ring (curcumin has an –OH group on both plored to control the fibroin protein conformations by precisely tuning of its aromatic rings) has also been shown to be important as the sub- its self-assembly process for various biomedical applications of silk stitution of the latter by a methoxy (-OCH3) group led to a drastic biomaterials.

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