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Insights into the mechanism of how Morin suppresses amyloid fibrillation of hen egg white lysozyme
International Journal of Biological Macromolecules 101 (2017) 321–325
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Insights into the mechanism of how Morin suppresses amyloid fibrillation of hen egg white lysozyme Xiaoying Chong a,1 , Luchen Sun a,1 , Yonghui Sun a , Lin Chang a , Alan K. Chang a , Xian Lu a , Xuejie Zhou a , Junqing Liu a , Bing Zhang b , Gary W. Jones c , Jianwei He a,∗ a
School of Life Science, Liaoning University, Shenyang 110036, China Experimental Center of Functional Subjects, China Medical University, 92 Bei Er Road, Heping District, Shenyang 110001, China c Centre for Biomedical Science Research, School of Clinical and Applied Sciences, Leeds Beckett University, City Campus, Leeds LS1 3HE, United Kingdom b
a r t i c l e
i n f o
Article history: Received 26 November 2016 Received in revised form 17 March 2017 Accepted 20 March 2017 Available online 21 March 2017 Keywords: Morin Hen egg white lysozyme Amyloid
a b s t r a c t This communication describes the inhibitory effect of Morin on the fibrillation of Hen Egg White Lysozyme (HEWL), a generic amyloid-forming model protein. This effect was dose-dependent and stronger than other small molecules we have tested previously. Spectrofluorometric and computational studies support a model suggesting that Morin inhibits amyloid fibril formation of HEWL by binding to the aggregation prone cleft region of the -domain of HEWL, thereby stabilizing the molecule in its native-like state. Interestingly, transmission electron microscopy observations suggest that, along with increases in Morin concentration, the observed amorphous aggregates became larger and morphologically different. We propose that following occupation of the binding cleft, excess Morin adheres and coats the HEWL protein surface, thereby minimizing the interaction between the protein surface and water molecules. © 2017 Elsevier B.V. All rights reserved.
1. Introduction A number of chronic degenerative diseases, including Alzheimer’s disease, Parkinson’s disease, type-2 diabetes and some coronary heart diseases are associated with formation of amyloid fibrils [1]. Highly ordered and -sheet rich abnormal conformations are typical characteristics of amyloid fibrils, which are formed following the aggregation of misfolded proteins [2,3]. Hen egg-white lysozyme (HEWL) is a structural homologue of human lysozyme, whose amyloidogenic variants correlate with the incidence of systemic amyloidosis [4]. The structure of HEWL consists of several ␣-helical and -sheet regions, which are designated as ␣ and -domains, respectively. A core structure termed the HEWL K peptide (GILQINSRW, residues 54–62 in the -domain of the cleft), isolated from trypsin-digested HEWL, is found to have the ability to self-assemble and is involved in fibril formation [5]. The search for new compounds that can interfere with the aggregation of amyloid-forming proteins is considered to be an important strategy in the development of potential new therapeutics [6]. Naturally occurring polyphenolic compounds are found
∗ Corresponding author. E-mail address: [email protected] (J. He). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2017.03.107 0141-8130/© 2017 Elsevier B.V. All rights reserved.
extensively in a variety of foods and herbal remedies, and are considered as promising pharmaceuticals against in the treatment of amyloid diseases [7]. Among the polyphenol class of plant flavonoids, Morin (2 , 3, 4 , 5, 7-pentahydroxyflavone) was originally isolated from the members of the Moraceae family and is a major component of many fruits, herbs and wine [8,9]. We have previously reported the inhibitory activity of Myricetin against HEWL fibril formation, in which Myricetin exhibited a stronger inhibition than the well-characterized polyphenol Quercetin [10]. In contrast to our previous studies using other polyphenols, we find the generation of irregular structural aggregates formed by the binding of Morin to HEWL, which support a novel and distinctive model for how this small molecule inhibits amyloid formation.
2. Materials and methods 2.1. Proteins and reagents Morin, thioflavin T (ThT), and 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HEWL was purchased from Solarbio (Beijing, China). Preparation of HEWL samples were performed as previously described [10].
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2.2. Spectrofluorometric studies and physical analysis of HEWL fibril formation Stock solution containing 1 mM ThT was prepared in phosphate buffer (50 mM Na2HPO4, 50 mM NaH2PO4, pH 7.0) and stock solution of ANS (0.4 mM) was prepared by dissolving ANS in PBS (pH 7.0) and were both stored at 4 ◦ C. ThT, ANS and Intrinsic fluorescence assay as well as transmission electron microscopy (TEM) analysis were performed as previously described [10]. The IC50 of Morin with respect to inhibition of HEWL fibril formation was determined using the equation Y = 100/(1 + 10(X−LogIC50) ) in Prism 5 (GraphPad Software, Inc, Avenida de la Playa, CA).
2.3. Molecular dynamic simulations and docking studies Molecular dynamics (MD) simulations were performed using GROMACS 4.0.7 program at the Supercomputer Center of Liaoning University on dual-core Pentium 2.8 G processor of Linux cluster [11]. The crystal structure of HEWL (PDB entry 1GXV) was down-
loaded from the Protein Data Bank (PDB) [12]. The 3D structure of Morin was obtained from Chem Spider database (http://www. chemspider.com/) [13]. Molecular dynamics (MD) simulations and the docking studies were performed by the method of He et al. [10].
3. Results and discussion 3.1. Spectrofluorometric studies of HEWL fibril formation and anti-fibrillogenic activity of Morin ThT fluorescence assay was carried out to monitor the kinetics of HEWL fibril formation. After a lag of about one day, the ThT fluorescence intensity of HEWL increased rapidly, reaching a plateau on day four (Fig. 1A). In contrast, the presence of Morin prolonged the fibrillation lag-phase and showed a significant decrease in ThT fluorescence over the amyloid fibril formation period. To quantitatively compare the differences in the extent of fibril formation among the lysozyme samples with different concentrations of Morin, the time-dependent data points obtained from ThT fluo-
Fig. 1. Effects of different concentrations of Morin on fibril formation by HEWL amyloid. (A) Kinetics of HEWL fibril formation in the absence (filled squares) or the presence of Morin as monitored by ThT fluorescence. The curves were obtained by fitting experimental data against a sigmoidal-type equation (shown in the Result section) associated with the nucleation-dependent pathway. (B) Dose-response curve plotting the plateau value of the ThT fluorescence of the HEWL sample against Morin concentration. (C) ANS fluorescence and (D) intrinsic fluorescence emission spectra of HEWL fibrils after seven days of incubation without or with Morin.
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Fig. 2. Transmission electron microscopic images of HEWL fibrils formed in the absence or presence of Morin after incubation at 65 ◦ C and pH 2.0 for 7 days or 12 days. HEWL fibrils formed after incubation for 7 days (A), 12 days (B); HEWL fibril in the presence of 10 M (C, D), 50 M (E), 100 M (F) Morin after a 12-day incubation period. Scale bars represent 1 m.
rescence measurements were fitted against a sigmoidal-type curve, which suggest correlation with the nucleation-dependent pathway and can be described by the following equation: y=
a 1 + e−k(x−xc )
Where y is the fluorescence intensity at time t, a the initial fluorescence intensity, b the final fluorescence intensity, x the incubation time, and Xc the time to reach 50% of maximal fluorescence. The values of four parameters, a, b, Xc and k were obtained by the nonlinear regression. The apparent growth rate constant for fibril growth is given by k [14]. Two key kinetic parameters, the lag times and the growth rates of different samples were respectively determined and listed as follows: 1.02 ± 0.15 d and 43.15 ± 2.24 for the sample of HEWL alone, 1.74 ± 0.20 d and 19.85 ± 1.52 for the sample of HEWL incubated with 10 M Morin and 2.63 ± 0.58 d and 7.68 ± 1.23 for the sample of HEWL incubated with 100 M Morin. Our results revealed that the inhibition of amyloid fibril formation was dependent upon the concentrations of Morin tested. Moreover, compared to the inhibitory effect of Myricetin investigated in our previous study, Morin exhibited a stronger inhibitory effect (Fig. 1A and data not shown) [10]. By calculating the percentage of protection from the ThT curve obtained from HEWL alone and constructing a logarithmic concentration response curve, the median effective concentration (IC50 value) of Morin with respect to its inhibition of HEWL fibril formation was determined to be 13.1 M (Fig. 1B). ANS is a fluorescence dye that has high affinity for the hydrophobic surfaces of proteins. Once ANS binds to the hydrophobic surface of a protein, the intensity of the light emission is increased resulting in a red shift of the fluorescence emission maximum [15]. The intensity of ANS fluorescence exhibited by HEWL fibrils showed a prominent increase in intensity and a blue shift from 515 to 473 nm when compared to native HEWL (Fig. 1C). In contrast, the addition of Morin to HEWL led to a large reduction in ANS fluorescence intensity in conjunction with a red shift (Fig. 1C). This result indicated that, under fibril-forming conditions, Morin could prevent the exposure of accessible hydrophobic regions of HEWL by stabilizing
the native structure of HEWL. Interestingly, a significant decrease in ANS fluorescence intensity was observed when Morin was added to HEWL at a concentration between 10 M and 100 M (Fig. 1C). This decrease in fluorescence suggested that exposure of a large hydrophobic region on the surface of the protein molecule was largely prevented when concentrations of Morin reached greater than 10 M. To further study the effect of Morin on the conformational changes of HEWL the intrinsic fluorescence of HEWL in the absence or presence of Morin was investigated. Compared to native HEWL, HEWL fibrils displayed a reduced intensity in intrinsic fluorescence accompanied by a slight red shift from 344 to 355 nm (Fig. 1D). This indicates that the local environment of the tryptophan residues in the HEWL fibril has become more accessible to solvent during the process of fibril formation, resulting in the quenching of fluorescence by water [16,17]. However, the fluorescence intensity of HEWL gradually decreased upon the addition of Morin, and 10 M Morin induced a significant change in fluorescence intensity, corresponding to an IC50 value of 13.1 M (Fig. 1D). This result indicated that the local environment of the tryptophan residues in the HEWL fibril became more accessible to solvent during the process of fibril formation, resulting in the quenching of fluorescence by water [18,19]. The IC50 values for a number of inhibitory molecules of lysozyme amyloid have been reported. Several are considered to have potent inhibitory effects against the fibrillation of amyloidogenic HEWL including bis(indolyl)aryl methanes [20], erythrosine B [21], nordihydroguaiaretic acid [22] and myricetin [10], which exhibit IC50 values of 12.3, 13.3, 26.3 and 28.2 M, respectively. The IC50 value of Morin was determined to be 13.1 M, indicating Morin has a significant effect on inhibition of HEWL fibril formation compared to other characterized small molecule inhibitors. 3.2. Physical analysis of anti-fibrillogenic activity of Morin The morphology of HEWL fibers and/or aggregates in the absence or presence of Morin is shown in Fig. 2A–F. In the absence of Morin, HEWL displayed long and unbranched immature amyloid
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Fig. 3. (A) Panoramic view showing the binding mode between Morin and HEWL at the binding energy of −5.85 kcal/mol. (B) Residues of interaction between Morin and HEWL at binding energy of −5.85 kcal/mol (green dots, H-bonds). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
fibrils (Fig. 2A) or mature amyloid fibrils (Fig. 2B) with reticular structure. HEWL incubated with 10 M Morin exhibited similar structure but had fewer fibrils compared to HEWL incubated without Morin (Fig. 2C). Additionally, many smaller aggregates, several nanometers in diameter, were also observed amongst the fibrils (Fig. 2D). Interestingly, HEWL formed amorphous aggregates with diameters ranging from 0.1 to 0.2 m when the concentration of Morin was increased to 50 M (Fig. 2E). Less fibrils and more amorphous aggregates were observed when the concentration of Morin increased to 100 M (Fig. 2F). Of particular note is that the amorphous aggregates consisting of Morin and HEWL became larger with increased Morin concentration. The spheroidal-like aggregates were found in a freely existing state and were not adhered or associated with fibers. 3.3. Computational analysis of the binding between Morin and HEWL To identify the binding site at the atomic level, in silico studies were carried out using molecular dynamics (MD) simulations and docking studies. The interacting residues of the three most probable binding patterns and properties resulting from the binding were determined and according to the best binding model, Morin was located in an area between the ␣-domain and the -domain (Fig. 3A). Such efficient binding of Morin to HEWL is probably a major contributing factor to the effective inhibition of fibrillogenesis. The interactions between Morin and the relevant residues of HEWL consisted of van der Waals interaction and hydrogen bonding. The predicted Morin-binding site on HEWL at the lowest binding energy of −5.85 kcal/mol was clustered around residues that included Glu35, Asn46, Asp52, Gln57, Asn59, Trp62 and Trp63, Ala107, Trp108 and Val109. Among these, Ala107, Trp108 and Val109, all of which are hydrophobic residues, are located in the ␣domain whereas most of the other residues are located in or around the K peptide of the -domain (Fig. 3B). In addition, a mutagenesis study has shown that Trp62 is crucial for HEWL to form fibrils [23]. Taken together, the result suggested that the interaction between Morin and both the ␣-domain and -domain of HEWL plays a key role in the suppression of HEWL fibril formation by potently reinforcing the protein-ligand complex, so as to stabilize the native structure of HEWL. Of particular note is the interaction between Morin and Trp62 as well as Trp108, the two dominant emitters of intrinsic fluorescence in native HEWL, which could explain the quenching of intrinsic fluorescence [24]. Our findings here, particularly the ANS results, suggest a possible two-step mechanism for Morin’s inhibition of HEWL fibrillation.
We proposed that after Morin was mixed with HEWL and, is present at a low concentration, only a very small proportion of Morin molecules will be available to interact directly with HEWL in a sitespecific manner and will result in a greater exposure of the partially unfolded protein surface, in turn, would lead to self-association and fibrillation. On the other hand, when Morin is present in excess at higher concentrations, it would interact with the protein surface freely, and in doing so, Morin would displace water molecules, thereby reducing the interaction between the HEWL and water molecules. Choudhary et al. recently proposed a possible mechanism for osmolytes’ inhibitory effects on insulin fibrillation. In the presence of osmolytes, two processes will happen: direct polar interaction of osmolytes with insulin and preferential hydration of the protein surface. Both these factors inhibit fibrillation of the protein and a certain degree of amorphous aggregation of the partially folded molecules is thus possible [25]. We propose a similar mechanism to explain the concentration dependence for the mode of action of Morin binding to the surface of HEWL and influencing amyloid fibril formation (Fig. 1C). Consequently, the amorphous aggregates of the partially folded proteins increased in size with the increasing concentration of Morin (Figs. 2E, F & S4) Acknowledgements This work was supported by grants from National Natural Science Foundation of China (No. 31670103) and partially sponsored by Innovation Team Project (No. LT2015011) from the Education Department of Liaoning Province. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2017. 03.107. References [1] C.M. Dobson, Nature 426 (2003) 884–890. [2] M.B. Pepys, Annu. Rev. Med. 57 (2006) 223–241. [3] K.W. Tipping, P. van Oosten-Hawle, E.W. Hewitt, S.E. Radford, Trends Biochem. Sci. 40 (2015) 719–727. [4] M.B. Pepys, G.M. Hirschfield, G.A. Tennent, J.R. Gallimore, M.C. Kahan, V. Bellotti, P.N. Hawkins, R.M. Myers, M.D. Smith, A. Polara, A.J. Cobb, S.V. Ley, J.A. Aquilina, C.V. Robinson, I. Sharif, G.A. Gray, C.A. Sabin, M.C. Jenvey, S.E. Kolstoe, D. Thompson, S.P. Wood, Nature 440 (2006) 1217–1221. [5] Y. Sugimoto, Y. Kamada, Y. Tokunaga, H. Shinohara, M. Matsumoto, T. Kusakabe, T. Ohkuri, T. Ueda, Biochem. Cell Biol. 89 (2011) 533–544. [6] B. Cheng, H. Gong, H. Xiao, R.B. Petersen, L. Zheng, K. Huang, Biochim. Biophys. Acta 1830 (2013) 4860–4871. [7] M. Stefani, S. Rigacci, Int. J. Mol. Sci. 14 (2013) 12411–12457. [8] R. Kapoor, P. Kakkar, PLoS One 7 (2012) e41663. [9] K.F.S. Ricardo, T.T. d. Oliveira, T.J. Nagem, A.S. d. Pinto, M.G.A. Oliveira, J.F. Soares, Braz. Arch. Biol. Technol. 44 (2001) 263–267. [10] J. He, Y. Wang, A.K. Chang, L. Xu, N. Wang, X. Chong, H. Li, B. Zhang, G.W. Jones, Y. Song, J. Agric. Food Chem. 62 (2014) 9442–9449. [11] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J. Chem. Theory Comput. 4 (2008) 435–447. [12] M. Refaee, T. Tezuka, K. Akasaka, M.P. Williamson, J. Mol. Biol. 327 (2003) 857–865. [13] C.Y. Chen, PLoS One 6 (2011) e15939. [14] L. Nielsen, R. Khurana, A. Coats, S. Frokjaer, J. Brange, S. Vyas, V.N. Uversky, A.L. Fink, Biochemistry 40 (2001) 6036–6046. [15] G.V. Semisotnov, N.A. Rodionova, O.I. Razgulyaev, V.N. Uversky, A.F. Gripas, R.I. Gilmanshin, Biopolymers 31 (1991) 119–128. [16] C. Duy, J. Fitter, Biophys. J. 90 (2006) 3704–3711. [17] K. Russo, E. Di Stasio, G. Macchia, G. Rosa, A. Brancaccio, T.C. Petrucci, Biochem. Biophys. Res. Commun. 274 (2000) 93–98. [18] H.M. Rawel, D. Czajka, S. Rohn, J. Kroll, Int. J. Biol. Macromol. 30 (2002) 137–150. [19] P. Suryaprakash, R.P. Kumar, V. Prakash, Int. J. Biol. Macromol. 27 (2000) 219–228. [20] H. Ramshini, B. Mannini, K. Khodayari, A. Ebrahim-Habibi, A.S. Moghaddasi, R. Tayebee, F. Chiti, Eur. J. Med. Chem. 124 (2016) 361–371.
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[24] T. Imoto, L.S. Forster, J.A. Rupley, F. Tanaka, Proc. Natl. Acad. Sci. U. S. A. 69 (1972) 1151–1155. [25] S. Choudhary, N. Kishore, R.V. Hosur, Sci. Rep. 5 (2015) 17599.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Insights into the mechanism of how Morin suppresses amyloid fibrillation of hen egg white lysozyme Xiaoying Chong a,1 , Luchen Sun a,1 , Yonghui Sun a , Lin Chang a , Alan K. Chang a , Xian Lu a , Xuejie Zhou a , Junqing Liu a , Bing Zhang b , Gary W. Jones c , Jianwei He a,∗ a
School of Life Science, Liaoning University, Shenyang 110036, China Experimental Center of Functional Subjects, China Medical University, 92 Bei Er Road, Heping District, Shenyang 110001, China c Centre for Biomedical Science Research, School of Clinical and Applied Sciences, Leeds Beckett University, City Campus, Leeds LS1 3HE, United Kingdom b
a r t i c l e
i n f o
Article history: Received 26 November 2016 Received in revised form 17 March 2017 Accepted 20 March 2017 Available online 21 March 2017 Keywords: Morin Hen egg white lysozyme Amyloid
a b s t r a c t This communication describes the inhibitory effect of Morin on the fibrillation of Hen Egg White Lysozyme (HEWL), a generic amyloid-forming model protein. This effect was dose-dependent and stronger than other small molecules we have tested previously. Spectrofluorometric and computational studies support a model suggesting that Morin inhibits amyloid fibril formation of HEWL by binding to the aggregation prone cleft region of the -domain of HEWL, thereby stabilizing the molecule in its native-like state. Interestingly, transmission electron microscopy observations suggest that, along with increases in Morin concentration, the observed amorphous aggregates became larger and morphologically different. We propose that following occupation of the binding cleft, excess Morin adheres and coats the HEWL protein surface, thereby minimizing the interaction between the protein surface and water molecules. © 2017 Elsevier B.V. All rights reserved.
1. Introduction A number of chronic degenerative diseases, including Alzheimer’s disease, Parkinson’s disease, type-2 diabetes and some coronary heart diseases are associated with formation of amyloid fibrils [1]. Highly ordered and -sheet rich abnormal conformations are typical characteristics of amyloid fibrils, which are formed following the aggregation of misfolded proteins [2,3]. Hen egg-white lysozyme (HEWL) is a structural homologue of human lysozyme, whose amyloidogenic variants correlate with the incidence of systemic amyloidosis [4]. The structure of HEWL consists of several ␣-helical and -sheet regions, which are designated as ␣ and -domains, respectively. A core structure termed the HEWL K peptide (GILQINSRW, residues 54–62 in the -domain of the cleft), isolated from trypsin-digested HEWL, is found to have the ability to self-assemble and is involved in fibril formation [5]. The search for new compounds that can interfere with the aggregation of amyloid-forming proteins is considered to be an important strategy in the development of potential new therapeutics [6]. Naturally occurring polyphenolic compounds are found
∗ Corresponding author. E-mail address: [email protected] (J. He). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2017.03.107 0141-8130/© 2017 Elsevier B.V. All rights reserved.
extensively in a variety of foods and herbal remedies, and are considered as promising pharmaceuticals against in the treatment of amyloid diseases [7]. Among the polyphenol class of plant flavonoids, Morin (2 , 3, 4 , 5, 7-pentahydroxyflavone) was originally isolated from the members of the Moraceae family and is a major component of many fruits, herbs and wine [8,9]. We have previously reported the inhibitory activity of Myricetin against HEWL fibril formation, in which Myricetin exhibited a stronger inhibition than the well-characterized polyphenol Quercetin [10]. In contrast to our previous studies using other polyphenols, we find the generation of irregular structural aggregates formed by the binding of Morin to HEWL, which support a novel and distinctive model for how this small molecule inhibits amyloid formation.
2. Materials and methods 2.1. Proteins and reagents Morin, thioflavin T (ThT), and 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HEWL was purchased from Solarbio (Beijing, China). Preparation of HEWL samples were performed as previously described [10].
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2.2. Spectrofluorometric studies and physical analysis of HEWL fibril formation Stock solution containing 1 mM ThT was prepared in phosphate buffer (50 mM Na2HPO4, 50 mM NaH2PO4, pH 7.0) and stock solution of ANS (0.4 mM) was prepared by dissolving ANS in PBS (pH 7.0) and were both stored at 4 ◦ C. ThT, ANS and Intrinsic fluorescence assay as well as transmission electron microscopy (TEM) analysis were performed as previously described [10]. The IC50 of Morin with respect to inhibition of HEWL fibril formation was determined using the equation Y = 100/(1 + 10(X−LogIC50) ) in Prism 5 (GraphPad Software, Inc, Avenida de la Playa, CA).
2.3. Molecular dynamic simulations and docking studies Molecular dynamics (MD) simulations were performed using GROMACS 4.0.7 program at the Supercomputer Center of Liaoning University on dual-core Pentium 2.8 G processor of Linux cluster [11]. The crystal structure of HEWL (PDB entry 1GXV) was down-
loaded from the Protein Data Bank (PDB) [12]. The 3D structure of Morin was obtained from Chem Spider database (http://www. chemspider.com/) [13]. Molecular dynamics (MD) simulations and the docking studies were performed by the method of He et al. [10].
3. Results and discussion 3.1. Spectrofluorometric studies of HEWL fibril formation and anti-fibrillogenic activity of Morin ThT fluorescence assay was carried out to monitor the kinetics of HEWL fibril formation. After a lag of about one day, the ThT fluorescence intensity of HEWL increased rapidly, reaching a plateau on day four (Fig. 1A). In contrast, the presence of Morin prolonged the fibrillation lag-phase and showed a significant decrease in ThT fluorescence over the amyloid fibril formation period. To quantitatively compare the differences in the extent of fibril formation among the lysozyme samples with different concentrations of Morin, the time-dependent data points obtained from ThT fluo-
Fig. 1. Effects of different concentrations of Morin on fibril formation by HEWL amyloid. (A) Kinetics of HEWL fibril formation in the absence (filled squares) or the presence of Morin as monitored by ThT fluorescence. The curves were obtained by fitting experimental data against a sigmoidal-type equation (shown in the Result section) associated with the nucleation-dependent pathway. (B) Dose-response curve plotting the plateau value of the ThT fluorescence of the HEWL sample against Morin concentration. (C) ANS fluorescence and (D) intrinsic fluorescence emission spectra of HEWL fibrils after seven days of incubation without or with Morin.
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Fig. 2. Transmission electron microscopic images of HEWL fibrils formed in the absence or presence of Morin after incubation at 65 ◦ C and pH 2.0 for 7 days or 12 days. HEWL fibrils formed after incubation for 7 days (A), 12 days (B); HEWL fibril in the presence of 10 M (C, D), 50 M (E), 100 M (F) Morin after a 12-day incubation period. Scale bars represent 1 m.
rescence measurements were fitted against a sigmoidal-type curve, which suggest correlation with the nucleation-dependent pathway and can be described by the following equation: y=
a 1 + e−k(x−xc )
Where y is the fluorescence intensity at time t, a the initial fluorescence intensity, b the final fluorescence intensity, x the incubation time, and Xc the time to reach 50% of maximal fluorescence. The values of four parameters, a, b, Xc and k were obtained by the nonlinear regression. The apparent growth rate constant for fibril growth is given by k [14]. Two key kinetic parameters, the lag times and the growth rates of different samples were respectively determined and listed as follows: 1.02 ± 0.15 d and 43.15 ± 2.24 for the sample of HEWL alone, 1.74 ± 0.20 d and 19.85 ± 1.52 for the sample of HEWL incubated with 10 M Morin and 2.63 ± 0.58 d and 7.68 ± 1.23 for the sample of HEWL incubated with 100 M Morin. Our results revealed that the inhibition of amyloid fibril formation was dependent upon the concentrations of Morin tested. Moreover, compared to the inhibitory effect of Myricetin investigated in our previous study, Morin exhibited a stronger inhibitory effect (Fig. 1A and data not shown) [10]. By calculating the percentage of protection from the ThT curve obtained from HEWL alone and constructing a logarithmic concentration response curve, the median effective concentration (IC50 value) of Morin with respect to its inhibition of HEWL fibril formation was determined to be 13.1 M (Fig. 1B). ANS is a fluorescence dye that has high affinity for the hydrophobic surfaces of proteins. Once ANS binds to the hydrophobic surface of a protein, the intensity of the light emission is increased resulting in a red shift of the fluorescence emission maximum [15]. The intensity of ANS fluorescence exhibited by HEWL fibrils showed a prominent increase in intensity and a blue shift from 515 to 473 nm when compared to native HEWL (Fig. 1C). In contrast, the addition of Morin to HEWL led to a large reduction in ANS fluorescence intensity in conjunction with a red shift (Fig. 1C). This result indicated that, under fibril-forming conditions, Morin could prevent the exposure of accessible hydrophobic regions of HEWL by stabilizing
the native structure of HEWL. Interestingly, a significant decrease in ANS fluorescence intensity was observed when Morin was added to HEWL at a concentration between 10 M and 100 M (Fig. 1C). This decrease in fluorescence suggested that exposure of a large hydrophobic region on the surface of the protein molecule was largely prevented when concentrations of Morin reached greater than 10 M. To further study the effect of Morin on the conformational changes of HEWL the intrinsic fluorescence of HEWL in the absence or presence of Morin was investigated. Compared to native HEWL, HEWL fibrils displayed a reduced intensity in intrinsic fluorescence accompanied by a slight red shift from 344 to 355 nm (Fig. 1D). This indicates that the local environment of the tryptophan residues in the HEWL fibril has become more accessible to solvent during the process of fibril formation, resulting in the quenching of fluorescence by water [16,17]. However, the fluorescence intensity of HEWL gradually decreased upon the addition of Morin, and 10 M Morin induced a significant change in fluorescence intensity, corresponding to an IC50 value of 13.1 M (Fig. 1D). This result indicated that the local environment of the tryptophan residues in the HEWL fibril became more accessible to solvent during the process of fibril formation, resulting in the quenching of fluorescence by water [18,19]. The IC50 values for a number of inhibitory molecules of lysozyme amyloid have been reported. Several are considered to have potent inhibitory effects against the fibrillation of amyloidogenic HEWL including bis(indolyl)aryl methanes [20], erythrosine B [21], nordihydroguaiaretic acid [22] and myricetin [10], which exhibit IC50 values of 12.3, 13.3, 26.3 and 28.2 M, respectively. The IC50 value of Morin was determined to be 13.1 M, indicating Morin has a significant effect on inhibition of HEWL fibril formation compared to other characterized small molecule inhibitors. 3.2. Physical analysis of anti-fibrillogenic activity of Morin The morphology of HEWL fibers and/or aggregates in the absence or presence of Morin is shown in Fig. 2A–F. In the absence of Morin, HEWL displayed long and unbranched immature amyloid
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Fig. 3. (A) Panoramic view showing the binding mode between Morin and HEWL at the binding energy of −5.85 kcal/mol. (B) Residues of interaction between Morin and HEWL at binding energy of −5.85 kcal/mol (green dots, H-bonds). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
fibrils (Fig. 2A) or mature amyloid fibrils (Fig. 2B) with reticular structure. HEWL incubated with 10 M Morin exhibited similar structure but had fewer fibrils compared to HEWL incubated without Morin (Fig. 2C). Additionally, many smaller aggregates, several nanometers in diameter, were also observed amongst the fibrils (Fig. 2D). Interestingly, HEWL formed amorphous aggregates with diameters ranging from 0.1 to 0.2 m when the concentration of Morin was increased to 50 M (Fig. 2E). Less fibrils and more amorphous aggregates were observed when the concentration of Morin increased to 100 M (Fig. 2F). Of particular note is that the amorphous aggregates consisting of Morin and HEWL became larger with increased Morin concentration. The spheroidal-like aggregates were found in a freely existing state and were not adhered or associated with fibers. 3.3. Computational analysis of the binding between Morin and HEWL To identify the binding site at the atomic level, in silico studies were carried out using molecular dynamics (MD) simulations and docking studies. The interacting residues of the three most probable binding patterns and properties resulting from the binding were determined and according to the best binding model, Morin was located in an area between the ␣-domain and the -domain (Fig. 3A). Such efficient binding of Morin to HEWL is probably a major contributing factor to the effective inhibition of fibrillogenesis. The interactions between Morin and the relevant residues of HEWL consisted of van der Waals interaction and hydrogen bonding. The predicted Morin-binding site on HEWL at the lowest binding energy of −5.85 kcal/mol was clustered around residues that included Glu35, Asn46, Asp52, Gln57, Asn59, Trp62 and Trp63, Ala107, Trp108 and Val109. Among these, Ala107, Trp108 and Val109, all of which are hydrophobic residues, are located in the ␣domain whereas most of the other residues are located in or around the K peptide of the -domain (Fig. 3B). In addition, a mutagenesis study has shown that Trp62 is crucial for HEWL to form fibrils [23]. Taken together, the result suggested that the interaction between Morin and both the ␣-domain and -domain of HEWL plays a key role in the suppression of HEWL fibril formation by potently reinforcing the protein-ligand complex, so as to stabilize the native structure of HEWL. Of particular note is the interaction between Morin and Trp62 as well as Trp108, the two dominant emitters of intrinsic fluorescence in native HEWL, which could explain the quenching of intrinsic fluorescence [24]. Our findings here, particularly the ANS results, suggest a possible two-step mechanism for Morin’s inhibition of HEWL fibrillation.
We proposed that after Morin was mixed with HEWL and, is present at a low concentration, only a very small proportion of Morin molecules will be available to interact directly with HEWL in a sitespecific manner and will result in a greater exposure of the partially unfolded protein surface, in turn, would lead to self-association and fibrillation. On the other hand, when Morin is present in excess at higher concentrations, it would interact with the protein surface freely, and in doing so, Morin would displace water molecules, thereby reducing the interaction between the HEWL and water molecules. Choudhary et al. recently proposed a possible mechanism for osmolytes’ inhibitory effects on insulin fibrillation. In the presence of osmolytes, two processes will happen: direct polar interaction of osmolytes with insulin and preferential hydration of the protein surface. Both these factors inhibit fibrillation of the protein and a certain degree of amorphous aggregation of the partially folded molecules is thus possible [25]. We propose a similar mechanism to explain the concentration dependence for the mode of action of Morin binding to the surface of HEWL and influencing amyloid fibril formation (Fig. 1C). Consequently, the amorphous aggregates of the partially folded proteins increased in size with the increasing concentration of Morin (Figs. 2E, F & S4) Acknowledgements This work was supported by grants from National Natural Science Foundation of China (No. 31670103) and partially sponsored by Innovation Team Project (No. LT2015011) from the Education Department of Liaoning Province. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2017. 03.107. References [1] C.M. Dobson, Nature 426 (2003) 884–890. [2] M.B. Pepys, Annu. Rev. Med. 57 (2006) 223–241. [3] K.W. Tipping, P. van Oosten-Hawle, E.W. Hewitt, S.E. Radford, Trends Biochem. Sci. 40 (2015) 719–727. [4] M.B. Pepys, G.M. Hirschfield, G.A. Tennent, J.R. Gallimore, M.C. Kahan, V. Bellotti, P.N. Hawkins, R.M. Myers, M.D. Smith, A. Polara, A.J. Cobb, S.V. Ley, J.A. Aquilina, C.V. Robinson, I. Sharif, G.A. Gray, C.A. Sabin, M.C. Jenvey, S.E. Kolstoe, D. Thompson, S.P. Wood, Nature 440 (2006) 1217–1221. [5] Y. Sugimoto, Y. Kamada, Y. Tokunaga, H. Shinohara, M. Matsumoto, T. Kusakabe, T. Ohkuri, T. Ueda, Biochem. Cell Biol. 89 (2011) 533–544. [6] B. Cheng, H. Gong, H. Xiao, R.B. Petersen, L. Zheng, K. Huang, Biochim. Biophys. Acta 1830 (2013) 4860–4871. [7] M. Stefani, S. Rigacci, Int. J. Mol. Sci. 14 (2013) 12411–12457. [8] R. Kapoor, P. Kakkar, PLoS One 7 (2012) e41663. [9] K.F.S. Ricardo, T.T. d. Oliveira, T.J. Nagem, A.S. d. Pinto, M.G.A. Oliveira, J.F. Soares, Braz. Arch. Biol. Technol. 44 (2001) 263–267. [10] J. He, Y. Wang, A.K. Chang, L. Xu, N. Wang, X. Chong, H. Li, B. Zhang, G.W. Jones, Y. Song, J. Agric. Food Chem. 62 (2014) 9442–9449. [11] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J. Chem. Theory Comput. 4 (2008) 435–447. [12] M. Refaee, T. Tezuka, K. Akasaka, M.P. Williamson, J. Mol. Biol. 327 (2003) 857–865. [13] C.Y. Chen, PLoS One 6 (2011) e15939. [14] L. Nielsen, R. Khurana, A. Coats, S. Frokjaer, J. Brange, S. Vyas, V.N. Uversky, A.L. Fink, Biochemistry 40 (2001) 6036–6046. [15] G.V. Semisotnov, N.A. Rodionova, O.I. Razgulyaev, V.N. Uversky, A.F. Gripas, R.I. Gilmanshin, Biopolymers 31 (1991) 119–128. [16] C. Duy, J. Fitter, Biophys. J. 90 (2006) 3704–3711. [17] K. Russo, E. Di Stasio, G. Macchia, G. Rosa, A. Brancaccio, T.C. Petrucci, Biochem. Biophys. Res. Commun. 274 (2000) 93–98. [18] H.M. Rawel, D. Czajka, S. Rohn, J. Kroll, Int. J. Biol. Macromol. 30 (2002) 137–150. [19] P. Suryaprakash, R.P. Kumar, V. Prakash, Int. J. Biol. Macromol. 27 (2000) 219–228. [20] H. Ramshini, B. Mannini, K. Khodayari, A. Ebrahim-Habibi, A.S. Moghaddasi, R. Tayebee, F. Chiti, Eur. J. Med. Chem. 124 (2016) 361–371.
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