- Email: [email protected]
Effect of Curcumin on the metal ion induced fibrillization of Amyloid-β peptide
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Short Communication
Effect of Curcumin on the metal ion induced fibrillization of Amyloid-b peptide Rona Banerjee ⇑ Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, Uttaranchal, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Curcumin can reduce the b-sheet
The reduction in b-sheet content of Amyloid-b peptide occurs in presence of Curcumin as a function of time. 30
Aβ A β+Curcumin (30 min) A β+Curcumin (6hrs) A β+Curcumin (24 hrs)
20 -1
([θ] mdeg cm dmol )
10
2
content of Ab peptide in time dependent manner. Metal ions like Cu(II) and Zn(II) cause Ab fibrillization. Curcumin can inhibit metal ion induced fibrillization and aggregation of the peptide.
0 -10 -20 -30 200
210
220
230
240
250
260
Wavelength (nm)
a r t i c l e
i n f o
Article history: Received 20 May 2013 Received in revised form 29 August 2013 Accepted 18 September 2013 Available online 27 September 2013 Keywords: Curcumin Ab Metals Fibrillization CD AFM
a b s t r a c t The effect of Curcumin on Cu(II) and Zn(II) induced oligomerization and protofibrillization of the amyloid-beta (Ab) peptide has been studied by spectroscopic and microscopic methods. Curcumin could significantly reduce the b-sheet content of the peptide in a time dependent manner. It also plays an antagonistic role in b-sheet formation that is promoted by metal ions like Cu(II) and Zn(II) as observed by Circular Dichroism (CD) spectroscopy. Atomic force microscopic (AFM) images show that spontaneous fibrillization of the peptide occurs in presence of Cu(II) and Zn(II) but is inhibited on incubation of the peptide with Curcumin indicating the beneficial role of Curcumin in preventing the aggregation of Ab peptide. Ó 2013 Elsevier B.V. All rights reserved.
Introduction The fibrillation and aggregation of misfolded amyloid-beta (Ab) peptide leads to the formation of senile plaques that are the pathological hallmarks in Alzheimer’s disease (AD). Ab peptide has a very high binding affinity to divalent metal ions like Zn(II) and ⇑ Tel.: +91 7895635142; fax: +91 1332 27 3560. E-mail addresses: [email protected], [email protected] 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.064
Cu(II) [1,2] that are found in excess in the AD brain and can act as agonist in the aggregation of Ab. The Fenton reaction involving the redox active metal generate the reactive oxygen species (ROS) causing severe oxidative stress and promotes protein aggregation [3,4]. Curcumin, a polyphenolic phytochemical obtained from the rhizome of Curcuma longa, is a potential anti-inflammatory, antioxidant and neuroprotective agent [5,6]. Curcumin has also been demonstrated to inhibit the aggregation of Ab peptide in vitro
799
R. Banerjee / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800
30 25 20
Curcumin (99% pure) from Merck (Germany) and synthetic human Amyloid-b (1-42) peptide from Genscript (USA) were procured and used without any further modification. Salts and buffers were procured from Himedia and the solutions were prepared in Millipore water. Concentrated stock solution of Curcumin was prepared in spectroscopic grade acetone and was diluted to desired concentration using 10 mM phosphate buffer of pH 7.4. The stability of Curcumin was checked before every experiment using absorption spectroscopy. The peptide stock solution was prepared in 10 mM phosphate buffer saline (PBS) of pH 8 and stored at 20 °C. Circular Dichroism (CD) studies were performed by Chirascan spectropolarimeter (Applied Photophysics, USA) using quartz cuvettes of 1 mm path length. The peptide samples were prepared in 10 mM phosphate buffer of pH 7.4 and the experiments were carried out at a temperature of 25 °C. Atomic Force microscopy (AFM) has been employed to obtain information about the morphology of the peptide. The concentration of peptide was kept very low (10 lM) to ensure uniform spreading of the samples on the cover slips in thin monolayers for better visualization. The images were acquired by the Atomic Force Microscope (AFM, NT-MDT model NTEGRA T5-150) in the semi-contact mode at ambient air (RH 50%) and temperature (23 °C) with a silicon cantilever at its resonance frequency five successive scans for each image. Results and discussion The CD spectroscopic studies show the effect of Curcumin on the secondary structure of Ab peptide (Fig. 2) as a function of time. Incubation of the peptide with equimolar amount (50 lM) of Curcumin induces a significant decrease in the b-sheet content of the peptide over an incubation period of 0–24 h showing a reduction in [h] value (mean residual molar ellipticity) at 220 nm. The appearance of isodichroic point around 210 nm for Curcumin containing samples imply an equilibrium between b-sheet and the unstructured coil structure of the peptide.
2
5 0 -5 -10 -15 -20 -25 -30 200
210
220
230
240
250
260
Wavelength (nm) Fig. 2. CD spectra of Ab peptide (—), Ab with equimolar amount of Curcumin after 30 min ( ), 6 h (- - ) and 24 h of incubation (- - -).
On addition of equimolar amount (20 lM) of CuCl2 and ZnCl2 separately in the buffer containing 20 lM peptide, the [h] values around 218 nm were increased (Fig. 3) indicating a higher content of b-sheets, whereas, on addition of equimolar amount of Curcumin in the peptide solutions along with metal ions the reduction of [h] values signify a marked decrease in the same. It was also observed that the reduction of b-sheet by Curcumin is more pronounced for Cu(II) containing peptide sample than with Zn(II) which could be explained by higher binding affinity of Curcumin to Cu(II) than Zn(II) [11]. As no precipitation of the peptide occurred, therefore, it can be concluded that the reduction in the negative ellipticity value implies less content of b-sheets in the peptide for Curcumin containing samples where Curcumin could inhibit the b-sheet structure formation. The AFM images (Fig. 4) show the appearance of large oligomeric aggregates and protofibrillar structures of Ab peptide (10 lM) on incubation for 6 h at room temperature. For peptide treated with equimolar amount of Cu(II) in the buffer the presence
6 4 2 -1
Materials and methods
10
0
2
and in vivo [7]. By virtue of its chemical structure (Fig. 1) Curcumin can efficiently bind metal ions through its b-diketone moiety, and can prevent free radical generation by metal ions in solution [8,9]. In the aggregation process of the peptide, the soluble oligomers can act as the seeding factors for protofibril formation followed by matured fibrils and aggregates. The accumulation of the aggregates is considered as principally responsible for neurodegeneration [7]. Recent reports also emphazise on the neurotoxicity imparted by the high molecular weight oligomers that contain a substantial amount of b-sheet structure that accelerates the formation of protofibrils. Therefore, inhibitors of b-sheets could play a crucial role in preventing rapid oligomerization, fibrillization and cytotoxicity [10]. In this work, the effect of Curcumin on the secondary structure of the amyloid peptide and also the metal ion induced oligomerization and fibrillation have been investigated by biophysical methods.
[θ] mdeg cm dmol
Fig. 1. Chemical structure of Curcumin.
[θ] mdeg cm dmol
-1
15
-2 -4 -6 -8 200
220
240
260
280
300
Wavelength(nm) Fig. 3. CD spectra of Ab peptide showing the effect of free metal ions and that in combination with Curcumin. Ab in 10 mM PBS (—), Ab with 20 lM CuCl2 (- - -), with 20 lM ZnCl2 ( ) with 20 lM Curcumin and 20 lM CuCl2 (- - ), with 20 lM Curcumin and 20 lM ZnCl2 (- -).
800
R. Banerjee / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800
Fig. 4. AFM images acquired in semi-contact mode showing the oligomerization and fibrillization of (a) Ab peptide, Ab peptide treated with (b) Cu(II), (c) Zn(II), (d) Curcumin, (e) Cu(II) and Curcumin, (f) Zn(II) and Curcumin. The samples were incubated for 6hrs at room temperature.
of beaded fibrillar structures was more pronounced and with Zn(II) treated sample, large oligomeric assemblies was observed. This is evident of the agonistic role of Cu(II) and Zn(II) in promoting fibrillation and aggregation of the peptide. However, in the presence of equimolar amount of Curcumin, considering the particle morphology and size, no fibrillar structures were observed in the peptide, only small oligomeric nano-aggregates were detected. Absence of protofibrillar structures also indicate that Curcumin could effectively inhibit the Cu(II) and Zn(II) induced fibrillization of the peptide. As Curcumin is a potential metal chelator, it can compete with the peptide on complexing with metal ions thereby preventing peptide–metal complex formation that produces degenerative ROS, promoting the fibrillation and aggregation of Ab. CD results have shown that Curcumin efficiently reduces the b-sheet content of the peptide, which is the precursor for protofibrils and AFM imaging supports the observation as Curcumin could potentially inhibit fibrillation in vitro by arresting the polymerization of small oligomers. These observations also agree with the molecular dynamics simulation studies recently reported by Zhao et al. [12] suggesting that Curcumin could reduce b-sheet content of amyloid peptide without hindering the monomeric contacts. The present work also elucidates the novel property of Curcumin in inhibiting the metal ion induced b-sheet formation of Ab peptide. Conclusion Curcumin inhibits the seeding of fibrillation by preventing the peptide-metal complex formation with Cu(II) and Zn(II) as
observed by CD and AFM studies. Therefore, Curcumin, along with its many other beneficial properties can be considered as potential therapeutic agent for amyloidosis. Acknowledgements This work has been supported by the Department of Science and Technology, Govt. of India, under the SERC-Fast-track Young Scientist Scheme. References [1] X. Huang, C.S. Atwood, R.D. Moir, M.A. Hartshorn, J. Vonsattel, R.E. Tanzi, A.I. Bush, J. Biol. Chem. 272 (1973) 26464–26470. [2] C. Lin, H. Huang, Z. Jiang, Brain Res. Bull. 82 (2010) 235–242. [3] T. Rival, R.M. Page, D.S. Chandraratna, T.J. Sendall, E. Ryder, B. Liu, H. Lewis, T. Rosah, R. Hider, L.M. Camargo, M.S. Shearman, D.C. Crowther, D.A. Lomas, Eur. J. Neurosci. 29 (2009) 1335–1347. [4] D.G. Smith, R. Cappai, K.J. Barnham, Biochim. Biophys. Acta 1768 (2007) 1976– 1990. [5] B.B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Adv. Exp. Med. Biol. 595 (2007) 1–75. [6] S. Dong, Q. Zeng, E.S. Mitchell, J.X. Yale Duan, C. Li, J.K. Tiwari, Y. Hu1, X. Cao, Z. Zhao, PLoS ONE 7 (2012) 1–12. e31211. [7] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar, P. Chen, R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, J. Biol. Chem. 280 (2005) 5892–5901. [8] T. Ak, I. Gulcin, Chem. Biol. Interact. 174 (2008) 27–37. [9] T.M. Kolev, E.A. Velcheva, B.A. Stamboliyska, M. Spiteller, Int. J. Quantum Chem. 102 (2005) 1069–1079. [10] K. Onoa, M.M. Condrona, D.B. Teplowa, Proc. Natl. Acad. Sci. (USA) 106 (2009) 14745–14750. [11] L. Baum, A. Ng, J. Alzheimer’s. Dis. 6 (2004) 367–377. [12] L.N. Zhao, S.W. Chilu, J. Benoit, L.Y. Chew, Y. Mu, J. Phys. Chem. B 116 (2012) 7428–7435.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Short Communication
Effect of Curcumin on the metal ion induced fibrillization of Amyloid-b peptide Rona Banerjee ⇑ Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, Uttaranchal, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Curcumin can reduce the b-sheet
The reduction in b-sheet content of Amyloid-b peptide occurs in presence of Curcumin as a function of time. 30
Aβ A β+Curcumin (30 min) A β+Curcumin (6hrs) A β+Curcumin (24 hrs)
20 -1
([θ] mdeg cm dmol )
10
2
content of Ab peptide in time dependent manner. Metal ions like Cu(II) and Zn(II) cause Ab fibrillization. Curcumin can inhibit metal ion induced fibrillization and aggregation of the peptide.
0 -10 -20 -30 200
210
220
230
240
250
260
Wavelength (nm)
a r t i c l e
i n f o
Article history: Received 20 May 2013 Received in revised form 29 August 2013 Accepted 18 September 2013 Available online 27 September 2013 Keywords: Curcumin Ab Metals Fibrillization CD AFM
a b s t r a c t The effect of Curcumin on Cu(II) and Zn(II) induced oligomerization and protofibrillization of the amyloid-beta (Ab) peptide has been studied by spectroscopic and microscopic methods. Curcumin could significantly reduce the b-sheet content of the peptide in a time dependent manner. It also plays an antagonistic role in b-sheet formation that is promoted by metal ions like Cu(II) and Zn(II) as observed by Circular Dichroism (CD) spectroscopy. Atomic force microscopic (AFM) images show that spontaneous fibrillization of the peptide occurs in presence of Cu(II) and Zn(II) but is inhibited on incubation of the peptide with Curcumin indicating the beneficial role of Curcumin in preventing the aggregation of Ab peptide. Ó 2013 Elsevier B.V. All rights reserved.
Introduction The fibrillation and aggregation of misfolded amyloid-beta (Ab) peptide leads to the formation of senile plaques that are the pathological hallmarks in Alzheimer’s disease (AD). Ab peptide has a very high binding affinity to divalent metal ions like Zn(II) and ⇑ Tel.: +91 7895635142; fax: +91 1332 27 3560. E-mail addresses: [email protected], [email protected] 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.064
Cu(II) [1,2] that are found in excess in the AD brain and can act as agonist in the aggregation of Ab. The Fenton reaction involving the redox active metal generate the reactive oxygen species (ROS) causing severe oxidative stress and promotes protein aggregation [3,4]. Curcumin, a polyphenolic phytochemical obtained from the rhizome of Curcuma longa, is a potential anti-inflammatory, antioxidant and neuroprotective agent [5,6]. Curcumin has also been demonstrated to inhibit the aggregation of Ab peptide in vitro
799
R. Banerjee / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800
30 25 20
Curcumin (99% pure) from Merck (Germany) and synthetic human Amyloid-b (1-42) peptide from Genscript (USA) were procured and used without any further modification. Salts and buffers were procured from Himedia and the solutions were prepared in Millipore water. Concentrated stock solution of Curcumin was prepared in spectroscopic grade acetone and was diluted to desired concentration using 10 mM phosphate buffer of pH 7.4. The stability of Curcumin was checked before every experiment using absorption spectroscopy. The peptide stock solution was prepared in 10 mM phosphate buffer saline (PBS) of pH 8 and stored at 20 °C. Circular Dichroism (CD) studies were performed by Chirascan spectropolarimeter (Applied Photophysics, USA) using quartz cuvettes of 1 mm path length. The peptide samples were prepared in 10 mM phosphate buffer of pH 7.4 and the experiments were carried out at a temperature of 25 °C. Atomic Force microscopy (AFM) has been employed to obtain information about the morphology of the peptide. The concentration of peptide was kept very low (10 lM) to ensure uniform spreading of the samples on the cover slips in thin monolayers for better visualization. The images were acquired by the Atomic Force Microscope (AFM, NT-MDT model NTEGRA T5-150) in the semi-contact mode at ambient air (RH 50%) and temperature (23 °C) with a silicon cantilever at its resonance frequency five successive scans for each image. Results and discussion The CD spectroscopic studies show the effect of Curcumin on the secondary structure of Ab peptide (Fig. 2) as a function of time. Incubation of the peptide with equimolar amount (50 lM) of Curcumin induces a significant decrease in the b-sheet content of the peptide over an incubation period of 0–24 h showing a reduction in [h] value (mean residual molar ellipticity) at 220 nm. The appearance of isodichroic point around 210 nm for Curcumin containing samples imply an equilibrium between b-sheet and the unstructured coil structure of the peptide.
2
5 0 -5 -10 -15 -20 -25 -30 200
210
220
230
240
250
260
Wavelength (nm) Fig. 2. CD spectra of Ab peptide (—), Ab with equimolar amount of Curcumin after 30 min ( ), 6 h (- - ) and 24 h of incubation (- - -).
On addition of equimolar amount (20 lM) of CuCl2 and ZnCl2 separately in the buffer containing 20 lM peptide, the [h] values around 218 nm were increased (Fig. 3) indicating a higher content of b-sheets, whereas, on addition of equimolar amount of Curcumin in the peptide solutions along with metal ions the reduction of [h] values signify a marked decrease in the same. It was also observed that the reduction of b-sheet by Curcumin is more pronounced for Cu(II) containing peptide sample than with Zn(II) which could be explained by higher binding affinity of Curcumin to Cu(II) than Zn(II) [11]. As no precipitation of the peptide occurred, therefore, it can be concluded that the reduction in the negative ellipticity value implies less content of b-sheets in the peptide for Curcumin containing samples where Curcumin could inhibit the b-sheet structure formation. The AFM images (Fig. 4) show the appearance of large oligomeric aggregates and protofibrillar structures of Ab peptide (10 lM) on incubation for 6 h at room temperature. For peptide treated with equimolar amount of Cu(II) in the buffer the presence
6 4 2 -1
Materials and methods
10
0
2
and in vivo [7]. By virtue of its chemical structure (Fig. 1) Curcumin can efficiently bind metal ions through its b-diketone moiety, and can prevent free radical generation by metal ions in solution [8,9]. In the aggregation process of the peptide, the soluble oligomers can act as the seeding factors for protofibril formation followed by matured fibrils and aggregates. The accumulation of the aggregates is considered as principally responsible for neurodegeneration [7]. Recent reports also emphazise on the neurotoxicity imparted by the high molecular weight oligomers that contain a substantial amount of b-sheet structure that accelerates the formation of protofibrils. Therefore, inhibitors of b-sheets could play a crucial role in preventing rapid oligomerization, fibrillization and cytotoxicity [10]. In this work, the effect of Curcumin on the secondary structure of the amyloid peptide and also the metal ion induced oligomerization and fibrillation have been investigated by biophysical methods.
[θ] mdeg cm dmol
Fig. 1. Chemical structure of Curcumin.
[θ] mdeg cm dmol
-1
15
-2 -4 -6 -8 200
220
240
260
280
300
Wavelength(nm) Fig. 3. CD spectra of Ab peptide showing the effect of free metal ions and that in combination with Curcumin. Ab in 10 mM PBS (—), Ab with 20 lM CuCl2 (- - -), with 20 lM ZnCl2 ( ) with 20 lM Curcumin and 20 lM CuCl2 (- - ), with 20 lM Curcumin and 20 lM ZnCl2 (- -).
800
R. Banerjee / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 798–800
Fig. 4. AFM images acquired in semi-contact mode showing the oligomerization and fibrillization of (a) Ab peptide, Ab peptide treated with (b) Cu(II), (c) Zn(II), (d) Curcumin, (e) Cu(II) and Curcumin, (f) Zn(II) and Curcumin. The samples were incubated for 6hrs at room temperature.
of beaded fibrillar structures was more pronounced and with Zn(II) treated sample, large oligomeric assemblies was observed. This is evident of the agonistic role of Cu(II) and Zn(II) in promoting fibrillation and aggregation of the peptide. However, in the presence of equimolar amount of Curcumin, considering the particle morphology and size, no fibrillar structures were observed in the peptide, only small oligomeric nano-aggregates were detected. Absence of protofibrillar structures also indicate that Curcumin could effectively inhibit the Cu(II) and Zn(II) induced fibrillization of the peptide. As Curcumin is a potential metal chelator, it can compete with the peptide on complexing with metal ions thereby preventing peptide–metal complex formation that produces degenerative ROS, promoting the fibrillation and aggregation of Ab. CD results have shown that Curcumin efficiently reduces the b-sheet content of the peptide, which is the precursor for protofibrils and AFM imaging supports the observation as Curcumin could potentially inhibit fibrillation in vitro by arresting the polymerization of small oligomers. These observations also agree with the molecular dynamics simulation studies recently reported by Zhao et al. [12] suggesting that Curcumin could reduce b-sheet content of amyloid peptide without hindering the monomeric contacts. The present work also elucidates the novel property of Curcumin in inhibiting the metal ion induced b-sheet formation of Ab peptide. Conclusion Curcumin inhibits the seeding of fibrillation by preventing the peptide-metal complex formation with Cu(II) and Zn(II) as
observed by CD and AFM studies. Therefore, Curcumin, along with its many other beneficial properties can be considered as potential therapeutic agent for amyloidosis. Acknowledgements This work has been supported by the Department of Science and Technology, Govt. of India, under the SERC-Fast-track Young Scientist Scheme. References [1] X. Huang, C.S. Atwood, R.D. Moir, M.A. Hartshorn, J. Vonsattel, R.E. Tanzi, A.I. Bush, J. Biol. Chem. 272 (1973) 26464–26470. [2] C. Lin, H. Huang, Z. Jiang, Brain Res. Bull. 82 (2010) 235–242. [3] T. Rival, R.M. Page, D.S. Chandraratna, T.J. Sendall, E. Ryder, B. Liu, H. Lewis, T. Rosah, R. Hider, L.M. Camargo, M.S. Shearman, D.C. Crowther, D.A. Lomas, Eur. J. Neurosci. 29 (2009) 1335–1347. [4] D.G. Smith, R. Cappai, K.J. Barnham, Biochim. Biophys. Acta 1768 (2007) 1976– 1990. [5] B.B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Adv. Exp. Med. Biol. 595 (2007) 1–75. [6] S. Dong, Q. Zeng, E.S. Mitchell, J.X. Yale Duan, C. Li, J.K. Tiwari, Y. Hu1, X. Cao, Z. Zhao, PLoS ONE 7 (2012) 1–12. e31211. [7] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar, P. Chen, R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, J. Biol. Chem. 280 (2005) 5892–5901. [8] T. Ak, I. Gulcin, Chem. Biol. Interact. 174 (2008) 27–37. [9] T.M. Kolev, E.A. Velcheva, B.A. Stamboliyska, M. Spiteller, Int. J. Quantum Chem. 102 (2005) 1069–1079. [10] K. Onoa, M.M. Condrona, D.B. Teplowa, Proc. Natl. Acad. Sci. (USA) 106 (2009) 14745–14750. [11] L. Baum, A. Ng, J. Alzheimer’s. Dis. 6 (2004) 367–377. [12] L.N. Zhao, S.W. Chilu, J. Benoit, L.Y. Chew, Y. Mu, J. Phys. Chem. B 116 (2012) 7428–7435.