Experimental and Theoretical Insights into the Inhibition Mechanism

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Experimental and Theoretical Insights into the Inhibition Mechanism of Prion Fibrillation by Resveratrol and its Derivatives Lanlan Li, Yongchang Zhu, Shuangyan Zhou, Xiaoli An, Yan Zhang, Qifeng Bai, Yong-Xing He, Huanxiang Liu, and Xiao-Jun Yao ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00240 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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

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

Page 1 of 37

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

ACS Chemical Neuroscience

Experimental and Theoretical Insights into the Inhibition Mechanism of Prion Fibrillation by Resveratrol and its Derivatives Lanlan Li1, Yongchang Zhu2, Shuangyan Zhou1, 2, Xiaoli An1, Yan Zhang1, 2, Qifeng Bai1, Yong-Xing He3*, Huanxiang Liu2*, Xiaojun Yao1, 4* 1

State Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, P. R. China 2

3

4

School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China

School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China

State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, P. R. China

* Corresponding author Tel.: +86-931-891-2578 Fax: +86-931-891-2582 E-mail address: [email protected], [email protected], [email protected]

1

ACS Paragon Plus Environment


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

Abstract Resveratrol and its derivatives have been shown to display beneficial effects to neurodegenerative diseases. However, the molecular mechanism of resveratrol and its derivatives on prion conformational conversion is poorly understood. In this work, the interaction mechanism between prion and resveratrol as well as its derivatives were investigated using steady-state fluorescence quenching, Thioflavin T binding assay, western blotting and molecular dynamics simulation. Protein fluorescence quenching method and Thioflavin T assay revealed that resveratrol and its derivatives could interact with prion and interrupt prion fibrils formation. Molecular dynamics simulation results indicated that resveratrol can stabilize the PrP127-147 peptide mainly through π-π stacking interactions between resveratrol and Tyr128. The hydrogen bonds interactions between resveratrol and the PrP127-147 peptide could further reduce the flexibility and the propensity to aggregate. The results of this study can not only provide useful information about the interaction mechanism between resveratrol and prion, but also provide useful clues for further design of new inhibitors inhibiting prion aggregation.

Keywords: Prion, aggregation, resveratrol, ThT, molecular dynamics simulations

2


Page 2 of 37

Page 3 of 37

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


Introduction Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of infectious, fatal neurodegenerative disorders of the central nerve system (CNS) found in both humans and animals, including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, Creutzfeldt–Jakob disease (CJD) and Fatal Familial Insomnia (FFI) in humans. All these diseases are associated with the misfolding and aggregation of endogenously expressed prion protein, which converts from the α-helix rich cellular prion protein (PrPC) into a β-sheet-rich pathological, infectious isoform termed PrPSc 1-4. There are enough evidences indicate that pathological PrPSc can act both as a template for the conformational conversion of PrPC and as a neurotoxic agent leading to neuronal dysfunction and even cell death 1, 5. Based on this conversion mechanism, several strategies have been proposed for the treatment of prion diseases, including stabilization of PrPC and blocking the conversion of PrPC to PrPSc using small molecules, peptides or antibodies

6-8

. The compounds that do not target at PrPC or

PrPSc were also investigated at cellular level 9. However, none of these compounds have been proven successfully in clinical trials to date. Therefore, it is still urgent to develop and discover new compounds that can effectively inhibit prion misfolding and aggregation. So far, many compounds such as dextran sulfate, pentosan polysulfate, suramin, 2-ami-nothiazoles, dendritic polyamines Congo red and quinacrine have been evaluated for their anti-prion activities using in vitro cell models in prion disease 10-13. 3



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

Page 4 of 37

The detailed molecular mechanism of these active compounds remains uncovered and a majority of these compounds were failed to demonstrate improvement in TSE patients

14-16

. The evaluation of the anti-prion activity of natural or pharmaceutical

compounds already used in other diseases will be of interest to find more effective compounds

6, 17

. Recently, special attention has been paid to the compounds from

natural products owing to their structural diversity, low cytotoxicity and druggability features. Among them, polyphenols have attracted much attention. As an abundant active compound which can be easily extracted from various plants and foods, resveratrol (3,5,4’-trihydroxy-trans-stilbene) displays beneficial effects in heart cardiovascular disease

18

, cancer

19, 20

, type 2 diabetes

21

, and neural system

22, 23

.

Resveratrol can reduce Aβ40 levels of Alzheimer disease patients 24 and attenuate Aβ deposition in Alzheimer transgenic mouse

25

. There is also evidence that resveratrol

and its derivatives can directly interact with Aβ peptides by inhibiting and destabilizing the formation of Aβ fibrils

26

. Resveratrol can induce autophagy to

prevent prion-mediated neurotoxicity 27. However, the detailed interaction mechanism between resveratrol and prion protein still needs to be further investigated. In this study, the inhibiting activity of resveratrol and its derivatives on the fibrillation of prion protein were investigated by using multiple approaches. Firstly, endogenous steady-state fluorescence quenching was used to screen potential compounds that could bind and interact with prion protein. Then ThT assay was applied to follow the progress of amyloid fibril formation. Subsequently, western blotting was used to qualitatively describe the fibrillation inhibition ability of 4


Page 5 of 37

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


resveratrol and its derivatives. Since the transition characteristics of amyloid aggregates makes it quite challenging to capture and describe structural properties of the fibrillation process using conventional experiments. Molecular dynamics (MD) simulation can be used in study misfolding and aggregation behavior of various amyloid proteins 28-32. MD simulation can not only offer the structural details but also display the dynamic process of misfolding and aggregation of protein or peptides 33, 34. The simulation results will be useful to understand the molecular mechanism of resveratrol inhibiting prion aggregation and give valuable information for the design and discovery of more potent prion inhibitors.

Results and Discussion Characterization of the Interaction Between Prion and Resveratrol and Its Derivatives PrP117-231 protein was expressed in E.coli. 12% SDS-PAGE was used to detect the protein expression (Fig. S1). The insoluble prion protein was then subjected to on-column refolding and used in the following assays. Concentration dependent fluorescence quenching of seven resveratrol compounds (Fig. 1) on prion protein were determined firstly. According to the fluorescence titration, three compounds including resveratrol (denoted as JZ-II) were demonstrated to quench prion fluorescence in a concentration dependent manner. The maximum emission at 345 nm of each concentration was fitted to the One Site Specific Binding Model in GraphPad Prism 6 software to obtain the relations of fluorescence quenching abilities and concentrations (Fig. 2). As can be seen from Fig. 2a, fluorescence quenching of prion protein by 5



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

Page 6 of 37

compounds JZ-II and JZ-XIII quickly saturated at low concentrations (

Experimental and Theoretical Insights into the Inhibition Mechanism

Recommend Documents