Carnosine-LVFFARK-NH2 Conjugate: A Moderate Chelator but Potent

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Carnosine-LVFFARK-NH2 Conjugate: A Moderate Chelator but Potent Inhibitor of Cu2+-Mediated Amyloid #-Protein Aggregation Huan Zhang, Xiaoyan Dong, and Yan Sun ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00133 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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ACS Chemical Neuroscience

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Carnosine-LVFFARK-NH2 Conjugate: A Moderate Chelator but

2

Potent Inhibitor of Cu2+-Mediated Amyloid β-Protein Aggregation

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Huan Zhang, Xiaoyan Dong, and Yan Sun*

5

Department of Biochemical Engineering and Key Laboratory of Systems

6

Bioengineering of the Ministry of Education, School of Chemical Engineering and

7

Technology, Tianjin University, Tianjin 300354, China

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ACS Paragon Plus Environment

ACS Chemical Neuroscience 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

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ABSTRACT:

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Aggregation of amyloid-β (Aβ) protein stimulated by Cu2+ has been recognized as a

11

crucial element in the neurodegenerative process of Alzheimer's disease. Hence it is of

12

significance to develop bifunctional agents capable of inhibiting Aβ aggregation as

13

well as Cu2+-mediated Aβ toxicity. Herein, a novel bifunctional nonapeptide,

14

carnosine-LVFFARK-NH2 (Car-LK7) was proposed by integrating native chelator

15

carnosine (Car) and an Aβ aggregation inhibitor Ac-LVFFARK-NH2 (LK7). Results

16

revealed the bifunctionality of Car-LK7, including remarkably enhanced inhibition

17

capability on Aβ aggregation as compared to LK7 and a moderate Cu2+ chelating

18

affinity (KD=28.2 ± 2.1 µM) in comparison to the binding affinity for Aβ40 (KD=1.02 ±

19

0.13 µM). The moderate Cu2+ affinity was insufficient for Car-LK7 to sequester Cu2+

20

from Aβ40–Cu2+ species, but to form ternary Aβ40–Cu2+–Car-LK7 complexes.

21

Formation of the ternary complexes directed the aggregation into small unstructured

22

aggregates with little β-sheet structure. Car-LK7 also showed higher activity on

23

arresting Aβ40-Cu2+-catalyzed reactive oxygen species production than Car. Cell

24

viability assays confirmed the prominent protection activity of Car-LK7 against

25

Cu2+-mediated Aβ40 cytotoxicity; Car-LK7 could almost eliminate Aβ40 cytotoxicity at

26

an equimolar dose (cell viability increased from 59% to 99%). The research has thus

27

provided new insight into the design of potent bifunctional agents against

28

metal-mediated amyloid toxicity by conjugating moderate metal chelators and

29

existing inhibitors.

30

amyloid

β-protein;

31

KEYWORDS:

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Cu2+-chelator; inhibitor; peptide

aggregation;

2


reactive

oxygen

species;

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INTRODUCTION

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Fibrillation of amyloid β-protein (Aβ) in the brain has been recognized as the main

35

hallmark of Alzheimer’s disease (AD),1-2 and in vivo studies have revealed that

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redox-active Cu2+ interacts with Aβ species and produces reactive oxygen species

37

(ROS), which would enhance and amplify neuronal damage and further promoting

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neuronal death.3-5 Studies have indicated that metal chelators are able to reduce Aβ

39

burden, restore metal homeostasis, and improve cognition in mouse AD models.6-9

40

Hence, development of functional agents that inhibit Aβ fibrillation as well as chelate

41

metal ions, particularly Cu2+, has been considered as an effective strategy for AD

42

therapy and prevention. Up to now, however, only a few agents, such as clioquinol

43

(CQ) and 5,7-dichloro-2-((dimethylamino)methyl) 8-quinolinol (PBT2) are suitable

44

for clinical trials,7, 10 and the therapeutics are facing major failures due to adverse side

45

effects, such as subacute myelo-optic neuropathy.10-12 Hence, there is a great demand

46

to develop bifunctional agents that can inhibit Aβ and metal-mediated Aβ

47

aggregations, reduce Aβ-mediated cytotoxicity and decrease Cu2+-mediated ROS

48

production.

49

Recently, an endogenic water-soluble dipeptide, carnosine (β-alanyl-L-histidine,

50

Car) attracted the attention of researchers. Car is commonly present in the muscle and

51

brain tissues of humans at high levels,13 indicating its high biocompatibility. Moreover,

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Car is capable of chelating Cu2+ to regulate the metal ion dyshomeostasis in the body,

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thus protecting neurons against lipid peroxidation catalyzed by Cu2+.14 Moreover, Car

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is also an effective quencher of ROS by directly reacting with hydroxyl radicals (·OH)

55

and peroxyl radicals.15-16 The extensive biological activities of Car suggest that it

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might be a promising therapeutic agent for the treatment of AD involving metals and

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free radicals. Several groups have reported that Car is capable of inhibiting Aβ

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aggregation17-18 and Aβ-induced toxicity.19-20 However, the inhibitory effect of Car is

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quite limited, making it disable in dealing with the Aβ fibrillogenesis in the brain

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tissues of AD patients.17-18 Hence, if we can enhance the inhibitory capacity of Car, it

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would function as both the metal ion dyshomeostasis modulator and Aβ aggregation

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inhibitor in the brain. Given this, we have herein proposed to conjugate Car to a 3



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peptide inhibitor to improve its inhibitory capability on Aβ assembly.

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In a recent work of our group, a heptapeptide (Ac-LVFFARK-NH2), derived from

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the central hydrophobic Aβ sequence, has been reported to display effective inhibition

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against Aβ amyloidogenesis.21 Hence, we have herein designed a novel peptide

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sequence Car-LVFFARK-NH2 to make it have potential Cu2+-complexation and

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amyloid inhibition properties. The effects of Car-LK7 on Cu2+-mediated Aβ40

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aggregation, Cu2+-chelating ability of Car-LK7 and the arresting effect of Car-LK7

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on ROS production were extensively examined by using biophysical and biological

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assays. The results revealed remarkable inhibiting capability of Car-LK7 on

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Cu2+-mediated Aβ aggregation and cytotoxicity.

73 74

RESULTS AND DISCUSSION

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Inhibitory Effect of Car-LK7 on Aβ40 Aggregation. Thioflavin T (ThT)

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fluorescence assays were first performed to evaluate the inhibitory capability of

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Car-LK7 on Aβ40 fibrillation (Figure 1A). For pure Aβ40, a typical dramatic

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enhancement in the ThT fluorescence was observed after 72 h incubation due to the

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fibrillar aggregation of Aβ40. When it was co-incubated with Car, the ThT

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fluorescence was little affected by increasing its molar ratios to 1:5 (Aβ40:Car). In the

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presence of 10 equivalents of Car, only 20% ThT fluorescence reduction was

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observed (Figure 1A). This confirms that Car at the experimental conditions (25-250

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µM) had little inhibitory effect on Aβ40 fibrillization, consistent with literature data.18

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In the presence of LK7, the maximal inhibition occurred at 1:2 molar ratio of

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Aβ40:LK7 with a 42% reduction of ThT fluorescence intensity (FI) (Figure 1A). The

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inhibitory capacity of LK7 decreased with further concentration increase (Figure 1A).

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This was due to its increasing self-assembly property at high concentrations,21 as

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evidenced by a significant amount of self-aggregates observed under atomic force

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microscopy (AFM) (Figure S1A). In contrast, Car is highly soluble, so no Car

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aggregation was observed (Figure S1B). As a result, the Car-LK7 conjugate showed

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little aggregation tendency (Figure S1C). Thus, Car-LK7 decreased the ThT FI of

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Aβ40 with increasing concentration till 1:10 (Aβ40:Car-LK7) (Figure 1A). The result 4


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indicates that LK7 became a favorable inhibitor of Aβ fibrillation due to the

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conjugation of Car.

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Considering the maximum inhibitory capacity of LK7 at a molar ratio of 1:2

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(Aβ40:LK7), circular dichroism (CD) spectroscopy and AFM observations were

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conducted under the specific inhibitor concentration for comparison. As shown in

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Figure 1B, the CD spectrum of freshly solubilized Aβ40 displayed a single minimum

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around 202 nm, indicating a random coil conformation.22-23 The Aβ40 aggregates

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exhibited a single minimum around 218 nm, indicating the presence of secondary

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structure of β-sheets.24-25 When it was incubated with Car, Aβ40 aggregates displayed

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the similar CD spectrum to that of pure Aβ40, indicating an inappreciable effect of Car

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on Aβ40 conformational transitions. By contrast, LK7 and Car-LK7 significantly

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reduced the ellipticity at 218 nm in the CD spectra, and it affirmed that the β-sheet

105

reduction by Car-LK7 was more significant than LK7. It is notable that the peptide

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conformation may change in the presence of Aβ, thus affecting the quantitative

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accuracy of β-sheet structure in Aβ40 determined by subtracting the corresponding

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peptide spectrum from a peptide-Aβ40 co-incubation sample (See METHODS below).

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However, it is not possible to probe this change because the free peptides gave much

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lower CD signals than Aβ40. As seen in Figure S1D, even at the highest concentration

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(250 µM) used in the study, only LK7 presented the β-turn structure with a positive

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peak at 205 nm,26 while Car and Car-LK7 did not form any ordered structures. After

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72 h incubation, the ellipticity of LK7 (250 µM) at 205 nm was even lower than that

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of Aβ40 (25 µM) at 218 nm. These results demonstrated that the free peptides did not

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produce significant β-sheet structure during incubation (Figure S1D), and the

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influence of free peptides on β-sheet structure detection would be negligible. Because

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the total content of β-sheet structure in the inhibitor-treated Aβ40 was less than that of

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Aβ40 alone (Figure 1B), the β-sheet structure formed in Aβ40 in the inhibitor-treated

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Aβ40 should be less than that of the Aβ40 alone system, even if the peptide converted

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to β-sheet structure in the presence of Aβ40. This fully demonstrated the effectiveness

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of Car-LK7 on the inhibition of Aβ40 conformational transition to β-sheet structure.

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Figure 1C-1F shows the morphologies of Aβ40 assemblies corresponding to those 5



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detected by CD spectroscopy. Similar with literature result,27 Aβ40 aggregated and

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formed dense fibrillar networks (Figure 1C). When it was incubated with Car,

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inappreciable morphology change was observed (Figure 1D), consistent with ThT

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(Figure 1A) and CD (Figure 1B) experiments. Upon co-incubation with LK7, the

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amyloid fibrils appeared to be rather shorter but still serried (Figure 1E). Strikingly,

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Car-LK7 greatly blocked Aβ40 fibrillogenesis and only sparse fibrils were found

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(Figure 1F). These observations further confirmed the high potency of Car-LK7 on

130

inhibiting Aβ fibrillation.

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Moreover, the inhibitory effects of Car-LK7 on Aβ40 aggregation kinetics were

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explored by in situ ThT fluorescence (Figure S2A). Generally, the growth curve of

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amyloid fibrillogenesis follows a nucleation-dependent polymerization process, in

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which Aβ monomers nucleate into critical nuclei, followed by the formation of

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protofibrils and finally mature fibrils.28-29 As shown in Figure S2A, incubation of Aβ40

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with Car-LK7 effectively prolong the lag time, reflecting the ability of Car-LK7 to

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retard the nucleation of Aβ40. Also, the lag time increased with increasing Car-LK7

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concentration (Table 1). Compared to pure Aβ40, it is clear that the presence of 10

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equivalents of Car-LK7 prolonged the lag time of Aβ40 aggregation by three times.

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The strong inhibitory effect of Car-LK7 in a dose-dependent manner was also

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verified by CD spectroscopy and AFM measurements. The CD spectra indicate that

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β-sheet conformation characterized by the negative peak at 218 nm (Figure S2B)

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decreased with increasing Car-LK7 concentration. This implies the formation of

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off-pathway aggregates with distinctly different morphologies as observed by AFM

145

(Figure S3). Few fibrils and amorphous aggregates were observed at 10 equivalents of

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Car-LK7 (Figure S3D).

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The nonapeptide was further tested for its protective activity against the

148

cytotoxicity

of

Aβ40

aggregates

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate

150

dehydrogenase (LDH) release assays (Figure 2). In this work, LDH release assay was

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performed as the secondary backup to prevent the possible false-positive results in the

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MTT assay.30-31 As shown in Figure S4, the aged LK7 showed toxicity to the cells at 6


by

both

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high concentrations due to its self-assembly (Figure S1A).21 By contrast, almost

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similar cell viability (Figure S4A) and LDH leakages (Figure S4B) to the untreated

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control were obtained when Car or Car-LK7 was aged for 24 h at a concentration up

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to 50 µM, indicating that the biocompatibility of Car-LK7 was comparable to Car.

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Then, the protective effects of the three peptides on cultured cells were compared. By

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co-cultivation with Aβ40 aggregates for 24 h, the cell viability was about 67% of the

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control, and Car conferred almost no protection in the MTT assay (Figure 2A).

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Slightly better results were obtained in the LDH release assay (Figure 2B), which

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showed ∼8% reduction of LDH leakage when Aβ40 were treated with a high

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concentration of 50 µM Car (Aβ40:Car=1:10), consistent with the above

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anti-aggregation experiments (Figure 1). In the MTT assay, LK7 showed a moderate

164

protective effect at a molar ratio of 1:1 (Aβ40: LK7), and further concentration

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increase led to significant cell viability decrease (Figure 2A) due to the cytotoxicity of

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LK7 aggregates (Figure S4). By the LDH release assay (Figure 2B), co-treatment with

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LK7 could not reverse Aβ40-induced LDH leakages at all concentrations tested (5−50

168

µM), and in some cases, its inhibitory activity was even worse than that obtained in

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the MTT assay. This indicates that LK7 could not effectively protect the cells from

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Aβ40-induced cytotoxicity. When Car-LK7 was co-incubated with Aβ40, however, a

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dramatic enhancement in cell viability was observed, and co-incubating at 2

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equivalents of Aβ40, Car-LK7 almost completely prevented Aβ40-induced cell death in

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the MTT assay (Figure 2A). The parallel LDH release assay (Figure 2B) showed a

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trend similar to that of SH-SY5Y viability in the MTT assay. When different

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concentrations of Car-LK7 were added to Aβ40 solution, they all protected SH-SY5Y

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cells from Aβ40-induced apoptosis to some extent, and the protection effect was

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dose-dependent. Therefore, Car-LK7 demonstrated its high biocompatibility and

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neuroprotective effects.

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The above results demonstrated that Car-LK7 was a potent inhibitor against Aβ40

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aggregation and cytotoxicity. In other words, although Car had little inhibitory effect

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on Aβ40 fibrillogenesis, the conjugation of Car to LK7 significantly enhanced the

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inhibition effect of LK7 by suppressing the self-assembly of the heptapeptide. 7



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Chelation Effect of Car-LK7 Toward Cu2+. Since Tyr10 is located in the close

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proximity to the three histidine residues (H6, H13, H14) on Aβ, it can be affected by

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metal coordination.32-33 It was reported that tyrosine fluorescence was quenched by

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Cu2+, indicating that a structural change in Aβ conformation ensues following the

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metal ion binding.34 To evaluate the Cu2+-chelating ability of Car-LK7, the intrinsic

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fluorescence of Tyr10 in Aβ40 was measured. Figure 3 shows that the tyrosine

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fluorescence of Aβ40-Cu2+ complex was remarkably lower than pure Aβ40, indicating

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the binding of Cu2+. By incubation of Aβ40-Cu2+ with any of the three peptides (Car,

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LK7 and Car-LK7), the fluorescence intensity kept almost identical to that for Aβ40–

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Cu2+. This indicates that Car-LK7 was unable to sequester Cu2+ from the Aβ40-Cu2+

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complex.

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To further look into the Cu2+ chelation role of Car-LK7 in the presence of Aβ40,

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isothermal titration calorimetry (ITC) was performed. The titration curves are shown

196

in Figures S5 and S6, and the corresponding thermodynamic parameters obtained

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from the titrations are listed in Table 2. Results shown that the apparent dissociation

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constant (KD) for Cu2+ binding to Car-LK7 was 28.2 ± 2.1 µM, but only 1.02 ± 0.13

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µM for Cu2+ binding to Aβ40, suggesting that the binding affinity of Car-LK7 for Cu2+

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was much lower than that of Aβ40 for Cu2+. This suggests that Car-LK7 was a

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moderate Cu2+ chelator; the chelation was weak for disrupting the binding of Cu2+ to

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Aβ40. Similar dissociation constants of Cu2+ binding to Aβ40 under the same

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conditions were reported in literatures.35-36 However, in the presence of Car-LK7, the

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apparent binding affinity of Aβ40–Cu2+ became weaker (KD=12.4 ± 3.2 µM) (Table 2),

205

implying that Car-LK7 could interfere with Cu2+ binding to Aβ, despite the weak

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Cu2+–Car-LK7 interaction.

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It is known that Cu2+ binds to Aβ via three nitrogen atoms coming from the

208

N-terminus together with three histidine residues in the histidine-rich segment (Aβ6–14)

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and also to the oxygen atom coming from the carboxylate function of Asp1 or

210

Ala2.37-39 It is possible that Car-LK7 competes with Aβ40 for Cu2+ binding sites or

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directly interacts with Aβ40-Cu2+ complexes and displaces some of the Cu2+ binding

212

sites. However, in the presence of Aβ40, Car-LK7 could not completely occupy the 8


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four Cu2+ binding sites or strip Cu2+ from Aβ40-Cu2+ complexes, as can be concluded

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from the above ITC (Table 2) and tyrosine fluorescence results (Figure 3). Thus, the

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strong Aβ40–Cu2+ complexation and competitive Cu2+ chelation by Car-LK7 can

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occur simultaneously on the same Cu2+ ion, leading to the formation of ternary Aβ40–

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Cu2+-Car-LK7 complexes. Previous anti-aggregation studies (Figure 1) have shown

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that Car-LK7 could block amyloid fibrillization via specific LK7-Aβ interactions (the

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central hydrophobic core of Aβ21). Therefore, in the ternary complexes, Car-LK7

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simultaneously binds to Cu2+ and the hydrophobic region of Aβ40 via metal-chelation

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and LK7-Aβ interactions. Ternary complexation was previously reported in the

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inhibition

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(−)-epigallocatechin-3-gallate.40-41

of

metal-mediated

Aβ

aggregation

by

curcumin

and

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Inhibitory Effect of Car-LK7 on Cu2+-Mediated Aβ40 Aggregation. Previous

225

studies have proven that Cu2+ modulates Aβ aggregation pathways, leading to the

226

formation of more toxic Aβ species.42-44 Thus, it is important to investigate whether

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Car-LK7 possesses an inhibitory effect on Cu2+-mediated Aβ40 aggregation. Previous

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research has suggested that a trace of copper could be enough for initiating Aβ

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aggregation45 and the morphology of Aβ aggregates depended on the molar ratio of

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Aβ40:Cu2+.46-47 Hence, the ability of the inhibitors in inhibiting Cu2+-mediated Aβ

231

fibrillation was investigated at a sub-stoichiometric amount of Cu2+ (Aβ40:Cu2+=1:0.4),

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a condition that promotes amyloid aggregation and induces fibrillar aggregates.

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Figure 4 shows that the addition of Cu2+ remarkably reduced Aβ40 amyloidogenesis,

234

but still a strong ThT fluorescence remained (Figure 4A), with fibrillar aggregates

235

(Figure 4C) containing β-sheet conformations (Figure 4B), consistent with previous

236

data.48-49 When Car was added, the ThT fluorescence increased somewhat with its

237

concentration (Figure 4A), and the CD signal (Figure 4B) and Aβ fibrils (Figure 4D)

238

also showed slight increases. This implied that Car could chelate Cu2+ moderately, but

239

unable to inhibit Aβ40–Cu2+ amyloidogenesis. By the addition of LK7 or Car-LK7, the

240

ThT intensity of Cu2+-mediated Aβ40 aggregates decreased significantly (Figure 4A).

241

Particularly, Car-LK7 showed an overwhelming potency in inhibiting Aβ40–Cu2+

242

aggregation. In detail, the ThT fluorescence intensity of Aβ40–Cu2+ aggregates 9



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incubated with equimolar Car-LK7 was reduced by 87%, while it was only about 10%

244

with LK7. Even at higher concentrations, LK7 could only confer about 35% reduction

245

in ThT fluorescence (Figure 4A). Moreover, with an equivalent Car-LK7 to Aβ40,

246

almost no β-sheet conformation (Figure 4B) and Aβ40 fibrils (Figure 4F) were

247

detected, but small amorphous aggregates were observed (Figure 4F). Such changes

248

in aggregate structure and morphology imply that Car-LK7 redirected Aβ40–Cu2+

249

species into unstructured, off-pathway aggregates. In contrast, the LK7-treated group

250

maintained even rich β-sheet structure (Figure 4B) and large amount of Aβ40 fibrils

251

(Figure 4E). The results confirmed the intense inhibitory effect of Car-LK7 on the

252

Cu2+-mediated Aβ aggregation. Comparison to literature data also revealed that

253

Car-LK7 was superior to most of the recently reported peptide inhibitors, such as

254

GGHRYYAAFFARR (GR) (68% reduction with equimolar GR),46 RTHLVFFARK

255

(RK10) (80% reduction by 4 equivalents of RK10),48 and GHKSrVSrFSr (P6) (70%

256

reduction by 4 equivalents of P6).50

257

To gain further understanding of its disassembly capability on Cu2+-mediated Aβ40

258

aggregation, a reduced dosage of Car-LK7 was investigated (Figure 5). It is seen that

259

the fibrillation of Aβ40–Cu2+ upon co-incubation with sub-stoichiometric doses of

260

Car-LK7 was also greatly suppressed at a molar ratio of 1:0.2 (Aβ40:Car-LK7)

261

(Figure 5A). The decreases of β-sheet structure (Figure 5B) and fibrous aggregates

262

(Figures 5C and 5D) were also in accordance with the ThT data (Figure 5A). The

263

results demonstrated that even a sub-stoichiometric concentration of Car-LK7

264

inhibited Cu2+-mediated Aβ40 aggregation.

265

Furthermore, the dynamics of Cu2+-mediated Aβ40 aggregation without and with

266

Car-LK7 at different concentrations provided more evidence of Car-LK7 in inhibiting

267

Cu2+-mediated Aβ40 fibrillation (Figure S7). As listed in Table 3, the lag time of Aβ40–

268

Cu2+ aggregation (10.4 h, Table 3) was considerably shorter than that of pure Aβ40

269

(14.1 h, Table 1), indicating that Cu2+ could promote Aβ nucleation, as reported in the

270

literature.46, 51 By incubation of Aβ40–Cu2+ mixture with Car-LK7, the lag time was

271

significantly prolonged with increasing Car-LK7 concentration (Figure S7 and Table

272

3). When Car-LK7 and Aβ40 reached to a molar ratio of 5:1, Aβ40 nucleation was 10


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273

eventually completely inhibited. Thus, it can be concluded that Car-LK7 could

274

efficiently delay Aβ40–Cu2+ nucleation and inhibit fibril elongation in a

275

dose-dependent manner.

276

Based on the promising anti-aggregating properties of the nonapeptide described

277

above, we studied whether Car-LK7 could also exert neuroprotective effect against

278

Cu2+-triggered Aβ40 cytotoxicity. As determined by MTT assays (Figure 6A),

279

incubation of SH-SY5Y cells with 5 µM Aβ40 aggregates resulted in a reduction of

280

about 33% of cell viability. As for the Aβ40–Cu2+ samples, the cell viability was

281

further reduced than that treated with Aβ40, showing 41% reduction of cell viability,

282

similar to the previous reports that Cu2+ potentiated of Aβ neurotoxicity.5,

283

Dose-response analysis showed that while addition of Car-LK7 to Aβ40 resulted in

284

rapid increase in cell viability, Car and LK7 at the same concentrations displayed only

285

moderate effects on Aβ40 toxicity. For example, the maximum cell viabilities of 73%

286

and 77% were observed at 1:2 molar ratio of Aβ40/Car and Aβ40/LK7, respectively. By

287

contrast, when 0.2 equivalence of Car-LK7 to Aβ40 was added, the cell viability with

288

Aβ40–Cu2+ species remarkably increased to 80%. Increasing concentration of

289

Car-LK7 to 1:1 (Aβ:Car-LK7) achieved about 99% cell viability. The striking

290

difference suggests that Car-LK7 inhibited the formation of toxic Aβ40-Cu2+ species.

36, 50

291

In parallel, similar inhibitory effects of these peptides on Cu2+-Aβ40-induced

292

cytotoxicity were found in the LDH release assay (Figure 6B). As a control, the cell

293

viability did not increase in the LDH release assay until the molar ratio of Aβ40:Car

294

was increased to 1:2, similar to that in the MTT assay (Figure 6A). By contrast, LK7

295

achieved maximal inhibition against Cu2+-Aβ40-induced cytotoxicity at 1:1 molar ratio

296

of Aβ40:LK7, while at higher concentrations its inhibitory effect determined by the

297

LDH release assay (Figure 6B) was slightly lower than that by the MTT assay (Figure

298

6A). As for Car-LK7, it could protect SH-SY5Y cells from Cu2+-Aβ40-induced

299

apoptosis at an extremely low peptide concentration (i.e., 5 µM) (Figure 6B) and the

300

protection effect became more effective at higher concentrations, which is consistent

301

with the MTT results (Figure 6A). Thus, both assays confirm that Car-LK7 exhibited

302

the strongest inhibitory activity to prevent Cu2+-triggered Aβ40 cytotoxicity. By 11



303

comparison to other bifunctional peptide inhibitors (e.g. GR, RK10 and P6)46, 48, 50

304

reported in the literature, Car-LK7 also possessed higher potency on inhibiting

305

Cu2+-mediated Aβ40 cytotoxicity.

306

Effect on ROS Production. The generation of ROS is a common toxic feature in

307

many neurodegenerative diseases.52-53 It was previously reported that Aβ40 had strong

308

redox catalytic ability to promote ROS production when it was bound to Cu2+, thus

309

increasing cell oxidative stress and cell damage.51, 54 To test whether Car-LK7 could

310

rescue ·OH toxicity by directly reducing ROS, we evaluated the antioxidant capacities

311

of Car-LK7 by 7-OH-CCA fluorescence. The 7-OH-CCA fluorescence growth curve

312

shows the in situ production of ·OH. It can be seen in Figure S8, the 7-OH-CCA

313

fluorescence intensity incubated with Cu2+ alone increased sharply with time, the

314

maximum of which was set as 100%. Treatment with increasing concentrations of

315

Car/Car-LK7 markedly suppressed ROS production, albeit to a different extent. In

316

particular, Car-LK7 was shown to be more effective in preventing the Cu2+-induced

317

ROS production, evidenced by a lower fluorescence than Car at any test

318

concentrations (Figure S8). Figure 7 shows the fluorescence curve of Aβ–Cu2+

319

complexes. In the presence of Aβ40, the final fluorescence reduced to 38% and the

320

curve slope also became small, indicating decreased redox activity of Cu2+ when it

321

was bound to Aβ40. In this case, Car-LK7 also displayed superior antioxidant activity

322

over Car on Aβ40–Cu2+-catalyzed ROS production. It is seen that Car at the

323

experimental conditions (25-250 µM) showed no significant effect on the

324

fluorescence intensity (Figure 7A). However, when 5-fold molar excess of Car-LK7

325

was added, the fluorescence intensity decreased to the control level (Figure 7B),

326

implying the redox activity of Aβ–Cu2+ species was completely inhibited by Car-LK7.

327

Because LK7 was proven not to affect the ROS level,49 the prominent antioxidant

328

function of Car-LK7 should be ascribed to its specific binding to Aβ40–Cu2+ species

329

via both Car and LK7 moieties.

330

Mechanistic Discussion. Based on the above experimental results, a mechanistic

331

model was proposed to describe the bifunctional effects of Car-LK7 on

332

Cu2+-mediated Aβ40 aggregation and cytotoxicity (Figure 8). For the on-pathway 12


Page 12 of 39

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333

aggregation, Aβ40 monomers rapidly convert to β-sheet structure which is more

334

conducive to Aβ40 aggregation (Figure 1B). After that, Aβ40 aggregates into oligomers,

335

protofibrils and fibrils of high cytotoxicity (Figure 1C and Figure 2).

336

Sub-stoichiometric level of Cu2+ rapidly induces the aggregation of Aβ40 (Figure 8A),

337

followed by the formation of considerable fibrils (Figure 4C) and intense ROS (Figure

338

7), both of which are highly toxic. However, Car-LK7 simultaneously binds to Cu2+

339

(chelated on Aβ6-1437-39) and Aβ40 at the central hydrophobic core21 to form ternary

340

Aβ40–Cu2+–Car-LK7 complexes (Figure 8B). Thus, Car-LK7 can bind to the two

341

different regions of Aβ40, making Car-LK7 display more prominent potency on

342

regulating Aβ40–Cu2+ conformation (Figures 4B and 5B). Then, Car-LK7 completely

343

breaks the fatal pathway of Cu2+-associated Aβ40 aggregation (Figure 8B), redirected

344

Aβ40–Cu2+ species into small amorphous aggregates (Figure 4F) with little β-sheet

345

structure and low cytotoxicity (Figure 6). Besides, the formation of the ternary

346

complexes could almost deplete the catalytic activity of Aβ40–Cu2+ on ROS formation

347

(Figure 7B), which was also beneficial to keep the cells at high viability.

348 349

CONCLUSIONS

350

This work presented a nonapeptide, Car-LVFFARK-NH2, that encompassed moderate

351

affinity for Cu2+, but remarkably high activity on inhibiting Cu2+-mediated Aβ40

352

aggregation and cytotoxicity. The anti-aggregation studies verified that Car-LK7

353

effectively

354

accumulation, redirected toxic Aβ40/Aβ40–Cu2+ species into small unstructured

355

aggregates with little β-sheet structure and low cytotoxicity. Similar to the native Car,

356

Car-LK7 effectively prevented the production of ROS catalyzed by Cu2+ and Aβ40–

357

Cu2+ species. Comprehensive studies revealed that Car-LK7 plays multiple roles in

358

preventing Aβ40/Aβ40–Cu2+ aggregation and ROS production through ternary Aβ40–

359

Cu2+–Car-LK7 complexes, in which Car-LK7 simultaneously bind to Aβ40 and Cu2+

360

chelated on Aβ40. It was the ternary complexes that direct the aggregation into small

361

unstructured aggregates with little β-sheet structure and low cytotoxicity, making

362

Car-LK7 display prominent potency on inhibiting Cu2+-mediated Aβ40 aggregation

disturbed

the

aggregation

pathway

of

13


Aβ40/Cu2+-induced-Aβ40


Page 14 of 39

363

and ROS generation. Furthermore, Car-LK7 maintains high biocompatibility as

364

native Car does, making it promising for further development as a therapeutic agent

365

against Cu2+-associated Aβ40 aggregation, cytotoxicity and ROS production in AD.

366 367

METHODS

368

Materials. All the peptides (Aβ40, Car, LK7 and Car-LK7 with >95% purity) used

369

in this research were obtained from GL Biochem (Shanghai, China). CuCl2,

370

coumarin-3-carboxylic acid (3-CCA), ascorbate (Asc), dimethyl sulfoxide (DMSO),

371

2-[4-(2-hydroxyethyl)-1-piperazine]

372

1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), ThT and MTT were all purchased from

373

Sigma-Aldrich (St. Louis, MO). Human neuroblastoma SH-SY5Y cells were obtained

374

from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell

375

culture reagents including fetal bovine serum (FBS), Dulbecco’s modified Eagle’s

376

medium/Ham’s Nurtient Mixture F-12 (DMEM/F12) were purchased from Gibco

377

Invitrogen (Grand Island, NY, USA). All other chemical reagents of analytical grade

378

were available from local sources.

ethanesulfonic

acid

(HEPES),

379

Aβ Solution Preparation and Incubation. The initial Aβ40 powder was first

380

treated with HFIP at a concentration of 1.0 mg/mL. The solution was left undisturbed

381

for 2 h before a centrifugation for 20 min at 15,000 × g to remove the existing

382

aggregates.55-56 Then, HFIP was removed by freeze-drying using a vacuum

383

concentrator (Labconco, America). The lyophilized protein was stored in refrigerator

384

at -20 °C.

385

For Aβ aggregation experiments, the treated Aβ40 was dissolved in 20 mM NaOH

386

and 11-fold diluted in buffer A (20 mM HEPES, 100 mM NaCl, pH 7.4) to obtain a

387

monomeric Aβ40 (25 µM) without/with 1 to 10-fold concentrations of each peptide

388

(i.e., Car, LK7, and Car-LK7). The peptide, LK7 or Car-LK7, was first dissolved in

389

DMSO to form a stock peptide solution (1.1 mM), which was diluted to required

390

concentrations with the same buffer before use. The samples in the inhibition

391

experiments were composed of 25 µM Aβ40 and different concentrations (25, 50, 125,

392

and 250 µM) of a peptide inhibitor in the buffer A with 2.5% DMSO. The presence of 14


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393

DMSO increases the background of the signal of CD spectroscopy,57 and has also a

394

significant effect on the detection accuracy of ·OH production by the 3-CCA

395

fluorescence. Therefore, LK7 or Car-LK7 solution without DMSO was used to dilute

396

Aβ40 in the two experiments. Samples for Aβ40-Cu2+ complex were prepared similarly

397

with the above, with only difference in the addition of 10 µM Cu2+. Each sample was

398

incubated at 37 °C for 72 h with continuous shaking at 150 rpm.

399

ThT Fluorescence Assay. ThT fluorescence assay was conducted to quantify

400

amyloid fibrils in the absence and presence of inhibitors. In the assay, 200 µL of each

401

sample withdrawn from the above incubation was mixed uniformly with 2 mL ThT

402

solution (25 µM ThT in buffer A) in a quartz cell. ThT fluorescence intensity was

403

measured using an LS-55 fluorescence spectrometer (Perking Elmer, MA, USA) at

404

excitation and emission wavelengths of 440 and 480 nm, respectively.58 The FI of the

405

solution without Aβ40 was subtracted as background from each read with Aβ40. The FI

406

of Aβ40 incubated alone was set as 100% for comparison. Each measurement was

407

performed in triplicate and the average value was reported with its standard deviation.

408

Statistical analysis was performed by t-test, and P70

>70

50 µM

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Figures 762

763 764

Figure 1. (A) Normalized ThT fluorescence intensities of Aβ40 aggregates in different

765

inhibitors. Aβ40 concentration was 25 µM, and was co-incubated with each inhibitor at

766

molar ratios of 1:1, 1:2, 1:5 and 1:10 for 72 h. Data are expressed as percentage of

767

ThT fluorescence intensities; #p

Carnosine-LVFFARK-NH2 Conjugate: A Moderate Chelator but Potent

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