(AÎ²) Fibrillogenesis - ACS Publications - American Chemical Society

Subscriber access provided by READING UNIV

Article

A Tau Derived Hexapeptide AcPHF6 Promotes Beta-Amyloid (A#) Fibrillogenesis Tarek Mohamed, Sarbjeet Singh Gujral, and Praveen P. N. Rao ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00433 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 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 39 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

A Tau Derived Hexapeptide AcPHF6 Promotes Beta-Amyloid (Aβ) Fibrillogenesis Tarek Mohamed,† Sarbjeet Singh Gujral† and Praveen P. N. Rao*†

______________________________________________________________________________ *Corresponding author: Tel: +1 519 888 4567 ext: 21317; e-mail: [email protected] †School of Pharmacy, Health Sciences Campus, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

1 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

Page 2 of 39

A Tau Derived Hexapeptide AcPHF6 Promotes Beta-Amyloid (Aβ) Fibrillogenesis Tarek Mohamed,† Sarbjeet Singh Gujral† and Praveen P. N. Rao*† †School of Pharmacy, Health Sciences Campus, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 ______________________________________________________________________________ ABSTRACT: We studied the interactions of a tau derived hexapeptide AcPHF6 with beta-amyloid peptides Aβ40 and Aβ42 which reveals its unusual ability to promote Aβ fibrillogenesis. The results demonstrate that the N-acetylated and C-amidated AcPHF6 hexapeptide can cause significant acceleration in Aβ40 and Aβ42 fibril growth. Aggregation kinetic studies at pH 7.4 show that at 25 µM, AcPHF6 hexapeptide was able to cause ~2.3-fold increase in Aβ40 fibrillogenesis dramatically changing the aggregation kinetics. In addition, AcPHF6 peptide was able to reduce cellular toxicity mediated by Aβ40 and Aβ42 in hippocampal neuronal cell line (HT22). Computational studies suggest that the AcPHF6 peptide can act as an anchor and provides a hydrophobic surface for Aβ monomer to bind and undergo rapid fibrillogenesis to form less toxic fibrils and alter the aggregation kinetics. At the molecular level we propose a "dock-and-pack" mechanism where the AcPHF6 hexapeptide aggregates can stabilize the β-hairpin and promote rapid Aβ self-assembly and growth to form less toxic oligomers or fibrils. Our results have direct implications in designing novel peptide/peptidomimetics as novel pharmacological tools to study protein aggregation and potentially prevent Aβ-mediated toxicity in Alzheimer’s disease.

KEYWORDS: Alzheimer’s disease,

beta-amyloid, hexapeptide,

aggregation kinetics,

transmission electron microscopy, neuroprotection, molecular docking


Page 3 of 39 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 The complexity of Alzheimer’s pathogenesis has hindered the desperate need for promising treatment strategies. While the “how, why, what, where and when” aren’t fully understood, it is clear that the neurotoxic aggregates (beta-amyloid; Aβ and neurofibrillary tangles; NFTs) are the chief culprits in the Alzheimer’s disease (AD) pathology.1-8 The intricacies of Aβ fibrillogenesis is still a work in progress with many unanswered questions. One approach to prevent protein misfolding and aggregation process is the utilization of aggregation inhibitors that stabilize the aggregate building blocks (monomers, dimers, trimers or other higher order aggregates).9-13 These aggregation inhibitors typically act on various stages of amyloid aggregation kinetics either by delaying the lag phase or by affecting the growth or saturation phases.14, 15 Alternatively, molecules that promote the aggregation kinetics of beta-amyloid (Aβ) fibrillogenesis pathway are proposed to greatly reduce the load of more toxic Aβ oligomers and shorten their exposure time in vivo by driving the aggregation rapidly toward the less toxic Aβfibrils.16-19 Recently few studies have reported the discovery of molecules that can promote Aβ aggregation. For example, Bieschke and coworkers demonstrated that a small molecule can convert toxic oligomers to nontoxic fibrils and reduce neurotoxicity.16 In another study, Ryan and coworkers identified molecules capable of promoting Aβ aggregation suggesting their potential application in AD therapy.17 Interestingly, the polyphenol 1,2,3,4,6-penta-O-galloyl-βd-glucopyranose (PGG) was reported to enhance Aβ aggregation19 whereas Sonzini and coworkers reported the reduction in Aβ toxicity by increasing the rate of aggregation by a macrocyclic compound cucurbit[8]uril (CB[8]) which suggests that some molecules have the



Page 4 of 39

unique ability to remodel the Aβ aggregation kinetics.20 Another effort in this direction was the work by Dutta and coworkers who showed that the natural L-Aβ42 peptide exhibits greater aggregation propensity to form nontoxic fibrils in the presence of the corresponding mirrorimage D-Aβ42 peptide. This evidence further supports the investigation and development of potential AD therapies involving molecules capable of accelerating the Aβ fibrillogenesis to form nontoxic or less toxic fibrils.21 In this regard, we report the unusual observation where Aβ40 and Aβ42 fibrillogenesis were promoted by the N-acetylated and C-amidated hexapeptide AcPHF6 (MeCO-VQIVYKNH2) derived from the native tau-hexapeptide sequence 306VQIVYK311 (Figure 1), also known as PHF6. This peptide is known to undergo spontaneous aggregation on its own to form stericzipper assemblies and is used as a model to study aggregation inhibitors.22–24 Recent evidence suggests the role of Aβ in promoting tau pathogenesis although not much is known about their interactions.25–27 We investigated the effect of AcPHF6 on Aβ40 and Aβ42 aggregation kinetics using thioflavin T (ThT)-fluorescence monitoring, transmission electron microscopy (TEM) imaging, neuroprotection in hippocampal cells and computational studies. These investigations demonstrate the remarkable ability of AcPHF6 to promote both Aβ40/Aβ42 fibrillogenesis and reduce neuronal toxicity thereby providing hitherto unknown detail on their interactions at the molecular level. RESULTS AND DISCUSSION Effect of AcPHF6 hexapeptide on Aβ40 and Aβ42 aggregation The aggregation kinetics and subsequent morphology assessments were conducted at physiological and acidic pH (~7.4 and 5.6 respectively) settings considering the fact that acidic brain pH levels can promote Aβ-aggregation.28, 29 The aggregation kinetics at physiological pH


Page 5 of 39 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


(~7.4, Figure 2, Panels A and C) revealed a concentration- dependent, pro-aggregatory nature of AcPHF6 on both Aβ40/42. At 25 µM, AcPHF6 was able to dramatically increase the fibrillogenesis of both Aβ40 and Aβ42. In the absence of AcPHF6, at pH 7.4 Aβ40 alone exhibited a lag phase of ~8.2 h whereas in the presence of 25 µM of AcPHF6, the lag phase of Aβ40 aggregation was dramatically reduced to ~3.5 h with almost 2.3-fold acceleration in Aβ40 fibrillogenesis (Figure 2, Panel A). Furthermore, an exponential increase in growth phase was seen with a dramatic shift in the growth phase kinetics. In contrast, at 1 µM, AcPHF6 was able to prevent Aβ40 aggregation (48% inhibition at 24 h) and was a weak promoter of Aβ42 aggregation. At 5 µM, it exhibited almost no activity toward Aβ40 and was able to promote Aβ42 aggregation. At 25 µM AcPHF6 was able to spontaneously promote and accelerate Aβ42 aggregation and fibrillogenesis (Figure 2, Panel C). Similar observation was made when the aggregation kinetics were carried out at pH 5.6 for both Aβ40 and Aβ42 (Figure 3, Panels A and C). In order to rule out the possibility of fluorescence quenching and potential interference at high concentrations of AcPHF6, we conducted control experiments at 1, 5 and 25 µM of AcPHF6 alone in buffer at both pH 7.4 and 5.6; respectively. These studies showed that at all concentrations (1–25 µM), AcPHF6 exhibited minimum fluorescence interference (Supporting Information, Figure S1). Further affirmation of AcPHF6’s ability to promote Aβ fibrillogenesis was corroborated by transmission electron microscopy (TEM) studies (Figure 2, Panel B and D). At 25 µM, AcPHF6 was able to promote the fibril load when incubated with either Aβ40 or Aβ42. At this concentration, there was a significant increase in Aβ-AcPHF6 fibril assembly as can be seen with the accumulation of fibrillar structures (Figure 2, Panels B and D). The observations at acidic pH (~5.6, Figure 3, Panels A–C) revealed remarkable pro-aggregatory activity of AcPHF6 at 25 µM on both Aβ40/42 with a significant reduction in the lag phase and



Page 6 of 39

increases in the growth phase. The AcPHF6 was not able to induce aggregation at 1 µM but was able to promote aggregation at 5 µM. In contrast, it was inactive at both 1 and 5 µM toward Aβ42. The TEM images show that under acidic conditions, both Aβ40 and Aβ42 formed nonnative spherical aggregates or oligomers and not typical fibrillary species which is consistent with a previous report.29 The TEM image of AcPHF6 alone (25 µM) shows the formation AcPHF6 fibrils which are distinct from Aβ40 fibrils (Figure S2, Supporting Information). The AcPHF6 peptide was able to increase both Aβ40 and Aβ42 spherical oligomers at 25 µM (Figure 3, Panels B and D). This is a significant discovery which shows that the N-acetylated and Camidated AcPHF6 can promote the formation of spherical Aβ oligomers under acidic pH in complete contrast to its ability to form fibrils under physiological pH. In contrast, the Aβaggregation kinetic study in the presence of the uncapped/free native tau-hexapeptide (NH2VQIVYK-COOH) PHF6 did not exhibit a similar trend (Figure 4). The native peptide PHF6 was able to prevent Aβ40 aggregation at 5 and 25 µM respectively at pH 7.4. However at 1 µM, it was able to promote Aβ fibrillogenesis (Figure 4, Panel A) although this effect was not comparable to the significant and dramatic acceleration in fibril assembly seen with AcPHF6. Similarly at pH 5.6 no significant increases in Aβ40 aggregation was seen at 5 and 25 µM whereas at 1 µM, PHF6 was able to promote Aβ40 fibrillogenesis (Figure 4, Panel C). The aggregation kinetics data for Aβ42 clearly shows that PHF6 had no effect on Aβ fibrillogenesis (Figure 4, Panel B and D). In another interesting observation, we noticed that under acidic pH 5.6, the aggregation curves for Aβ (Figure 3 and Figure 4, Panel C and D) exhibit reduced fluorescence compared to same runs conducted at neutral pH 7.4. In addition, the aggregation kinetic trends vary for Aβ42 with no lag phase and no clear distinction between growth and saturation phase. These observations


Page 7 of 39 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


can be partly explained due to potential denaturation of Aβ under acidic conditions. Furthermore, Aβ42 is more hydrophobic and might be less soluble leading to some precipitation under acidic pH.30 To further assess the pro-aggregatory role of AcPHF6, we used six N-acetylated and C-amidated alanine mutants (AQIVYK, VAIVYK, VQAVYK, VQIAYK, VQIVAK and VQIVYA) and investigated their aggregation kinetic profile to evaluate their effect on Aβ40 and Aβ42 at two different pHs (7.4 and 5.6 respectively). The detailed kinetic plots are provided in the supporting information (Supporting Information, Figure S3–S6). In general, unlike AcPHF6, none of the alanine mutants produced consistent pro-aggregatory effect on both Aβ40/Aβ42 species under neutral and acidic pH conditions at 25 µM (Figure S3–S5: Supporting Information). At neutral pH, a wider range of activity (inhibitory to promotion) was observed with the Ala-mutant AQIVYK exhibiting maximum increase in Aβ40 fibrillogenesis (~57% increase, Fig. S3, Panel A) at 25 µM, while under acidic conditions, Ala-mutant VQIVYA exhibiting maximum increase of Aβ42 fibrillogenesis (Figure S6, Panel E). Unlike AcPHF6, none of the mutants were able to exhibit an exponential increase in the growth phase accompanied by a dramatic reduction in lag phase time (Figure S3–S6, Supporting Information). Effect of AcPHF6 hexapeptide on Aβ40 and Aβ42-induced cell death in mouse hippocampal (HT22) cells In the presence of 1, 5, 10 or 25 µM of AcPHF6 peptide alone, the HT-22 cells exhibited excellent viability (range 97–131%, Figure S7) indicating that AcPHF6 peptide alone was nontoxic. The mouse hippocampal cells (HT22)31,

32

were treated with Aβ40 (5 µM) alone

exhibited significant cell death (cell viability ~57%) after 24 h incubation (Figure 5, Panel A). Strikingly, incubating the HT22 cells with 1 µM of AcPHF6 provided significant neuroprotection



Page 8 of 39

(cell viability of 75.4%) and this trend continued at higher concentrations of AcPHF6 (5, 10 and 25 µM respectively) with maximum neuroprotection (~95%) seen at 25 µM, almost 1.6-fold increase in cell viability compared to Aβ40 control as shown in Figure 5. This demonstrates the ability of tau derived hexapeptide AcPHF6 to provide neuroprotection to Aβ40-induced cell death in HT22 cells. These studies were repeated using the more toxic Aβ42 peptide (Figure 5, Panel B) which shows that when incubated for 24 h, Aβ42 (5 µM) was able to cause a greater degree of cell death (cell viability ~28.5% ) compared to Aβ40 peptide. More significantly, coincubating HT22 cells with Aβ42 peptide in the presence of AcPHF6 (1–25 µM) led to a dramatic increase in cell viability (~40–44%, Figure 5, Panel B). This 1.5-fold increase in cell viability further supports the ability of AcPHF6 to provide neuroprotection in HT22 cells. These observations support with the in vitro Aβ40 and Aβ42 aggregation kinetics and TEM data observed which shows that at higher concentrations, AcPHF6 was promoting Aβ-fibrillogenesis by rapid conversion of more toxic oligomers and protofibrils to less toxic fibrils. It should also be noted that any direct comparison and correlation between in vitro Aβ aggregation kinetics and in vivo HT22 cell toxicity in the presence and absence of AcPHF6 is not appropriate since in vitro aggregation kinetic studies represent isolated system with known concentrations of Aβ peptides interacting with a known concentration of AcPHF6 hexapeptide whereas in a cell system the scenario is entirely different with membrane barriers that the AcPHF6 peptide needs to cross as well as the presence of all the cell machinery including ion-gated/voltage gated receptors, transporter and a plethora of biological molecules including enzymes and proteins.33, 34 Computational modeling of AcPHF6 hexapeptide on Aβ40 and Aβ42 peptides In order to understand the dramatic proaggregation properties of AcPHF6, we conducted computational experiments. Molecular docking studies of N- and C-capped AcPHF6 monomer


Page 9 of 39 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


and Aβ40 protofibril derived from Tycko’s structure35 suggests that the AcPHF6 (Ac-VQIVYKNH2) side chains were interacting with the HQKLVFFA region of Aβ40 in an antiparallel fashion (Figure 6, Panel A and B). The protonated lysine side chains at the interface of protofibril assembly underwent intermolecular polar interactions with lysine and the backbone amides of AcPHF6 peptide. This binding mode is similar to the steric-zipper assembly seen in the x-ray crystal structures of amyloidogenic hexapeptides.36 Previous studies have shown that the LVFFA region of Aβ was involved in interacting with the VQIVYK region of full length tau protein.26,

27

It is known that the tau derived hexapeptide

AcPHF6 undergoes rapid aggregation on its own.22, 23 This suggests that the consistent increase in fibrillogenesis observed for both Aβ40 and Aβ42 in the presence of higher ratio of AcPHF6 (1:5 ratio of Aβ:AcPHF6) is due to the interaction of AcPHF6 aggregates with the HQKLVFFA region of Aβ peptide which can stabilize the β-hairpin structure and promote Aβ self-assembly. Furthermore, modeling studies suggest that the binding of N- and C-capped AcPHF6 to the Nterminal HQKLVFFA region can expose the hydrophobic C-terminal to solvent environment which can promote rapid aggregation (Figure 6, Panel B). Previous studies have shown that small peptide sequences can provide hydrophobic surface and accelerate aggregation of proteins.33 In our case, we anticipated that AcPHF6, which contains N-acetyl and C-amide groups, would be less polar compared to PHF6 with free N- and C-terminals. Indeed the electron density maps obtained for AcPHF6 and PHF6 (Figure 7) unambiguously showed that AcPHF6 had a hydrophobic surface in contrast to PHF6 which suggests that unlike the polar PHF6, AcPHF6 aggregates can provide a hydrophobic surface for oncoming Aβ40/Aβ42 proteins and consequently accelerate Aβ fibrillogenesis.



Page 10 of 39

Our studies clearly demonstrate the ability of the N- and C-capped AcPHF6 hexapeptide to accelerate and promote Aβ fibrillogensis by reducing the lag phase and rapid promotion of growth phase. Promotion of fibrillogenesis by AcPHF6 was observed under both neutral and acidic pHs. Based on these studies, we have developed a mechanistic model where at higher concentrations, AcPHF6 β-sheets can stabilize the β-hairpin conformation of Aβ in solution and acts as a catalyst by promoting the rapid growth and assembly of mixed Aβ:AcPHF6 fibrils (Figure 8). Taking a cue from Wei and Ma’s work37 on Aβ-promoted full length tau protein aggregation, we propose that at higher concentrations, AcPHF6 uses a “dock-and-pack” approach where it provides a docking surface to the HQKLVFFA region of Aβ monomer and stabilizes the β-hairpin to facilitate Aβ self-assembly/packing and rapid fibrillogenesis to form mixed Aβ: AcPHF6 fibrils (Figure 8). This is further supported by a recent work which reports that human insulin aggregation is accelerated by a short β-sheet forming peptide LVEALYL which facilitates the conformational change in full length insulin and its subsequent aggregation.38 This evidence indicates that AcPHF6 β-sheets can act as an anchor and provide a hydrophobic surface for the Aβ peptide to anchor and undergo self-assembly to form nontoxic fibrils.

CONCLUSION Our studies show for the first time, the dual nature of the N-acetylated and C-amidated AcPHF6 toward Aβ fibrillogenesis where under physiological pH, it was able to promote the formation of fibrils whereas under acidic conditions, oligomer load tends to prevail. In addition, AcPHF6 was able to reduce Aβ40 and Aβ42 mediated toxicity in neuronal cells. These observations will have significant implications in our current understanding of Aβ fibrillogenesis and provides a novel


Page 11 of 39 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


peptide based tool to study protein aggregation and develop anti-AD therapies. Furthermore, current evidence has shown that it is virtually impossible to prevent Aβ aggregation39, and the best approach is to reduce the lifespan of toxic oligomers by directing the aggregation pathway toward minimally toxic route. In this context, combining a fibrillogenesis promoter with fibril selective anti-Aβ immunotherapy is a viable approach to reduce oligomer load and neurotoxicity.40, 41 Significantly, our study has shown for the first time the role of AcPHF6, in promoting the amyloid aggregation which has implications in the design of potential peptide/peptidomimetics as pharmacological tools to study protein misfolding, aggregation and develop treatments. METHODS Thioflavin-T (ThT) based Aβ aggregation kinetics. The effect of AcPHF6, PHF6 and its Ala-silenced mutants, on the aggregation kinetics of Aβ40 or Aβ42 was assessed using the thioflavin (ThT)-binding assay.42–44 The Aβ40 or Aβ42 (HFIP, rPeptide, Bogart, USA) was reconstituted in 1% ammonium hydroxide to 0.5 mg/mL, sonicated at room temperature for 5 minutes before diluting in 215 mM phosphate (Na2HPO4•7H2O) buffer (at pH ~ 7.4 or pH ~ 5.6) to 50 µM and stored in an ice-water bath. The AcPHF6 (Ac-VQIVYK-NH2, Celtek Peptides, Franklin, USA), PHF6 (NH2-VQIVYK-COOH) and six Ala-silenced mutants (Shanghai Bootech BioScience & Technology Co., Ltd, Shanghai, China) were reconstituted in Ultra-pure water, UPW (Cayman Chemical, Ann Arbor, USA) to 500 µM, sonicated at room temperature for 5 minutes before diluting further in UPW to 250, 50 or 10 µM stocks respectively. Solutions were stored in an ice-water bath. The ThT solution (15 µM) was prepared in UPW containing 50 mM glycine. The aggregation kinetics were monitored at neutral (~ pH 7.4) and acidic (~ pH 5.6) conditions in 215 mM phosphate buffer. The effect of varying concentrations (1-25 µM range) of



Page 12 of 39

AcPHF6, PHF6 and its Ala-mutants was monitored by adding 44 µL ThT, 19 µL phosphate buffer, 1 µL DMSO, 8 µL of either Aβ40 or Aβ42 (final concentration of 5 µM) and 8 µL of the appropriate hexapeptide using a Costar (black, clear-bottom) 384-well plate. The fluorescence was measured at 440 nm (excitation)/490nm (emission) at 5 minute intervals with 30 seconds of linear shaking (~ 730 cpm) prior to each readout over a 24 h incubation period at 37 °C. Results are an average of triplicate readings for two to three independent assays and analyzed with the appropriate ThT blank (44 µL ThT (in 50 mM glycine, 15 µM ThT, pH 7.4 or 5.6), 35 µL phosphate buffer (215 mM, pH 7.4 or 5.6), 1 µL DMSO) and individual hexapeptide controls (44 µL ThT, 27 µL buffer, 1 µL DMSO, 8 µL of Aβ40 or Aβ42 and 8 µL of appropriate hexapeptides respectively). The relative fluorescence intensity units (RFU) were corrected for ThTinterference before processing the aggregation kinetic plots. Transmission electron microscopy (TEM). The aggregation morphology of both Aβ40 and Aβ42 in the presence of AcPHF6 was assessed using TEM.45, 46 The Aβ morphology was carried out under both neutral (~ pH 7.4) and acidic (~ pH 5.6) conditions using 215 mM phosphate (NaHPO4•7H2O) buffer. The Aβ40 or Aβ42 (HFIP, rPeptide, Bogart, USA) was reconstituted in 1% ammonium hydroxide to 0.5 mg/mL, sonicated at room temperature for 5 minutes before diluting in 215 mM phosphate buffer (at either pH ~ 7.4 or pH ~ 5.6) to 50 µM and stored in an ice-water bath. The AcPHF6 (Ac-VQIVYK-NH2, Celtek Peptides, Franklin, USA) was reconstituted in Ultra-pure water, UPW (Cayman Chemical, Ann Arbor, USA) to 500 µM, sonicated at room temperature for 5 minutes before diluting further in UPW to 250, 50 or 10 µM stocks respectively. Solutions were stored in an ice-water bath. The following solutions were added to 96-well plate (clear, round-bottom) in duplicates; 158 µL buffer, 2 µL DMSO, 20 µL of Aβ40 or Aβ42, 20 µL of AcPHF6, along with Aβ40 or Aβ42 controls (178 µL buffer, 2 µL


Page 13 of 39 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


DMSO, 20 µL of Aβ40 or Aβ42). The plate was incubated at 37 °C for 24 h with continuous shaking (~ 730 cpm). After the incubation period, duplicates samples were combined and transferred to a 1.5 mL microcentrifuge tube and 30 µl from each tube was added to a Formvar, carbon-coated copper grid (400 mesh), then allowed to air-dry before re-applying a second aliquot and drying further. Buffer precipitates were rinsed off with 50 µL of UPW, twice, and left to air-dry before staining with 10 µL of 2% phosphotungstic acid (PTA) for 5 seconds before blotting dry with filter paper segments. Grids were left to air-dry for 12-24 h before imaging using a Philips CM 10 transmission electron microscope at 60 kV (Department of Biology, University of Waterloo) and micrographs were obtained using a 14-megapixel AMT camera. Effect of AcPHF6 hexapeptide on hippocampal HT22 cells in the presence of Aβ40 or Aβ42. The neuroprotective effect of AcPHF6 on cell death induced by either Aβ40 or Aβ42 (5 µM each) on mouse hippocampal HT22 cells was evaluated by using the dye 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) which gets reduced by the mitochondrial dehydrogenase to a formazan intermediate and which was monitored by measuring the UV absorption at 570 nm.32 The cell viability was monitored in the presence of increasing concentrations of AcPHF6 peptide. Initially the HT22 cells were plated at a density of 10,000 cells/mL into 96-well plate cell culture plates in full growth media consisting of 1:1 Dulbecco's modified eagle media (DMEM) and Ham's F12 with the addition of glutamate (2.5 mM), 10% fetal bovine serum (FBS) supplemented with 1% penicillin and streptomycin at 37 ºC, in 5% CO2 until 80% confluence. Cells were co-incubated for 24 h at 37 ºC with either Aβ40 or Aβ42 (5 µM each) and different concentration of AcPHF6 solutions (1, 5, 10 and 25 µM respectively) with no more than 1% DMSO. Control experiment with AcPHF6 alone (1, 5, 10 and 25 µM) in the absence of Aβ was also carried out. The media was exchanged with MTT



Page 14 of 39

solution (phenol red-free DMEM/F12 with 10% MTT), followed by 3 h incubation at 37 ºC. HT22 cells were then solubilized manually, with MTT reagent solution (10% Triton X-100 and 0.1 N HCl in anhydrous isopropanol) and the absorbance at 570 nm and 690 nm were read using a BioTek Synergy H1 plate reader. The absorbance value at 690 nm was subtracted from the value at 570 nm to account for the background signals. The final cell viability was calculated as percent reduction of MTT into formazan compared to control wells (untreated cells). The results were calculated as average % cell viability based on two independent experiments (n = 3). Molecular docking studies. The computational chemistry software Discovery Studio (DS) – Structure-Based-Design (SBD), version 4.0 from BIOVIA Inc. (San Diego, USA) was used to conduct molecular docking studies. The Aβ40 (Aβ9-40) NMR solution structure (pdb id: 2LMN) was used to build the Aβ-protofibril model consisting of a tetramer β-sheet assembly. The protofibril model was prepared using the macromolecules module in DS. The AcPHF6 hexapeptide was built using the coordinates of the native tau-hexapeptide VQIVYK was obtained from the protein data bank (pdb id: 3OVL), the bound ligand orange G was deleted and the N- and C-terminals were capped with an acetyl and an amide group respectively using the small molecules module in DS after which the AcPHF6 was subjected to smart minimizer protocol (200 steps, RMS gradient 0.1 kcal/mol) using CHARMm force field and an implicit solvent function generalized Born smooth switching function (GBSW) under SHAKE constraints. The molecular docking of native tau-hexapeptide to Aβ-protofibril was carried out by using the CDOCKER algorithm after defining a sphere of 25 Å radius at the tetramer interface comprising the the N-terminus. The CDOCKER algorithm included 2000 heating steps, 700K target temperature, 300K cooling temperature target with 5000 cooling steps and CHARMm force field. The output provided 10 different binding poses of AcPHF6 bound to Aβ-


Page 15 of 39 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


protofibrils that were ranked based on the CDOCKER energies, CDOCKER interaction energies in kcal/mol and number of polar and nonpolar interactions observed.

47, 48

Electrostatic potential

surface map for both PHF6 (pdb id: 3OVL) and AcPHF6 was calculated using the DelPhi atomic charge and radii in the simulation module of DS.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

Fluorescence aggregation kinetics data for AcPHF6, PHF6, AcPHF6 mutants, TEM image for AcPHF6 and HT-22 cell viability data for AcPHF6 peptide

AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID Tarek Mohamed: 0000-0001-5745-0916 Praveen P Nekkar Rao: 0000-0001-5703-8251 Author Contributions Notes The authors have no competing financial interest to declare ACKNOWLEDGEMENTS



Page 16 of 39

The authors would like to thank Ontario Mental Health Foundation (TM), NSERC-Discovery (RGPIN: 03830-2014), Canada Foundation for Innovation (CFI-JELF), Ontario Research Fund (ORF) and Early Researcher Award (ERA), Ministry of Research and Innovation, Government of Ontario (P. P. N.R), Canada for financial support of this research project. ABBREVIATIONS AD, Alzheimer’s disease; Aβ, beta-amyloid; AcPHF6, tau derived hexapeptide; ThT, thioflavinT; TEM, transmission electron microscopy; UPW, ultra-pure water; DMSO, dimethyl sulfoxide; HFIP,

hexafluoroisopropranol;

MTT,

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium

bromide; PTA, phosphotungstic acid; SBD, structure-based-design; CHARMm, Chemistry at Harvard macromolecular mechanics; DMEM, Dulbecco's modified eagle media; FBS, fetal bovine serum REFERENCES (1) Selkoe, D. J. (1994) Cell biology of the amyloid beta-protein precursor and the mechanism of Alzheimer’s disease. Ann. Rev. Cell Biol. 10, 373–403. (2) Hardy, J., and Selkoe, D. J. (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002, 297, 353–356. (3) LaFerla, F. M., Green, K. N., and Oddo, S. (2007) Intracellular amyloid-beta in Alzheimer’s disease. Nat. Rev. Neurosci. 8, 499–509. (4) Eisele, Y. S., Monteiro, C., Fearns, C., Encalada, S. E., Wiseman, R. L., Powers, E. T., and Kelly, J. W. (2015) Targeting protein aggregation for the treatment of degenerative diseases. Nat. Rev. Drug Discov. 11, 759–780. (5) Lee, V. M., Goedert, M. and Trojanowski, J. Q. (2001) Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24, 1121–1159.


Page 17 of 39 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


(6) Bloom, G. S. (2014) Amyloid-β and tau: the trigger and bullet in Alzheimer pathogenesis. JAMA Neurol. 71, 505–508. (7) Wang, Y., and Mandelkow, E. (2016) Tau in physiology and pathology. Nat. Rev. Neurosci. 17, 5–21. (8) Kayed, R., Head, E., Thompson, J. L., McIntire, T. Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003) Common structures of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489. (9) Doig, A. J. (2007) Peptide inhibitors of beta-amyloid aggregation. Curr. Opin. Drug Discov. Devel. 5, 533–539. (10) Yang, W., Wong, Y., Ng, O. T., Kwong, D. W., Ke, Y., Jiang, Z. H., Li, H. W., Yung, K. K., and Wong, M. S. (2012) Inhibition of beta-amyloid peptide aggregation by multifunctional carbazole-based fluorophores. Angew. Chem. Int. Ed. Engl. 51, 1804–1810. (11) Belluti, F., Rampa, A., Gobbi, S., and Bisi, A. (2013) Small molecule inhibitors/modulators of amyloid-β peptide aggregation and toxicity for the treatment of Alzheimer’s disease: a patent review (2010 - 2012). Expert Opin. Ther. Pat. 5, 581–596. (12) Godyn, J., Jonczyk, J., Panek, D., and Malawska, B. (2016) Therapeutic strategies for Alzheimer’s disease in clinical trials. Pharmacol. Rep. 68, 127–138. (13) Doig, A. J., and Derreumaux, P. (2015) Inhibition of protein aggregation and amyloid formation by small molecules. Curr. Opin. Struct. Biol. 30, 50–56. (14) Hamley, I. W. (2012) The amyloid beta peptide: a chemist’s perspective. Role in Alzheimer’s and fibrillization. Chem. Rev. 112, 5147–5192.



Page 18 of 39

(15) Nguyen, P., and Derreumaux, P. (2014) Understanding amyloid fibril nucleation and Aβ oligomer/drug interactions from computer simulations. Acc. Chem. Res. 47, 603–611. (16) Bieschke, J., Herbst, M., Wiglenda, T., Friedrich, R. P., Boeddrich, A., Schiele, F., Kleckers, D., Lopez del Amo, J. M., Gruning, B. A., Wang, Q., Schmidt, M. R., Lurz, R., Anwyl, R., Schnoegl, S., Fandrich, M., Frank, R. F., Reif, B., Gunther, S., Walsh, D. M., and Wanker, E. E. (2011) Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils. Nat. Chem. Biol. 8, 93–101. (17) Ryan, T. M., Friedhuber, A., Lind, M., Howlett, G. J., Masters, C., and Roberts, B. R. (2012) Small amphipathic molecules modulate secondary structure and amyloid fibril-forming kinetics of Alzheimer disease peptide Aβ(1-42). J. Biol. Chem. 287, 16947–16954. (18) Luo, L., Yu, C. H., Yu, H., Borstnar, R., Kamerlin, S. C., Graslund, A., Abrahams, J. P., and Warmlander, S. K. (2013) Cellular polyamines promote amyloid-beta (Aβ) peptide fibrillation and modulate the aggregation pathways. ACS Chem. Neurosci. 4, 454–462. (19) De Almeida, N. E., Do, T. D., Tro, M., LaPointe, N. E., Feinstein, S. C., Shea, J. E. and Bowers, M. T. (2016) Opposing effects of cucurbit[7]uril and 1,2,3,4,6-penta-Ogalloyl-β-d-glucopyranose on amyloid β25-35 assembly. ACS Chem. Neurosci. 7, 218–226. (20) Sonzini, S., Stanyon, H. F., and Scherman, O. A. (2017) Decreasing amyloid toxicity through an increased rate of aggregation. Phys. Chem. Chem. Phys. 19, 1458–1465.


Page 19 of 39 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


(21) Dutta, S., Foley, A. R., Warner, C. J. A., Zhang, X., Rolandi, M., Abrams, B., and Raskatov, J. A. (2017) Suppression of oligomer formation and formation of nontoxic fibrils upon addition of mirror-image Aβ42 to the natural L-enantiomer. Angew. Chem. Int. Ed. Engl. 56, 11505–11510. (22) Zheng, J., Liu, C., Sawaya, M. R., Vadla, B., Khan, S., Woods, R. J., Eisenberg, D. and Goux, W. J., Nowick, J. S. (2011) Macrocyclic β-sheet peptides that inhibit aggregation of tau-protein derived hexapeptide. J. Am. Chem. Soc. 133, 3144–3157. (23) Adamcik, J., Sanchez-Ferrer, A., Ait-Bouziad, Reynolds, N. P., Lashuel, H. A., and Mezzenga, R. (2016) Microtubule-binding R3 fragment from tau self-assembles into giant multistranded amyloid ribbons. Angew. Chem. Intl. Ed. Engl. 55, 618–622. (24) Mohamed, T., Hoang, T., Jelokhani-Niaraki, M., Rao, P. P. N. (2013) Tau-derivedhexapeptide 306VQIVYK311 aggregation inhibitors: nitrocatechol moiety as a pharmacophore in drug design. ACS Chem. Neurosci. 4, 1559–1570. (25) Miller, Y., Ma, B., and Nussinov, R. (2011) Synergistic interactions between repeats in tau protein and Aβ amyloids may be responsible for accelerated aggregation via polymorphic states. Biochemistry 50, 5172–5181. (26) Do, T. D., Economou, N. J., Chamas, A., Buratto, S. K. Shea, J. E., and Bowers, M. T. (2014) Interactions between amyloid-β and tau fragments promote aberrant aggregates: implications for amyloid toxicity. J. Phys. Chem. B. 118, 11220–11230. (27) Wray, S., and Noble, W. (2009) Linking amyloid and tau pathology in Alzheimer’s disease: the role of membrane cholesterol in Aβ-mediated tau toxicity. J. Neurosci. 29, 9665–9667.



Page 20 of 39

(28) Magnotta, V. A., Heo, H. Y., Dlouhy, B. J., Dahdaleh, N. S., Follmer, R. L., Thedens, D. R., Welsh, M. J., and Wemmie, J. A. (2012) Detecting activity-evoked pH changes in human brain. Proc. Natl. Acad. Sci. U. S. A. 109, 8270–8273. (29) Tipping, K. W., Karamanos, T. K., Jakhria, T., Iadanza, M. G., Goodchild, S. C., Tuma, R., Ranson, N. A., Hewitt, E. W., and Radford, S. (2015) pH-induced molecular shedding drives the formation of amyloid fibril derived oligomers. Proc. Natl. Acad. Sci. U. S. A., 112, 5691–5696. (30) Hortschansky, P., Schroeckh, V., Christopeit, T., Zandomeneghi, G., and Fandrich, M. (2005) The aggregation kinetics of Alzheimer’s β-amyloid peptide is controlled by stochastic nucleation. Protein Sci. 14, 1753–1759. (31) Martin, V., Almansa, E., Fabelo, N., and Diaz, M. (2006) Selective polyunsaturated fatty acids enrichment in phospholipids from neuronal-derived cell lines. J. Neurosci. Methods 153, 230–238. (32) Yang, S. H., Lee, D. K., Shin, J., Lee, S., Baek, S., Kim, J., Jung, H., Hah, J. M., and Kim, Y. (2016) Nec-1 alleviates cognitive impairment with reduction of A and tau abnormalities in APP/PS1 mice. EMBO Mol. Med. 9, 61–77. (33) Cha, M. Y., Han, S. H., Son, S. M., Hong, H. S., Choi, Y. J., Byun, J., and MookJung, I. (2012) Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction in apoptotic cell death. PLoS One. 7, e34929. (34) Brimson, J. M., Brimson, S. J., Brimson, C. A., Rakkahitawatthana, V., and Tencomnao, T (2012) Rhinacanthus nasutus extracts prevent glutamate and amyloidβ neurotoxicity in HT-22 mouse hippocampal cells: possible active compounds include lupeol, stigmasterol and β-sitosterol. Int. J. Mol. Sci. 13, 5074–5097.


Page 21 of 39 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


(35) Petkova, A. T., Yau, W. M., and Tycko, R. (2006) Experimental constraints on quaternary structure in Alzheimer’s beta-amyloid fibrils. Biochemistry 45, 498–512. (36) Colletier, J. P., Laganowsky, A., Landau, M., Zhao, M., Soriaga, A. B., Goldschmidt, L., Flot, D., Cascio, D., Sawaya, M. R., and Eisenberg, D. (2011) Molecular basis for amyloid-beta polymorphism. Proc. Natl. Acad. Sci. U. S. A. 108, 16938–16943. (37) Qi, R., Luo, Y., Wei, G., Nussinov, R., and Ma, B. (2015) Aβ “stretching and packing” cross-seeding mechanism can trigger tau protein aggregation. J. Phys. Chem. Lett. 6, 3276–3282. (38) Nault, L., Vendrely, C., Brechet, Y., Bruckert, F., and Weidenhaupt, M. (2013) Peptides that form β–sheets on hydrophobic surfaces accelerate surface-induced insulin amyloidal aggregation. FEBS Lett. 587, 1281-1286. (39) Giacobini, E., and Gold, G. (2013) Alzheimer disease therapy – moving from amyloid-β to tau. Nat. Rev. Neurol. 12, 677–686. (40) Liu, E., Schmidt, M. E., Margolin, R., Sperling, R., Koeppe, R., Mason, N. S., Klunk, W. E., Mathis, C. A., Salloway, S., Fox, N. C., Hill, D., Les, A. S., Collins, P., Gregg, K. M., Di, J., Tudor, I. C., Wyman, B. T., Booth, K., Broome, S., Yuen, E., Grundman, M., and Brashear, H. R., (2015) Amyloid-β 11C-PiB-PET imaging results from the 2 randomized bapineuzumab phase 3 AD trials. Neurology 85, 692– 700. (41) Slomski, A. (2015) Anti-β-amyloid treatment may be needed to be started early. JAMA 314, 1217.



Page 22 of 39

(42) Levine, H. (1993) Thioflavin T interaction with synthetic Alzheimer’s disease betaamyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2, 404– 410. (43) Levine, H. (1987) Stopped-flow kinectics reveal multiple phases of thioflavin T binding to Alzheimer beta (1-40) amyloid fibrils. Arch. Biochem. Biophys. 342, 306–316. (44) Stine, W. B., Jungbauer, L., Yu, C., and LaDu, M. J. (2011) Preparing synthetic Aβ in different aggregation states. Methods Mol. Biol. 670, 13–32. (45) Jan, A., Hartley, D. M., and Lashuel, H. A. (2011) Preparation and characterization of toxic abeta aggregates for structural and functional studies in Alzheimer’s disease research. Nat. Protoc. 5, 1186–1209. (46) Gras, S. L., Waddington, L. J., and Goldie, K. N. (2011) Transmission electron microscopy of amyloid fibrils. Methods Mol. Biol. 752, 197–214. (47) Rao, P. P. N., Mohamed, T., Teckwani, K., and Tin, G. (2015) Curcumin binding to beta amyloid: a computational study. Chem. Biol. Drug Des. 86, 813–820. (48) Mohamed, T., Shakeri, A., and Rao, P. P. N. (2016) Amyloid cascade in Alzheimer’s disease: recent advances in medicinal chemistry. Eur. J. Med. Chem. 113, 258–272.


Page 23 of 39 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


Figure 1. Sequence of AcPHF6 hexapeptide and Aβ40/42 highlighting the amyloidogenic (KLVFFA) core.



Page 24 of 39

Figure 2. Panels A and C show ThT-monitored 24 h aggregation kinetics of Aβ40 or Aβ42 (5 µM ) in the presence of 1, 5 or 25 µM of AcPHF6 at pH 7.4, 37 °C; Panels B and D show TEM images of Aβ40 or Aβ42 aggregation morphology at 24 h in the presence of 1, 5 or 25 µM of AcPHF6. Aggregation kinetics were monitored by ThT-fluorescence spectroscopy (excitation = 440 nm, emission = 490 nm). Results are average of two to three independent experiments in triplicate measurements. TEM images: scale 500 nm.


Page 25 of 39 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


Figure 3. Panels A and C show ThT-monitored 24 h aggregation kinetics of Aβ40 or Aβ42 (5 µM each) in the presence of 1, 5 or 25 µM of AcPHF6 at pH 5.6, 37 °C; Panels B and D show TEM images of Aβ40 or Aβ42 aggregation morphology at 24 h in the presence of 1, 5 or 25 µM of AcPHF6. Aggregation kinetics were monitored by ThT-fluorescence spectroscopy (excitation = 440 nm, emission = 490 nm). Results are average of two to three independent experiments in triplicate measurements. TEM images: scale 500 nm.



Page 26 of 39

Figure 4. Panels A and C show ThT-monitored 24 h aggregation kinetics of Aβ40 (Panels A and C) or Aβ42 (Panels B and D), 5 µM each in the presence of 1, 5 or 25 µM of PHF6 at pH 7.4 or 5.6 respectively, 37 °C; Aggregation kinetics were monitored by ThT-fluorescence spectroscopy (excitation = 440 nm, emission = 490 nm). Results are average of two to three independent experiments in triplicate measurements.


Page 27 of 39 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


Figure 5. (A) Effect of AcPHF6 (1, 5, 10 and 25 µM) on cell death induced by Aβ40 (5 µM) in HT22 cells after 24 h incubation at 37 ºC in the MTT assay; (B) Effect of AcPHF6 (1, 5, 10 and 25 µM) on cell death induced by Aβ42 (5 µM) in HT22 cells after 24 h incubation at 37 ºC in the MTT assay. The results are provided as average ± SD of two independent experiments (n = 3). 27 ACS Paragon Plus Environment


Page 28 of 39

*p< 0.01 compared to Aβ40 alone treatment group and *p< 0.05 compared to Aβ42 alone treatment group (one-way ANOVA followed by Bonferroni post hoc test).

Figure 6. (A) Top and side views of the AcPHF6 monomer (green stick cartoon) docked on the surface of Aβ40 protofibril assembly along the fibril axis. The amino acids of AcPHF6 are shown in green-box whereas the amino acids of Aβ protofibril interface are labeled. Hydrogen atoms are


Page 29 of 39 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


removed to enhance clarity; (B) Binding mode of AcPHF6 monomer (yellow) on the HQKLVFFA interface of Aβ40 protofibril shown at different angles.

Figure 7. Electrostatic potential surface of AcPHF6 (A) and PHF6 (B) respectively. Regions of low and high electron densities are color coded with red indicating regions of high electron density and blue indicating regions of low electron density.



Page 30 of 39

Figure 8. The proposed mechanism of AcPHF6 hexapeptide promoted Aβ fibrillogenesis.


Page 31 of 39 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


Figure 1. Sequence of AcPHF6 hexapeptide and Aβ40/42 highlighting the amyloidogenic (KLVFFA) core. 183x162mm (300 x 300 DPI)

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


Page 32 of 39

Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22



ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 ( C) 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

( D)


Page 34 of 39

Page 35 of 39 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


Figure 5. (A) Effect of AcPHF6 (1, 5, 10 and 25 µM) on cell death induced by Aβ40 (5 µM) in HT22 cells after 24 h incubation at 37 ºC in the MTT assay; (B) Effect of AcPHF6 (1, 5, 10 and 25 µM) on cell death induced by Aβ42 (5 µM) in HT22 cells after 24 h incubation at 37 ºC in the MTT assay. The results are provided as average ± SD of two independent experiments (n = 3). *p< 0.01 compared to Aβ40 alone treatment group and *p< 0.05 compared to Aβ42 alone treatment group (one-way ANOVA followed by Bonferroni post hoc test). 264x352mm (72 x 72 DPI)


( A) 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


Al a

( B)


Page 36 of 39

Page 37 of 39 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


Figure 7. Electrostatic potential surface of AcPHF6 (A) and PHF6 (B) respectively. Regions of low and high electron densities are color coded with red indicating regions of high electron density and blue indicating regions of low electron density. 502x166mm (96 x 96 DPI)



Figure 8. The proposed mechanism of AcPHF6 hexapeptide promoted Aβ fibrillogenesis. 184x101mm (96 x 96 DPI)


Page 38 of 39

Page 39 of 39 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



(AÎ²) Fibrillogenesis - ACS Publications - American Chemical Society

Recommend Documents