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Synthesis and Evaluation of Amyloid Beta-derived and Amyloid Beta-independent Enhancers of the Peroxidase-like Activity of Heme Amelie Wissbrock, Toni Kuehl, Katja Silbermann, Albert J. Becker, Oliver Ohlenschläger, and Diana Imhof J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01432 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on December 11, 2016

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Journal of Medicinal Chemistry

Synthesis and Evaluation of Amyloid Beta-derived and Amyloid Beta-independent Enhancers of the Peroxidase-like Activity of Heme

Amelie Wißbrock†‡, Toni Kühl†‡, Katja Silbermann†, Albert J. Becker#, Oliver Ohlenschläger0* and Diana Imhof†*

† Pharmaceutical Chemistry I, Pharmaceutical Institute, University of Bonn, Bruehler Str. 7, 53119 Bonn, Germany #

Institute of Neuropathology, University Hospital Bonn, Sigmund Freud Str. 25, 53105 Bonn

0 Leibniz Institute on Aging – Fritz Lipmann Institute, Beutenbergstr. 11, 07745 Jena, Germany ‡ equal contribution

KEYWORDS Alzheimer’s disease • Amyloid β • Regulatory heme • Peroxidase activity • LDL

ACS Paragon Plus Environment

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ABSTRACT

Labile heme has been suggested to have an impact in several severe diseases. In the context of Alzheimer’s disease (AD), however, decreased levels of free heme have been reported. Therefore, we were looking for an assay system that can be used for heme concentration determination. From a biochemical point of view the peroxidase activity of the Abeta-heme complex seemed quite attractive to pursue this goal. As a consequence a peptide which is able to increase the read-out even in the case of a low heme concentration is favorableThe examination of Aβ- and non-Aβ-derived peptides in complex with heme revealed that the peroxidase-like activity significantly depends on the peptide sequence and length. A 23mer His-based peptide derived from human fatty acyl-CoA reductase 1 in complex with heme exhibited a significantly higher peroxidase activity than Aβ(40)-heme. Structural modelling of both complexes demonstrated that heme binding via a histidine can be supported by hydrogen bond interactions of a basic residue nearby the propionate carboxyl function of protoporphyrin IX. Furthermore, the interplay of Aβ-heme and the lipoprotein LDL as a potential physiological effector of Aβ was examined.


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INTRODUCTION Alzheimer’s disease (AD) is one of the most serious neurodegenerative diseases. Although the disease has already been described in 1906, there is still a lack of information to provide adequate and sufficient medical treatment.1 One of the characteristic biomarkers in the pathology of AD is the plaque formation of amyloid β (Aβ) peptides that is mainly related to the longer peptide forms Αβ(1-40) and Aβ(1-42).2 While initially Aβ aggregation was thought to be the crucial starting point for AD development (amyloid cascade hypothesis), it is now discussed whether it is the cause or a consequence within the disease progression.3,4 A strong connection between iron-related metabolic dysfunctions, usually observed as typical cytopathological symptoms of AD, and Aβ has recently been demonstrated by Atamna et al. with the direct association of heme (iron(II/III) protoporphyrin IX) to Aβ.5 Interestingly, rodents, which do not develop AD, possess the Aβ-peptide (roAβ) and also form Aβ aggregates. However, roAβ differs from the human version in three amino acids (Arg5, Tyr10, His13). These three residues were shown to be crucial for high affinity heme binding and the subsequent development of an enhanced peroxidase-like activity indicating a potential key role of heme binding to Aß within AD.6 The interaction of heme with Aβ might be part of the heme’s nature as a regulatory molecule. Transient heme-protein interactions have gained increased attention recently. This temporary effect has been associated with protein regulation and is meanwhile accepted to be mediated by so-called regulatory heme (free or labile heme) binding to heme-regulatory motifs (HRM) in proteins.7 Up to now several heme-regulated proteins have been described.8 These proteins are part of different physiological processes, for instance transcription, translation, protein degradation and cell cycle regulation, as can be exemplified with p53,9 Per2,10 ALAS1 and ALAS2.11–13 The participation of heme in pathophysiological processes is indicated in


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diseases such as porphyrias,14 anemias,15 cerebral vasospasm, but also in cancer16 and in the neurodegenerative Alzheimer’s and Parkinson’s disease.7,8,17,18 Heme metabolism in AD is altered,5 and the direct interaction of heme and Aβ seems responsible for the functional role of heme in AD patients. This is of particular importance in AD due to the excess of Aβ peptides which are able to bind two heme molecules each.6,19 Aβ shows an enhancement of the catalytic activity of free heme upon Aβ-heme complex formation that is thought to lead to oxidative damage.19 The complex is able to oxidize neurotransmitters as e.g. levodopa and serotonin in vitro, the latter is being critically discussed within AD because of its role in cognition.19–22 Furthermore, heme is suggested to be an endogenous inhibitor of Aβ(40/42) aggregation.5,23 Besides neurotransmitter it has recently been shown that Aβ-heme is able to oxidize ferrocytochrome c (Cyt c(II)), that in general is done by the cytochrome c peroxidase (CCP). Meaning that Aβ-heme does not only possess a peroxidase-like activity but a CPP-like one, too.24 Another issue is the so-called ‘functional heme deficiency’ described by Atamna et al. that results from Aβ-heme association and, in turn, from a decreased cellular concentration of unbound heme. Consequently, heme metabolism will be disturbed and oxidative stress and dysfunctions of mitochondrial complex IV might appear.5,19,20 In addition, it has been shown that heme itself is able to oxidize lipoproteins.25 Whether or not heme takes part in the enhanced oxidation of lipoproteins observed in AD is not yet clear.26 It has been debated that oxidized lipoproteins might be suitable as potential biomarkers for dementia. However, a detailed insight into the biological relevant interactions and the molecular processes of the lipoproteins with other molecules is needed to evaluate the suitability as a diagnostic marker. As a consequence of Aβ-heme association, several studies have focused on the cause and the molecular mechanism of its catalytic activity. Currently, it is proposed that heme binds Aβ(1-40)


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and Aβ(1-42) (herein referred to as Aβ(40/42) if both forms are meant), respectively, via amino acids His13 or His14.27–31 Gosh et al. recently suggested the dominance of a high-spin mono Hisbound complex under physiological conditions.31 However, they also found a concentrationdependent equilibrium between the high-spin and the low-spin complex, with the latter representing a bis-His-bound species in excess of Aβ.31 The sequence of Aβ(40/42) highlighting all possible heme-coordinating amino acids is depicted in Figure 1. As a consequence of the intense studies on the features of the Aβ-heme complex and the transient interaction of regulatory heme with peptides and proteins, the question arises whether or not such a peroxidaselike activity is found for other protein/peptide-heme complexes as well. In recent years, numerous novel heme-regulated proteins have been identified,7,8 which are involved in highly diverse and important cellular processes. Most importantly, ROS formation by an individual complex might play a critical role in the development of pathological conditions such as inflammatory processes. It is for this particular reason that it is necessary to focus more on a possible catalytic activity of other heme-protein complexes. In this study, we demonstrate that heme in complex with different peptides apart from Aβ possesses an increased peroxidase activity. A set of diverse peptides different in kind and number of heme-coordinating amino acids and, in turn, different binding affinities has been evaluated with regard to their potential to enhance the catalytic activity of heme using UV/Vis spectroscopy.32–35 The activity of these complexes to degrade hydrogen peroxide and oxidize the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was assessed in comparison to the complexes of heme with Aβ-derived peptides as well as full-length Aβ(1-40). Our results indicate that heme complexes with peptide or protein sequences other than Aβ may possess a seriously high, potentially pathophysiological critical peroxidase activity, too. The importance of an acidic


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environment was observed to be crucial within the experiments implying that the physiological relevance of the peroxidase activity might be restricted to regions within the organism that are indicative of a low pH. We found a 23mer peptide that exhibits an even higher peroxidase activity than Aβ in complex with heme and perspectively can be used as a diagnostic tool to determine concentrations of free heme in patient samples. Finally, we found that the presence of the lipoprotein LDL did not influence the peroxidase activity of the Aβ-heme complex. However, if the complex is not formed already, LDL appears to be able to prevent Aβ-heme complex formation by interacting with the heme molecule, thereby reducing its potential to interact with Aβ.


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RESULTS Aβ β-derived peptides confirm heme binding via His13/14 The capacity of heme to bind to Aβ(1-40) and to a set of six Aβ-derived peptides was examined using UV/Vis spectroscopy (Figure 1). The pathological relevant forms Aβ(1-40) and Aβ(1-42) only differ in the two C-terminal amino acids alanine and isoleucine that both do not serve as heme axial ligands. An interaction with the heme molecule is not probable here due to the large distance to the site-H. Consequently it may be hypothesized that the heme-binding mechanism of these two peptides is rather similar. For our studies, Aβ(1-40) was chosen due to its lower aggregation potential as found in comparative studies with both peptides. For Aβ(1-40) a heme binding affinity of 0.10 ± 0.07 µM (λ~414 nm) was determined by UV/vis spectroscopy confirming the results of Atamna et al. who discuss a KD of 0.14 ± 0.06 µM for Aβ(1-42) (Figure 1).6 Peptides 1-4 are sequence stretches of nine amino acids,33 each containing one of the four possible heme axial ligands (His or Tyr) in the central position. Peptides 5 and 6 consist of 10 and 18 amino acids since further residues (e.g. Arg5) were proposed to have an impact on binding behavior and/or enzymatic activity of the formed complex (Supporting information, Figure S1, Table S1).25,28–30,36 For peptides 1 and 2 containing His6 and Tyr10, no heme binding was observed implying that neither His6 nor Tyr10 function as the axial ligand in the complex. In contrast, peptides 3 and 4, which include His13 and His14, showed a bathochromic shift of the Soret band to λ ~416 nm and λ ~421 nm, respectively, upon heme addition. The determination of binding constants was not possible due to the low absorbance of these peptide-heme complexes. In case of the longer peptides 5 and 6, however, heme binding was quantifiable. Peptide 5 displayed a weak interaction with a KD value of 80.70 ± 4.00 µM (λ~421 nm), while peptide 6 exhibited a significant higher affinity of 0.59 ± 0.05 µM


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(λ ~415 nm) (Figure 1). If compared directly, then either His13 or His14 appear to be the hemecoordinating amino acid in peptides 3-6, yet sequence length and composition seem to have a strong impact on heme-binding efficiency. These results are in good agreement with previous studies on Aβ-heme interaction27,29,30 as well as our own findings for different classes of hemebinding peptides.32–35 Peptides 2 and 5 are similar except for the additional N-terminal arginine in decapeptide 5. Because no significant effect was observed upon heme addition to peptide 2, the basic residue arginine obviously had a positive effect on heme binding in the situation occurring. The shortened sequence stretches 1-6 bind none or only one molecule of heme each, regarding the best fit possible for KD determination. In contrast, one peptide Aβ(1-40) interacts with two to three molecules of heme. A hydrophobic interaction of Aβ’s site-L with a second heme molecule has been foundd before.6 Our studies also show a strong indication that at least two molecules of heme interact with one Aβ molecule. However, further studies as e.g. structural analysis by NMR are needed to obtain detailed information on Aβ-heme binding behavior.

Heme-induced peroxidase-like activity is strongly pH-dependent Free heme itself shows a marginal enzymatic activity against peroxide substrates.5 The reaction catalyzed by heme is the Fenton reaction which results in the formation of hydroxyl radicals and subsequently the oxidation of a substrate,38 such as levodopa and serotonin,19,21 TMB19 or 2,2′azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid)diammonium salt (ABTS).39 In our set-up TMB served as the substrate. Upon oxidation, TMB forms a blue-colored charge-transfer complex that can be detected at 652 nm.40 Because physiological heme concentrations are described to be in the range of 0.1-1 µM,7,25,41 a heme concentration of 1 µM was chosen for all


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measurements. To analyze the impact of the pH and different substrate concentrations on the reaction, three different set-ups were applied: i.) a commercial TMB substrate kit (TMB: 1.66 mM, H2O2: 6.5 mM, pH 5.0, Thermo Scientific) based on a citrate buffer (approach I), ii.) a selfmade citrate buffer-based system (TMB: 1.66 mM, H2O2:163.2 mM, pH 5.0) (approach II), and iii.) a phosphate buffer-based system (TMB: 20.8 mM, H2O2: 163.2 mM, physiological pH 7.4) (approach III). When using approach I with a self-established protocol and set-up (approach I*), i.e. the same substrate concentrations as given in the manufactured assay kit (approach I) with freshly prepared solutions, the observed activity of heme alone was reduced to approximately ~60 % compared to the result obtained with approach I (Supporting information, Figure S2). Mass spectrometry analysis of the H2O2-solution of the commercial assay kit (approach I) revealed additional components, whose nature could not be unequivocally identified. However, while performing approach II using citrate buffer at pH 5.0, it was realized that H2O2 is not stable for the reaction progress for more than 10 minutes, which further supports the presence of e.g. stabilizers for H2O2 in the kit system. In our opinion, this observation has to be taken into account when using the available assay kit, in particular if considering possible interactions of unknown ingredients of test kits with heme and/or heme-peptide/protein-complexes leading to false positive and/or negative results. In case of approach II the H2O2 concentration was app. 25-fold increased from 6.5 mM to 163.2 mM which, in general, led to a higher product formation as seen from increased absorbance after 400 seconds from ~0.06 (approach I) to ~0.15 (approach II). The use of the same substrate concentrations in a phosphate buffer-based system was not possible due to lack of detectable substrate transformation at neutral pH. This was tested with the aim to approximate physiological conditions as far as possible enabling perspective measurements of free heme


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concentrations in actual patient samples. Due to the low concentrations of free heme in the organism (nM range in healthy individuals) an enhancement of the heme signal is necessary to obtain detectable data. The latter might be realized by the utilization of a peptide-induced peroxidase activity. An increase of the applied TMB concentration to 20.8 mM TMB and a concentration of 163.2 mM H2O2 (approach III) leads to low product formation that is, nevertheless, detectable and gives an impression of what might happen in the organism at neutral pH. However, our following peptide studies indicate that this approach can only be used for specific peptides as described in the following section. A clear pH-dependence of the reaction occurring in approaches I and II compared to III was observed. It is well known that an acidic environment is optimal for the Fenton reaction to occur.42,43 Aβ(1-42) peptides have been found in low-pH organelles such as lysosomes and endosomes.44,45 An acidic environment is also suggested to be a critical factor for Aβ-fibril formation suggesting that a low pH may play a role in AD development. Therefore, the peroxidase activity of the Aβ-heme complex that is significantly higher at low pH compared to neutral milieu might be even more critical in cell compartments with a low or decreased pH in AD.46

Analysis of the Aβ β-heme activity through studying the role of individual amino acids The sequence of Aβ(40/42) possesses several features similar to well-known heme peroxidases. For example, heme binding is mediated by a histidine, while an additional distal histidine and also a distal arginine are present.47,48 To obtain more information on the possible impact of single residues within the 40mer Aβ-peptide the catalytic activity of heme-incubated Aβ-derived peptides 1-6 was studied and compared to full-length Aβ(1-40). The aggregation state of Aβ(40) within the respective set-ups was tested using a Thioflavin T fluorescence-based


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assay system (Supporting information, Figure S3). Analysis revealed that no Aβ(40) aggregates are to be expected in the described experiments. However, due to the fact that aggregation of short oligomers cannot be detected with the ThT assay, we cannot exlude the formation of low molecular weight species of Aβ(40) aggregates with certainty for the experimental set-up used herein. Peptides (7-19, Table 1) were examined as aforementioned using the approaches I-III. The assays were performed with peptide concentrations ranging from 0.1-1 µM using a constant heme concentration of 1 µM. In general, the activity increased with increasing peptide concentrations. The following comparison of the peptide-heme complexes is based on a 1:1 peptide-heme ratio. Required controls such as buffer-peptide and TMB-peptide solutions confirmed that the reaction only proceeds when heme or heme-peptide complex is present. None of the chosen peptides showed an activity without heme. All data obtained for the following evaluation of peptide-heme complexes were normalized against the activity observed for unbound heme (heme alone). Therefore, the values given can be regarded as a direct comparison of the activities of peptide-bound and unbound heme. Aβ(1-40) itself showed no peroxidase activity in approaches I-III which is in agreement with earlier results.19 An Aβ(1-40) concentration of 0.5 µM is required to observe a significant effect upon heme addition (1 µM) in the performed experiments (Supporting information, Figure S3). In this case, the activity in systems I or II is ~500 %, while the activity of the complex at physiological pH is only ~170 % (Figure 2A). This discrepancy between approaches I and II compared to III again demonstrates the obvious pH dependency. Peptides 1-6 were employed to obtain further information on the role of Aβ sequences specificities for the peroxidase-like activity upon complex formation with heme (Figure 2A). In general, a considerable reduction of


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the activity was observed for peptides 1-6 compared to Aβ(1-40). In the acidic environment of assays I and II an increase of activity to 110-180 % compared to unbound heme was monitored for the different peptide-heme complexes with two exceptions, i.e. for peptide 1 no significant enhancement was observed, while for peptide 3 an increase to ~265 % (approach II) was found. These results and the data described below led us to conclude that the sequence length of Aβderived nonapeptides 1-4 and the decapeptide 5 is not sufficient to significantly increase the heme’s peroxidase activity upon complex formation. The 18mer peptide 6 (Aβ(1-18)) showed an activity of ~160-200 % in I and II. However, the activity of the complex is much lower compared to the parent Aβ(1-40)-heme complex. This is in agreement with the previous finding that the Aβ(1-16)-heme complex only possesses a minor catalytic activity.49 Due to the fact that binding affinity of peptides 5 and 6 as well as peroxidase activity of these sequences is significantly reduced in all studies performed, we believe that a different situation occurs for the shorter, conformationally more flexible sequences compared to the full-length, structurally more restricted Aβ(1-40). In addition, peptides 1-5 bind heme with a rather low affinity that might also explain the effects observed (see above). These results provide a first insight into the sequence requirements (e.g. length). In order to find a suitable candidate that prospectively might be used in a diagnostic set up, the catalytic activity of various peptides in complex with heme was evaluated as described in the following section.

Non-Aβ β -peptides possess a peroxidase activity in complex with heme Further peptides (7-19) originating from earlier investigations on heme binding to motifs from heme-regulated proteins (Table 1, Supporting information, Table S2)32–35 were included in this study and tested in the same way as described above. At first, peptides 7-10 were examined for


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the following reasons. Nonapeptide 7 contains only alanine residues and was used as a negative control. In addition, nonapeptides containing a central coordinating amino acid, i.e. Tyr (8), Cys (9) or His (10), surrounded by four Ala on either side were studied in order to find out about their effect on the activity, since none of these peptides bound heme.33–35 None of the peptides (7-10) showed a significant increase of the heme's peroxidase activity as compared to Aβ(1-40) (Supporting information, Figure S4), an observation that correlates with the lack of hemebinding capacity.34,35 All further measurements of peptides 11-19 were performed under the same conditions as for the Aβ-derived peptides and the controls to allow for a direct comparison, however, due to relevance the results obtained with approach II are discussed in more detail (Figure 2B) compared to those from systems I and III (Supporting information, Supporting Figure S5) The heme-binding behavior of peptides 11-19 (good heme binders) has been determined in earlier studies (Table 1).32–35 As observed for the Aβ-derived peptides 1-6, no general correlation between heme-binding affinity and peroxidase activity was found for the non-Aβ peptides. Peptides 11, 12 and 17 are Cys-based heme-binding peptides.33,34 The nonapeptides 11 and 12 showed a similar effect, i.e. a reduced activity (~80-90 %) (Figure 2B, Supporting information, Figure S5). A decrease of catalytic activity to less than 100 % can be explained by the formation of a heme-peptide complex having no effect on the peroxidase activity. The different activities in system I compared to II might again be generated by additional compounds in the commercial test system I as aforementioned (Figure 2B, Supporting information, Figure S5). The Cys-based 23mer peptide 17, derived from human heme-binding dipeptidyl peptidase 8,33 showed an increased peroxidase activity ~200 % in all approaches indicating an enhancing effect of the peptide sequence (Figure 2B, Supporting information, Figure S5). Compared to the following


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studies, however, the selected Cys-based peptides revealed only a small or no potential to increase the heme’s peroxidase activity. Heme-binding nonapeptides 13 and 14 as well as 23mer peptide 18 were chosen as His-based heme-binding peptide candidates.32,35 Heme-complexes with peptides 13 and 14 exhibited a slightly enhanced activity (13: ~195 %, 14: ~150 %) (Figure 2B). For both, 13 (KD 0.99 +/- 0.21 µM) and 14 (KD 0.43 +/- 0.23 µM), a strong heme binding was observed in previous UV/Vis studies.35 Peptide 14 displayed a second absorption maximum that might imply a hexacoordinated complex or a mixed state of penta- and hexacoordination.35 This effect supposedly leads to a reduction of the complex’s peroxidase activity due to steric hindrance for the substrate molecule. Surprisingly, peptide 18, a 23mer sequence stretch deduced from the human fatty acyl-CoA reductase 1,32 showed a higher peroxidase activity than Aβ(1-40) in complex with heme, especially in assay system II (>1000 %) (Figure 2B, Supporting information, Figure S5). Within our studies, this peptide was by far the most active candidate. For this reason, the sequence was examined in more detail, i.e. by mutational studies as discussed in the following section. Further experiments were also performed with Tyr-based peptides 15, 16 and 19. The nonapeptides 15 and 16 act in a similar way as described for Cys- and His-based nonapeptides 11-14. They displayed only a minor increase of the peroxidase activity to 140-195 %, except of peptide 16 which revealed an activity of ~325 % (Figure 2B, Supporting information, Figure S5). Within all nonapeptides examined this was the highest effect observed for a heme-peptide complex. If analyzing the sequence of peptide 16 one might hypothesize that the distal histidine is favorable for an increased substrate conversion upon complex formation with heme. Further studies are needed to draw a final conclusion here. Finally, 23mer peptide 19, a sequence stretch


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of the human DNA-directed RNA polymerase II subunit, showed a peroxidase activity of ~200 % (Figure 2B). Thus, the activity of the peptide-heme complex behaves in a similar way as described for the nonapeptides (e.g. 13-15).

Distal histidines affect heme-binding affinity but not necessarily catalytic activity We found peptide 18 to be able to enhance the heme’s peroxidase activity tenfold in the acidic assay system II, while the activity of the Aβ(1-40)-heme complex was increased only six-fold compared to free heme. Considering sequence length 23mer peptide 18 is missing 17 amino acids compared to Aβ(1-40). According to our results, a peptide length larger than 9 or 10 amino acids is highly favorable for peroxidase activity. On the other hand, sequence length above a critical limit might not be of great importance as soon as a distinct number of specific residues is present, i.e. ca. 10 < Xaa < 25 (Xaa, amino acid). It is probable that specific residues have a positive impact on the formation of a catalytically active complex. This might be further intensified by structural elements provoked by these amino acids. Heme binding to peptide 18 is expected to be mediated via the central histidine. The proximal tyrosine residues (and possibly the tryptophan residue) can support the enzyme-like activity by providing an electron as described earlier for the Aβ(40/42)-heme complex.27,47 As it is well known that the O-O bond cleavage of H2O2 in heme peroxidases is often mediated by a distal histidine in the protein’s sequence, we were interested in the role of the histidine residues within peptide 18, too.50 To obtain more information about the role of these histidines, four different Ala mutants of peptide 18 (peptides 20-23) were generated. In 20 (His8Ala), 21 (His19Ala) and 23 (His12Ala) one individual histidine was replaced by an alanine, while in


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peptide 22 both distal histidines were substituted (His8Ala and His19Ala) (Supporting information, Table S3). UV/Vis studies were performed in the same way as for the Aβ-derived peptides. Binding behavior dramatically changed with the loss of the central histidine in peptide 23 (Figure 3, Supporting information, Figure S6). While peptide 18 displayed a binding affinity to heme of 5.28 +/- 0.95 µM (λ ~416 nm), peptide 23 showed an affinity of 19.84 +/- 4.11 µM (λ ~416 nm). Therefore, the central histidine is most probably the heme coordinating amino acid. Data analysis further revealed that possibly two peptides bind to one heme molecule according to the best fit available (Figure3). Interestingly, the loss of one or both distal histidine in each peptide 20 (KD 3.00 +/- 1.19 µM, λ ~416 nm), peptide 21 (KD 1.89 +/- 0.72 µM, λ ~416 nm) and peptide 22 (KD 0.88 +/- 0.94 µM, λ ~414 nm) compared to 18 led to a minor affinity increase. This suggests that all three histidines residues are able to interact with heme. When all histidines are present, a competitive situation between the binding residues is conceivable and, in turn, might result in a slightly reduced affinity for the single interactions. With this knowledge all peptides were tested for their peroxidase activity in complex with heme using the self-established protocol of assay system II. As mentioned above peptide 18 showed an activity increase to ~1000 % compared to unbound heme. Peptides 20-22 exhibited a weak activity reduction to ~700-800 % compared to 18 (Supporting information, Figure S7). These results also suggest that the individual histidine residues do not possess an exclusive and specific role for the enzymatic activity. Indeed, a different situation occurs for peptide 23 that is missing the central histidine and is characterized by the distinct loss of binding affinity. The significant reduction of TMB conversion by the peptide 23-heme complex might be a consequence of the reduced complex formation with heme.


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Indeed, further studies are required to investigate heme binding to peptide 18 on the protein level. Peptide 18 is a sequence stretch derived from the fatty acyl-CoA reductase 1 that is located in the peroxisomal membrane.32 The suggested heme-binding sequence reaches into the peroxisomal lumen. Several biological facts might be taken into account when thinking about a heme interaction and a possible peroxidase-like activity on the protein level. Heme is transported into the peroxisome.51 The general surrounding with an acidic pH in the peroxisome and the availability of a large amount of H2O2 do further support the possibility of a heme-protein complex that exhibits a physiologically critical peroxidase-like activity. However, the molecule’s nature as a transmembrane protein as well as the presence of several H2O2-transforming enzymes may hamper simple investigation of the actual situation in the cell. Structure modeling of heme binding to Aβ β(1-40) and peptide 18 Without any doubts, structural elements of proteins and also peptides play a crucial role for all kind of interactions with other molecules. Our data suggest that a distinct peptide length between ca. 10 and 25 amino acids is at least required to form a heme-peptide complex that might possess an enhanced peroxidase activity. We assume that this is a result of specific structural requirements of the peptide that are needed and might not be formed by the shorter nonapeptides. Previous studies demonstrated that Aβ(40/42) in a membrane-mimicking environment is characterized by an α-helical structure between positions 8 or 15 to 24/25 and 28-36/3852–54 and can undergo reversible β-to-α conformational transitions depending on solvation conditions.55 The proposed heme binding site resides near the N-terminal region of the helix structure. This prompted us to model the binding situation for the observed interactions of Aβ(1-40) based on the PDB coordinates 1IYT deposited by Crescenzi et al..56 Assuming the integrity of the αhelical conformation, Figure 4A shows that binding of heme via His13 can be supported by a


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hydrogen bond between the Lys16 side chain ε-amino proton and the propionate carboxyl group. In contrast, a stronger binding network can be formulated for heme binding at His13 when including the side chain of Arg5 (Figure 4B). Here, the guanidinium protons can establish a hydrogen bond network including the carboxyl group of one of the heme propionates as well as the oxygen of a water molecule which might also serve as sixth ligand of the hexacoordinated heme as proposed earlier.30 Our study comprised three 23mer peptides (17-19), among which peptide 18 appeared to be the most promising candidate. Unfortunately, as for Aβ, the heme-peptide 18 complex excluded a structure determination due to solubility issues. Therefore, in order to obtain a structural insight of peptide 18 we applied computational modeling, too. As no template structure was available, the program PEP-FOLD, an approach for de novo prediction of peptide structures from amino acid sequences, was employed.57,58 In all derived model structures representing the best clusters of predicted folds (Supporting Information, Figure S8, Table S3) residue His12 was located in an α-helical-like segment, while for other parts of the peptide coil or sheet predictions were also observed. Figure 5 displays two possible heme-binding modes based on the predicted helical peptide conformation. Both scenarios allow to bind heme via the central His12 and to further stabilize the complex by interactions between the propionates to the side chains of either His8 or His19. The water molecule as ligand for the iron hexacoordination in its free accessible distal position is susceptible for a transient replacement by a second peptide unit.

Impact of lipoprotein LDL on the catalytic activity of the Aβ β-heme complex


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The occurrence of high levels of lipoproteins in the brain of AD patients, which can easily be oxidized, has attracted increasing interest regarding lipid peroxidation in context with AD.59 Nowadays, it is not clear whether lipid peroxidation is a specific process or an epiphenomenon in AD. A direct interaction of the pathological critical peptide Aβ and different lipoproteins, e.g. the high density lipoprotein HDL and the low density lipoprotein LDL, has been shown earlier.60–62 In addition, in 1998 Roher et al. found that increased levels of LDL appear to lead to higher levels of Aβ(1-42) independent of the pathophysiological critical apoE genotype.63 It was also proposed that Aβ is secreted as part of lipoproteins.64 However, the examination of human plasma samples revealed that the preponderant part of physiological Aβ seems to preferably bind to albumin and only a minor part (~5 %) binds to lipoproteins like HDL and LDL.62 Besides the link between Aβ and LDL, heme is also able to interact with LDL and, as a consequence, leads to the oxidation of the lipoprotein.25,65 Oxidized LDL (ox-LDL) is cytotoxic, and it is suggested to be another potentially critical feature in the process of AD.26,66,67 Heme binds LDL in a nanomolar range and is considered as more efficient in oxidizing the protein than Fe3+ ions.65 LDL as well as HDL appear to interact with heme faster than heme-scavenging protein hemopexin and albumin.68 In this context it is suggested that the lipoproteins function as the ‘initial heme scavenger’ and that the heme moiety can be transferred from LDL/HDL to e.g. hemopexin later on. If no transfer is possible the oxidation of lipoproteins takes place.68 Ox-LDL was suggested as a promising biomarker for traumas related to oxidative damage.69 In addition, it was also proposed that ox-LDL might be used as a peripheral marker within dementia, since several studies revealed significant increased levels of ox-LDL in this disease.69 To find out whether the presence of LDL has an impact on Aβ-heme complex formation, complex stability and consequently the observed peroxidase-like activity, additional studies in


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consideration of LDL were performed herein. LDL was added to Aβ, heme and the Aβ-heme complex before and after complex formation. The peroxidase assay was performed as described above using approach II. 1 µM of Aβ, heme or Aβ-heme complex was added to the substrate mixture of TMB/H2O2. A ratio of 1:100 (LDL: Aβ, heme or Aβ-heme), i.e. 0.01 µM LDL was chosen. The reason for this is that heme was described to bind LDL in highly different ratios65 that is easy conceivable due to the enormous discrepancy in the size of the single molecules (Figure 6). As expected neither LDL alone nor the combination of Aβ and LDL (negative controls) possessed a significant peroxidase activity towards TMB/H2O2. When LDL was added to heme and to the preformed Aβ-heme complex immediately before the performed assay, i.e. without incubation time, the peroxidase activity was similar as found in the respective mixtures without LDL (Figure 6, Supporting information, Figure S9). UV-spectra revealed that the mixture of heme and LDL showed a bathochromic Soret shift to ~429 nm (∆λ = nm) that is typical for the interaction of porphyrins and lipoproteins (Supporting information, Figure S9).65 Surprisingly, heme binding to LDL did not seem to inhibit the catalytic activity of heme towards TMB/H2O2 according to our data. The Aβ-heme complex revealed a maxima shift to ~420 nm in phosphate buffer independent of the addition of LDL (Supporting information, Figure S9). Our results indicate that the complex if formed prior to LDL exposure is stable for at least 15 minutes in the presence of LDL (Figure 6, Supporting information, Figure S9). Another possibility is an interaction of Aβ-heme with LDL that leads to a complex formed of the three compounds that retains peroxidase activity. However, UV/Vis spectra did not show a maxima shift upon LDL addition to Aβ-heme over 30 minutes. This observation might be interpreted as an indicator for the stability of the preformed Aβ-heme complex in the presence of LDL. Within our studies it


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was not possible to clarify between the two options mentioned. When all three compounds were added at the same time without prior incubation the substrate conversion rate was significantly reduced compared to Aβ-heme. A similar result was obtained when the mixture was incubated before it was added to the TMB/H2O2 substrate mixture (Figure 6). These results suggest that heme binds to LDL and Aβ leading to LDL-heme and Aβ-heme complex formation. However, eventually this results in a reduced concentration of the enzymatically active Aβ-heme complex. To specify the potential interactions of the individual compounds, LDL was pre-incubated with heme or Aβ before the missing third compound was added, and the catalytic activity was measured. When LDL was pre-incubated with Aβ the peroxidase activity observed was similar to the situation that occurred when all three molecules were added at the same time (Figure 6). If Aβ interacts with LDL, complex formation with heme did not seem to be disturbed and the heme molecule appeared to interact with LDL and Aβ. Again, a reduced concentration of the catalytic active Aβ-heme complex was assumed to be the consequence. Interestingly, a peroxidase activity that equals the autocatalytic activity of heme alone was observed when Aβ was added to preincubated heme and LDL. This observation points to a binding of heme to LDL and, as a consequence, Aβ was not able to form an enzymatically active complex with heme any longer.

DISCUSSION The present study demonstrates the importance of an acidic environment for the hememediated Fenton reaction to occur considering the detection of the peroxidase-like activity of Aβ-heme. The great differences between the substrate oxidation at pH 5 compared to physiological pH 7.4 have to be taken into account when interpreting a potential physiological context of catalytically active heme-peptide/protein complexes. In addition, the pH and a


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possible impact on e.g. proteins of patient samples have to be considered when planning a perspective preparation of the samples. Our data revealed that heme binding to peptides/proteins is necessary to obtain an enhanced peroxidase-like activity but the intensity of the effect did not directly correlate with the heme-binding affinity. The significantly enhanced peroxidase activity was exclusive to distinct peptide-heme complexes that seem to fulfill specific sequence requirements. The peroxidase-like activity of all Aβ-derived peptides was much lower compared to full-length Aβ(1-40). For Aβ(1-40) the heme-iron is coordinated via a histidine while a tyrosine and also an arginine are suggested to be responsible for the activity observed. Even though the shortened Aβ(1-18)sequence did not show a dramatic loss of heme-binding affinity, the catalytic activity of Aβ(118) (peptide 6), containing all amino acids of proposed relevance, was not one fourth as good as the activity of Aβ(1-40). These findings taken together imply the existence of specific structural elements within full-length Aβ(1-40), most probable the described helical structure, that are responsible for the formation of a catalytically active complex. The investigation of a series of non-Aβ-derived heme-binding peptides revealed a 23mer peptide (18), derived from human fatty acyl-CoA reductase 1, which in complex with heme exhibited an even higher peroxidase-like activity compared to the Aβ-heme complex. Mutational studies showed that heme binding of this peptide is mediated via the central histidine residue. A direct sequence comparison with Aβ(1-40) revealed the presence of one(Aβ) and two (18) distal histidines and also the occurrence of a tyrosine near the coordinating amino acid in both peptides (Supporting information, Figure S9alt). The peroxidase activity of Aβ-heme was reported to be significantly supported by the presence of an arginine.27 Peptide 18 does not contain an arginine, however, the question arises whether another basic residue, namely the present lysine, may fulfil


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the role of the above mentioned arginine. Nevertheless, we focused on the potential role of the distal histidines, a sequence feature that is well known from heme peroxidases. Our studies demonstrate that the distal histidines of peptide 18 have only a minor impact on the activity of the corresponding complexes formed. If the catalytic activity cannot be attributed to these residues, most probably one or more tyrosine residues are the crucial amino acids besides the coordinating histidine for the peroxidase activity within peptide 18. Peptide 18 is of great interest regarding its potential as a possible enhancer of the heme “signal” that might be detected in the form of the complexes’ catalytic activity. On the other hand, the investigation of a set of nonapeptides revealed that probably a peptide length of ca. 10 < Xaa < 25 (Xaa, amino acid) is required to observe any activity in addition to a distinct sequence composition. Peptides 13 and 14 are histidine-based nonapeptides, and in case of peptide 14 additionally a tyrosine and an arginine are present within the sequence. However, the peptide did not show a great enhancement of the heme’s peroxidase activity. Taken these observations together, this again points to the formation of specific structural elements that are possibly related to effects derived from conformational aspects, i.e. secondary structure elements might promote orientation and placement of the substrates. The structural models of Aβ and peptide 18 allow a direct comparison of the two heme-bound peptides. In both molecules the binding of the heme moiety via a histidine can be supported by hydrogen bond interactions of a basic residue nearby to the propionate carboxyl function of the protoporphyrin IX. In proximity to the stabilized complex in peptide 18 as well as in Aβ(1-40) the helix accommodates a tyrosine residue (cf. Figure 4C,D and Figure 5A,B). A rotation of the χ1 torsion angle of these tyrosines would allow to bring the hydroxyl function in vicinity of the heme ring and to functionally contribute in the catalytic process. Our data form the basis that enables the evaluation and possibly the synthesis of an ideal


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compound for the detection of regulatory (free) heme via a catalytically active complex with heme. Further studies focused on the potential impact of the lipoprotein LDL on the formation and the stability of the Aβ-heme complex. Our data suggest that Aβ-heme does not dissociate if formed prior to the occurrence of LDL (Figure 6). Heme binding to LDL has been described several times.25,65,70 A comparative study determined binding constants of 49 +/- 5 nmol/l for the heme/LDL-binding, while heme/HDL-binding was in the range of 291 +/- 26 nmol/l.65 It has been shown further that heme from distinct hemoproteins (e.g. hemoglobin) is transferred to LDL, which in turn is oxidized by the heme molecule.70 In contrast, hemopexin that binds heme with an exceptional high affinity (98% HPLCpurity.

Amino acid analysis For determination of peptide content, the peptides were hydrolyzed in 6 N HCl at 110 °C for 24 h. Afterwards amino acid analysis was performed on an Eppendorf Amino Acid Analyser LC 3000 that is based on an ion exchange chromatography. An external standard (Laborservice Onken) was used for comparison.

Mass spectrometry An ESI (electrospray) micrOTOF-Q III (quadrupole - time-of-flight) system (Bruker Daltonics GmbH) connected to a Dionex Ultimate 3000 LC (Thermo Scientific) was used for mass detection (LC/MS). Elution of the peptides was performed by an EC 100/2 Nucleoshell RP18 column (C18 Reversed Phase, 100 x 2 mm, 2.7 µm particle size, 90 Å pore size). . Detailed information for individual peptide masses can be found in the supporting information.

Determining heme-peptide interaction and binding affinities


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Heme-binding to Aβ and peptides 1-6 as well as peptides 20-23 was studied by UV/Vis spectroscopy as described before.33 20 µM peptide was incubated with various concentrations (0.2-40 µM) of hemin in 100 mM Hepes-buffer (pH 7.0) and measured at 300-600 nm.

Peroxidase activity of heme and peptide-heme complexes Peroxidase activity of heme and peptide-heme complexes was studied under different assay conditions. In general, 42 µM of hemin was incubated with peptide (4.2 - 42 µM) for 30 minutes in PBS-buffer. A TMB/H2O2 mix was used as the substrate. Systems were based on H2O2 in citrate buffer (pH 5) or phosphate buffer (pH 7.4) in concentrations of 6.5 mM or 163.2 mM. TMB was used as 1.66 mM or 163.2 mM solution dissolved in 0.12 N HCl. For comparison a commercially available TMB substrate kit was used in addition. 10 µl of incubated peptide-heme or buffer-heme solution was added to 200 µl of substrate mix (1:1) and measured at 652 nm. As controls all peptides have been applied to only buffer and to the specific TMB/H2O2-mix without heme. All data has been normalized against the activity of free heme.

Aβ β(40) aggregation state: Thioflavin T (ThT) spectroscopic assay. A Thioflavin T-based system was applied to study Aβ aggregation in our experimental set-up. Thioflavin was purchased from Sigma Aldrich GmbH (Schnelldorf, Germany) and dissolved in PBS buffer pH 7.4 (0.8 mg ThT T in 50 ml buffer). In general, 1980 ml of ThT solution was mixed with 22 µl of the respective sample, mixed and measured. Fluorescence intensity was recorded using the following parameters: excitation at 440 nm (slit width 5 nm) and emission at 482 nm (slit width 10 nm). Measurements were carried out using a JASCO FP-8300 fluorescence spectrometer (JASCO Germany GmbH, Gross-Umstadt, Germany).


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Impact of LDL on Aβ β -heme peroxidase-activity and complex formation LDL was dissolved in PBS buffer on ice for 24 hours. Protein concentration was determined by absorption at 280 nm using 432970 (ExPASy ScanProsite, without signal peptide) as the molar extinction coefficient. The solution was diluted to a final concentration of 0.44 µM. Solutions of hemin and Aβ (44 µM) were produced in PBS. The peroxidase activity was performed as described above using 163.2 mM H2O2 in citrate buffer and 1.66 mM TMB. Due to the addition of LDL a total volume of 220 µl in the peroxidase assay was used. Absorption was measured over a time period of 15 minutes at 652 nm. In general, the Aβ-heme complex was prepared as explained earlier. Hemin and Aβ, hemin and LDL and Aβ and LDL were incubated for 30 minutes before the third compound was added immediately before performing the peroxidase assay. In addition samples with all three compounds without and with incubation time (30 min) were used. Structure modeling of heme binding to Aβ β(1-40) and peptide 18. Aβ(1-40) structure models were generated using the coordinates of Crescenzi et al.1 for Aβ(142) deposited at the PDB under accession code 1IYT. Backbone and side chain angles were extracted from the PDB structure. Torsion angles of residues 8-38 were translated into tight angle constraints for structure calculations by the program CYANA version 3.97 by adding +/0.5° to the original values. Residues 1-7 and 39-40 as well as the side chain torsions of the heme interacting residues under consideration were not restrained in the structure calculation. Heme binding was established by 17 additional upper and 13 lower limit distance constraints defining the metal to Nε2 distances as well as the distances to the protoporphyrin IX unit.2 Hydrogen bonding of the propionate side chains was considered by addition of Hδ1/Nδ1 to O2D upper and


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lower distance limits of 2.0/3.0, 1.8/2.8, respectively. As for heme in binding processes often a conserved out-of-plane distortion known as ruffling is observed, which e.g. was found to be modulated by a stronger binding to an axial histidine ligand,3 we employed a ruffled hemeresidue in the CYANA structure calculations. Presented structures were selected of the group of 5 % best out of 100 calculated CYANA structures. Figures were produced using MOLMOL.4 For inclusion of the water ligand in the calculations the following new CYANA residue library entry was generated (Supporting Table S3). No template structure was available for peptide 18. The presented peptide 18 model was generated with the program PEP-FOLD, an approach for de novo prediction of peptide structures from amino acid sequences.5,6 Representatives of the predicted 5 best clusters are shown in Figure S8. The best-ranked model (Figure S8A) was used for modeling of the heme binding analogous to the approach described above for Aβ(40).

ASSOCIATED CONTENT Supporting information: UV spectra of heme incubated Aβ-derived peptides and peptide 18 mutants. Peroxidase activity of heme incubated negative controls and peptide 18 mutants. Analytical data of all peptides synthesized within this study. Heme binding affinities of peptides used in this study derived from earlier investigations. Description of the molecular modeling of Aβ(1-40) and peptide 18. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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*Prof. Dr. Diana Imhof, Pharmaceutical Chemistry I, Pharmaceutical Institute, University of Bonn, Bruehler Str. 7, 53119 Bonn, Germany. Phone: +49-(0)228-7360258, e-mail: [email protected] *Dr. Oliver Ohlenschläger, Leibniz Institute on Aging – Fritz Lipmann Institute, Beutenbergstr. 11, 07745 Jena, Germany, Phone +49-(0)3641-656219, e-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources The presented experiments were funded by the Deutsche Forschungsgemeinschaft within FOR1738 (to O.O. and D.I.). ACKNOWLEDGEMENT Financial support by Deutsche Forschungsgemeinschaft within FOR1738 (to O.O. and D.I.) is gratefully acknowledged. The authors thank H. Henning Brewitz and Vera Hegebecker for technical support. ABBREVIATIONS Aβ, Amyloid beta; TMB, 3,3′,5,5′-tetramethylbenzidine; LDL, low density lipoprotein; ESI, electrospray ionization; HPLC, high performance liquid chromatography; TLC, thin layer chromatography;

polyacrylamide

gel

electrophoresis;

PyBOP,

benzotriazol-1-yl-

oxytripyrrolidinophosphonium hexafluorophosphate. ANCILLARY INFORMATION


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Authors will release the atomic coordinates and experimental data upon article publication. Aβ(1-40) structure models were generated using the coordinates of PDB accession code 1IYT

REFERENCES

(1)

Alzheimer, A. Über Eine Eigenartige Erkrankung Der Hirnrinde. Allg. Zeits. Psychiatry Psych. Med. 1907, 64, 146–148.

(2)

Hamley, I. W. The Amyloid Beta Peptide: A Chemist’s Perspective. Role in Alzheimer's and Fibrillization. Chem. Rev. 2012, 112 (10), 5147–5192.

(3)

Hardy, J.; Allsop, D. Amyloid Deposition as the Central Event in the Aetiology of Alzheimer’s Disease. Trends Pharmacol. Sci. 1991, 12 (10), 383–388.

(4)

Hardy, J. The Amyloid Hypothesis for Alzheimer’s Disease: A Critical Reappraisal. J. Neurochem. 2009, 110 (4), 1129–1134.

(5)

Atamna, H.; Frey, W. H. A Role for Heme in Alzheimer’s Disease: Heme Binds Amyloid Beta and Has Altered Metabolism. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (30), 11153– 11158.

(6)

Atamna, H.; Frey, W. H.; Ko, N. Human and Rodent Amyloid-Beta Peptides Differentially Bind Heme: Relevance to the Human Susceptibility to Alzheimer’s Disease. Arch. Biochem. Biophys. 2009, 487 (1), 59–65.

(7)

Zhang, L. Heme Biology : The Secret Life of Heme in Regulating Diverse Biological Processes. World Sci. Singapore; Hackensack, NJ 2011.

(8)

Kühl, T.; Imhof, D. Regulatory FeII/III Heme: The Reconstruction of a Molecule’s Biography. ChemBioChem 2014, 15 (14), 2024–2035.

(9)

Shen, J.; Sheng, X.; Chang, Z.; Wu, Q.; Wang, S.; Xuan, Z.; Li, D.; Wu, Y.; Shang, Y.; Kong, X.; Yu, L.; Li, L.; Ruan, K.; Hu, H.; Huang, Y.; Hui, L.; Xie, D.; Wang, F.; Hu, R. Iron Metabolism Regulates p53 Signaling through Direct Heme-p53 Interaction and Modulation of p53 Localization, Stability, and Function. Cell Rep. 2014, 7 (1), 180–193.

(10)

Yang, J.; Kim, K. D.; Lucas, A.; Drahos, K. E.; Santos, C. S.; Mury, S. P.; Capelluto, D. G. S.; Finkielstein, C. V. A Novel Heme-Regulatory Motif Mediates Heme-Dependent Degradation of the Circadian Factor Period 2. Mol. Cell. Biol. 2008, 28 (15), 4697–4711.


33


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 34 of 49

(11)

Lathrop, J. T.; Timko, M. P. Regulation by Heme of Mitochondrial Protein Transport through a Conserved Amino Acid Motif. Science 1993, 259 (5094), 522–525.

(12)

Munakata, H., Sun, J. Y., Yoshida, K., Nakatani, T., Honda, E., Hayakawa, S.; Furuyama, K., Hayashi, N. Role of the Heme Regulatory Motif in the Heme-Mediated Inhibition of Mitochondrial Import of 5-Aminolevulinate Synthase. J. Biochem. 2004, 136 (2), 233– 238.

(13)

González-Domínguez, M.; Freire-Picos, M. A.; Cerdán, M. E. Haem Regulation of the Mitochondrial Import of the Kluyveromyces Lactis 5-Aminolaevulinate Synthase: An Organelle Approach. Yeast 2001, 18 (1), 41–48.

(14)

Puy, H.; Gouya, L.; Deybach, J. Porphyrias. Lancet 2010, 375 (9718), 924–937.

(15)

Sankaran, V. G.; Weiss, M. J. Anemia: Progress in Molecular Mechanisms and Therapies. Nat. Med. 2015, 21, 221–230.

(16)

Ye, W.; Zhang, L. Heme Controls the Expression of Cell Cycle Regulators and Cell Growth in HeLa Cells. Biochem. Biophys. Res. Commun. 2004, 315 (3), 546–554.

(17)

Schipper, H. M. Heme Oxygenase-1: Role in Brain Aging and Neurodegeneration. Exp. Gerontol. 2000, 35 (6-7), 821–830.

(18)

Cuadrado, A.; Rojo, A. I. Heme Oxygenase-1 as a Therapeutic Target in Neurodegenerative Diseases and Brain Infections. Curr. Pharm. Des. 2008, 14 (5), 429– 442.

(19)

Atamna, H.; Boyle, K. Amyloid-Beta Peptide Binds with Heme to Form a Peroxidase: Relationship to the Cytopathologies of Alzheimer’s Disease. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (9), 3381–3386.

(20)

Atamna, H. Heme Binding to Amyloid-Beta Peptide: Mechanistic Role in Alzheimer’s Disease. J. Alzheimers. Dis. 2006, 10 (2-3), 255–266.

(21)

Mukherjee S., Seal M., D. S. G. Kinetics of Serotonin Oxidation by Heme-Aβ Relevant to Alzheimer’s Disease. J. Biol. Inorg. Chem. 2014, 19 (8), 1355–1365.

(22)

Geldenhuys, W. J.; der Schyf, C. J. Role of Serotonin in Alzheimer’s Disease: A New Therapeutic Target? CNS Drugs 2011, 25 (9), 765–781.

(23)

Howlett, D.; Cutler, P.; Heales, S.; Camilleri, P. Hemin and Related Porphyrins Inhibit Beta-Amyloid Aggregation. FEBS Lett. 1997, 417, 249–251.

(24)

Igarashi, J.; Kitanishi, K.; Martinkova, M.; Murase, M.; Iizuka, A.; Shimizu, T. The Roles of Thiolate-Heme Proteins, Other than the P450 Cytochromes, in the Regulation of HemeSensor Proteins. Acta Chim. Slov. 2008, 55(1) 67–74.


34

Page 35 of 49

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


(25)

Balla, G.; Jacob, H. S.; Eaton, J. W.; Belcher, J. D.; Vercellotti, G. M. Hemin: A Possible Physiological Mediator of Low Density Lipoprotein Oxidation and Endothelial Injury. Arter. Thromb. 1991, 11 (6), 1700–1711.

(26)

Draczynska-Lusiak, B.; Doung, A.; Sun, A. Y. Oxidized Lipoproteins May Play a Role in Neuronal Cell Death in Alzheimer Disease. Mol. Chem. Neuropathol. 1998, 33 (2), 139– 148.

(27)

Lu N., Li J., Tian R., P. Y. Y. Key Roles of Arg(5), Tyr(10) and His Residues in AβHeme Peroxidase: Relevance to Alzheimer’s Disease. Biochem. Biophys. Res. Commun. 2014, 452 (3), 676–681.

(28)

Zhou, Y.; Wang, J.; Liu, L.; Wang, R.; Lai, X.; Xu, M. Interaction between Amyloid-Beta Peptide and Heme Probed by Electrochemistry and Atomic Force Microscopy. ACS Chem. Neurosci. 2013, 4 (4), 535–539.

(29)

Pramanik, D.; Dey, S. G. Active Site Environment of Heme-Bound Amyloid Β Peptide Associated with Alzheimers Disease. J. Am. Chem. Soc. 2011, 133, 81–87.

(30)

Pramanik, D.; Ghosh, C.; Mukherjee, S.; Dey, S. G. Interaction of Amyloid Β Peptides with Redox Active Heme Cofactor: Relevance to Alzheimer’s Disease. Coord. Chem. Rev. 2013, 257 (1), 81–92.

(31)

Ghosh, C.; Seal, M.; Mukherjee, S.; Ghosh Dey, S. Alzheimer’s Disease: A Heme–Aβ Perspective. Acc. Chem. Res. 2015, 48 (9), 2556–2564.

(32)

Kühl, T.; Sahoo, N.; Nikolajski, M.; Schlott, B.; Heinemann, S. H.; Imhof, D. Determination of Hemin-Binding Characteristics of Proteins by a Combinatorial Peptide Library Approach. ChemBioChem 2011, 12 (18), 2846–2855.

(33)

Kühl, T.; Wißbrock, A.; Goradia, N.; Sahoo, N.; Galler, K.; Neugebauer, U.; Popp, J.; Heinemann, S. H.; Ohlenschläger, O.; Imhof, D. Analysis of Fe(III) Heme Binding to Cysteine-Containing Heme-Regulatory Motifs in Proteins. ACS Chem. Biol. 2013, 8 (8), 1785–1793.

(34)

Brewitz, H. H.; Kühl, T.; Goradia, N.; Galler, K.; Popp, J.; Neugebauer, Ute Ohlenschläger, O.; Imhof, D. Role of the Chemical Environment beyond the Coordination Site: Structural Insight into Fe(III) Protoporphyrin Binding to Cysteine-Based HemeRegulatory Protein Motifs. ChemBioChem 2015, 16 (15), 2216–2224.

(35)

Brewitz, H. H.; Goradia, N.; Schubert, E.; Galler, K.; Kühl, T.; Syllwasschya, B.; Popp, J.; Neugebauer, U.; Hagelueken, G.; Schiemann, O.; Ohlenschläger, O.; Imhof, D. Heme Interacts with Histidine- and Tyrosine-Based Protein Motifs and Inhibits Enzymatic Activity of Chloramphenicol Acetyltransferase from E. Coli. Biochim. Biophys. Acta Gen. Subj. 2016, 1860 (6), 1343–1353.


35


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 36 of 49

(36)

Lu, N.; Li, J.; Tian, R.; Peng, Y. Key Roles for Tyrosine 10 in Aβ − Heme Complexes and Its Relevance to Oxidative Stress. Chem. Res. Toxicol. 2015, 28 (3), 365–372.

(37)

Yuan, C.; Gao, Z. Aβ Interacts with Both the Iron Center and the Porphyrin Ring of Heme: Mechanism of Heme’s Action on Aβ Aggregation and Disaggregation. Chem. Res. Toxicol. 2013, 26 (2), 262–269.

(38)

Josephy, P. D.; Eling, T.; Mason, R. P. The Horseradish Peroxidase-Catalyzed Oxidation of 3,5,3’,5'-Tetramethylbenzidine. Free Radical and Charge-Transfer Complex Intermediates. J. Biol. Chem. 1982, 257 (7), 3669–3675.

(39)

Qin, Y.; Zhang, L.; Ye, G.; Zhao, S. Homogeneous Label-Free Colorimetric Strategy for Convenient Bleomycin Detection Based on Bleomycin Enhanced Fe(II)–H2O2–ABTS Reaction. Anal. Methods 2014, 6, 7973–7977.

(40)

Yin, J.; Cao, H.; Lu N., Li J., Tian R., P. Y. Y. Self-Assembly into Magnetic Co3O4 Complex Nanostructures as Peroxidase. J. Mater. Chem. 2012, 22 (2), 527–534.

(41)

Sassa, S. Why Heme Needs to Be Degraded to Iron, Biliverdin IXalpha, and Carbon Monoxide? Antioxid. Redox Signal. 2004, 6 (5), 819–824.

(42)

Strlič, M.; Kolar, J.; Pihlar, B. The Effect of Metal Ion, pH and Temperature on the Yield of Oxidising Species in a Fenton-like System Determined by Aromatic Hydroxylation. Acta Chim. Slov. 1999, 46 (4), 555–566.

(43)

Neyens, E.; Baeyens, J. A Review of Classic Fenton’s Peroxidation as an Advanced Oxidation Technique. J. Hazard. Mater. 2002, 98 (1-3), 33–50.

(44)

Koo, E. H.; Squazzo, S. L. Evidence That Production and Release of Amyloid BetaProtein Involves the Endocytic Pathway. J. Biol. Chem. 1994, 269 (26), 17386–17389.

(45)

Yamazaki, T.; Koo, E. H.; Selkoe, D. J. Trafficking of Cell-Surface Amyloid Beta-Protein Precursor. II. Endocytosis, Recycling and Lysosomal Targeting Detected by Immunolocalization. J. Cell Sci. 1996, 109, 999–1008.

(46)

Su, Y.; Chang, P. T. Acidic pH Promotes the Formation of Toxic Fibrils from BetaAmyloid Peptide. Brain Res. 2001, 893 (1-2), 287–291.

(47)

Battistuzzi, G.; Bellei, M.; Bortolotti, C. A.; Sola, M. Redox Properties of Heme Peroxidases. Arch. Biochem. Biophys. 2010, 500 (1), 21–36.

(48)

Nicolis, S.; Casella, L.; Roncone, R.; Dallacosta, C.; Monzani, E. Heme-Peptide Complexes as Peroxidase Models. C. R. Chim. 2007, 10 (4-5), 380–391.


36

Page 37 of 49

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


(49)

Thiabaud G., Pizzocaro S., Garcia-Serres R., Latour J. M., Monzani E., C. L. Heme Binding Induces Dimerization and Nitration of Truncated Β-Amyloid Peptide Aβ16 Under Oxidative Stress. Angew. Chem. Int. Ed. 2013, 52 (31), 8041–8044.

(50)

Zhao, J.; de Serrano, V.; Dumarieh, R.; Thompson, M.; Ghiladi, R. a; Franzen, S. The Role of the Distal Histidine in H2O2 Activation and Heme Protection in Both Peroxidase and Globin Functions. J. Phys. Chem. B 2012, 116 (40), 12065–12077.

(51)

Soga, O.; Kinoshita, H.; Ueda, M.; Tanaka, a. Evaluation of Peroxisomal Heme in Yeast. J. Biochem. 1997, 121 (1), 25–28.

(52)

Coles, M.; Bicknell, W.; Watson, A. A.; Fairlie, D. P.; Craik, D. J. Solution Structure of Amyloid Beta-peptide(1-40) in a Water-Micelle Environment. Biochemistry 1998, 37 (31), 11064–11077.

(53)

Crescenzi, O.; Tomaselli, S.; Guerrini, R.; Salvadori, S.; D’Ursi, A. M.; Temussi, P. A.; Picone, D. Solution Structure of the Alzheimer Amyloid β-Peptide (1-42) in an Apolar Microenvironment: Similarity with a Virus Fusion Domain. Eur. J. Biochem. 2002, 269 (22), 5642–5648.

(54)

Jarvet, J.; Danielsson, J.; Damberg, P.; Oleszczuk, M.; Gräslund, A. Positioning of the Alzheimer Aβ(1-40) Peptide in SDS Micelles Using NMR and Paramagnetic Probes. J. Biomol. NMR 2007, 39 (1), 63–72.

(55)

Kumar, S. T.; Leppert, J.; Bellstedt, P.; Wiedemann, C.; Fändrich, M.; Görlach, M. Solvent Removal Induces a Reversible Β-to-Α Switch in Oligomeric Aβ Peptide. J. Mol. Biol. 2016, 428 (2), 268–273.

(56)

Crescenzi, O.; Tomaselli, S.; Guerrini, R.; Salvadori, S.; D’Ursi, A. M.; Temussi, P. A.; Picone, D. Solution Structure of the Alzheimer Amyloid Beta-Peptide (1-42) in an Apolar Microenvironment: Similarity with a Virus Fusion Domain. Eur. J. Biochem. 2002, 269 (22), 5642–5648.

(57)

Shen, Y.; Maupetit, J.; Derreumaux, P.; Tufféry, P. Improved PEP-FOLD Approach for Peptide and Miniprotein Structure Prediction. J. Chem. Theory Comput. 2014, 10 (10), 4745–4758.

(58)

Thévenet, P.; Shen, Y.; Maupetit, J.; Guyon, F.; Derreumaux, P.; Tufféry, P. PEP-FOLD: An Updated de Novo Structure Prediction Server for Both Linear and Disulfide Bonded Cyclic Peptides. Nucleic Acids Res. 2012, 40 (W1), W288–W293.

(59)

Kontush, A. Lipid Peroxidation and Alzheimer’s Disease: Key Role of Amyloid-Β. Oilseeds fats, Crop. Lipids 2006, 13 (1), 46–53.

(60)

Koudinov, A.; Matsubara, E.; Frangione, B.; Ghiso, J. The Soluble Form of Alzheimer’s Amyloid Beta Protein Is Complexed to High Density Lipoprotein 3 and Very High


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

Page 38 of 49

Density Lipoprotein in Normal Human Plasma. Biochem. Biophys. Res. Commun. 1994, 205 (2), 1164–1171. (61)

Koudinov, A. R.; Koudinova, N. V; Kumar, A.; Beavis, R. C.; Ghiso, J. Biochemical Characterization Of Alzheimers Soluble Amyloid Beta. Biochem. Biophys. Res. Commun. 1996, 223 (3), 592–597.

(62)

Biere, A. L.; Ostaszewski, B.; Stimson, E. R.; Hyman, B. T.; Maggio, J. E.; Selkoe, D. J. Amyloid Β-Peptide Is Transported on Lipoproteins and Albumin in Human Plasma. J. Biol. Chem. 1996, 271 (51), 32916–32922.

(63)

Kuo, Y. M.; Emmerling, M. R.; Bisgaier, C. L.; Essenburg, a D.; Lampert, H. C.; Drumm, D.; Roher, a E. Elevated Low-Density Lipoprotein in Alzheimer’s Disease Correlates with Brain Abeta 1-42 Levels. Biochem. Biophys. Res. Commun. 1998, 252 (3), 711–715.

(64)

Koudinov, A. R.; Koudinova, N. V. Alzheimer’s Soluble Amyloid Beta Protein Is Secreted by HepG2 Cells as an Apolipoprotein. Cell Biol. Int. 1997, 21 (5), 265–271.

(65)

Camejo, G.; Halberg, C.; Manschik-Lundin, a; Hurt-Camejo, E.; Rosengren, B.; Olsson, H.; Hansson, G. I.; Forsberg, G. B.; Ylhen, B. Hemin Binding and Oxidation of Lipoproteins in Serum: Mechanisms and Effect on the Interaction of LDL with Human Macrophages. J. Lipid Res. 1998, 39 (4), 755–766.

(66)

Bassett, C. N.; Neely, M. D.; Sidell, K. R.; Markesbery, W. R.; Swift, L. L.; Montine, T. J. Cerebrospinal Fluid Lipoproteins Are More Vulnerable to Oxidation in Alzheimer’s Disease and Are Neurotoxic When Oxidized Ex Vivo. Lipids 1999, 34 (12), 1273–1280.

(67)

Sun, A. Y.; Draczynska-Lusiak, B.; Sun, G. Y. Oxidized Lipoproteins, Beta Amyloid Peptides and Alzheimer’s Disease. Neurotox. Res. 2001, 3 (2), 167–178.

(68)

Larsen, R.; Gouveia, Z.; Soares, M. P.; Gozzelino, R. Heme Cytotoxicity and the Pathogenesis of Immune-Mediated Inflammatory Diseases. Front. Pharmacol. 2012, 3 MAY.

(69)

Chang, Y.-T.; Chang, W.-N.; Tsai, N.-W.; Huang, C.-C.; Kung, C.-T.; Su, Y.-J.; Lin, W.C.; Cheng, B.-C.; Su, C.-M.; Chiang, Y.-F.; Lu, C.-H. The Roles of Biomarkers of Oxidative Stress and Antioxidant in Alzheimer’s Disease: A Systematic Review. Biomed. Res. Int. 2014, 2014, 1–14.

(70)

Grinshtein, N.; Bamm, V. V.; Tsemakhovich, V. A.; Shaklai, N. Mechanism of LowDensity Lipoprotein Oxidation by Hemoglobin-Derived Iron. Biochemistry 2003, 42 (23), 6977–6985.

(71)

Satoh, T.; Satoh, H.; Iwahara, S.; Hrkal, Z.; Peyton, D. H.; Muller-Eberhard, U. Roles of Heme Iron-Coordinating Histidine Residues of Human Hemopexin Expressed in


38

Page 39 of 49

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


Baculovirus-Infected Insect Cells. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (18), 8423– 8427. (72)

Pramanik, D.; Mukherjee, S.; Dey, S. G. Apomyoglobin Sequesters Heme from Heme Bound Ab Peptides. Inorg. Chem. 2013, 52 (19), 10929–10935.

(73)

Koradi, R.; Billeter, M.; Wüthrich, K. MOLMOL: A Program for Display and Analysis of Macromolecular Structures. J. Mol. Graph. 1996, 14 (1), 51–55.


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TABLES Table 1. Heme-binding peptides used for peroxidase assay.

No. 7 8 9 10 11 12 13 14 15 16 17 18 19

peptide sequence AAAAAAAAA AAAAYAAAA AAAACAAAA AAAAHAAAA AIRRCSTFQ TPILCPFHL FKAAHKHVR AAHYHTYER WELDYFQWK HPFPYIWKA SGGLPAPSDFKCPIKEEIAITSG NVNLTSNHLLYHYWIAVSHKAPA VRMDTLAHVLYYPQKPLVTTRSM

KD-value (µM) n.b. n.b.32,35 n.b.32,34 n.b.32,35 0.50 ± 0.2034 0.60 ± 0.4033,34 0.99 ± 0.2135 0.83 ± 0.3335 n.sat.35 0.33 ± 0.2532,35 1.42 ± 0.2432,33 1.40 ± 0.1332 0.51 ± 0.2732

Soret band (nm) 432 368 421 417 376 423 369 419 430

n.b., not binding; n.sat., not saturated


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FIGURE LEGEND Figure 1. Top: Sequence of Aβ(40/42) and Aβ-derived peptides 1-6. Aβ sequence is divided into a hydrophilic site (site-H) and lipophilic site (site-L). Possible heme axial ligands are in red, and the arginine that is suggested to affect the peroxidase-like activity of Aβ-heme in green. Bottom: Differential spectra of heme incubated Aβ(1-40) and Aβ-derived peptide 6 (Aβ(1-18)).

Figure 2. Peroxidase activity [%] of heme-incubated Aβ-derived peptides 1-6 in approach II (A) and peptides 11-19 (B). All data included were normalized against the activity of unbound heme (100 %, blue line). The dashed, green line shows the activity of the Aβ(1-40)-heme complex (~500 %). All peptides were measured in approach I-III.

Figure 3. Change of absorbance of peptides 18 and 20-23 upon heme incubation at ~416 nm (maximum). Peptide mutant 23 (His12Ala) shows a significant loss of heme binding affinity compared to wild-type peptide 18.

Figure 4. Structural models of potential Aβ(1-40)-heme binding situations involving residues H13/K16 (A,C) or R5/H13 (B,D). The respective coordinating residues are given in dark blue, while the heme ring is colored in green with the iron center in orange, water in red, Y10 in magenta. Only heavy atom positions for other amino acids are depicted. Close-ups (C,D) were included to visualize the potential bonding network (yellow). The ribbon diagram indicating the helix orientations were generated with MolMol.73


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Figure 5. Structural model of peptide 18 with binding scenario His8/His12 (A) and His12/His19 (B). Coloring as in Figure 5.

Figure 6. Molecular size of the applied molecules LDL, Aβ and heme in direct comparison. Schematic overview of the suggested molecular processes in the presence of LDL. Top: LDL and pre-incubated Aβ-heme are added to the assay. Center: Same situation occurs when LDL, Aβ and heme are added at the same time (0 and 30 min) as well as when Aβ and LDL are preincubated before the addition of heme. Bottom: LDL and heme are incubated before the presence of Aβ impairing the Aβ-heme complex formation.


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Figure 21.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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TABLE OF CONTENTS GRAPHIC


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Synthesis and Evaluation of Amyloid Î² Derived ... - ACS Publications

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