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Stabilization of discordant helices in amyloid fibril-forming proteins

¹ Department of Molecular Biosciences, Swedish University of Agricultural Sciences, The Biomedical Centre, S-751 23 Uppsala, Sweden
² Department of Medical Biochemistry and Biophysics and
³ Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden

Reprint requests to: Jan Johansson, Department of Molecular Biosciences, Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575, S-751 23 Uppsala, Sweden; e-mail: jan.johansson{at}vmk.slu.se; fax: 46-18-550762.

(RECEIVED September 17, 2003; FINAL REVISION December 2, 2003; ACCEPTED January 20, 2004)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Several proteins and peptides that can convert from -helical to -sheet conformation and form amyloid fibrils, including the amyloid -peptide (A) and the prion protein, contain a discordant -helix that is composed of residues that strongly favor -strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but A apparently lacks such interactions. The helical propensity of the A discordant region (K₁₆LVFFAED₂₃) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the -helical content of A(12–28) and reduces aggregation and fibril formation of A(1–40), A(12–28), A(12–24), and A(14–23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on A secondary structure or fibril formation. These data suggest that in free A, the discordant -helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.

Abbreviations: A, amyloid -peptide • AD, Alzheimer’s disease • APP, amyloid precursor protein • ASA, accessible surface area • CD, circular dichroism • PrP, prion protein • TFE, trifluoroethanol • ThT, thioflavin T

Keywords: -helix; protein structure; protein aggregation; conformational disease

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03442404.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Amyloid fibrils formed from specific proteins are associated with ~20 different diseases, including Alzheimer’s disease (AD; amyloid -peptide [A] forms fibrils), prion diseases (prion protein [PrP] forms fibrils), hereditary systemic amyloidosis (lysozyme mutants form fibrils), and familial amyloid polyneuropathy (transthyretin forms fibrils; Kelly 1998). Amyloid fibrils are composed of polypeptide chains in -strand conformation, which form -sheets running perpendicular to the long axis of the fibril (Serpell 2000). The morphology and molecular architecture of amyloid fibrils are apparently very similar, although they are formed from proteins with widely different native structures, sizes, and localization (Dobson 1999). Moreover, under partly denaturing conditions, fibrils can be formed from almost any protein, suggesting that the polypeptide backbone is a main determinant of the fibril structure (Fändrich et al. 2001; Srisailam et al. 2003). Although many polypeptide sequences thus can form fibrillar -sheet structure under certain conditions, the ability to do so under physiological conditions is apparently limited to a small number of proteins. Out of ~1300 nonhomologous proteins with experimentally determined three-dimensional structures, PrP, A, and ~30 other proteins were recently found to contain -helices composed of amino acid sequences that are strongly predicted to form -strands, called discordant helices (Kallberg et al. 2001). This observation raises the possibility that conflicts in structural preferences localized to comparatively short linear polypeptide regions can underlie fibril formation. In agreement with these results, it was recently found that the discordant helix of PrP is frustrated in its helical state (Dima and Thirumalai 2002). Four of the 10 proteins with the longest discordant helices (11 residues) have been analyzed, and all form fibrils. It has also been observed that by mutating the amino acid sequences of the discordant helices of A and lung surfactant protein C, resulting in predicted helical structures instead, fibril formation can be abrogated (Kallberg et al. 2001; Hosia et al. 2002). Because discordant helices are made from amino acid sequences that appear intrinsically unsuited for helix formation, it is likely that these helices require additional interactions for stabilization. In this study, we analyzed the 38 longest discordant helices found previously (Kallberg et al. 2001) regarding their native local environments, and found that all of them except A interact with polypeptide segments or lipid membranes.

The 39–43 residues-long (preferentially 40 or 42 residues) A is invariably present in amyloid plaques found in association with AD, and formation of A fibrils is thought to be part of the cause of this devastating disease (Selkoe 2000). The postmortem amounts of A in the cerebral cortices of AD patients correlate with the disease progression, indicating that A is a rational therapeutic target (Näslund et al. 2000). A is generated by proteolytic cleavages of a large transmembrane protein, the amyloid precursor protein (APP). The transmembrane helix of APP is predicted to start at a position corresponding to residue 29 in A. Cleavage by -secretase generates the N terminus of the A part and leaves behind a C-terminal membrane-associated stub, which is cleaved by -secretase, generating free A. Cleavage by -secretase C-terminally of residue 16 in A generates a nonamyloidogenic peptide (Selkoe 1999; Esler and Wolfe 2001). Hereditary forms of early onset AD are associated with point mutations in three regions of APP. Two of these regions are located in close vicinity of the N- and C-terminal ends of A, and are likely associated with elevated production of A(1–40) and/or the more amyloidogenic variant A(1–42). The third region covers positions Ala 21, Glu 22, and Asp 23 of A, and the pathogenic mechanisms associated with mutations of these residues are not entirely understood (Haass and Steiner 2001).

The secondary structure of A(1–40/42) peptides in aqueous solution is mainly disordered (Riek et al. 2001), whereas in the presence of 20%–40% (v/v) trifluoroethanol (TFE), a significant portion of helical structure (~15%–30%) is found (Barrow and Zagorski 1991; Soto et al. 1995; Sticht et al. 1995). NMR structure determinations of A(1–40) and A(1–42) in SDS micelles revealed an -helix covering positions 15–36 with a kink located at positions 25–27 (Coles et al. 1998), or two helices covering positions 10–24 and 28–42, respectively (Shao et al. 1999). In 40% (v/v) TFE two helices, covering positions 15–23 and 31–35, were found (Sticht et al. 1995). A(1–42) is more prone to aggregate and form plaques than A(1–40). This is not reflected in different conformations of the monomeric peptides (Riek et al. 2001), but may be attributed to the presence of two additional unpolar residues (Ile–Ala) at the C-terminal end, making the A(1–42) less soluble in aqueous solvents (Jarrett et al. 1993). The unpolar C-terminal region of A, which emanates from the predicted transmembrane part of APP, thus possibly influences plaque formation by decreasing A solubility and increasing peptide–peptide contacts. However, A(1–28) also forms fibrils (Kirschner et al. 1987; Tjernberg et al. 1996), indicating that the hydrophobic C-terminal region of A is not essential for fibril formation. Several lines of evidence indicate that a region centering around positions 17–20 is important for A fibril formation. In A(1–40), positions 16–20 were found to be involved in formation of A intermolecular contacts and fibril formation, and removal of A positions 14–23 prevents fibril formation (Tjernberg et al. 1996, 1999). In A(1–42), destabilization of a helix covering residues 11–24, in particular residues 17–24, is critical for -helix -strand conversion and fibril formation (Janek et al. 2001). The discordant helix of A covers residues K₁₆LVFFAED₂₃. Herein we found that residue replacements in this region and compounds that stabilize A-helical conformation reduce fibril formation.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Native environments of discordant helices
The interactions with surrounding amino acid residues were determined for each residue of the 38 longest discordant helices within their native proteins. For this purpose, we used the relative accessibility as a measure of the degree of shielding imposed by surrounding residues. The result shows that the helices fall into one of three categories (Fig. 1). One group represents helices that are predominantly buried with exception of the terminal regions (Fig. 1A). A second group represents helices with periodic variations in their residue accessibilities. The periodicity corresponds to three to four residues, and ~50% of the residues in these helices are buried (Fig. 1B), corresponding to helices located at the protein surface. For both these groups, it is concluded that the local protein environment stabilizes the helices. A third group represents helices that are apparently exposed along their entire length (Fig. 1C). These six helices would be expected to adopt a -strand conformation as predicted from their sequences. However, in three cases (1bct [PDB] , bacteriorhodopsin; 1spf [PDB] , surfactant-associated protein C; and 1bl1 [PDB] , parathyroid hormone receptor), the helices are buried in a lipid membrane in their native environment, and in two cases (1aa0 [PDB] , fibritin deletion mutant; and 2ifo [PDB] , inovirus major coat protein), the discordant helices are involved in oligomer formation. Interactions with surrounding lipids or other polypeptides caused by oligomerization are expected to stabilize these helices. The only remaining case of the 38 studied discordant helices for which no local interactions are apparent is thus 1ba6 [PDB] (human A). Possibly, lack of stabilization of helical A contributes to its tendency to form -sheet aggregates and fibrils. We therefore investigated whether stabilization of the discordant A-helix, by residue replacements or by addition of ligands, affects fibril formation.

View larger version (35K): [in this window] [in a new window]   Figure 1. Surface accessibility of discordant helices. The relative solvent-accessible surface of each discordant helix residue in the respective protein calculated as described in Materials and Methods. For each protein, the PDB code and the trivial name are given (see Kallberg et al. 2001 for details). (A) Helices with mainly buried residues. (1aur [PDB] ) Carboxylesterase, (1b2v [PDB] ) heme-binding protein A, (1b5e [PDB] ) dCMP hydroxymethylase, (1b8o [PDB] ) purine nucleoside phosphorylase, (1cpo [PDB] ) chloroperoxidase, (1ggt [PDB] ) coagulation factor XIII, (1h2a [PDB] ) hydrogenase, (1iab [PDB] ) astacin, (1jkm [PDB] ) brefeldin A esterase, (1lml [PDB] ) leishmanolysin, (1mhd [PDB] ) smad MH1 domain, (1mnm [PDB] ) transcription factor MVM1 (middle of helix is not discordant and not plotted), (1mty [PDB] ) methane monooxygenase (middle of helix is not discordant and not plotted), (1nom [PDB] ) DNA polymerase , (1noz [PDB] ) DNA polymerase, (1qut [PDB] ) lytic transglycosylase Slt35, (1sra [PDB] ) osteonectin, (1tah [PDB] ) lipase, (1tca [PDB] ) lipase B, (1vns [PDB] ) chloroperoxidase (middle of helix is not discordant and not plotted), (1wer [PDB] ) Ras-GTPase-activating domain of p120GAP, (2sqc [PDB] ) squalene-hopene cyclase, (3aig [PDB] ) adamalysin II, (3pte [PDB] ) transpeptidase. (B) Helices with at least two exposed residues (accessibility ratio >0.3) separated by buried residues. The first buried residue in each helix is aligned. (1b10 [PDB] ) Prion protein, (1cv8 [PDB] ) staphopain, (1ecr [PDB] ) replication terminator protein, (1kpt [PDB] ) killer toxin, (1pbv [PDB] ) sec7 domain of exchange factor ARNO, (1wer [PDB] ) Ras-GTPase-activating domain of p120GAP, (2erl [PDB] ) pheromone Er-1, (2occ [PDB] ) cytochrome C oxidase. (C) Helices with mainly exposed residues. (1aa0 [PDB] ) Fibritin deletion mutant, (1ba6 [PDB] ) amyloid -peptide (A), (1bct [PDB] ) bacteriorhodopsin, (1bl1 [PDB] ) parathyroid hormone receptor, (1spf [PDB] ) surfactant-associated protein C, (2ifo [PDB] ) inovirus major coat protein.

A discordant helix stabilization by residue replacements

View larger version (22K): [in this window] [in a new window]   Figure 2. Removal of the A discordant nature prevents fibril formation. (A) Sequences and predicted secondary structures for A(12–28) and A(12–28; V18A/F19A/F20A). Blue cylinders (bottom) represent the experimentally determined central helix of A; the central yellow strand or blue cylinder represents -strand or helical structure, respectively, predicted by the PHD method in which the figures give the reliability indexes (range 1–9); and the top line E or H represents extended or helical structure, respectively, predicted using the Chou-Fasman algorithm (see Kallberg et al. 2001 for details). In contrast to the native sequence, a helical structure is predicted for the sequence containing the V18A/F19A/F20A replacements. (B) Residual molar ellipticity at 222 nm as a function of TFE content for A(12–28) (open squares) and A(12–28; V18A/F19A/F20A) (filled triangles). (C) ThT fluorescence of solutions containing 100 M A(12–28) (open squares) or A(12–28; V18A/F19A/F20A) (filled triangles) during 14 d of incubation in 10 mM sodium phosphate buffer (pH 6.0) at 37°C.

A discordant helix stabilization by ligands

View larger version (27K): [in this window] [in a new window]   Figure 3. KAD increases the helical content of A(12–28). CD spectra of 100 µM A(12–28) in 40% aqueous TFE (open squares) and after addition of 1 mM KAD tripeptide (upper graph) or 1 mM AAA (lower graph), after subtraction of the contribution from respective tripeptide. The residual molar ellipticity is expressed in kilodegrees centimeters squared per decimole.

View larger version (137K): [in this window] [in a new window]   Figure 4. KAD and AAA effects on A fibril formation. Electron micrographs of fibrillar material formed from 100 µM A(1–40) in the presence of 1 mM AAA (A) or KAD (B).

View larger version (22K): [in this window] [in a new window]   Figure 5. Reduction of A fibril formation by tripeptides. Here 100 µM A(14–23), A(12–24), or A(1–40) in sodium phosphate-buffered saline was incubated for 3 d at 37°C either alone or in the presence of 1 mM each indicated tripeptide. After incubation, the solutions were centrifuged at 20,000g and the number of fibril bundles in the pellet was estimated by electron microscopy. Unless indicated otherwise, the tripeptides used have free N and C termini. The mean values and standard deviations of three independent experiments are shown.

View larger version (16K): [in this window] [in a new window]   Figure 6. Reduction of A(14–23) fibril formation by tri- and tetrapeptides. Here 100 µM A(14–23) was incubated in the presence of 1 mM the indicated tri- or tetrapeptides, and amounts of fibrils formed were determined as described for Figure 5.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effects on A(1–40) aggregation of KAD, AAA, and KFFE. Here 100 µM A(1–40) was incubated in phosphate-buffered saline in the presence of 1 mM KAD, AAA, or KFFE at 37°C. At the indicated time points, aliquots of the solutions were withdrawn and centrifuged at 20,000g for 20 min, and the relative amounts of A(1–40) peptide in the supernatants were determined by amino acid analysis. The values shown are the mean of duplicate samples.

View larger version (23K): [in this window] [in a new window]   Figure 8. Phospho-L-serine increases helical content and reduces fibril formation. CD spectra of 100 µM acetyl-A(12–28)-amide in 30% aqueous TFE alone (open squares), and after addition of 1 mM phospho-L-serine (filled triangles, A) or 1 mM phosphoethanolamine (filled circles, B). Spectra in the presence of phosphocholine or phosphoglycerol were virtually identical to that of A(12–28). (C) ThT fluorescence of solutions containing 100 µM A(12–28) alone (open squares), A(12–28) plus 1 mM phospho-L-serine (filled triangles), or A(12–28) plus 1 mM phosphoethanolamine (filled circles) during 14 d of incubation at 37°C.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

The A discordant helix, covering positions 16–23, differs from the other helices now studied in that it lacks apparent stabilizing interactions. This likely reflects that A is a proteolytic fragment of APP. A positions 16–23 show helical structure in the presence of membrane-mimicking solvents or detergents, suggesting that this region is helical also in membrane-associated APP. However, for liberated A in aqueous solution, mainly unordered conformation is detected by spectroscopic methods (Serpell 2000; Riek et al. 2001). In the absence of helix-stabilizing interactions, the inherent propensity of the A discordant region to form -sheet structure is expected to contribute to the ability to form fibrils. Mutating Lys 16, Leu 17, and Phe 20 to Ala changes the secondary structure propensity of A(1–28) so that the discordant nature is abolished (Kallberg et al. 2001) and prevents fibril formation (Tjernberg et al. 1996). Like-wise, the replacement Val18Ala in A(1–40) increases the helical content and reduces the capacity to form fibrils (Soto et al. 1995). However, although optimal stabilization of helical A by the addition of TFE prevents fibril formation, partial stabilization of helical structure of A instead apparently accelerates formation of -sheet aggregates and fibrils (Fezoui and Teplow 2002). Furthermore, transient formation of -helix structure prior to formation of -sheet structure and fibrils has been observed, suggesting that partially helical forms of A may be on-pathway to fibril formation (Kirkitadze et al. 2001). These studies were performed with A peptides containing the hydrophobic C-terminal part encompassing residues 29–40/42, which forms helical structure in membrane-mimicking solvents, including TFE (Serpell 2000). Our results indicate that stabilization of the A-helix around residues 16–23, as judged by analysis of A(12–28), A(14–23), and A(12–24), reduces aggregation and fibril formation (Figs. 2 , 3, 5, 6, and 8). Further studies are needed to understand the respective involvement of the central and C-terminal regions of A in fibril formation.

In an unbiased search for sequence determinants of A amyloidogenesis (Wurth et al. 2002), 18 of 36 variants of A(1–42) with reduced tendency to aggregate contained replacements in the region covering residues 17–19. In several cases, the reduction in A aggregation could be rationalized as a result of increased solubility due to the replacements. Of interest in relation to the present investigation, it was found that V18A and F19L mutations reduced aggregation, which would not have been predicted based on effects on solubility only (Wurth et al. 2002). Reduced aggregation as a result of these replacements may be an effect of the increased helical propensity of the A discordant region, similar to the effects observed by V18A/F19A/F20A replacements (Fig. 2).

The ligand-induced effects indicate that an increase in A discordant helix stability reduces fibril formation. Thus, KAD tripeptide and phospho-L-serine, which increase the helical occupancy of A(12–28) (Figs. 3, 8), reduce fibril formation and aggregation (Figs. 4–8), whereas structurally related compounds with no effects on secondary structure do not affect fibril formation. In -helical conformation, the separation of the -carbons of Lys 16 and Glu 22/Asp 23 is 9–11 Å. In the tripeptide KAD, the positively and negatively charged side chains are separated by ~11 Å, and the charge separation in phospho-L-serine is ~7–8 Å. Thus, dipolar compounds with charge separation that matches the charge separation in the A discordant helix appear to stabilize the helix. However, the lack of effects of phosphoethanolamine and phosphocholine, with similar charge separation as phospho-L-serine, indicate that other factors are also important.

Finally, the effects of phospho-L-serine now observed suggest that membrane phospholipid head-groups can interact with the A discordant helix. Phospholipid membranes have been shown to give variable effects on A fibril formation, with plasma, endosomal, and lysosomal membrane enhancing fibril formation and Golgi membrane preventing it (Waschuk et al. 2001). Modulation of membrane fluidity by cholesterol affects A membrane insertion and fibril formation. At low cholesterol content, A was located in the surface region in mainly -sheet conformation, whereas at high cholesterol concentrations, A became membrane-inserted in helical conformation and fibril formation was reduced (Ji et al. 2002). It remains to be investigated how membrane phospholipid head-groups interact with A discordant helix, and to what extent such interactions influence the structure of the A region in membrane-associated APP.

Conclusions
Discordant helices, composed of residues with a high -strand propensity, are found in A, PrP, and other amyloid-forming proteins. In most cases, such helices appear to be stabilized by surrounding protein and lipid environments, but for free A, no such interactions are found. Addition of ligands that stabilize the discordant A -helix reduces A aggregation and fibril formation in vitro.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Calculation of accessible surface area
The solvent-accessible surface of each discordant helix residue, in the context of their respective protein, was calculated using the program ICM (Molsoft). The calculations were performed for the same experimentally derived structures as those used for the secondary structure predictions (Kallberg et al. 2001). The relative accessibility for individual residues is derived by dividing their solvent-accessible surface by the standard residue accessibilities calculated for Gly–X–Gly tripeptide segments. A residue is considered buried if the relative accessibility ratio is 0.05, and exposed if the relative accessibility ratio is 0.3.

Peptides and chemicals
Synthetic peptides corresponding to human A positions 1–40 (amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVG SNKGAIIGLMVGGVV), 12–28, 12–24, 14–23, and A(12–28) with V18A/F19A/F20A replacements were purchased from Research Genetics or from Interactiva. A(1–40) was purified by reversed-phase HPLC over a C18 column, using a linear gradient of acetonitrile running into 0.1% trifluoroacetic acid for elution. The purified peptide was lyophilized, stored at –20°C, and dissolved shortly before experiments. The tri- and tetrapeptides were synthesized and purified by reversed-phase HPLC (>70% purity) by Interactiva. Phospho-L-serine and phosphoethanolamine were from Fluka, and phosphoglycerol and phosphocholine chloride were from Sigma.

Circular dichroism (CD) spectroscopy
For analysis of secondary structure by CD spectroscopy, A(12–28) or A(12–28; V18A/F19A/F20A) was dissolved at 100 µM concentration in 0%, 30%, 40%, or 70% TFE in 10 mM sodium phosphate buffer (pH 7.0). CD spectra between 180 and 260 nm of A(12–28) or A(12–28; V18A/F19A/F20A) peptides, and of mixtures containing A(12–28) plus 1 mM of tripeptides, phos-pho-L-serine, phosphoethanolamine, phosphocholine, or phosphoglycerol were recorded at 20°C with 2 sec response time, 2 data points/nm, and scan speed 20 nm/min using an AVIV Model 62DS Spectropolarimeter. Spectra of the tripeptides KAD and AAA in 40% aqueous TFE were subtracted from the spectrum of A(12–28) mixed with the corresponding tripeptide. -Helical contents were calculated from the residual molar ellipticity at 222 nm (Barrow et al. 1992).

Analysis of A fibril formation and aggregation
Fibril formation and aggregation of A(1–40) and fragments covering positions 12–28, 12–24, or 14–23, in the absence and presence of oligopeptides or phospho-compounds were determined. For determination of relative abundance and morphology of fibrils by electron microscopy, the A peptides (100 µM) were incubated for 3 d at 37°C in phosphate-buffered saline (50 mM sodium phosphate/150 mM NaCl at pH 7.4) in the presence or absence of 1 mM of the ligands, and then centrifuged at 20,000g for 20 min. For each experiment, control A samples and those incubated with ligands were divided from the same initial A solution. For analyses of fibrils, the pellets were suspended in a small volume of water by low-energy sonication for 5 sec. Aliquots of 8 µL were placed on electron microscopy grids covered by a formvar film. Excess fluid was withdrawn after 30 sec, and after air-drying, the grids were negatively stained with 2% uranyl acetate in water. The stained grids were examined and photographed in a Philips CM120TWIN electron microscope operated at 80 kV. For an evaluation of the amount of material in the different specimens, the grids (50 mesh) were first scanned at low magnification and the number of larger fibril bundles per grid square counted. The specimens were subsequently examined at high magnification to judge the size of the fibril bundles, the presence of smaller fibril aggregates, and the morphology of the individual fibrils.

Thioflavin T (ThT) fluorescence was used to quantify fibril formation of A(12–28), A(12–28; V18A/F19A/F20F), and of A(12–28) plus 1 mM of ligands after 0, 3, 7, and 14 d incubation. The A peptides were dissolved to 100 µM concentration in 10 mM sodium phosphate buffer (pH 6.0) and incubated at 37°C. At the indicated time points, the peptide solutions were mixed with ThT solution in a 1 : 1 ratio, giving an end concentration of ThT of 10 µM. Fluorescence measurements with excitation wavelength 442 nm and emission at 485 nm were made within 1 min after mixing peptide and ThT.

Aggregation of A(1–40) in the presence of KAD, AAA, or KFFE was studied by determining A contents in 20,000g supernatants at different time points after solubilization. The A(1–40) contents were determined by amino acid analysis of duplicate samples.

	Acknowledgments

 
We are grateful to the Swedish Research Council, the Hans Sigrist Foundation, the Swedish Heart-Lung Foundation, and the King Gustaf V 80th Birthday Fund for support.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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