Dimeric structure of transmembrane domain of ... - Wiley Online Library

FEBS Letters 586 (2012) 1687–1692

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Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment Kirill D. Nadezhdin, Olga V. Bocharova, Eduard V. Bocharov ⇑, Alexander S. Arseniev Division of Structural Biology, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS, Str. Miklukho-Maklaya 16/10, Moscow 117997, Russian Federation

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Article history: Received 28 February 2012 Revised 12 April 2012 Accepted 30 April 2012 Available online 11 May 2012 Edited by Christian Griesinger Keywords: Alzheimer’s disease Amyloid precursor protein Transmembrane domain Dimerization NMR spectroscopy Spatial structure

a b s t r a c t Some pathogenic mutations associated with Alzheimer’s disease are thought to affect structuraldynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln686-Lys726 was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy. The APP membrane-spanning a-helix Lys699-Lys724 self-associates in a left-handed parallel dimer through extended heptad repeat motif I702X3M706X2G709X3A713X2I716X3I720X2I723, whereas the juxtamembrane region Gln686-Val695 constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-b peptide. Structured summary of protein interactions: APPjmtm and APPjmtm bind by comigration in gel electrophoresis (View interaction) APPjmtm and APPjmtm bind by nuclear magnetic resonance (View interaction). Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Neurodegenerative Alzheimer’s disease (AD) is the one of leading causes of death among the elderly, especially in developed nations [1]. This kind of dementia progresses with time and is usually symptomatically characterized by irritability, mood swings and memory loss. Physiological analysis of AD patients reveals accumulation of neurofibrillary tangles and amyloids in the brain leading to failure of nerve impulse transmission and ultimately neuron death [1]. The amyloid deposits are formed by amyloid-b peptide (Ab), which is a product of sequential proteolytical cleavage of the type I single-pass membrane glycoprotein called amyloid precursor protein (APP) [1–3]. Ab was recently shown to possess powerful antimicrobial activity and, possibly, to be a component of innate immunity in the mammalian nervous system [4]. Nevertheless, the excessive accumulation of Ab in the brain is believed to play a primary role in the pathogenesis of AD [1]. Proteolytical processing of APP occurs in two main stages. After catalytic cleavage of the APP ectodomain by membrane-anchoring Abbreviations: AD, Alzheimer’s disease; APP, amyloid precursor protein; Ab, amyloid b-peptide; TM, transmembrane; JM, juxtamembrane; APPjmtm, APP686–726 fragment; DPC, dodecylphosphocholine; NOE, nuclear overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single-quantum correlation ⇑ Corresponding author. E-mail address: [email protected] (E.V. Bocharov).

b-secretase a 99-residues, C-terminal fragment C99 is released and then processed in membrane by presenilins, which are responsible for the proteolytic activity of heteromeric c-secretase complex. The c-secretase sequentially cleaves the membrane part of C99 at the so-called e- and c-sites, yielding a set of Ab peptides [1–3]. Alternative processing of APP by membrane-anchoring a-secretase results in release of an 83-residue C-terminal fragment C83, truncated on a cation-binding domain of Ab, with subsequent synthesis of an innocuous p3 peptide via the c-secretase cleavage [1– 3]. The a-secretase cleavage occurs primarily on the cell surface in the cholesterol-poor bulk plasma membrane, whereas b- and csecretase proteolyses occur primarily in cholesterol-rich lipid rafts, most often following internalization of the protein into cholesterol-rich acidic endosomes [5]. The length of Ab varies from 38 to 43 residues, with the majority of Ab40 and Ab42 peptides in typical proportion of 10:1 [1,2]. Inherited mutations in transmembrane (TM) and juxtamembrane (JM) region of APP or in the catalytic site of presenilins induce an increase in Ab42 production, thus causing familial early-onset AD forms [2]. In comparison with Ab40, longer Ab42 peptide has two additional hydrophobic residues on its C-terminus and tends to aggregate rapidly forming oligomers, which are especially toxic to neurons [1,2]. Familial mutations in the APP TM region presumably affect its structure and lateral mobility in membranes; and alter proteolytic processing with the c-secretase complex. The

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.04.062

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appearance of data about possibility of APP to dimerize via specific TM helix–helix interactions [5,6] suggests a new insight on the effects of these familial mutations, implying that they could influence the APP self-association in the membrane. Despite some progress in structural-dynamic studies, only computational models of the dimeric TM domain of APP and its mutant pathogenic forms exist to date, based on the modeling studies alone or in combination with experimental data, e.g. obtained using solid-state NMR spectroscopy [7]. Recently, the secondary structure and interaction with cholesterol of the presumably dimeric C99 fragment were characterized directly by means of NMR methods [5]. The monomeric spatial structure and interaction with cholesterol analog of a recombinant APPjmtm peptide corresponding to the APP686–726 fragment including the intact TM domain with adjacent N-terminal JM region (without cationbinding domain of Ab was investigated by us previously using heteronuclear NMR spectroscopy in a membrane mimetic environment [8]. Now we describe high-resolution structure of the APP TM domain in the dimeric state. 2. Materials and methods 2.1. Preparation of NMR samples of APPjmtm in a membrane mimetic environment The 15N- and 15N/13C-labeled and unlabeled sample of the recombinant peptide APPjmtm (GSQ686KLVFFAEDDVGSNK726 GAIIGLMVGGVVIATVIVITLVMLKKK ) were produced in Escherichia coli and purified as described [9]. (Additional N-terminal residues Gly-Ser remained after hybrid protein cleavage with thrombin protease.) Peptide identity, purity and dimerization were confirmed by mass spectrometry (Supplementary Fig. S1), SDS– PAGE (Supplementary Fig. S2) and by analyzing of 1H/15N-HSQC NMR spectra (Fig. 1A) of the labeled peptides. Two APPjmtm samples were prepared: uniformly 15N-labeled and 1:1 mixture of unlabeled and 15N/13C-labeled peptide (so-called ‘‘isotopic-heterodimer’’). The peptide’s powder of both sample species was first dissolved in 1:1 trifluoroethanol–water mixture with addition of deuterated DPC (d38, 98%, CIL), phosphate buffer and EDTA, then kept for several minutes in an ultrasound bath and lyophilized. After that dried 15N-labeled and isotopic-heterodimer samples were dissolved in 350 ll of H2O (with addition 5% of D2O) and D2O, respectively. To attain uniformity of the micelle size, several freeze–thaw cycles (heating to 40–45 °C) were carried out, followed by storage in an ultrasound bath in order to achieve complete transparency of the solution. The APPjmtm concentration of the 15N-labeled and isotopic-heterodimer samples was 0.9 and 1.5 mM, respectively, at the peptide/DPC molar ratio of 1:50, pH 6.2, 20 mM phosphate buffer, 1 mM EDTA. Both samples were placed in Shigemi NMR tubes with glass plunger. 2.2. NMR spectroscopy and spatial structure calculation NMR spectra were acquired at 45 °C on 600 and 800 MHz AVANCE III spectrometers (Bruker BioSpin, Germany) equipped with the pulsed-field gradient triple-resonance cryoprobes. 1H, 13 C, and 15N resonances of APPjmtm were assigned with CARA software [10] using two- and three-dimensional heteronuclear experiments [11]: 1H/15N-HSQC, 1H/15N-TROSY, 1H/13C-HSQC, 1H/15NHNHA, 1H/13C/15N-HNCA, 1H/13C/15N-HN(CO)CA, 1H/13C/15NHNCO, 1H/13C-TOCSY-HSQC, 13C- and 15N-edited NOESY-HSQC (recorded on 600 and 800 MHz spectrometers, respectively). To characterize intramolecular dynamics the effective rotation correlation times sR were calculated for individual amide groups of 15N-labeled APPjmtm from the ratio of transverse and longitudinal 15N-

Fig. 1. Heteronuclear NMR spectra of APPjmtm solubilized in an aqueous suspension of DPC micelles at the peptide/detergent ratio of 1/50, 45 °C and pH 6.2. (A) 1 H/15N-HSQC spectrum acquired for the 15N-labeled APPjmtm sample is shown with the 1H–15N backbone and side-chain resonance assignments. Distinct crosspeaks for dimeric and monomeric states are labeled with ‘‘d’’ and ‘‘m’’, respectively. The investigation of the dimer-monomer equilibrium revealed an enhancement of the APPjmtm dimerization when the micelle occupied by more than one peptide (the case of ‘‘saturated’’ micelle [26]), which corresponds to the P/D values more than 1/70 (see Supplementary Fig. S3). (B) Strips from three-dimensional 15N,13C F1-filtered/F3-edited-NOESY spectrum [15] acquired for the ‘‘isotopic-heterodimer’’ APPjmtm sample are shown with assignments of intermonomeric NOE cross-peaks. The residual diagonal peaks are observed as paired peaks.

relaxation rates as described in [12]. Exchange rates between water and HN protons were analyzed by detection of peaks in CLEANEX spectrum [11] recorded for 15N-labeled APPjmtm sample, and observed cross-peaks were treated as an indication of water exposed amide groups. Dimeric spatial structure of APPjmtm was calculated with CYANA program [13] based on torsion angle restraints estimated from the chemical shift values with standard protocol of TALOS + program [14] and intra- and inter-monomeric


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Fig. 2. NMR data for the APPjmtm embedded into DPC micelles at P/D = 1/50, 45 °C, pH 6.2. (A) Interproton NOE connectivities observed in the 1H/15N-NOESY-HSQC spectrum acquired with 40-ms mixing time. (B) Water accessibility of the backbone amide groups of APPjmtm. Filled and open circles indicate amide protons for which fast and medium exchange with water were observed, respectively. For residues without any circles ether no peak was presented in CLEANEX spectrum [11] or corresponding peak was not uniquely assigned due to spectral overlapping. (C) Secondary 13Ca chemical shifts of APPjmtm are given by the difference between the actual chemical shift and typical random-coil chemical shift for a given residue. Pronounced positive or negative Dd(13Ca)h–c values indicate a helical structure or an extended conformation of a protein [11]. (D) Secondary 1HN chemical shifts of the APPjmtm dimer. The Dd(1HN)h–c value strongly depends on the length of the corresponding hydrogen bond, so the local increase in Dd(1HN)h–c indicates shortened hydrogen bond. The 0.5 ppm variation observed for Dd(1HN)h–c with the step of 4 residues along the TM-helix implies the periodic 0.2 Å length alteration of the helical OCi HNi+4 hydrogen bonds [19]. (E) Difference between 1HN chemical shifts of the dimeric and monomeric APPjmtm states. Downfield 1HN chemical shifts (positive Dd(1HN)d–m values) assume shortened hydrogen bonds, and upfield shifts (negative Dd(1HN)d–m values) denote elongated ones upon the dimerization of the TM-helices. Observed variation of Dd(1HN)d–m is rather small and do not exceed 0.1 ppm corresponding to the length variations of helical hydrogen bonds of 0.05 Å at most [19] that can not be detected from NOE data. (F) 15N-relaxation data of the APPjmtm amide groups are presented by local effective rotation correlation times sR. The sR values corresponding to the APPjmtm dimeric state are shown in dark grey, whereas others obtained for distinct monomer cross-peaks or overlapping dimer-monomer crosspeaks in 1H/15N-HSQC spectrum (Fig. 1A) are highlighted in light grey. Sequential c-secretase cleavage sites and mutations associated with familial forms of Alzheimer’s disease [1–3] are indicated by arrows at the bottom of the APPjmtm amino acid sequence.

NOE distance restraints derived through the analysis of threedimensional 15N- and 13C-edited NOESY and 15N,13C-F1-filtered/ F3-edited-NOESY spectra [11,15] acquired for the 15N-labeled and ‘‘isotopic-heterodimer’’ APPjmtm samples. Detailed procedure

of spatial structure calculation is described in Supplementary data. Hydrophobic properties of the a-helices in the APPjmtm dimer were calculated using the molecular hydrophobicity potential (MHP) approach [16] as described in Supplementary data. The con-

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tact area between the dimer subunits was calculated using the DSSP program [17] as a difference between the accessible surface areas of APPjmtm residues in the monomeric and dimeric states. The APPjmtm dimer structures were visualized with MOLMOL [18]. 3. Results and discussion 3.1. APPjmtm undergoes a slow monomer–dimer transition in the micellar environment Heteronuclear NMR 1H/15N-HSQC spectrum of APPjmtm (Fig. 1A) solubilized in an aqueous suspension of DPC micelles at the peptide/detergent (P/D) ratio of 1/50 (45 °C, pH 6.2) revealed at least 18 more cross-peaks than at the P/D ratio of 1/120 and than was expected from the amino acid sequence. It was suggested that

new ensemble of peaks is induced by self-association of APPjmtm in saturated micelles. This hypothesis was later confirmed by the analysis of 15N,13C-F1-filtered/F3-edited-NOESY spectrum acquired at the P/D ratio of 1/50 that reveals the characteristic NOE connectivities for a dimeric structure of APPjmtm (Fig. 1B). At such P/D value the volume ratio in the cross-peaks pairs was about 60/40 in favor of the dimeric state. At higher P/D values the dimer population is increased rapidly (see Supplementary Fig. S3), but precipitation of the peptide occurs within 1 h. Since the sample precipitation does not happen during 1–2 weeks when the P/D ratio is about 1/50, this value has been chosen as a trade-off between the sample life time and relatively high population of the APPjmtm dimeric form that allows to record high quality NMR spectra for assignment and structure calculation. As the minimal distinguishable chemical shift difference between signals of two states in

Fig. 3. Spatial structure of the APPjmtm dimer. (A) The 12 NMR structures of the APPjmtm dimer are superimposed on backbone atoms of the TM-helices (Lys699-Lys724)2. Alternative superposition on backbone atoms of the nascent JM-helix Gln686-Val695 is shown for one dimer subunit after a gap (yellow lines) between loop residues Gly696 and Ser697. Heavy atom bonds are shown only. Backbone is highlighted in black, side-chains are painted in blue and magenta for different dimer subunits. (B) Schematic representation of the APPjmtm dimer spatial structure. Cylinders illustrate the JM- and TM-helices; the last have a kink near the paired residues Gly708-Gly709. (C) Hydrophobic and hydrophilic properties of the APPjmtm dimer interface. At left, the hydrophobicity map of the molecular surface of the APPjmtm TM-helix with contour isolines encircling hydrophobic regions with high values of molecular hydrophobicity potential (MHP) [16] (see Supplementary data). The map is presented in cylindrical coordinates associated with the TM-helix. The helix–helix packing interface is indicated by violet points. Residues composing characteristic dimerization GG4-like motifs G700X3G704GX3G708 (unemployed) and G709X3A713 (employed) are highlighted in green and red, respectively. Residues, mutations of which are associated with familial forms of Alzheimer’s disease, are indicated by yellow ovals. At right, the hydrophobic and hydrophilic (polar) surfaces of the TM-helix of the APPjmtm dimer subunit colored in yellow and green, respectively, according to MHP.


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1

H/15N-HSQC spectrum is 20 Hz, the monomer–dimer transition is a slow process (on the millisecond timescale).

APPjmtm can be explained by slight stretching of the TM-helix upon dimerization.

3.2. Dimeric structure of APPjmtm TM region

3.3. Nascent helical structure of APPjmtm JM region

Pattern of intra-monomeric NOE connectivities (Fig. 2A) along with distribution of positive secondary 13Ca chemical shifts, Dd(13Ca)h–c Fig. 2C, revealed distinct helical structure along the APPjmtm TM span in the micellar environment. Six upper-limit inter-monomeric NOE connectivities per each dimer subunit were assigned unambiguously (Fig. 1B, Supplementary Table S1 and Fig. S4). Since only one set of signals can be seen in 1H/15N-HSQC spectra for APPjmtm in the dimeric state, indicating that its conformation is symmetrical on the NMR timescale, the identified intraand inter-monomeric NOE connectivities as well as torsion angle restraints derived from the chemical shifts were symmetrized in order to provide spatial structure of the dimer (Fig. 3A). The overview of the input data, statistics and parameters for the ensemble of 12 structures of the APPjmtm dimer with the lowest target function is represented in Supplementary Table S2. The atomic coordinates and experimental restraints were deposited in the Protein Data Bank under accession code PDB ID: 2loh. The 38 Å TM-helix (residues 699–724) of APPjmtm consists of the uncharged region Gly700-Leu723 flanked by positively charged Lys699 and Lys724, which presumably interact as anchors with negatively charged phosphate groups of detergent heads (Fig. 3 and Supplementary Fig. S5). There is a 17 ± 7° kink of the TM-helix near the paired residues Gly708-Gly709 (Fig. 3B). The concavity with a weakly polar surface formed by residues Gly700, Gly704, Gly708 and Gly709 (Fig. 3C) covers the N-terminal half of the TM-helix. This apparently results in the observed (i + 4) periodicity of the secondary 1HN chemical shift, Dd(1HN)h–c (Fig. 2D), indicating [19] that amide groups of these glycine residues form shorter hydrogen bonds as compared with the residues located on the opposite side of the TM-helix. In the C-terminal part of the TM-helix, besides backbone helical hydrogen-bonding, the side-chain hydroxyl OcH groups of Thr714 and Thr719 form hydrogen bonds with the backbone carbonyl groups of Val710 and Val715 (according to d(1HOc) values and local NOE pattern). In the micellar environment, the APPjmtm TM-helices (Lys699Lys724)2 associate in a left-handed parallel dimer via extended heptad repeat [20] motif I702X3M706X2G709X3A713X2I716X3I720X2I723 with the distance d between helix axes of 8.3 ± 0.5 Å and the contact surface area of 630 ± 60 Å2. In C-terminal part (Gly709-Lys724)2 of the dimer, where spatial structure is determined more precisely, the helix–helix crossing angle h is equal to 26 ± 4°, whereas in the N-terminal part (Lys699-Gly708)2 the h value amounts to 60 ± 11°. The left-handed TM-helix pairing is mostly stabilized along the heptad repeat by van-der-Waals contacts of large hydrophobic side-chains of isoleucine, methionine, valine, and leucine residues, while slightly polar interactions of residues G709X3A713 having small side-chains (as part of so-called GG4-motif [21]) are observed in the middle of the helix–helix interface (Fig. 3C) where helices are maximally close. The almost identical NMR spectra for monomeric and dimeric forms of APPjmtm indicate that the overall structure of two subunits does not undergo significant changes upon the helix–helix interaction. Nevertheless, a variation of differences between dimeric and monomeric 1HN chemical shifts, Dd(1HN)d–m (Fig. 2E), observed with the step of 3–4 residues along the N-terminal half of the TM-helix could be explained by small periodical changes in helical hydrogen bond lengths [19]. So, the Dd(1HN)d–m variation, which is more pronounced for Met706 and Gly709 situated exactly in the helix–helix interface, can be interpreted as slight bending of Ile702-Ile712 region in consequence of the dimerization process. Additionally, positive Dd(1HN)d–m values near the C-terminus of

According to local effective rotation correlation times sR estimated for amide groups of APPjmtm (Fig. 2F), its N-terminal JM region is more mobile in comparison with the TM-helix. Measured 3 JHnHa coupling constants of about 5–7 Hz indicate that this region does not form a tight secondary structure. Nevertheless, dihedral angle constrains predicted from the 1H, 13C, and 15N chemical shifts and a few detected daN(i,i + 3) NOE connectivities (Fig. 2A) suggest that Gln686-Val695 region forms a short nascent helix (Fig. 3A, B and Supplementary Fig. S5), possibly undergoing helix–coil transitions. CLEANEX experiment [11] reveals that amide protons from the region Leu688-Gly696 is less water-accessible than the adjacent residues (Fig. 2B), probably due to effective shielding from solvent caused by hydrogen-bonding within the nascent helix or/and by deepening this region into DPC micelle mediated by hydrophobic side-chains of Leu688-Val689. This is in reasonable agreement with the previously solved APPjmtm monomeric structure (at pH 4.6) [8] and with the investigations of C99 fragment (at pH 6.5) [5]. Potential salt bridge between side-chains of Lys699 and Glu693 or Asp694 could stabilize V695GSN698 turn, the latter having been shown important for aggregation of Ab and fibril formation [22,23]. Available NMR data do not allow defining conformation of this region, resulting in an ambiguity in position of nascent JM-helix with respect to the TM-helix (Fig. 3A, B). Intriguingly, the changes of backbone and side-chain proton chemical shifts upon the TM-helix dimerization are also pronounced in the N-terminal JM region of APPjmtm (Fig. 2E), indicating that the dimerization affects this region in spite of its apparent mobility. 3.4. Dimerization alternatives and sequential proteolysis of APP TM domain Understanding the possible dimeric structure of APP and its proteolytic derivatives in the membrane would provide a relevant insight into the molecular mechanisms underlying the sequential cleavage by c-secretase resulting in variation of Ab levels. Currently, two alternative modes of parallel helix–helix interactions employing different characteristic GG4-motifs [21] have been proposed for the APP TM domain self-association on the basis of computational searches and scanning mutagenesis accomplished by FRET studies [7,24,25]. The first of them, a right-handed TM dimeric structure mediated by two consecutive GG4-motifs G700X3G704GX3G708 is in good agreement with the recent solidstate NMR studies of oligomerization of APP TM fragment Glu693Lys735 reconstituted into DMPC:DMPG (10:3) lipid bilayers [7]. Alternatively, present study shows that being embedded into the DPC micelle, another APP TM fragment Gln686-Lys726 self-associates in a symmetric left-handed dimer via GG4-motif G709X3A713 encompassing the c-secretase cleavage sites (yielding Ab40 and Ab42, see Fig. 2). It is interesting to note that several familial AD mutations are situated exactly in the TM helix–helix interface identified in the DPC micelles (Figs. 2 and 3C). Overall, spatial structure of APP transmembrane domain found in DPC micelles might prove helpful for explaining the appearing biochemical data. However, the structural alternatives suggested by different arrays of experimental data are not necessarily mutually exclusive. Moreover, all the various dimerization motifs can be functionally relevant in different circumstances, such as local lipidic composition of the membrane, surface pH, or interaction with other membrane-bound proteins, especially since our present knowledge of APP functions is not exhaustive. The observed kink of the TM-helix can serve for adapting APP TM domain structure to the membrane

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thickness [23], allowing for different dimerization modes to be realized in appropriate conditions. The sequential c-secretase cleavage (occurring in the vicinity of lipid polar heads) can initiate transitions between various dimerization modes, or even cause new modes to appear as a result of sinking of the N-terminal JM region, which contains an additional GG4-motif G696AIIG700, into the membrane. Thus, the conformational diversity of APP appears to require detailed investigation in different conditions, and not only for the wild and mutated proteins, but also for their proteolytic derivatives. This information can be valuable for the development of novel therapeutic agents, such as small molecules or TM peptides stabilizing certain conformation of APP in membrane for controlling Ab production. Acknowledgements

[8]

[9]

[10] [11]

[12]

[13]

This work was supported by the Russian Foundation for Basic Research (11-04-01795), the Program of the Russian Academy of Science ‘‘Molecular and Cellular Biology’’, the Russian Funds Investment Group, the Federal Target Program ‘‘Research and development in priority fields of Russian scientific and technological complex in 2007–2013’’ (16.512.11.2079). Bocharov E.V. thanks personally Beirit K.A. for financial support. The authors are grateful to Ziganshin R.Kh. for the MS measurements.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012. 04.062.

[15]

[16]

[17]

[19] [20]

[21]

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