Tài liệu Báo cáo khoa học: Oxidation inhibits amyloid fibril formation of transthyretin ppt

Oxidation inhibits amyloid fibril formation of transthyretin Simin D. Maleknia 1 , Nata ` lia Reixach 2 and Joel N. Buxbaum 2 1 School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, NSW, Australia 2 Division of Rheumatology Research, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA, USA Protein oxidation has been implicated in a wide range of diseases, and ageing [1–4]. Reactive oxygen species (ROS) contribute to processes that induce irreversible structural damage and alter protein activity. Oxygen- containing radicals, in particular the hydroxy radical, react with proteins through hydrogen abstraction, addition and elimination reactions at both the amino acid side chains and backbone amide bonds to produce oxidized, degraded, and cross-linked proteins [2,5,6]. The oxidized cross-linked products and protein aggre- gates have been identified as insoluble proteins in many diseased tissues including amyloid fibrils [7,8]. We are investigating the role of amino acid side chain oxidation in amyloid assemblies by comparing the kinetics of fibril formation of native and oxidized proteins. Interactions between amino acid side chains help to stabilize protein structures and control folding and the assembly of complexes [9,10]. The nature of amino acid side chain bonds and their thermodynamic stabil- ity direct the formation of secondary structure in pro- teins [11,12], and these types of information are useful in predicting misfolding or aggregation events in rela- tion to disease [13,14]. Oxidation of amino acids may alter their tertiary structure contacts, and oxidation can be used as a facile method of investigating the Keywords amyloid fibril; footprinting; radical probe mass spectometry; reactive oxygen species; transthyretin Correspondence S. D. Maleknia, School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, NSW 2052, Australia E-mail: s.maleknia@unsw.edu.au (Received 11 July 2006, revised 28 Septem- ber 2006, accepted 9 October 2006) doi:10.1111/j.1742-4658.2006.05532.x The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on pro- tein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing pro- tein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril forma- tion, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by com- paring the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxi- dative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril forma- tion for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by  76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures. Abbreviations ROS, reactive oxygen species; TTR, transthyretin. 5400 FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS residues and interactions that are critical in stabilizing protein structures and folding. The amyloidoses are a group of protein-misfolding diseases that result from deposition of proteins nor- mally soluble under physiological conditions [15–18]. These include Alzheimer’s disease, Creutzfeldt–Jakob disease, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy and senile systemic amy- loidosis. Transthyretin (TTR) is a homotetrameric plasma protein associated with the transport of thyrox- ine and vitamin A [19]. Deposition of the wild-type (WT) protein has been associated with senile systemic amyloidosis [20], and more than 80 TTR variants have been linked to familial amyloidotic polyneuropathy and familial amyloidotic cardiomyopathy when depos- ition occurs in peripheral nerve and heart, respectively [21]. The kinetics of fibril formation of TTR and its variants have been the subject of many studies [22–24], and TTR makes an ideal model system for investi- gating the effects of protein oxidation. Although the onset of amyloidogenesis is not well understood, in vitro studies suggest that the molecular mechanism of amyloid fibril formation is based on dis- sociation of the tetrameric protein into its monomeric subunits, which, upon misfolding, self-assemble to form insoluble fibrils [25,26]. Further studies have shown that mutant proteins with modified disulfide bonds are more susceptible to fibril formation, suggest- ing that tetramer dissociation may not be the rate- limiting step in fibril kinetics [27]. Moreover, mutations of single amino acids alter the kinetics of fibril forma- tion. For example, familial mutations in which valine at position 30 has been substituted with methionine (V30M) or leucine at position 55 has been replaced with proline (L55P) increase fibril formation kinetics [28,29]. Accordingly in this study, we investigated the effects of protein oxidation by comparing the kinetics of fibril formation of WT and V30M TTR mutant with their oxidized counterparts. Results and Discussion Reactions of proteins with ROS induce predominantly covalent modification of amino acid side chains [2,5,6]. The amino acids methionine, cysteine, phenylalanine, tyrosine, tryptophan, proline, histidine, leucine and lysine are most susceptible to reactions with ROS [5,6,30,31]. When reactions are restricted to millisecond timescales, limited oxidation of amino acid side chains occurs without structural damage. This limited oxida- tion method, termed radical probe mass spectometry [6,30], has been utilized for probing protein structure [32], folding [33] and interactions [34,35]. As the reac- tion timescale increases, backbone cleavage and aggre- gation reactions occur [6], resulting in the possibility of structural damage [36]. The dose-dependent oxidation method has been applied to the study of protein stabil- ity and the onset of oxidative damage [36]. The present study expands the utility of radical probe mass specto- metry in investigating side chain interactions that are critical in stabilizing protein assemblies. Oxidized proteins for this study were prepared by reaction with hydrogen peroxide [37] in a timescale range of 10 min to 12 h. Oxidation of WT and V30M TTR proteins in these timescales increased their molecular masses by 80 Da, indicating that up to five oxygen atoms were incorporated into the protein structure. Electrospray mass spectometry (ESI-MS) analysis also revealed that, after reaction with hydrogen peroxide, these proteins were nearly all oxidized (i.e. oxidized samples did not contain unre- acted proteins). To verify that this level of oxidation did not disturb the tetrameric structure of TTR, glu- taraldehyde cross-linking reactions were performed for WT and V30M TTR and their oxidized forms. Products of cross-linking reactions were analyzed by gel electrophoresis (data not shown). The unoxidized and oxidized proteins contained similar cross-linking products, and a dominant band of 55 kDa signified that tetrameric structures of WT and V30M TTR were preserved after oxidation. These results suggest that oxidation in these timescales did not alter the structure of TTR significantly, and the oxidized pro- teins maintained tetrameric structures. In vitro fibril formation of TTR was performed to compare the effects of amino acid side chain oxidation. Structural transitions of proteins to amyloid fibrils can be followed under laboratory conditions by exposing the folded protein to mildly denaturing conditions such as low pH or elevated temperatures [28]. TTR can be converted into amyloid fibrils through a pH-mediated tetramer-dissociation step. The in vitro mechanism of fibril formation is believed to involve tertiary structural changes at low pH resulting in the formation of mono- meric amyloidogenic intermediates that can self-assem- ble into fibrils [21,26]. Oxidation of amino acid side chains is used in this study to facilitate generation of new TTR variants, and the kinetics of fibril formation of these oxidized proteins reveal the amino acid inter- actions that are critical in the onset of amyloido- genesis. The rates of amyloid fibril formation for WT and V30M TTR and their oxidized forms were monitored by turbidity measurement at 330 nm and 400 nm. These absorbance measurements detect both fibrils and aggregates [24]. The results of measurements at 330 S. D. Maleknia et al. Oxidation inhibits amyloid fibril formation of TTR FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS 5401 and 400 nm in this study were similar, and therefore only the 330-nm data are discussed here. The kinetics of fibril formation for the unoxidized proteins and oxidized proteins resulting from the 12-h reaction with hydrogen peroxide are shown in Fig. 1. Fibril growth was followed as a function of time for up to 14 days. These results show that both the unoxidized and oxid- ized proteins could form fibrils. The absorbance meas- urements (Fig. 1) show the normal pattern of an initial exponential fibril growth over the 5-day period fol- lowed by a slower growth period as a function of time. As the concentration and buffers for all samples were similar and the oxidized samples did not contain signi- ficant amounts of unreacted protein, differences in tur- bidity measurements reflect the effects of amino acid side chain oxidation on fibril growth kinetics. Oxida- tion had a dramatic affect on initial rates (slopes of tangent lines to experimental curves up to t ¼ 24 h) of fibril growth for both WT and V30M TTR. Larger effects on the kinetics of fibril formation were seen for oxidized V30M TTR compared to the unoxidized V30M TTR than for oxidized WT TTR compared to unoxidized WT TTR, consistent with the fact that in V30M TTR there is one more methionine available for oxidation than in WT TTR. While fibril growth progressed over the 14 days, oxi- dation inhibited the extent of fibril formation overall for both the WT and V30M TTR proteins. The extent of fibril formation can be calculated as the percentage of the turbidity (absorbance at 330 nm) of the oxidized proteins divided by the turbidity of the unoxidized proteins. Oxidation reduced fibril growth of the WT protein by  76% after 1 day to  60% after 14 days. In the case of V30M TTR protein, oxidation reduced fibril growth by 90% after 1 day and 74% after 14 days. After 1 day of incubation, 60% of the unoxi- dized V30M TTR was in the supernatant, whereas 80% of the oxidized protein was in the supernatant. After 3 days of incubation, the values were 27% for the unoxidized V30M TTR and 44% for the oxidized protein. These data show that the decrease in turbidity is not due to different properties of the fibril formed by oxidized relative to unoxidized protein, rather the differences observed reflect true inhibition of fibril formation. A similar effect was observed for both the WT and V30M TTR when they were reacted with ROS on shorter timescales. The percentages of fibril formation over time for V30M TTR are compared in Fig. 2 for unoxidized and oxidized proteins from reactions with hydrogen peroxide for 10 min and 1 h. These results show that shorter reaction times of 10 min are suffi- cient to inhibit the growth of fibrils, although the extent is somewhat smaller; for example, after 1 day, inhibition of fibril formation decreased from 90% for the 1 h oxidation treatment to 84% for the 10 min oxi- dation preparation. Oxidation of amino acid side chains follows their order of solvent accessibility when oxidative reactions are performed in millisecond timescales [6,30–36]. The reaction time influences the level of oxidation at each reactive residue. The site of oxidation of amino acid side chains was investigated after proteolysis by mass spectometry sequencing. Methionine residues are highly reactive and oxidize readily in the presence of ROS [5,6,37]. The WT contains methionine at posi- tions )1 (methionine resulting from the recombinant preparation) and 13. V30M TTR contains an additional methionine at position 30 [38]. These methionine residues were highly oxidized to their mono-oxidized and di-oxidized forms. The oxidation of Met13 can be explained by an accessible surface area of 22.8 A ˚ 2 [solvent accessible surface area calcu- lated for V30M TTR monomer (Protein Data Bank entry1TTC) and based on the percentage of the maximum possible exposure of the C-terminal Glu127 350 300 250 200 150 100 50 0 0.0 0.1 0.2 0.3 0.4 0.5 incubation time (h) A 330 nm V30M TTR V30M TTR Oxidize d WT TTR WT TTR Oxidized Fig. 1. Kinetics of fibril formation monitored at 330 nm for WT TTR, V30M TTR and their oxidation products after reaction with hydro- gen peroxide for 1 h. 0 25 50 75 100 336120 72246 incubation time (h) % fibril formation 60 min oxidation10 min oxidationunoxidized Fig. 2. Percentage of TTR fibril formation over time for V30M TTR and its oxidized forms from reaction with hydrogen peroxide for 10 min and 1 h. Absorbance measurements (A 330 ) for each dataset normalized to absorbance of unoxidized V30M TTR on day 14. %Fibrils ¼ [A 330nm (oxidized) ⁄ A 330nm (unoxidized)] x 100. Oxidation inhibits amyloid fibril formation of TTR S. D. Maleknia et al. 5402 FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS residue]. However, Met30 is not solvent accessible and was completely oxidized [39]. Oxidation of the methionine residues to their mono- oxidized and di-oxidized forms was confirmed by mass spectometry sequencing. Figure 3 shows post-source decay sequencing mass spectra for the di-oxidized (after reaction with ROS) and unoxidized tryptic pep- tides covering residues 23–35 for V30M TTR. The protonated di-oxidized tryptic peptide is observed at m/z 1430.5. Oxidation of the methionine residue is verified, as C-terminus fragment ions from y 5 (MHVFR) to y 8 (NVAMHVFR) are shifted by 32u, indicating the addition of two oxygen atoms on this methionine residue. The y 1 to y 4 remain unchanged, signifying that the C-terminal HVFR portion of this peptide was not oxidized. The N-terminal fragment ions b 3 to b 8 remain unchanged, indicating that the GSPAINVA portion is not oxidized, and (b 10 +32) and (b 11 +32) ions signify that oxidation is exclusive to the methionine residue. These results confirm that the methionine residues of WT TTR and V30M TTR are highly reactive toward oxidative modification. The inhibition effects of fibril formation for these oxidized proteins are intriguing and show that side chain oxidation can be used as a method of inducing mutations in protein sequences to investigate amino acids that are critical in preserving a protein’s structure and stability [36]. Interestingly, in vitro studies of a 17-residue peptide showed that replacement of methi- onine residues with their oxidized forms eliminated fibril formation [40]. In the case of TTR, dissociation of the tetramers into monomers is believed to be a pre- liminary and limiting step of the fibril formation pro- cess [26]. This inhibition of fibril formation seen in the oxidized proteins suggests that they are more stable than the unoxidized forms. Whereas changing the valine residue at position 30 to methionine increases the amyloidogenesis of TTR [28,29], oxidation of the methionine is shown here to partially inhibit fibril growth. The amino acid side chain oxidation may have Fig. 3. Post-source decay sequencing mass spectra for (top) di-oxidized and (bottom) unoxidized tryptic peptides showing the oxidation of methionine after reaction of V30M TTR with ROS. S. D. Maleknia et al. Oxidation inhibits amyloid fibril formation of TTR FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS 5403 altered tertiary contacts in a manner that stabilized the oxidized tetramers. We speculate that oxidation may have introduced new tertiary contacts that stabilized the folded monomeric structure of the oxidized pro- teins and inhibited the formation of the unfolded monomer, which has been proposed [25,26] to be a prerequisite for fibril growth. Together these effects caused a delay in the onset of amyloid fibril formation. Alternatively, the inhibition of fibril formation may purely be the result of an increase in solubility of oxid- ized proteins [6,41]. Limited oxidation increases the hydrophilicity of proteins as determined by their elu- tion times from hydrophobic columns [6,31,32]. On the basis of liquid chromatography ⁄ ESI-MS analysis under similar conditions, oxidized TTR proteins were eluted  40 s faster than their unoxidized forms, indicating an increase in their hydrophilicity. These results show that amino acid side chain oxida- tion can be used as a method of investigating regions of proteins that are critical in the onset of amyloid forma- tion. This study reveals that domains neighboring methionine residues are critical in the formation of fibril assemblies. These oxidation reactions are being followed in shorter timescales to possibly distinguish between the oxidation of Met13 and Met30 in order to more accu- rately define the key residues of amyloid fibril inhibition. The timescales of reactions with hydrogen peroxide are limiting, yet ROS can be generated by an electrospray discharge source [30] that has been shown to generate a high flux of ROS on millisecond timescales for studies of protein structures [6,30–36]. Alternatively, other mutant proteins could be designed to further investigate the effect of fibril formation by substituting amino acids neighboring methionine residues. Studies revealing the onset and growth of amyloid fibrils are necessary to understand the pathological con- ditions that lead to many diseases. Valuable information can be gained on why certain mutants have a greater propensity to form fibrils or to inhibit fibrils in compar- ison with their respective native proteins. Identifying protein sequences or domains that are critical in preser- ving protein stability and function should provide opportunities for prevention and treatment of diseases. Experimental procedures Two variants of WT TTR and V30M TTR were selected for this study. These proteins were expressed in an Escherichia coli system as described elsewhere [29]. The proteins were purified by gel-filtration chromatography on a Superdex 75 column (Amersham Biosciences, Uppsala, Sweden) in 10 mm sodium phosphate buffer (pH 7.6) ⁄ 100 mm KCl ⁄ 1mm EDTA. Oxidized proteins were pre- pared by allowing the proteins (35 lm) to react with hydrogen peroxide (reagent-grade; 30 mgÆmL )1 ; Sigma Chemicals, St Louis, MO, USA) at a concentration of 2.7% peroxide. The oxidation reactions were performed at pH 7.6 in a timescale range of 10 min to 12 h. The oxid- ized proteins were then purified from the hydrogen perox- ide reagent through extensive buffer exchange [10 mm phosphate buffer (pH 7.6) ⁄ 100 mm KCl ⁄ 1mm EDTA] with centriprep devices with 10-kDa filters (Millipore, Bill- erica, MA, USA). The concentrations of all protein solu- tions were adjusted to 10 lm with the sodium phosphate buffer at pH 7.6 based on A 280 . The proteins were ana- lyzed by liquid chromatography ⁄ ESI-MS to verify their molecular masses and extent of oxidation. Proteins were also digested with trypsin, and post-source decay sequen- cing experiments identified the site of amino acid side chain modification. Kinetics of amyloid fibril formation Chemical cross-linking was performed to check that the tetrameric structure of proteins was preserved after the oxi- dation reactions. Glutaraldehyde (25%) was added to pro- tein solutions (10% v ⁄ v), and incubated for 4 min. The reaction was quenched by the addition of NaBH 4 (7% in 0.1 m NaOH). The samples were analyzed by 1D SDS ⁄ PAGE, and protein bands were visualized with Coomassie blue stain. 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FASEB J 2, 2591–2595. 42 Hammarstrom P, Jiang X, Hurshman AR, Powers ET & Kelly JW (2002) Sequence-dependent denatura- tion energetics: a major determinant in amyloid disease diversity. Proc Natl Acad Sci USA 99, 16427–16432. Oxidation inhibits amyloid fibril formation of TTR S. D. Maleknia et al. 5406 FEBS Journal 273 (2006) 5400–5406 ª 2006 The Authors Journal compilation ª 2006 FEBS . showing the oxidation of methionine after reaction of V30M TTR with ROS. S. D. Maleknia et al. Oxidation inhibits amyloid fibril formation of TTR FEBS Journal. including amyloid fibrils [7,8]. We are investigating the role of amino acid side chain oxidation in amyloid assemblies by comparing the kinetics of fibril formation