Nitration of Tyrosine Residue Y10 of Aβ1-42 Significantly Inhibits Its Aggregation and Cytotoxicity.

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Nitration of tyrosine residue Y10 of A#1-42 significantly inhibits its aggregation and cytotoxicity Jie Zhao, Jinming Wu, Zhen Yang, Hailing Li, and Zhonghong Gao Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00447 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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Chemical Research in Toxicology

Nitration of tyrosine residue Y10 of Aβ1-42 significantly inhibits its aggregation and cytotoxicity

Jie Zhaoa, Jinming Wua, Zhen Yangb, Hailing Lia,c, Zhonghong Gaoa,c a. School of Chemistry and Chemical Engineering, Hua Zhong University of Science and Technology, Wuhan 430074, People’s Republic of China. b. Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77004, United States c. Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, Wuhan, 430074, P.R. China

E-mail address:[email protected] ABSTRACT Amyloid-β plaques and oxidative stress are the major hallmarks of Alzheimer's disease. Our previous study found that: heme-Aβ complex enhanced the catalytic effect of free heme on protein tyrosine nitration in the presence of hydrogen peroxide (H2O2), nitrite (NO2-). Y10 in Aβ could be the first target to be nitrated. We also found that nitration of Aβ1-40 significantly decreased its aggregation. However, a contrary report showed that nitration of Aβ1-42 by peroxynitrite enhanced its aggregation. To rule out the interference of peroxynitrite caused Aβ oxidation, we used synthetic Y10 nitrated Aβ1-42 to study the influence of Y10 nitration on Aβ1-42’s aggregation and cytotoxicity in this study. We confirmed that Aβ1-42 could be nitrated in the presence

ACS Paragon Plus Environment


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of H2O2, NO2- and heme by dot blotting. CD spectroscopy showed an increase of βsheet structure of Aβ1-42 and its mutants. Thioflavin T (ThT) flourescence assay revealed that both nitration and chlorination significantly inhibited Aβ1-42 fibril formation. TEM and AFM observations of Aβ peptide aggregates further confirmed that Y10 modification inhibited Aβ1-42 fibril formation. The cytotoxicity study of native and modified Aβ peptides on SH-SY5Y cells revealed that nitration of Aβ1-42 remarkably decreased the neurotoxicity of Aβ1-42. Based on these results, we hypothesized that nitration of Y10 may block the π-π stacking interactions of Aβ1-42 so that inhibit its aggregation and neurotoxicity. More importantly, considerable evidence suggested that the levels of nitrite plus nitrate significantly decreased in brain of AD patients. Thus, we believe that these findings would be helpful for further understanding the function of Aβ in AD. Keywords : Alzheimer's disease, Aβ1-42 , aggregation and neurotoxicity, nitration 1.

Introduction

β-amyloid protein, a main protein component of senile plaques, has been found to play a key role in the onset and development of AD.1,2 Aβ is produced from cleavage of the amyloid precursor protein (APP) by β- and γ- secretases,3,4 and the two main common forms of Aβ are Aβ1-40 and Aβ1-42.5 Another key hallmark in AD is the oxidative stress. Interestingly, many evidences consider that oxidative stress is associated with the Aβ generation, and it could increase Aβ production by increasing activity of β- and γ- secretases.6,7 Protein tyrosine nitration is a protein post-translation modification through adding a nitro group to the 3-position of


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phenolic ring of tyrosine.11 Recently, it was found that Aβ could bind to heme to form Aβ-heme complex and significantly increased the peroxidase activity of free heme.8 Furthermore, we found that this Aβ-heme complex led to the increase of protein tyrosine nitration in the presence of H2O2 and NO2-.9 This reaction may play an important role to the fact that the nitration levels of many important proteins are significantly increased in AD brain.10 There is a tyrosine residue, Y10, in the sequence of Aβ peptide, which might be prone to nitration. Our recent study indicated that nitration of Y10 in Aβ1-40 significantly decreased the aggregation of Aβ1-40 and reduced its neurotoxicity. We also hypothesized that nitration of Y10 in Aβ1-40 was a compensatory reaction against oxidative/nitrative stress and Aβ aggregation.12 However, a large number of researches indicated that it differs a lot between Aβ1-40 and Aβ1-42 in toxicities, physiological functions and aggregation mechanism, despite a slight differences in their sequences.13 Aβ1-42 is more prone to oligomerization and fibril formation than the more abundantly produced Aβ1-40 peptide. Moreover, Aβ1-42 exhibits notably higher neurotoxicity than Aβ1-40. Hence, Aβ1-42 was regarded as the most important pathogenic species of AD.14-18 Additionally, the structural model of Aβ1-42 fibril is characterized by S-shaped three β-strand regions encompassing residues 12-18 (β1), 24-33 (β2) and 36-40 (β3). It is distinct from the structure model of Aβ40 fibril with β-loop-β motifs.19,20 Recently, Kummer et al. reported that nitration of Y10 critically enhanced Aβ1-42 aggregation and plaque formation.21 However, they used peroxynitrite to induce tyrosine nitration, which introduced oxidation to Aβ peptide that could not rule out the influence of Aβ oxidation. It needs to be further



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studied whether tyrosine nitration would affect the aggregation and function of Aβ1-42 or not. In this work, we used synthetic Aβ1-42Y10(3N)T (Aβ1-42NT) to study the impact of nitration of Y10 on the aggregation and function of Aβ1-42. It was found that nitration of Y10 significantly decreased Aβ aggregation and cytotoxicity. This study suggests a new role of tyrosine nitration in AD. 2.

Experiments

2.1. Materials Aβ peptides (Aβ1-42, Aβ1-42NT and Aβ1-42Y10(3Cl)T (Aβ1-42Cl )) were synthesized by ChinaPeptides (Shanghai, China). 3,3’,5,5’-tetramethylbenzidine (TMB), dimethyl sulfoxide (DMSO), Thioflavin T (ThT), hexafluoroisopropanol (HFIP), Hemin (Ferriprotoporphyrin

IX

chloride)

and

rabbit

polyclonal

antibody

against

3-nitrotyrosine (3-NT) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Carlsbad, CA, USA). All solvents and other reagents were the highest purity and commercially available. 2.2. Preparation of Monomer Aβ Aβ peptides and the mutants were dissolved in HFIP at 0.2 mM concentration and incubated at 4 oC overnight. Then, the HFIP was evaporated off under nitrogen gas, and the film was lyophilized overnight to remove residual solvent. Finally, the peptides were re-dissolved in DMSO to a concentration of 4 mM and stored at -20 oC as stock solution.


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2.3. Dot Blot Immunoassay To determine whether the tyrosine residues in Aβ1-42 could be nitrated, 50 µM Aβ1-42 was incubated with 2 µM heme, 500 µM NO2-, 500 µM H2O2 in 50 mM phosphate buffer (PB, pH 7.4) at 37 oC for 60 min. Then, 3 µl of sample was transferred to nitrocellulose membranes. A rabbit polyclonal antibody against 3-nitrotyrosine was employed to detect the nitrated Aβ peptide in this assay. 2.4. UV-Vis Absorption Spectroscopy For this experiment, 10 µM heme was mixed with 20 µM Aβ peptides (Aβ1-42 or Aβ1-42NT) in 50 mM PB (pH 7.4) at 37 oC for 15 min. Then, the spectra was recorded on a UV 2550 spectrophotometer (Shimadzu Co, Japan) using a 0.5 cm width cuvette at room temperature. 2.5. Peroxidase Activity Measurement For this assay, TMB was employed to measure peroxidase activity of heme-Aβ (Aβ1-42 or Aβ1-42NT) complex. The activity was made by monitoring the increase of TMB oxidation product absorbance at 652 nm (ε652=3.9×104 M-1cm-1).22 Reactions were initiated by the addition of heme or heme-Aβ, The reaction mixtures contained 3 mM H2O2, 0.42 mM TMB, 1 µM heme or heme-Aβ in 50 mM citric acid buffer (pH 5.0). 2.6. Thioflavin T Fluorescence Measurement The stock solutions of Aβ peptides (include Aβ1-42, Aβ1-42NT and Aβ1-42Cl ) were diluted to 50 µM with 50 mM PB (pH 7.4). Then the samples were incubated at 37 oC for series of time intervals (12, 24, 36 and 48 hours). The degree of aggregated Aβ



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peptides was determined by using ThT, which specifically bound to fibrous structures.23 For this assay, 80 µl incubated sample was mixed with 720 ul ThT of 10 µM and shaken for 1 min. Measurements were performed at an excitation of 450 nm (slit width =5 nm) and an emission of 482 nm (slit width = 10 nm) on RF5301 spectrofluorometer (Shimadzu Co, Japan). All of the experiments were performed at least three times. 2.7. Transmission Electron Microscopy Aβ peptides (include Aβ1-42, Aβ1-42NT and Aβ1-42Cl ) (50 µM) was incubated at 37 oC for 0 and 36 hours for compared groups. Then 30 µl of the sample was loaded onto a carbon-coated Formvar 200 mesh copper grid for 5 min. Excess solvent was carefully removed, and the grid was washed twice with water to remove the residual salt. At last, the grid was stained with 5% uranyl acetate for 5 min and air-dried. Images were obtained using a transmission electron microscope (HITACHI H-7000FA ) at an accelerating voltage of 100 kV. 2.8. Atomic Force Microscopy Aβ aggregates were deposited on freshly cleaved mica. After 5 min, the mica was washed twice with deionized water and dried under a gentle nitrogen stream. Images were obtained in tapping mode with a silicon tip under ambient condition by using a SPM 9700 instrument (Shimadzu Co, Japan). The scanning frequency was 1Hz. At least four regions of the mica surface were examined to ensure that similar structures existed throughout the sample. 2.9. Circular Dichroism


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Circular dichroism (CD) spectra were carried out by wavelength scanned from 260 to 200 nm using a JASCO-810 (Tokyo, Japan) spectropolarimeter at room temperature in a 1 mm path length quartz cell. Generally, a 2 nm bandwidth, a scan speed of 100 nm/min and a 1 s response were used. Each spectrum was the average of three scans. 2.10. Cell Viability SH-SY5Y cells were cultured in DEME media supplemented with 10% (v/v) fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained in a humidified atmosphere of 95% air and 5% carbon dioxide (CO2) at 37 oC. Thiazolyl blue tetrazolium bromide (MTT) was used to evaluate cell viability. Cells were incubated with 10 µM oligomeric Aβ peptides (include Aβ1-42, Aβ1-42NT and Aβ1-42Cl) at 37 oC for 48 h in serum-free medium.24 Afterward, the cells were treated with MTT for 4 h at 37 °C, and then they were lysed in DMSO at room temperature. The product was quantified by measuring the absorbance at 570 nm using a microplate reader. Cell viability was calculated as the percentage of untreated control. 2.11. Statistical Analysis All experiments were carried out at least in triplicate. Results were expressed as mean ± SEM. The student's t test was used for statistical analyses, and p < 0.05 was considered significant. 3.

Results

3.1. Nitrotyrosination of Aβ1-42 It has been demonstrated that Aβ could bind to heme and enhance its peroxidase



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activity. It is also generally accepted that Aβ peptides binds to heme through the H13 residue, which is close to the Y10.25 So Y10 in Aβ1-42 may subject to the －

heme-induced nitration in the presence of H2O2 and NO2 . To support this speculation, Aβ1-42 was treated in different systems. Figure 1 showed that Aβ1-42 exhibited significant nitration in the presence of heme, H2O2 and NO2-. It suggests that Y10 in Aβ1-42 is a potential nitration target in vitro.

Figure 1. Nitration of Aβ1-42 at Y10. Aβ1-42 was incubated with or without heme−H2O2−NO2- system for 60 min in 50 mM phosphate buffer (pH 7.4) at 37oC, then 3 µl of sample was transferred to nitrocellulose membranes. A rabbit polyclonal antibody against 3-nitrotyrosine was employed to detect the nitrated Aβ peptide. The same concentration of Aβ1-42NT was used as a positive control. 3.2. Effect of tyrosine modification on the binding of Aβ1-42 to heme and the peroxidase activity of Aβ1-42-heme complex Many researches found that nitration of tyrosine could increase the bulkiness and


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hydrophilicity of tyrosine; and function of many important proteins were altered by protein tyrosine nitration as well.10,11 Considering Aβ peptide binds to heme through the H13 residue8 along with the fact that Y10 is close to the binding site, it is straightforward to detect the influence of tyrosine nitration on the binding of Aβ1-42 to heme. It has been found that the UV-Vis absorption spectrum of heme can be changed by binding to Aβ.25 Thus, UV-Vis spectrum was employed to study the interaction between heme and Aβ1-42 and Aβ1-42NT. We can see in Figure 2 that all tested Aβ peptides induced a significant increase in the absorbance of the Soret band and the weak absorbance band around 530 nm upon binding to heme; there was no significant differences among the absorption wavelength and intensity of the Soret band of Aβ1-42 and Aβ1-42NT. These observations indicate that the binding of Aβ1-42 to heme is almost unchanged when tyrosine residue is nitrated. It has been found that the peroxidase activity of heme was significantly increased upon binding to Aβ1-42.26 Since Y10 is close to the active site of the complex, we proposed that nitration of tyrosine might change the peroxidase activity of the Aβ-heme complex. We used TMB to measure the peroxidase activities of Aβ-heme and Aβ1-42NT-heme. We found all the complexes have an obvious enhancement in peroxidase activity compared with free heme. However, the differences among the two samples were slight (Figure 3). These results indicate that there is no significant effect on the binding of Aβ1-42 to heme and the peroxidase activity of Aβ1-42-heme complex when the tyrosine is nitrated in Aβ1-42.



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Figure 2. Absorption spectra of Aβ1-42-heme and mutant-heme complex. 10 µM heme was mixed with 20 µM Aβ1-42 or Aβ1-42NT in 50 mM phosphate buffer (pH 7.4), and the complex was incubated at 37 oC for 15 min. Absorption spectra was recorded using 50 mM phosphate buffer (pH 7.4) as a control.

Figure 3. Effect of tyrosine nitration on the peroxidase activity of heme-Aβ1-42


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complex. The assay mixture contained 3 mM H2O2, 0.42 mM TMB, 1 µM heme or heme-Aβ in 50 mM citric acid buffer (pH 5.0). The reaction was initiated by adding heme or heme-Aβ. Peroxidase activity was evaluated by TMB. The values were the absorption subtracted from that at 0 min and were presented as the mean ± SEM of three independent experiments. 3.3. CD studies of the aggregation of Aβ1-42 CD spectroscopy was used to study the secondary structure of Aβ peptides. As shown in Figure 4, there is a negative peak around 217 nm in all tested samples at 0 h, indicating a β-sheet structure.34 When the incubation time increased, the negative CD values at 217 nm correspondingly increased, indicating the increase of β-sheet structure. Meanwhile, we found that all the samples have an significant increase of β-sheet content. This result suggests that tyrosine modification may have no effect on the formation of β-sheet of the Aβ1-42.



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Figure 4. Secondary structural analysis of Aβ1-42 and mutants aggregates using CD spectroscopy. 30 µM Aβ1-42 or mutants was incubated at 37 oC in 10 mM phosphate buffer (pH 7.4) for 0 or 36 h. 3.4. Effect of tyrosine modification on the aggregation of Aβ1-42 ThT is a fluorescent dye that can specifically bind to fibrous structures.23 Thus, the effect of tyrosine modification on the aggregation of Aβ1-42 was determined by using ThT binding assay. We can see from Figure 5 that the tyrosine modified peptides showed lower average fluorescence intensities during the incubation of 48 hours compared with Aβ1-42. This implies that tyrosine modification can significantly decrease the aggregation of Aβ1-42. It can also be seen that tyrosine nitration exhibits greater ability in inhibiting Aβ1-42 aggregation than tyrosine chlorination, since the flourescence intensity of Aβ1-42NT is lower than that of Aβ1-42Cl.


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To visually confirm the influence of tyrosine modification on the aggregation of Aβ1-42, TEM and AFM were used to image the morphological differences between Aβ1-42 and mutants fibrils. TEM images showed time-evolution of the sample morphology (Figure 6 A (36h)). It was obvious that no significant aggregates were observed at the beginning. After incubation for 36 hours at 37 oC, Aβ1-42 aggregated to form numerous long, thick, rod-like and cross-linked mature amyloid fibrils. On the contrary, less amyloid fibrils were observed in the images of mutant peptides after incubated for 36 h. Moreover, no cross-linked fibrils were found. We used AFM to further check the aggregates, and found that the fiber of Aβ1-42 was short and numerous after 36 h incubation (Figure 7). This observation is similar to the results obtained from the TEM images. It reveals that Aβ1-42 is easy to form fibrillar seeds, which could accelerate the aggregation of Aβ. These results indicate that tyrosine modification can significantly inhibit the aggregation of Aβ1-42.

Figure 5. Aggregation of Aβ1-42 determined by ThT flourescence assay. The relative



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aggregation degree of each sample was evaluated by thioflavin T fluorescence intensity at 482 nm. Each samples contained 50 µM Aβ1-42 or mutants was incubated at 37 oC in phosphate buffer（pH 7.4）for time intervals at 0 h，12 h，24 h，36 h and 48 h. Each point represents the average of triplicate experiments. The values are the mean ± SEM.

Figure 6. Negative-stained TEM images of Aβ1-42 and mutants. Peptide (50 µM) was incubated at 37 oC in 50 mM phosphate buffer (pH 7.4) for 0 or 36 h. A: Aβ1-42; B: Aβ1-42Cl; C: Aβ1-42NT. All samples were negatively stained with 5% uranyl acetate.


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Figure 7. AFM images of Aβ1-42 and mutants. The peptides morphology after 0 or 36 h incubation in 50 mM phosphate buffer (pH 7.4) at 37 oC analyzed by AFM. A: Aβ1-42; B: Aβ1-42Cl; C: Aβ1-42NT. The scale bar is 1 µm. Peptides concentration is 50 µM. 3.5. Effect of Tyrosine Modification on Aβ1-42 Cytotoxicity To determine the effect of tyrosine modification on the neurotoxicity of Aβ1-42, we exposed SH-SY5Y neuroblasoma to different Aβ1-42 peptides for 48 h, and cell viability was evaluated by MTT assay. The results showed that Aβ1-42 significantly increased the percentage of dead cells by 37%, whereas the mutant peptide displayed lower neurotoxicity (Aβ1-42NT 0%, Aβ1-42Cl 10%) (Figure 8). Cytotoxicity was directly correlated with the aggregation level of the samples. In addition, the cell morphology of the control group and Aβ1-42NT treatment group was similar. And the cells showed round cell bodies and clear neurites under an inverted phase contrast microscope. Cells would change their neuron-like morphology, lose their neuritic processes, and become rounded upon subjected to Aβ1-42, and eventually compromise



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their viability. Correspondingly, a lot of rounded cells were observed with the treatment of Aβ1-42, while Aβ1-42Cl showed moderate influence on cell morphology. This result implies that Aβ1-42 is more toxic than the mutants, and the neurotoxicity of Aβ1-42NT is the lowest. This result implied that tyrosine modification can remarkably reduce the neurotoxicity of Aβ1-42.


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Figure 8. Effects of Aβ1-42 and mutants on cell viability and cell morphology. (A): Cytotoxicity of Aβ1-42 or mutants to SH-SY5Y neuroblastoma. The cells were treated



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with 10 µM aggregated Aβ1-42 or mutants for 48 h before MTT assay. Cell viability was determined using MTT assay and was shown as a percentage of the untreated cells. Cell viability represents mean ± SEM (n=3). ** p < 0.01 and * p < 0.05 compared with the control; # p< 0.05 compared with the Aβ1-42 group (B). Representative photomicrographs of SH-SY5Y cells incubated with different peptides for 48 h are shown a): Control; b): Aβ1-42; c): Aβ1-42Cl; d): Aβ1-42NT. 4.

Discussion and Conclusion

Our Previous research results showed that tyrosine nitration significantly decreased the aggregation of Aβ1-40. Moreover, Aβ1-40NT showed lower cytotoxic effects than the wild-type Aβ1-40 on the SH-SY5Y cells. Therefore, we speculated that nitration of Aβ1-40 might be an Aβ detoxicant process and a compensatory reaction to nitrative stress.12 However, Aβ has two main isoforms (Aβ1-40 and Aβ1-42), and they displayed distinct clinical, biological, and biophysical behavior. Aβ1-42 was believed to be more directly relate to AD.14-18 In addition, it was reported that nitration of Y10 by peroxynitrite significantly enhanced amyloid β aggregation and plaque formation.21 But the nitrated Aβ obtained by treating Aβ with peroxynitrite is complex, since dityrosine product between two Aβ and oxidation of the peptide backbone products may also occur apart from tyrosine nitration. In order to ascertain the effect of tyrosine nitration on the structure and function of Aβ1-42, we chose synthetic Aβ1-42NT as the subject of this research. We firstly confirmed that the tyrosine residue in the Aβ1-42 was easy to be nitrated by heme−H2O2−NO2- system (Figure 1). Then we tested the binding of Aβ1-42


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peptides to heme and the peroxidase activities of heme-Aβ complex. UV-Vis spectrum of Aβ1-42-heme complex revealed that tyrosine nitration had little effect on the binding between heme and Aβ1-42 (Figure 2), even though Y10 is close to the binding site. Similarly, the peroxidase activity of Aβ1-42-heme complex exhibited no changes compared to mutant-heme complex (Figure 3). These results imply that formation of Aβ1-42-heme complex forms would induce inevitable nitration of heme bonded or free Aβ1-42 in the presence of H2O2 and NO2-, and Aβ1-42 nitration will happen under certain pathological event. Nextly, we further tested the aggregation of nitrated Aβ1-42. The results of ThT binding assay indicated that Y10 nitration could significantly decrease the aggregation of Aβ1-42. The observation from AFM and TEM images confirmed this result. In addition, we found that wild Aβ1-42 tended to aggregate into cross-linked amyloid (Figure 6 A（36h)). Numerous short fibers were observed in the AFM image of Aβ1-42, which was similar to other reports in the literature.34,35 This result indicates that Aβ1-42 has big capability of nucleation, which would result in lower monomer concentration and shorter fibers and more cross-linked structure. On the contrary, there was much less non-cross-linked amyloid structure in the TEM and AFM images of the mutant, and the Aβ1-42NT seemed to form long fibrils. This result indicates nitration of Y10 can inhibit the seed formation and thus decrease the aggregation of Aβ1-42. It is well known that Aβ peptides undergoes a conformational transition from a predominately random coil structure to a β-sheet-rich form, and then monomers attach to each other to form larger complex.36,37 Thus, there are two possible ways to decrease the aggregation of Aβ1-42. One way is to inhibit the



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conformational transition, and the other is to block the interaction between monomers. In order to study the reason how tyrosine nitration inhibits Aβ1-42 aggregation, we used CD to study the effect of tyrosine nitration on the secondary structure of the Aβ1-42. It is apparent that all peptides tended to increase of β-sheet content with the increase of incubation time (Figure 4). So results in Figure 4 clearly supported that tyrosine nitration has little effect on the forming of β-sheet. Taken together, we suggest that tyrosine nitration probably inhibit the aggregation of Aβ1-42 through blocking intermolecular interaction. Interestingly, considerable evidence supported that interstrand aromatic interactions play a critical role in the formation of Aβ assemblies.27 We found that Aβ1-40 (Tyr10Gly) showed lower aggregation compared to the wild-type Aβ1-40 after the site-directed mutant.28 Hence, we hypothesized that tyrosine play an important role in self-assembly processes of Aβ through forming π-π stacking interactions, and tyrosine nitration may inhibit this interaction and decrease the aggregation of Aβ1-42. To confirm whether Y10 modification can reduce the aggregation of Aβ1-42, we used Y10 chlorinated Aβ1-42 (Aβ1-42Cl), as a comparison. Tyrosine chlorination is also a posttranslational modification under oxidative stress.29 The results showed that both tyrosine nitration and chlorination could inhibit the aggregation of Aβ1-42, and there was no notable difference in morphology between Aβ1-42NT and Aβ1-42Cl. In addition, both tyrosine nitration and chlorination could change the hydrophility and increase the bulkiness of the residue. Since nitro-group is larger than Cl-group, it was found that the nitration of Y10 seemed to exhibit appreciably greater ability to inhibit


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the aggregation of Aβ1-42 compared to chlorination of Y10. This result suggested that the steric hindrance may play the major role in this case through blocking the interaction between two Aβ1-42 molecules (Figure 9).

Figure 9. Proposed mechanism of the effect of tyrosine nitration on aggregation. The aggregation of Aβ1-42 is initiated by a conformational change from random coil or α-helix into a β-strand. The three β-sheets encompassing residues 12-18(β1), 24-33(β2) and 36-40(β3). Then, the Aβ1-42 molecular attacks each other to form oligomer, protofibrils and fibrils. Y10 may play an important role in Aβ1-42 aggregation through forming π-π stacking interaction. When Y10 is nitrated, the adding nitro group would interfere this interaction and prevent Aβ1-42 from aggregating into fibrils. Finally, the cytotoxicities of Y10 modified Aβ were test by MTT assay. It’s well known that Aβ accumulation can induce neuronal death.30 It is interesting to find that tyrosine nitration can remarkably reduce the toxicity of Aβ1-42. Since Aβ toxicity is closely related to its aggregation, this result is consistent with the results obtained from the aggregation experiments. This result further confirms that tyrosine nitration



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is beneficial to neurons. However, our results are different from that reported by Kummer et al, in which they found that nitration of Aβ1-42 could significantly accelerate its aggregation.21 We think the difference between the result of Kummer et al and ours is the nitrated Aβ1-42 used. In their studies, peroxynitrite was used as a nitrating agent. It's well known that peroxynitrite is a strong oxidant, which not only promotes tyrosine nitration but also generates other byproducts such as protein oxidation and dityrosine. These byproducts may lead to false positive results. We confirmed that tyrosine nitration could significantly decrease the aggregation of Aβ1-42 and reduce its neurotoxicity. But the physiological significance of tyrosine nitration remained unclear. Interestingly, it was reported that the levels of nitrate in the cerebrospinal fluid of AD patients notably decreased as compared with controls.31 Miranda et al found that the NO2- plus NO3- levels significantly decreased in frontal, temporal, and occipital cortices in AD brains as compared with young people. More importantly, a remarkable decrease of nitrite was observed in the frontal cortex of AD patients as compared with age-matched controls.32 And reduced serum levels of n NO2- and NO3- were also present in AD.33 But the mechanism by which decreased nitrite and nitrate levels affect the pathogenesis of AD remains unclear. Our results may lead to the hypothesis that the decrease of nitrite will cause the decrease of nitrated Aβ that lead to the increase of Aβ aggregates and AD. In conclusion, our results provide a reasonable explanation for the link between the decreased nitrite and nitrate levels and AD. We found that the peroxidase


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activity of heme could be increased by binding to Aβ, and the increased peroxidase activity of heme made Aβ could be easily nitrated in the presence of NO2- and H2O2. As a result, the formation of nucleation is deceased and the toxicity of Aβ is reduced. Hence, we propose the role of Aβ nitration in antagonist AD pathogenesis: when neurons suffer oxidative stress, the cells would generate Aβ peptides together with nitric oxide (NO) against the reactive oxygen species (ROS); NO would firstly reacts with ROS to form nitrite, and nitrite will further react with ROS and Aβ peptides to form nitrated Aβ; the nitrated Aβ will not aggregate into fibers and can be cleaned in some way at last (Figure 10). Thus tyrosine nitration in Aβ may be a protection mechanism for neurons. These findings may open a new avenue in understanding the physiological function of Aβ and be helpful to the diagnosis, prevention and treatment of Alzheimer' disease.

Figure 10. Proposed link between the decreased level of nitrite plus nitrate and AD: ROS increases the generation of Aβ, and Aβ will form oligomer and cause neuron damage. However, when there is enough NO present, NO will first reacts with ROS to form nitrite, and nitrite will further react with ROS and Aβ peptides to form nitrated Aβ. The nitrated Aβ will not aggregate into fibers and can be cleaned in some way at



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last. AUTHOR INFORMATION *Corresponding Author Address: The Huazhong University of Science and Technology, 1037 Luoyu Rd., Hongshan Qu, Wuhan Shi, Hubei Sheng, China, 430074; Tel.: 86-027-87543532. Fax: 86-027-87543632. E-mail: [email protected]. Funding This work was supported by a grant from the National Natural Science Foundation of China (31170808). Notes The authors declare no competing financial interest. ABBREVIATIONS Aβ, amyloid β peptide; ThT, thioflavin-T; TEM, transmission electron microscopy; AD,

Alzheimer’s

disease;

TMB,

3,3′,5,5′-tetramethylbenzidine;

PB,

phosphate-buffered; HFIP, hexafluoroisopropanol; CD, Circular dichroism; AFM, atomic force microscopy; Reference (1) Wasling, P., Daborg, J., Riebe, I., Andersson, M., Portelius, E., Blennow, K., Hanse, E., and Zetterberg, H. (2009) Synaptic retrogenesis and amyloid-beta in Alzheimer's disease. J. Alzheimer's Dis. 16, 1-14. (2) Blennow, K., de Leon, M. J., and Zetterberg, H. (2006) Alzheimer's disease. J.-Lancet. 368, 387-403.


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173x69mm (300 x 300 DPI)


nitrative stress and Aβ aggregation?

Elimination of cooperativity in aspartate transcarbamylase by nitration of a single tyrosine residue.

Nitration of tyrosine in three cobra neurotoxins.

Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding.

Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56δ stabilizes its antiapoptotic activity.

Reducing TDP-43 aggregation does not prevent its cytotoxicity.

Tubulin tyrosine nitration regulates microtubule organization in plant cells.

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Evidence for a tyrosine residue at the active site of phosphoglucomutase and its interaction with vanadate.

Structural studies and cytotoxicity assays of "aggregation-prone" IAPP(8-16) and its non-amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin.

The reactive tyrosine residue of human serum albumin: characterization of its reaction with diisopropylfluorophosphate.

Metal-catalyzed protein tyrosine nitration in biological systems.

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy.

Amyloid-β(1-42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity.

Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation.

Mass spectrometric analysis of protein tyrosine nitration in aging and neurodegenerative diseases.

Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity.

Effect of curcumin analogs onα-synuclein aggregation and cytotoxicity.

Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration.

Alterations in connexin 43 during diabetic cardiomyopathy: competition of tyrosine nitration versus phosphorylation.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration.

Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel complex.

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke.