Copper binding induces nitration of NPY under nitrative stress

Article Cite This: Chem. Res. Toxicol. 2018, 31, 904−913

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Copper Binding Induces Nitration of NPY under Nitrative Stress: Complicating the Role of NPY in Alzheimer’s Disease Huixian Ye, Hailing Li,* and Zhonghong Gao*

Chem. Res. Toxicol. 2018.31:904-913. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/23/18. For personal use only.

Hubei Key Laboratory of Bioinorganic Chemistry and Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China ABSTRACT: Neuropeptide Y (NPY) is a 36 amino acid peptide that regulates a multitude of physiological functions in the central nervous system and has been shown to be involved in Alzheimer’s disease (AD). A change in copper homeostasis is a remarkable feature of AD, and the dysregulation may contribute to toxicity in neural cells. Moreover, it has been shown that copper could interact with many neuropeptides and result in catalyzing the production of reactive oxygen species, which may lead to peptide oxidation. Besides, copper could also catalyze protein tyrosine nitration under oxidative stress, and there are two tyrosine residues playing an important role in NPY. Therefore, it is also likely that copper has an action on NPY and potentially influences its functions through tyrosine nitration. In this paper, the studies of the interaction of copper with NPY and the copper-catalyzed NPY nitration were performed. The electrochemical techniques, UV−vis spectroscopy, mass spectrometry, and fluorescence titration, have been applied to show that copper can interact with NPY to form a Cu-NPY complex with a conditional dissociation constant of 0.021 μmol/L, and the binding promotes the generation of •OH. Dot blotting results reveal that NPY can be nitrated upon binding with copper under nitrative stress. Furthermore, liquid chromatography-mass spectrometry (LC-MS) identify that the tyrosine residues in NPY are all nitrated during the nitration process, which will cause the inactivation of NPY shown by our previous study. This study supports the hypothesis that copper has a close correlation with NPY and implicates the peptide in AD. These data may provide a new insight into understanding the pathology and pathogenesis of AD.

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NPY toward receptors reduction or even loss.10,11 Functionally, this peptide is involved in a diverse range of biological activities like regulation of energy homeostasis, blood pressure, body temperature, and circadian rhythms.7,12 It has a physiological role as a neurotransmitter and a modulator of cardiovascular physiology and appetite behavior.13 A considerable amount of study has shown that, in the central nervous system (CNS), the cerebral NPY has a vital role in exerting anxiolytic activity in response to stress,14,15 preventing the development of depression,16,17 and reducing epileptiform activity in the hippocampus by inhibition of glutamate release.18,19 Protein tyrosine nitration (PTN) is a post-translational modification occurring under the nitrative stress resulting in the addition of a nitro group (NO2) to the ortho position of phenolic hydroxyl group.20 The incorporation of a nitro group into protein tyrosine residues changes the phenol groups pKa and hydrophobicity and increases the bulkiness of the residue.21 It will affect enzymatic activity and impede the phosphorylation of tyrosine and also may change the structure of protein, thereby rendering a protein inactive.20,22 In biological conditions, it represents a pathological event that is associated with neurodegenerative diseases,23 such as

INTRODUCTION Neuropeptide Y (NPY) is a highly conserved 36 amino acid peptide (Figure 1 for the primary structure of human NPY)

Figure 1. Amino acid sequence of human neuropeptide Y.

that belongs to a family of structurally related bioactive peptides consisting of NPY, peptide YY (PYY), and pancreatic polypeptide (PP).1,2 It is largely present in the central and peripheral neurons and has been demonstrated to be the one of most abundant neuropeptides within the brain.3 The biological activity of NPY lies with its ability to bind and activate G protein-coupled receptors via the Gαi signaling pathway.4,5 NPY predominantly binds to neuropeptide Y receptor subtypes Y1, Y2, Y4, and Y5 and displays equally high affinities for all four receptors.6,7 The structure of the peptide bound to their receptors is not clear. However, extensive structure−activity relationship studies have provided the evidence that the C-terminal segment is crucial for the binding of NPY to its receptors.8,9 The mutation or modification of amino acid residue in this region will result in the affinity of © 2018 American Chemical Society

Received: May 18, 2018 Published: August 6, 2018 904

DOI: 10.1021/acs.chemrestox.8b00128 Chem. Res. Toxicol. 2018, 31, 904−913

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Chemical Research in Toxicology

might be useful to understand the pathology of AD and develop new therapeutic strategies.

amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Elevated levels of nitrated proteins have been reported in AD brains,23 demonstrating the prevalent occurrence of PTN with this disease. Intriguingly, it has been shown that NPY is implicated in neurodegenerative disease,24,25 such as Parkinson’s disease, Huntington’s disease, and especially AD. The involvement of NPY in AD has been defined throughout the years. Specifically, the number of NPY was found to be significantly reduced with respect to NPY receptors alteration in the brain of AD patients.26 It has also been reported that the level of NPY in plasma and cerebrospinal fluid was reduced during the progression of AD.27,28 More recently, a neuroprotective role of NPY against neuronal damages caused by Aβ plaques and tangles has been found in the AD transgenic model.29 In addition, recent studies indicated that NPY could increase the survival of SH-SY5Y neuroblastoma cells exposed to toxic concentrations of Aβ peptide fragment 25−35 (Aβ25−35) probably by influencing the synthesis and the release of nerve growth factor (NGF).30,31 These results imply that NPY has a potential role in the pathogenesis and pathophysiology of AD. Considering the abundance of tyrosine in NPY and the ubiquitous of PTN in AD, we hypothesized that the role of NPY in AD might be influenced through tyrosine nitration. Copper is one of the most prevalent biological transition metals by all organisms from bacteria to humans,32 and high concentrations of brain copper can be an important factor in the pathogenesis of many neurodegenerative disorders.33 For AD, one notable characteristic is disrupted copper ion homeostasis. The levels of Cu2+ in AD brain are 400% higher than that of a healthy brain, with the Cu2+ level at a 0.4 mM high concentration.34 Copper can catalyze the generation of reactive oxygen species (ROS) such as hydroxyl radicals and reactive nitrogen species (RNS) in the presence of nitrite via Fenton reaction, and this will lead to the oxidative and nitrative damage of many biological targets.32,33 For complexes with peptides, it was found to be still an excellent Fenton catalyst, even better than heme for the formation of hydroxyl radicals, leading to tyrosine nitration in polypeptides in the presence of nitrite.33 In AD, high concentration copper ions are able to bind to many peptides linked with the AD progression, for example, NKA,35 neuropeptide γ,32 Aβ peptide,36 etc. The level of oxidative stress in local environment is significantly increased upon copper binding to them,32,35,36 and in the presence of nitrite, this will result in tyrosine nitration, thereby affecting the structure and activity of related peptides or proteins, which are believed to contribute to AD symptoms. It has been revealed that the presence of histidine in peptides or proteins is common in such copper-binding proteins, and His is known as a copper binding site.37 This site can be observed everywhere, for example, Aβ peptide binds copper via the histidine imidazole nitrogen.38 Inspection of the sequences of NPY, it highlights the presence of a histidine residue at position 26. Thus, NPY also has the potential to bind with copper. Such binding may increase oxidative stress and induce important tyrosine nitration of NPY in the present of nitrite, thereby attenuating the biological activity of NPY. However, no work has been reported on the association of copper with NPY and the likely consequences of the binding. In this paper, we present that copper can indeed bind to NPY, and the binding induces the important tyrosine nitration under nitrative stress, which has been validated to counteract the bioactivity of NPY in our previous study.39 This study

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EXPERIMENTAL PROCEDURES

Materials. Human neuropeptide Y was synthesized by ChinaPeptides (Shanghai, China). The peptides were purified by HPLC to >95% and confirmed by ESI-MS. Antibodies for immunoblotting were a rabbit polyclonal antibody against 3-nitrotyrosine (3-NT) and horseradish peroxidase-conjugated Immuno-Pure goat antirabbit IgG, from Sigma-Aldrich and Pierce, separately. Unless specified, all other chemicals and materials were of the highest purity commercially available. Deionized water from a Milli-Q system (Millipore, Billerica, MA, USA) was used for solution preparation. Sample Preparation. A NPY peptide stock solution was prepared by dissolving it in ultrapure water to a stock concentration of 0.2 mM and stored in the dark at −20 °C. The peptide stock solutions were then diluted to desired concentration according to the experiment, and all other reagents were prepared freshly. Voltammetry. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed using a CHI 830C electrochemical workstation (Shanghai Chenhua, China) in a threeelectrode system with a saturated calomel electrode as the reference electrode, platinum wire as counter electrode, and glassy carbon as the working electrode. The glassy carbon electrode was polished with fine Al2O3 powder (0.05 μm) and then successively sonicated in ethanol and deionized water for 1 min. 0.2 M KCl in ultrapure water (18.2 MΩ) was used as the supporting electrolyte solution. Twenty μM copper(II) in the absence or presence of NPY was detected with 0.1 V s−1 sweep rate for CV and an amplitude of 0.05 V, a pulse width of 0.2 s, and a pulse period of 0.5 s for DPV. The electrolyte solutions were bubbled with high-purity nitrogen gas for 15 min to remove the oxygen dissolved prior to the experiments, and all experiments were carried out at room temperature. UV−vis Spectroscopy. The titration of NPY with Cu(II) by UV−vis was conducted by continuously adding aliquots of copper ion stock solutions (1 mM) every 5 min into 50 μM NPY solutions, and the absorption spectra were recorded on a UV 2600 spectrophotometer (Shimadzu Co., Japan) at room temperature in the 300−800 nm range using a quartz cuvette of 1 cm path length. Mass Spectrometry. ESI-MS was carried out on a micrOTOF II mass spectrometer (Bruker, Germany) equipped with an electrospray source. A syringe pump was used to infuse the solution to the atmospheric pressure ionization source with a gastight syringe at a flow rate of 4.0 L/min. Spectra were acquired in the 50−3000 m/z range with positive ion mode, setting a 1.5 kV capillary voltage, and ionization/desolvation conditions were optimized to allow conservation of the produced protonated or cationized complexes over the solvent desolvation. Samples were prepared by diluting NPY stock solution to a final concentration of 20 μM in a methanol/water 1:1 (v/v) solvent mixture (pH 6.0). The Cu(II) ion was added with a peptide:metal ion ratio of 1:5. All masses were calculated using charge-state ruler tools provided with Bruker Daltonics DataAnalysis software of the MS instrument, and all of the various charged molecular ions, corresponding to the same peptide or metal-peptide complex, were presented. Fluorescence Spectroscopy. Fluorescence spectra were recorded on a RF5301 spectrofluorimeter (Shimadzu Co., Japan). For the detection of intrinsic tyrosine fluorescence of NPY, the emission spectra were collected at an excitation of 275 nm in the range of 290− 400 nm, and the excitation and emission slit were set as 5 and 10 nm, independently. The binding constant and binding stoichiometry of Cu2+ with NPY were all investigated by fluorescence titration.40,41 For the determination of binding constant, the titration of NPY with copper was performed in a 1 cm path length quartz cell by keeping the peptide at a fixed concentration of 10 μM and then manually adding corresponding aliquots of stock solution of CuCl2 to the 800 μL peptide solution at 5 min time intervals. At least three independent titrations were carried out, and all the titration data were recorded at 905


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Chemical Research in Toxicology room temperature. The obtained titration data were analyzed to get the binding constant as before42 by fitting to following formula:

F = F0 − −

sequences of copper ability to interact with NPY were observed based on voltammetric assay, and the results are presented in Figure 2. As shown in Figure 2A, the potentials of

0.5 × (F0 − F∞) × ([A] + [Cu 2 +] + Kd [A]

([A] + [Cu 2 +] + Kd)2 − 4 × [A] × [Cu 2 +] )

(1)

where F, F0, and F∞ are the fluorescence intensities of the peptide in the presence, the absence, and the saturation of copper ions, and [A] and [Cu2+] are the total concentrations of the peptide and Cu2+, and Kd is the dissociation constant of Cu(II)-NPY complex. For the binding stoichiometry study, the titrations were conducted by fixing the concentration of NPY at 5 μM, whereas Cu2+ ion concentration was varied from 1 μM to 15 μM and the titration data were processed and plotted using origin software. Coumarin Assay for Hydroxyl Radical Production. The level of hydroxyl radical production by NPY in the absence or presence of Cu2+ was measured by coumarin fluorescence assay as described in previous literatures.43,44 The reagents were added to yield a final concentration of 20 μM NPY, 0.5 mM H2O2, 0.5 mM NaNO2, 100 mM coumarin, and CuCl2 varying from 10 to 40 μM. The reaction mixtures were then incubated at 37 °C for 24 h. The fluorescence of all samples were monitored at an excitation wavelength of 390 nm and emission wavelength of 460 nm. Data are presented as mean ± SD of three independent experiments. Dot Blotting Immunoassay. Dot blotting for detecting peptide nitration was performed as described in previous work.43 Briefly, 40 μM NPY was incubated with 40 μM Cu(II) and 0.5 mM H2O2 in the presence or absence of 0.5 mM NaNO2 at 37 °C for 24 h. For the nitration kinetic study, the samples were prepared in the same concentrations but for different time points (i.e., 0.5, 2, 4, 6, 12, and 24 h). Then, 5 μL of samples was transferred to nitrocellulose membranes and immunoblotted with an antibody against 3-nitrotyrosine (1:1000, Sigma). Thereafter, the antibody was detected employing a goat antirabbit IgG secondary antibody (1:3000, Sigma) conjugated to horseradish peroxidase. Finally, The ECL system (Pierce) was applied to measure the immunoreactivity by chemiluminescence. In Gel Digestion and Mass Spectrometry Analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to identify nitrated sites of tyrosine in NPY as described previously.39 In brief, NPY (200 μM) was incubated with 0.5 mM H2O2 and 0.5 mM NO2− in the presence of an equivalent molar of Cu(II) ions at 37 °C for 24 h. The samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Thereafter, the peptide bands were excised manually, and the gel slice was subjected to tryptic digestion in gel at 37 °C for 16 h. At the completion of digestion, the supernatant containing tryptic peptide fragments were transferred, extracted with another 50 μL 50% acetonitrile solution containing 0.1% trifluoroacetic, and submitted to mass spectrometry analysis. The analyses were performed with a 1100 series LC/MSD XCT ion trap mass spectrometer (Agilent). The trypsin-digested peptide fragments were separated on a HPLC system with mobile phase comprised of solvent A (0.1% trifluoroacetic acid in deionized water) and solvent B (100% acetonitrile) and eluted by using an increasing linear gradient of solvent B from 5 to 40% for 45 min with a 0.5 mL/min flow rate. Tyrosine nitrated fragments were identified by an increase of 45 Da in the mass of product ions in the MS/MS spectra.

Figure 2. Cyclic (A) and differential pulse (B) voltammograms of copper (20 μM) in the absence or presence of equimolar NPY in 0.2 M KCl under oxygen-free conditions.

electrochemical processes are 0.166 and 0.092 V, corresponding to oxidation potential and reduction potential. The values obtained in this potential range (−0.2−0.6 V) are referred to CuII/ CuI conversion as reported by Brzyska.46 The CV data revealed that the reduction peak potential in the presence of NPY was shifted toward more negative value (about 80 mV). It means that reduction of CuII in the presence of NPY is more difficult compared with free CuII ions. In the meantime, oxidation of CuI to CuII in the presence of the peptide was shifted to more positive potential (to 0.21 V approximately). Moreover, the peak current in the presence of NPY was also lowered during the reduction as well as oxidation processes, which are commonly observed when a peptide is bound to copper.46,47 These results indicate that copper can bind with NPY, forming a NPY-CuII complex altering electrochemical property of free copper. This is further confirmed by DPV, showing lower peak current and higher peak potential due to the combination of copper with NPY (Figure 2B). UV−vis spectroscopy is a conventional method to probe copper-peptide interactions, as it allows the characterization of a new electronic absorption peak associated with resulting copper-NPY complexes. Therefore, we also investigated the interaction between Cu(II) and NPY using UV−vis absorption spectroscopy. The UV−vis titrations were performed to validate this interaction and to try to observe the binding stoichiometry of copper with NPY. The titration of NPY with Cu(II) in aqueous solution is shown in Figure 3. As Cu(II) is titrated into a NPY solution, the electronic absorption of NPY in the visible region is increased with the continuous addition

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RESULTS Interaction of Copper(II) with NPY. Voltammetry is a convenient way to monitor subtle changes in redox properties of the electroactive species reflecting their interactions and has been widely used in previous studies.39,45−47 Herein, we first apply cyclic and differential pulse voltammetry to follow the changes of CuII-related electrode reaction processes in the absence and presence of NPY. The electrochemical con906


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Figure 3. Titration of NPY with Cu(II) by UV−vis spectroscopy. Dotted lines correspond to the addition of 0−1.8 equiv of Cu(II), and the solid red line represents the addition of 3.0 equiv of Cu(II). Inset shows the vis signal intensity at ∼427 nm plotted as a function of equivalents of Cu(II).

of copper ions and a new band at ∼427 nm emerges slightly, which indicated coordination to the peptide. The inset in Figure 3 shows the vis signal intensity at ∼427 nm plotted as a function of equivalents of Cu(II). The absorption at ∼427 nm changes slightly and tends to reach a plateau after 1 equiv of Cu(II) has been added. These results further validate the interaction between NPY and Cu(II) and seem to indicate a probably 1:1 stoichiometry in aqueous solution for the Cupeptide complex. Copper Binding to NPY. Binding and binding properties of proteins and peptides with metal ions like Cu2+ and Zn2+ can be studied by ESI-MS.48−51 To gain insight into the interaction and stoichiometry between Cu(II) and NPY observed from electrochemical and UV−vis spectroscopy experiments, the ESI-MS experiments were performed. As shown in Figure 4, it exhibits an array of multiply charged ions, which are the typical spectra of ESI-MS. As the theoretical average molecular mass of synthetic NPY is 4272.6, the peaks at m/z 855.2, 1069.0, 1425.0, and 2137.0 in the spectrum without copper ions (Figure 4A) can be attributable to NPY peptides with five, four, three, and two protons attached, respectively. By contrast, in the presence of copper, some new signals at m/z 867.8, 1084.5, 1445.6, and 2168.4 appear in the spectrum (Figure 4B), which corresponds to the attachment of one copper ion, and no more copper ions bound to these species were observed. However, the multiply protonated MHnn+ molecules of NPY are also displayed. This result indicates that some peptide still remained free of copper and the remainder of the peptide was associated with a single copper ion in the presence of copper. These results reveal a significant copper binding to NPY at pH 6.0 and a 1:1 Cu:NPY molar ratio, confirming the results obtained from electrochemical and UV−vis spectroscopy tests. Affinity and the Stoichiometry of Cu(II) Binding Determined by Intrinsic Fluorescence Titration. Intrinsic fluorescence titration is a common method to determine the binding and affinity of copper with peptide.40,42,52,53 Generally, the intrinsic fluorescence absorption of peptides originates from Tyr, Trp, and Phe residues. However, the full-length NPY sequence highlights only the presence of five tyrosine residues at positions 1, 20, 21, 27, and 36. Thus, the intrinsic fluorescence of NPY is mainly due to the sole Tyr upon excitation at 275 nm. For the reason for its paramagnetic character, Cu(II) can quench the tyrosine residues fluores-

Figure 4. ESI mass spectra of 20 μM NPY in methanol/H2O (1:1): (A) without Cu(II) ions and (B) with 100 μM Cu(II) ions.

cence when binding to NPY. A representative spectrum of Cu(II) titration of NPY is shown in Figure 5A. It could be observed that the addition of CuCl2 led to the decrease in the intrinsic fluorescence of NPY and the Tyr emission band remained unchanged. It is easy to understand that the large degree of peptide signal quenching seen in titration measurement is due to the complex formation with Cu2+. As shown in Figure 5B, fitting the fluorescence data at 305 nm to eq 1 app resulted in an apparent dissociation constant (Kapp d ) value, Kd = 0.893 ± 0.095 μmol/L. The conditional dissociation constant Kcond (buffer and competitor-independent stability d constants) can be yielded from the apparent dissociation constant using the correction equation:54 cL i zyz log Kd = log Kdcond + C , where C = logjjj1 + βCuL × −pH + pKa z 1 + 10 k {

(2)

where CL is the total concentration of Hepes, Ka is the deprotonization constant of the buffer, and βCuL is the dissociation constant for the Cu(II)-Hepes complex. The value of C, calculated for 50 mM Hepes at pH 7.4, is equal to 1.625. So the Kcond value for Cu(II)-NPY calculated from our d data is 0.021 ± 0.002 μmol/L, and this value reveal a high affinity of copper toward NPY. To further study the binding stoichiometry of Cu(II) with NPY under physiological pH, we performed an additional fluorescence titration over a wide range of ligand concentrations, with the copper to NPY ratio varying from 1:5 to 3:1. As shown in Figure 5C, the fluorescence intensity of NPY can be also nearly completely quenched with the number of Cu(II) equivalents added, similar to the result shown in Figure 5B, and the Tyr fluorescence quenching curve was found to be divided into three parts from this data set: (1) a linearity part from 0 to 1 equiv of Cu(II), (2) a second linearity part with a 907


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copper(II) binding affinity have been even shown to generate levels of ROS in the presence of ambient O2,55 which can result in peptide oxidation and inactivation. To obtain the information on the ability of NPY bound to Cu(II) ions to produce hydroxyl radical, the coumarin fluorescence assay was performed in the presence of H2O2. The results of our investigation are illustrated in Figure 6. As can be seen from

Figure 6. Coumarin fluorescence assay for detection of hydroxyl radicals content for different samples incubated at 37 °C for 24 h. The values were subtracted by those of coumarin control and plotted as mean ± SD, n = 3. *p < 0.01 compared with the NPY-H2O2 group; #p < 0.01 compared with the respective Cu(II)-NPY-H2O2 group.

Figure 6, there was no significant difference in hydroxyl radical production by NPY in the absence of Cu(II) compared with only H2O2 system. The addition of CuCl2 led to notable hydroxyl radical generation in NPY-H2O2 systems, and the level of •OH increased as the Cu(II) ion to peptide ratio increased. This result is similar what has been observed for Cu(II)-Aβ systems,44 which indicated the redox activity of the copper-peptide complex. The control experiments demonstrated that neither H2O2 nor NPY facilitated the formation of •OH without the Cu(II)-NPY complexes. It should be noted that when NaNO2 was added, the fluorescence intensity decreased dramatically in the Cu(II)-NPY-H2O2 systems. In previous study, it had been shown that hydroxyl radical could oxidize nitrite to •NO2 with a high rate constant (k = 1.0 × 1010 M−1·s−1).56 Consequently, the significantly lower level of hydroxyl radicals detected in samples is most likely by the formation of nitrogen dioxide, which can induce tyrosine nitration.33 These results imply that the binding of NPY to copper can increase the oxidative stress in the presence of hydrogen peroxide and nitrite and may result in the peptide oxidation and nitration. Tyrosine Nitration of NPY. We have already demonstrated that the binding of NPY to Cu(II) ions could promote the generation of hydroxyl radicals and the addition of nitrite would lower its content, which may be attributed to the formation of •NO2 by •OH oxidation. It is widely accepted that tyrosine nitration occurs in the presence of either peroxynitrite or nitrogen dioxide (•NO2). Besides, it has been shown that copper catalyzed the nitration process by nitrite oxidation.33 Therefore, the tyrosine residues in NPY are likely to be nitrated in the presence Cu(II), H2O2, and nitrite. To confirm this, dot blotting was performed to detect the tyrosine nitration in above-mentioned system. As shown in Figure 7A, significant tyrosine nitration of NPY was observed in the Cu(II)-H2O2-NO2− system, whereas no tyrosine

Figure 5. (A) Representative fluorescence spectra of 10 μM NPY in 50 mM Hepes (pH 7.4) in the presence of increasing copper(II) concentrations (2−22 μM). (B) Fluorescence intensity at 305 nm of NPY obtained in (A) as a function of added copper. (C) Binding isotherms for the interaction of NPY (5 μM) with Cu2+ in a wide range of molar ratios (1:5 to 3:1) in 50 mM Hepes buffer. The fluorescence titration curve is divided into three portions. Inset shows the first two portions of the titration curve, with slope change occurring at 1 Cu(II) equivalent of the peptide.

lower slope value between 1 and 2 equiv of Cu(II), and (3) a slightly decrease after 2 equiv of Cu(II). Two break points at 1 and 2 Cu(II) equivalents can be well-observed in this titration curve, which appeared in similar study on Cu(II) binding to peptide.41 According to the Alies et al. deep investigation on Cu(II) affinity for Alzheimer’s peptide by tyrosine fluorescence quenching,41 the emergence of two break points in the titration curve can be attributed to the formation of Cu(NPY) complex and Cu2(NPY) complex. This result indicates that NPY has two binding sites for copper ions, and it can bind with Cu(II) in 1:1 stoichiometric ratio under physiological pH, whereas in the presence of high copper ions, the dinuclear complex is also likely to form. Formation of •OH by Cu(II)-NPY Complex under Oxidative Stress. Some peptides with inherently high 908


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nitration was detected in the absence of nitrite, indicating that NO2− could be indeed oxidized to •NO2, which accounted for the lower level of hydroxyl radicals when nitrite was added into the Cu(II)-NPY-H2O2 system. Moreover, we even found that the tyrosine nitration of NPY occurred in 0.5 h though not obvious, and the remarkable nitration level could be detected after 4 h incubation (Figure 7B), which was also monitored by Qiao et al. in similar nitrating system.33 This result suggests that NPY easily undergoes nitration damage under oxidative stress upon binding with copper ions. Identification of Nitration Site(s). Selective nitrotyrosination of different sites of peptides or proteins is linked to differential conformational changes and functional alterations of the affected proteins.57 As for NPY, Tyr1 and Tyr36 have been shown to be extremely important in binding and activating neuropeptide receptors. The absence or mutation of them in NPY will result in loss of receptor affinity, inducing dysfunction of these receptor-related responses.58,59 Interestingly, it has been shown that five tyrosine residues in NPY are all found to be nitrated in the heme-H2O2-NO2− system in our previous work.39 Moreover, Tyr36 nitration of NPY can destroy the active monomeric conformation of the peptide and thereby counteract its bioactivity. To elucidate the biological significance of NPY bound to copper ions, we also applied the LC-MS/MS to identify the specific nitrated sites. The LC-MS/ MS spectra of tryptic digestion derived four peptide fragments containing tyrosine, that is, Y1PSK, Y20Y21SALR, HY27INLITR, and Y36, and are shown in Figure 8, including the masses of the deduced sequence from each peptide fragment and indicated by the corresponding b and y ions. As illustrated in Figure 8A, the location of the nitration modified tyrosine, as well as supportive sequence information, could be readily determined from b ions. The b-series, at m/z 164.1, 261.0, and 348.7 for

Figure 7. (A) Nitration of NPY. NPY was incubated with Cu(II) and H2O2 in the presence or absence of NaNO2 at 37 °C for 24 h, and then 5 μL of samples is transferred to nitrocellulose membranes. A 3nitrotyrosine antibody was used to detect the level of tyrosine nitration in NPY. The same concentration of NPY(3N) (Tyr36 replaced by 3-nitrotyrosine) was taken as a positive control. (B) Kinetic study of NPY nitration. The level of tyrosine nitration was detected after NPY had been incubated with Cu(II) and H2O2 in the presence of nitrite for different times at 37 °C.

Figure 8. Identification of the nitrated sites of tyrosine in NPY with HPLC-MS/MS spectroscopy. Tyrosine residues in the trypsin-digested peptide fragments Y1PSK (A), Y20Y21SALR (B), HY27INLITR (C), and Y36 (D) were found to be significantly nitrated when NPY was incubated with Cu(II)-H2O2-NO2− system (final concentration: 200 μM NPY, 200 μM Cu(II), 0.5 mM H2O2 and 0.5 mM NaNO2) at 37 °C for 24 h. The text refers to the b and y product ions of respective fragments, and the putative nitrated peaks are identified by an increase in the mass of 45 Da, corresponding b* and y*. 909


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Chemical Research in Toxicology b1−b3, respectively, was identical for unmodified peptide sequences, and with b1, b2, and b3 ions increased by 45 Da to m/z 209.0, 306.0, 393.4, indicating the nitrated peptide sequence. The m/z 860.7 for the peptide fragment Y20Y21SALR (Figure 8B) clearly identified it as dinitrated peptide. This assignment was also supported by the corresponding m/z of b series and y series ions. Even though the low intensity at m/z = 1028 was not observed, the major fragmentation ions in the mass spectrum of this portion (Figure 8C), obviously the b2*−b4*, b7*, and m/z at 1073.3, were virtually consistent with those from authentic H-nitroY27INLITR. The MS/MS spectra of peptide fragment Y36 (Figure 8D, the parent ions 181.1 and 226.1 Da) indicated that Tyr36 was nitrated as well for the mass increased by 45 Da. These mass spectra corroborated that the five tyrosine residues in NPY were also all involved in nitration under Cu(II)-H2O2NO2− system. This result implies that the biological activity of NPY might be also influenced via important tyrosine residues nitration upon binding with high levels of copper ions in AD brain.

aqueous solution. We postulate that the histidine in NPY is most likely to mediate this interaction for the reason that the amino acid residue is known as a copper binding site, which is commonly observed in copper-binding peptides or proteins.67−69 Furthermore, we determined the dissociation constant of NPY-Cu(II) complex using fluorescence spectroscopy, a method based on intrinsic fluorescence quenching by a quencher, which is generally used in literature.42,52 The Kd value calculated from our data (Figure 5B) using a 1:1 binding model for NPY-Cu(II) complexes at pH 7.4 was 0.893 μmol/ L. It has been revealed that various buffers used for the fluorescence experiment may differ in the affinity value of copper toward peptide.42 Hepes as a useful buffer for biochemical studies was therefore used for our tests. It has been believed to be innocent in metal ion complexation. This notion, while true for many metal ions,70 does not hold for Cu(II). Studies have shown that the Hepes buffer can associated with Cu2+ to form a 1:1 complex with the stability constant of 1.66 × 103 mol/L,54 it was therefore necessary to take its influence on binding equilibrium into account by calculating the conditional dissociation constant. Consequently, we applied correction formula (eq 2) to get an independent buffer value, and this value was 0.021. Interestingly, the value in 50 mM Hepes is close to that of Cu(II)-Aβ40.42 It was well-documented that the binding between copper and Aβ facilitated its aggregation and mediated the generation of ROS in AD.71 It played a crucial role in the pathogenesis and pathophysiology of AD. Accordingly, this value of copper binding to NPY illustrated that the interaction between copper and NPY also could not be ignored considering the neuroprotective role of NPY in AD. The association of NPY with copper may also destroy its function in the brain and result in the development of AD symptoms. This was verified by our following study that copper binding to NPY significantly induces the production of ROS (Figure 6), resulting in tyrosine nitration in the presence of NO2− (Figure 7), and all five tyrosine residues were identified to be nitrated under nitrative stress (Figure 8). Among them, Tyr1 and Tyr36 are critical for binding and activating for neuropeptide receptors; the absence or modification of them in NPY will affect the affinity of it toward the receptor subtypes, resulting in receptor-related response dysfunction.72 Besides, Tyr36 nitration of NPY would reduce its biological activity as shown by our previous work.39 Therefore, we can easily know that the bioactivity of NPY will be also influenced through Tyr36 nitration upon binding with copper. Taken together, we believe that the close correlation between copper and NPY will complicate the role of NPY in AD progression, and the possible process can be simply reflected in Figure 9. In present study, we verified the hypothesis that there is a direct link between copper and NPY, preliminarily determined the affinity of copper toward NPY, and underplayed the consequence of copper binding inducing tyrosine nitration. However, coordination sites to copper in NPY are not solved, and the influence of copper binding on the active structure of NPY is still unknown. It is widely acknowledged that oxidative modification of peptides or proteins by ROS is implicated in normal aging and in the progression of a variety of physiological disorders and diseases.73−75 We can easily understand that NPY will undergo oxidative damage when exposed to high levels of hydroxyl radicals generated by copper

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DISCUSSION NPY is an important peptide hormone that is undergoing extensive investigation for its physiological and pathological functions in central nervous and peripheral nervous systems. Great progress has been made in determining the role of this peptide in the regulation of food intake, body temperature, blood pressure, vasoconstriction, adipogenesis, depression, bone formation, anxiety, pain, drug addiction, epilepsy, circadian rhythms, and cognition.16,60−63 Now, the new functions for it in the pathogenesis of neurodegenerative disorders have drawn great interest for most researchers, and its role in neurogenesis and neuroprotection has been extensively reported in papers.25 Recently, a growing body of studies showed that NPY is involved in AD, 26−28,64 characterized by the accumulation of amyloid fibrils and carbonylation and nitration,65,66 and NPY can prevent the neural cells from the toxic effect of β-amyloid peptides, exerting a neuroprotection effect in AD.29−31 However, our new findings of this work imply that the bioactivity of NPY may be also counteracted by the interaction of it with copper. The interaction between NPY and copper was first probed in this work by electrochemistry (CV, DPV). The result showed that NPY binding with copper lowered the peak current and shifted the reduction peak potential to a more negative value and the oxidation peak potential to positive value (Figure 2). It is similar to the electrochemical behavior of copper complexing with other peptides as reported in literature,46 indicating the redox activity of copper was restricted to some extent by the binding. The UV−vis result (Figure 3) further validated this interaction with a new absorption band at ∼427 nm, which were considered as d−d transition of resulting complex. Moreover, the intensity at 427 nm plotted as a function of Cu(II) equivalents seems to imply a 1:1 binding ratio. However, the stoichiometry is difficult to ascertain because of the lack of distinct points after one Cu(II) equivalent, where the absorption intensity stays unchanged. Therefore, this binding was further evidenced by the MS signals at m/z 867.8, 1084.5, 1445.6, and 2168.4 in the ESI-MS spectrum (Figure 4), which indicates the attachment of one copper ion to NPY. No more copper ions bound to a NPY species were observed. It illustrates that copper can indeed associate with NPY to form a molar ratio 1:1 complex in 910



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REFERENCES

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Figure 9. Schematic plot of the process of association of copper and NPY that is likely complicated in the AD brain. Copper binding with NPY enhances oxidative stress, thereby inducing important tyrosine in NPY nitration in the presence of nitrite, which potentially impedes the biological functions of NPY.

binding to it. Thus, it is also worthy to investigate the effect of metal-catalyzed oxidation on the structure and bioactivity of NPY. Overall, with the increasing studies showing that NPY is involved in AD, much effort needs to be made to ascertain the molecular mechanism between them. In conclusion, this study for the first time supports the hypothesis that copper has an action on NPY. The binding increases the oxidative stress and induces the important tyrosine nitration of NPY in the presence of nitrite, thereby likely attenuating the function of NPY in AD. These data may give a novel insight into understanding the pathology of AD. Certainly, the possible implications of this finding in AD pathophysiology need to be explored more.

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhonghong Gao: 0000-0002-7878-9801 Funding

This work was sponsored by the National Natural Science Foundation of China (nos. 31770866 and 31570810) and the Natural Science Foundation of Hubei Scientific Committee (2016CFA001). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank The Analytical and Testing Center of Huazhong University of Science and Technology for its help with the LCMS/MS analysis.

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ABBREVIATIONS NPY, neuropeptide Y; AD, Alzheimer’s disease; LC-MS, liquid chromatography-mass spectrometry; PYY, peptide YY; PP, pancreatic polypeptide; CNS, central nervous system; PTN, protein tyrosine nitration; ALS, amyotrophic lateral sclerosis; PD, Parkinson’s disease; NGF, nerve growth factor; ROS, reactive oxygen species; RNS, reactive nitrogen species; CV, cyclic voltammetry; DPV, differential pulse voltammetry 911


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Copper binding induces nitration of NPY under nitrative stress

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