NAP Neuroprotective Peptide and its Analogs: Simultaneously Copper and Iron Binding and Reduction

ANCUTA-VERONICA LUPAESCU1, ION SANDU2,3, BRINDUSA ALINA PETRE1,4, LAURA ION1, CATALINA-IONICA CIOBANU5, GABI DROCHIOIU1* 1Alexandru Ioan Cuza University, Faculty of Chemistry, 11 Carol I Blvd., 700506 Iasi, Romania 2Alexandru Ioan Cuza University of Iasi, Arheoinvest Interdisciplinary Platform, Scientific Investigation Laboratory, 11 Carol I Blvd., 700506 Iasi, Romania 3Rmanian Inventors Forum, 3 Sf. Petru Movila Str., Bloc L11, III/3, 700089 Iasi, Romania 4Center for Fundamental Research and Experimental Development in Translation Medicine -TRANSCEND, Regional Institute of Oncology, 2-4 General Henri Mathias Berthelot, 700483 Iasi, Romania 5Alexandru Ioan Cuza University, Faculty of Chemistry, Research Department, 11 Carol I Blvd., 700506 Iasi, Romania

All living beings operate with transition metals such as copper or iron in order to conduct their basic metabolic processes. Their importance can be noticed even at the synaptic level. For example, iron ions are needed for gene expression, the synthesis of neurotransmitters, myelination or respiratory management of the brain, while copper ions are an important cofactor for antioxidant enzymes, neurotransmitter biosynthesis and mitochondrial respiration [1][2][3].
Neurodegenerative diseases are a critical worldwide health concern that affects the nervous system. These pathologies are associated with ageing and share features such as selective neuronal death, protein aggregation, oxidative stress, mitochondrial dysfunction, transition metal accumulation and inflammation [4]. However, evidence of a link between transition metals and neuronal death has been noticed in many neurodegenerative disorders such as Alzheimer 's (AD), Parkinson's (PD) and Huntington's (HD) diseases [5][6][7]. Moreover, statistically significant differences between copper and zinc concentration in serum and saliva samples were observed at patients with oral cancer and oral potentially malignant disorders, suggesting a pathological role in those disease progression [8]. The accumulation of metals was observed in the brain of patients with neurodegenerative diseases. Thus, altered metal homeostasis can be one of the factors causing those disorders. Besides, redox active metals such as cooper and iron carry pro-oxidant properties that induce oxidative stress by generating reactive oxygen species (ROS) [9,10]. Most of the reactions involving redox active metals are related to Fenton chemistry that includes a series of reactions that initiates with hydrogen peroxide and leads to the formation of highly unstable radicals that affect biological macromolecules [5]. The disproportionate production of ROS species causes protein, DNA or phospholipids oxidations that mirrors in functional alterations [11,12]. Thus, metal-binding proteins involved in the metal transport and distribution (copper transporter protein 1 and ATP7A, transferrin, transferrin receptor, DMT1) at synaptic level could have influence on the modified metal homeostasis in the brains of patients with neurodegenerative diseases [4].
Neuroprotective strategies against ROS-mediated damages in order to prevent oxidative stress and disease progression have been elaborated in numerous AD researches [13][14][15][16][17]. Some of them successfully targeted mitochondria known as a major source of ROS. However, no crucial improvement was observed at AD patients [18].
Peptide-based drugs have emerged as a major class of therapeutics. NAP (NAPVSIPQ) is a small active fragment of activity-dependent neuroprotective protein (ADNP) essential for brain formation [19]. Studies involving cell culture reveled the neuroprotective proprieties of NAP peptide against toxicity associated with the beta-amyloid peptide, N-methyl-D-aspartate, electrical blockade, the envelope protein of the AIDS virus, dopamine, H 2 O 2 , nutrient starvation and zinc overload [20]. Also, NAP can be delivered to the brain via intranasal or intravenous administration were it provides nerve cell protection at very low concentrations providing new solutions for the formulation of neuroprotective drugs for the treatment of AD [21]. Studies involving metal-NAP interaction displayed abnormal metal reduction for copper and iron using cysteine-mutant NAPC [22,23].
This study focuses on the interaction of Cu 2+ and Fe 3+ ions with neuroprotective peptide NAP and its analogs. An interesting phenomenon consisting in metal ion reduction followed by the formation of complexes with peptides was found within this research. MS analyses suggest a protective role of NAP-based peptides, since they are capable of capturing metal ions.

Experimental part Materials and methods Reagents
All chemicals were of analytical grade and were used as received without any further purification. All solutions were prepared using milliQ-grade water (18. Instruments. A Vibra HT analytical balance (Japan) was used to weigh the solid chemicals. The peptide samples were incubated at room temperature using an Eppendorf Thermomixer compact purchased from Germany, vortexed using a Vortex BioCote (UK) and centrifuged using a Sprout centrifuge (USA). For sample lyophilization, an ALPHA 1-2 LO Freeze Dry System was used.
MALDI-ToF MS analysis was performed on a Bruker Ultraflex MALDI ToF/ToF mass spectrometer operated in positive reflectron mode and equipped with a pulsed nitrogen UV laser (λ max 337 nm).
Peptide synthesis. The peptides were synthesized using the solid phase Fmoc/tBu Solid Phase Synthesis (SPPS) method. The peptide syntheses were described elsewhere [22,23]. In brief, the synthesis of peptides was carried out manually in a linear fashion on a Fmoc-Gln(Trt)-Wang Resin in a fritted plastic syringe. Crude peptides resulted by cleavage from the solid support using the standard TFA: TIS: H 2 O solution (95: 2.5: 2.5). Following precipitation and lyophilization, the products were characterized by analytical RP-HPLC and MALDI-ToF mass spectrometry and purified by semi preparative RP-HPLC, when necessary.
Metal-peptide complexes. The solutions of peptides (8 mM) and those of metal ions (80 mM) were prepared using milliQ-grade water. Metal complexes of NAP-like peptides were obtained by mixing the corresponding octapeptides with metal solution at pH 7 at a 1: 10 peptide to metal ratio. Then, the resulted solutions were incubated at 350 rpm overnight at room temperature.
MS study. MALDI-ToF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) mass spectra were recorded in positive reflectron mode using a Bruker Ultraflex MALDI ToF/ToF mass spectrometer. The samples were spotted onto a 384-spot target plate of the MALDI-ToF instrument according to the dried-droplet method. Each sample and matrix solution (2,5-dihydroxybenzoic acid) were mixed on the target and allowed to dry in the ambient air. For spectra processing, the Bruker´s FlexAnalysis 3.4 software was used.
Computer programs. The exact mass (monoisotopic mass) was calculated using the application web ChemCalc [24]. Also, this application allows predicting the isotopic distribution graph and calculating peptide fragmentation.

Results and discussions
In this study, five NAP-like peptides, previously synthesized [25], were incubated over night at room temperature in aqueous medium with Cu 2+ and Fe 3+ ions. The interaction and their affinity of these ions toward NAPlike peptides were investigated using the MALDI ToF mass spectrometer. It is well-known that cysteine and histidine side chains are potential anchors for metal ions [26].
The characteristics of NAP-like peptides investigated in our study are presented in table 1. In principle, these peptides have similar structures, the only modification occurring in the 5 SIP 7 (Ser-Ile-Pro) active center [21]. More precisely, all peptides have similar sequence, except the replacement of the fifth amino acid residue, serine, with glycine (NAPG), alanine (NAPA), histidine (NAPH) or tyrosine (NAPY).
The positive-ion matrix-assisted laser desorption/ ionization time-of-flight (MALDI ToF) mass spectra of the NAP-based peptides after incubation with metal ions (Cu 2+ or Fe 3+ ) are given in figure 1 and 2. At first glance, it can be clearly seen that both metal ions obeyed reduction upon binding to NAP peptides. All peptides displayed signals attributed to molecular ions [M+H] + and their adducts with sodium ([M+Na] + ) and potassium ([M+K] + ). Another signal, assigned to deamidated peptide can be spotted in all spectra at -16 m/z ([M+H-16] + ). As seen in table 1, all NAP peptides contain C-terminal glutamine (Q), which is susceptible to rearrangements that lead to peptide deamidation and pyroglutamic acid formation [27]. However, this photochemical process occurs only during the peptide ionization under the laser light source [25].
The most intense peak observed in the MALDI ToF spectra of copper incubated samples ( fig. 1) Table 2 shows the theoretical and experimental values of the main peaks in the mass spectra. The experimental m/z values displayed by the MALDI ToF mass spectrometer were in best agreement with theoretical data obtained using the ChemCalc program. In this study we showed that two noxious metal ions can bind to anti-amyloid peptides. Heavy metals, including copper and iron, are environmental contaminants that cannot be degraded or destroyed [10,29]. Their biological effect differs from that of organic pollutants such as dinitrophenols [30,31]. Decontamination of dinitrophenol derivatives can be done by yeast suspensions, since they can live anaerobically [32,33]. Thus, heavy metal altered metal homeostasis can be one of the factors causing many pathologies. In addition, redox active metals carry prooxidant properties that induce oxidative stress by ROS. These reactive oxygen species causes protein, DNA or phospholipids oxidations. Previously, we investigated the interaction of heavy metal ions with Aβ peptides associated with Alzheimer disease using glycyl-Ltryptophan, which interact with Fe 3+ and Cu 2+ ions that decrease its fluorescence intensity at 350 nm [7].
In this research, we performed mass spectrometric measurements to investigate heavy metal binding to peptides and proteins, since this technique is an important analytical approach utilized for quantitative and qualitative determination, structure and chemical properties elucidation [34,35]. Due to its wide range of applications, MS has become an indispensable tool for analyzing biomolecules [36]. Proteomics research took advantage of the development of soft ionization techniques such as electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) capable of easily transforming biomolecules into ions [37]. MALDI mass spectrometry implies the use of a matrix capable of absorbing the laser energy and creates ions from large molecules with minimal fragmentation. Thus, MALDI technique is capable to ionize complexes while using less solvent than electrospray ionization techniques [38]. The MALDI ionization is commonly used for the analysis of high molecular weight biological compounds, but has also been used for transition metal complexes analysis [39].
We investigated here NAP analogs containing glycine, alanine, histidine and tyrosine residues instead serine in the native NAP sequence, since cysteine is well-known ligand for copper and iron [22,26,41]. On using ESI ion-trap MS measurements, iron binding to Aβ(1-40) peptide associated with Alzheimer disease was demonstrated [42]. Our results using MALDI ToF MS have thus confirmed Fe 2+ ion binding to peptides, and not Fe 3+ ion. It is therefore possible that Fe 2+ ion, but Fe 3+ ion, to bind to Aβ , which means that further research is necessary to confirm this hypothesis. Besides, ESI-MS results seem to be in good agrement with those obtained by MALDI-ToF MS [41,43]. Our studies on copper and iron interaction with neuropeptides are in line with the literature data [44]. Copper binding to peptides and proteins was also studied in order to find new methods for protein determination [45]. Indeed, protein determination is still a difficult task and novel assays were proposed [46,47]. However, the main result of our study is redox metal binding to NAP-like peptides only under their reduced form.

Conclusions
In this work we have demonstrated the reduction of Fe 3+ and Cu 2+ ions to Fe 2+ and Cu + followed by their binding to some newly synthesized NAP-like peptides. This ubiquitous phenomenon may suggest a double protective role for NAP peptide: i) metal scavenger and ii) potential therapeutic agent against the ROS metal-mediated cascade. Since the Fenton oxidation chemistry is a general feature for all neurodegenerative disorders, chelation  therapy constitutes a possible treatment against heavy metal accumulation. Our results identify native NAP and its glycine (NAPG), alanine (NAPA), histidine (NAPH) and tyrosine (NAPY) modified peptides as therapeutic strategies for metal chelating. However, different peptides bind differently iron and copper ions. Further research is needed to understand the complex relationship of NAPlike peptides and metal ions involved in Alzheimer disease or other neurodegenerative pathologies.