Maria Chiara
Monti
,
Luigi
Margarucci
,
Alessandra
Tosco
,
Raffaele
Riccio
and
Agostino
Casapullo
*
Dipartimento di Scienze Farmaceutiche e Biomediche, Università degli Studi di Salerno, Via Ponte don Melillo, 84084, Fisciano, Italy. E-mail: casapullo@unisa.it; Fax: +39 089 969602; Tel: +39 089 969243
First published on 7th July 2011
Oleocanthal (OLC) is a phenolic component of extra-virgin olive oil, recently supposed to be involved in the modulation of some human diseases, such as inflammation and Alzheimer. In particular, OLC has been shown to abrogate fibrillization of tau protein, one of the main causes of Alzheimer neurodegeneration. A recent interpretation of this mechanism has been attempted on the basis of OLC reactivity with the fibrillogenic tau hexapeptide VQIVYK and SDS-PAGE of OLC/tau incubation mixtures, suggesting that covalent modification events modulate tau fibrillization. In this paper we report a detailed mass spectrometric investigation of the OLC reactive profile with both tau protein fibrillogenic fragment K18 and propylamine in biomimetic conditions. We show that K18 is prone to be covalently modified by OLC through Schiff base formation between the ε-amino group of lysine residues and OLC aldehyde carbonyls. Moreover, as expected from its de-structured conformation, K18 shows a non-selective modification profile, reacting with several lysine residues to give cyclic pyridinium-like stable adducts. These data give new insights on the mechanism of inhibition of tau fibrillization mediated by OLC.
Inside the secoiridoid family of olive oil constituents, oleocanthal (OLC, Fig. 1), the dialdehydic form of (−)-deacetoxy-ligstroside aglycon responsible for the bitter taste of olive oil, has been recently supposed to interfere within important pathways of relevant human diseases, such as inflammation and Alzheimer (AD).14,15
Fig. 1 Chemical structure of (−)-oleocanthal. |
Recently, a role of OLC in the alteration of oligomeric structure of soluble Aβ peptides and tau protein, both involved in AD, has been reported.15 In particular, OLC increases the immunoreactivity of soluble Aβ species, when assayed with both sequence and conformation specific Aβ antibodies, protecting neurons from the synapto-pathological effects of Aβ assemblies.16 Moreover, since the aggregation of tau protein strongly correlates with the clinical progression of the AD, it seemed likely that inhibition or reversal of tau aggregation could protect the affected neurons.17
Tau is a microtubule-associated protein, particularly abundant in the neuronal axons, working as a stabilizer of the microtubules (MTs) by a direct interaction promoted by a microtubule-binding domain (MBD),18 thus modulating the plasticity of cytoskeleton. The MBD is located in the C-terminal half of the protein and is composed of three- or four-repeat structures (3RMBD or 4RMBD, respectively), with each repeat peptide (R1–R4) consisting of 31 or 32 amino acids; these four single-repeat structures have relatively similar and conserved amino acid sequences.19,20 Tau is a highly soluble protein with a random conformation in aqueous solution, and hardly shows any tendency to assemble under physiological conditions.21 In the AD patients, however, tau dissociates from axonal microtubules and abnormally aggregates to form an insoluble paired helical filament (PHF), which is implicated in neurodegeneration.22,23 Since the amount of tau aggregates has been correlated with neuron loss and the severity of dementia, the analysis of its self-assembly mechanism and the discovery of lead compounds capable of reducing the PHF formation24–28 could provide the needed information to develop an effective method to slow down the neurodegenerative process.
It has been recently reported that two VQIXXK motifs in the MT binding region, named PHF6 (from V306 to K311) and PHF6* (from V275 to K280), are responsible for the development of β-sheet structure and tau fibril formation.21
In 2009, Li and co-workers demonstrated that OLC and several analogues abrogate tau fibrillization, by means of ThT fluorescence, sedimentation analyses and EM experiments, freezing the protein in its unfolded state.15 They also assessed, on the basis of the OLC reactivity with N-Bz-lys-OMe and the fibrillogenic hexapeptide PHF6, the key role of the two OLC aldehyde groups in the covalent modification of the PHF6 lysine (K133), mediated by a Schiff base formation, and consequently their potential relevance in the inhibitory activity of OLC. However, only a few suggestions on the exact molecular mechanism of interaction between the native protein and OLC were reported, essentially based on the SDS-PAGE analysis of OLC-K18 mixtures and low resolution MALDI-MS analysis.15 These data, in our opinion, are not fully adequate to infer the exact mechanism of action of OLC and the punctual reaction site on the entire native tau protein, since Li and co-workers pointed out OLC reaction pathway using the short PHF6 peptide which cannot be representative of a complex protein system. Thus, following our previous studies on bioactive aldehyde-containing natural products,29–33 we investigated OLC-tau protein interaction in pseudo-physiological conditions by high resolution MS approach. Since the longest tau isoform T40 was not suitable for MS evaluations, we used its K18 fragment that, as previously discussed, contains all four MT-binding domains representing the integral core element of the pathological tau filament responsible for the fibrillization process.34 Our work was based on the MS characterization of the reaction profile of OLC with K18 and the identification of residues involved in the covalent modification of the polypeptide. The following analysis of OLC reactivity with propylamine in relevant bio-mimetic conditions, by 1D/2D NMR, LC-ESIMS and tandem MS in real time, gave additional support to the proposed mechanism of ligand-protein interaction. Our results point towards a non-selective covalent modification of K18 in which the two OLC carbonyl groups react with different tau lysine residues to give cyclic pyridinium-like stable adducts.
The mixture of un-reacted and modified protein upon incubation at 4 °C and NaBH4 reduction was analyzed by RP-HPLC on a Phenomenex Proteo column (250 × 2.0 mm) by means of a linear gradient from 20% to 70% aqueous acetonitrile containing 0.1% TFA, over 50 min at 0.150 ml min−1. The elution profile was monitored at 220 and 280 nm. The fractions were collected and analyzed on a MALDI micro MX™ (Waters Co, Milford Massachusetts, USA) in reflectron positive ion mode, using α-cyano-4-hydroxycinnamic acid (10 mg ml−1) dissolved in H2O–CH3CN (50/50 v/v) 0.1% TFA as matrix, in a m/z range of 10000–30000.
A3: ESIMSm/z 326.3 [M]+; ESIMS/MSm/z 121.5;.1H NMR δ (CDCl3, 600.13 MHz) 8.73 (H–1, bs), 8.46 (H–3, d, J = 6.3.Hz), 7.79 (H–4, d, J = 6.3 Hz), 3.92 (H–6, bs), 6.80 (H–9, dd, J = 17.4 and 11.5 Hz), 5.73 (H–10a, d, J = 11.5 Hz), 5.94 (H–10b, d, J = 17.4 Hz), 4.18 (H–1′a, m), 4.22 (H–1′b, m), 2.80 (H–2′, t, J = 7.0 Hz), 7.05 (H–4′/8′, d, J = 8.0 Hz), 6.76 (H–5′/7′, d, J = 8.0 Hz); 4.46 (H–1′′, t, J = 7.3 Hz), 1.99 (H–2′′, sext., J = 7.3 Hz), 0.98 (H–3′′, t, J = 7.3 Hz); 13C NMR δ (CDCI3, 150.9 MHz) 140,1 (C–1), 141,4 (C–3), 128,0 (C–4), 150.0 (C–5), 37.5 (C–6), 171.9 (C–7), 127.4 (C–8), 138.4 (C–9), 122.3 (C–10), 65.2 (C–1′), 34.3 (C–2′), 129.5 (C–3′), 130.1 (C–4′/8′), 115.4 (C–5′/7′), 154.1 (C–6′), 61.9 (C–1′′), 23.4 (C–2′′), 11.0 (C–3′′)
The assessment of the mechanism of OLC-K18 interaction between OLC and K18 consisted of the following steps: (a) structural analysis of the ligand-protein complexes and (b) characterization of the OLC reaction profile with propylamine in bio-mimetic conditions. To this end, we employed an experimental protocol based on the combination of NMR spectroscopy, mass spectrometry and classical protein chemistry (proteolytic digestion, RP-HPLC).
The time course LCMS analysis of the reaction at 4 °C (see spectra in Fig. 2A) showed the presence of two main peaks, already after 5 min, corresponding to the unmodified K18 and mono-modified K18 species with a mass increment of 268.5 ± 1.3 Da, that increased at 272 ± 0.8 Da and 274 ± 0.6 Da after reduction with NaBH4 and NaBD4 respectively (Table 1). Mechanistic considerations, attempted on the basis of this data set, pointed towards two possible reaction pathways, both mediated by Schiff base intermediates formed between lysine ε-amino group(s) on K18 and the OLC aldehyde carbonyls.
Mw after OLC treatment | |||||
---|---|---|---|---|---|
Measured K18 Mw | Measured Mw | ΔMw | Reducing Agent | Temperature | Time |
13812.2(±1.0) | 14080.7(±1.3) | 268.5 | none | 4 °C | 5′;15′,60′,180′,300′ |
13812.5(±0.9) | 14084.6(±0.8) | 272.1 | NaBH4 | 4 °C | 5′;15′,60′,180′,300′ |
13812.4(±1.5) | 14086.4(±0.6) | 274.0 | NaBD4 | 4 °C | 5′;15′,60′,180′,300′ |
13812.2(±1.0) | 14080.8(±1.3) | 268.6 | none | 37 °C | 5′; |
13812.5(±0.9) | 14084.8(±0.8) | 272.3 | NaBH4 | 37 °C | 5′; |
13812.4(±1.5) | 14086.7(±0.6) | 274.3 | NaBD4 | 37 °C | 5′; |
13812.7(±0.8) | 13901.8(±0.8) | 89.1 | none | 37 °C | 15′,60′,180′,300′ |
13812.3(±1.1) | 13904.3(±0.9) | 92.0 | NaBH4 | 37 °C | 15′,60′,180′,300′ |
13812.8(±1.2) | 13906.9(±0.6) | 94.1 | NaBD4 | 37 °C | 15′,60′,180′,300′ |
Fig. 2 After addition of NaBH4 deconvoluted mass spectra of K18 incubated in presence of OLC. The spectra were recorded at different times (for convenience only three time points are shown) and different temperatures 4 °C (A) and 37 °C (B). |
In the first path (Scheme 1A), OLC cross-links two different K18 lysine residues, giving rise to a double imine reversible macrocyclic system (ΔMW of 268.5 Da) easily reduced in presence of NaBH4 or NaBD4 (Table 1). In a second hypothesis (Scheme 1B), a single lysine ε-amino group reacts with either of OLC carbonyls to give a stable iminium six-member ring adduct (ΔMW of 269 Da), that is converted to a cyclic amine15 with NaBH4.
Scheme 1 Possible reaction mechanisms for the covalent modification of K18 by OLC at 4 °C. Mass increments were measured by LC-ESIMS. Panel A shows the reaction mechanism between OLC and two different lysine residues on a unique K18-protein. Panel B reports the reaction between a single lysine ε-amino group and both OLC carbonyls to give an iminium six-member ring. |
When the reaction was carried out at 37 °C, a more complex pathway has been observed, as shown in Fig. 2B. As a matter of fact, we monitored the same K18 adduct observed at 4 °C, after 5 min (ΔMW of 268.5 Da), that evolved after 15 min to a species with a mass increment of 89.5 Da, subsequently reduced with NaBH4 (ΔMW of 92.1 Da) or NaBD4 (ΔMW of 94.2 Da), which remained unchanged even after 5 h (Fig. 2B and Table 1).
We suggested a plausible mechanism for this new evidence (Scheme 2), in which the early forming species with a mass increment of 268.5 Da turns into a more stable rearranged adduct giving rise to the loss of 4-hydroxy-phenylethyl acetate.
Scheme 2 Proposed mechanism for the covalent modification of K18 protein by OLC at 37 °C. |
Finally, we determined the sites of covalent modifications on K18 in the reaction with OLC, either at 4 °C or 37 °C. The reaction mixtures, treated with NaBH4, were digested with trypsin and analyzed by MALDI-MS. This experiment enlightened on the occurrence of the mechanism of K18 covalent modification reported in Scheme 1.
Indeed, as expected for an intrinsically disordered polypeptide, we found several lysine residues modified by OLC both at 4 °C and 37 °C (see ESI, Fig. S1†). Besides, the early reaction at 4 °C evolved by a cross-link modification mechanism (Scheme 1A), since we detected in the tryptic mixture only fragments containing two peptides cross-linked to OLC. Increasing the time, the reaction equilibrium shifted to the mechanism depicted in Scheme 1B, as we monitored only single peptides modified by OLC on a single lysine residue.
Since the 1,5-dialdehyde moiety of OLC is prone to undergo attack by nitrogenous nucleophiles giving rise to a complex web of multiple chemical equilibria and an array of possible intermediates, as already suggested by Li and co-workers,15 we decided to give a firm evidence of the reaction pathways proposed above. On this basis, we analyzed the covalent reactivity of OLC towards the small nucleophile propylamine (PA), in relevant bio-mimetic conditions, following the time course of the reaction by 1D/2D NMR and MS. Thus, we applied two parallel sets of HPLC-ESIMS and NMR experiments: in the first step we followed the course of the reaction monitoring by LCMS the intermediates formation, in different conditions and after addition of NaBH4. Then, we applied 1D and 2D NMR to track the reaction course and characterize the final product(s).
The interpretation of the LCMS and NMR data supported the reaction pathway depicted in Scheme 3, in which two key intermediates (A1 and A2) and the final product A3 can be postulated. OLC promptly reacted with PA producing, after 3 min, a main species at m/z 346.3 (OLC + PA – H2O), matching a Schiff base formation between PA and one of the OLC aldehyde carbonyls. The most reactive aldehyde function was identified tracking the reaction course by NMR. Proton spectra were recorded after addition (every 10 min) of little amounts of PA to a CD3CN solution of OLC in the NMR tube (see Materials and methods for details). The reduction and final disappearance of the signal at δ 9.64 (H–3) (see ESI, Fig. S2†) supported the formation of the imine intermediate A1. This species, through the isobar intermediate A1*, evolved to the adduct A2 (m/z 328.3, 15 min incubation time), that slowly turned into two species at m/z 326.3 and 148.2 (30 min incubation time). The species at m/z 326.3 was isolated and unequivocally characterized by 1D and 2D NMR (see Materials and methods) as the aromatic N-alkyl pyridinium A3 and consequently the other compound as A4 species, arising from the rearrangement of A2 (Scheme 3). This picture was confirmed by LCMS analysis of the reaction after addition of NaBH4 (Scheme 4). The reduction of the imineA1 gave the amineB1 that, after cyclization and dehydration, evolved into the imonium species B2, rapidly reduced to the cyclic alkyl-amine B3.
Scheme 3 Reaction mechanism between OLC and propylamine. |
Scheme 4 Reaction mechanism between OLC and propylamine in presence of NaBH4. |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1fo10064e |
This journal is © The Royal Society of Chemistry 2011 |