Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Early stages of salmon calcitonin aggregation: Effect induced by ageing and oxidation processes in water and in the presence of model membranes
Introduction
Salmon calcitonin (sCT) is a 32-amino acid peptide, pharmacologically active in the treatment of postmenopausal osteoporosis, Paget's disease and hypocalcaemia [1]. Human calcitonin (hCT) acts in calcium homeostasis, lowering the serum calcium concentration and inhibiting osteoclastic bone reabsorption [2]. The releasing of hCT is under hormonal regulation.
Both native hCT and sCT are characterised by a primary sequence with a disulphide bond between Cys1 and Cys7 and a random coil secondary structure in water solution [3]. As shown by many authors, the presence of organic solvents, like methanol and trifluoroethanol, favours the α-helix secondary structure [4], while the presence of dimethyl sulfoxide or SDS induces the β-sheet conformation [3], [5], [6]. Nevertheless, the protein shows conformational reversibility after substitution of the organic solvent with water [4].
Both hCT and sCT have the tendency to aggregate in vitro, forming protofibrils and fibrils very similar to those observed in the Alzheimer's disease β-amyloid aggregation. The propensity to aggregate is higher for the human form, while sCT aggregates slowly. This is the reason for the preferential use of sCT as therapeutic agent [7], [8]. The conformational ad ultrastructural stability of calcitonins is time-, concentration-, temperature- and solvent-dependent. The comprehension of these factors is of large interest in pharmaceutical preparations because of the decrease of hypocalcemic activity related to the aggregation processes and conformational instability [9], [10].
Extracellular proto-hCT amyloid like fibrils and plaques were observed in vivo in thyroid medullary carcinoma [11], while hCT aggregates were found in plasma and urine of normal subjects [12].
Arvinte et al. studied in vitro the hCT aggregation process as a function of time and peptide concentration and hypothesized an aggregation mechanism. In their opinion, the fibrillation process can be explained by the same double nucleation mechanism, proposed earlier for gelation of Sickle Cell Haemoglobin (a protein forming amyloid-like fibrils in vitro). In this process, hCT molecules modify their secondary structure from the random-coil, occurring in water, to a mixture of α-helical and β-sheet structure occurring in initial aggregation states conformed at first as granules and then evolving to protofibrils [7]. Bauer et al. performed an interesting morphological and structural investigation by TEM, showing that the protofibril aggregation leads to polymorphism of fibrillar supramolecular assemblies [13]. The same author showed by transmission-FTIR spectroscopy that hCT aggregation process is associated with the increase in α and β components in the secondary structure [14]. Finally, X-ray diffraction experiments demonstrated that the sCT mature fibrils, grown in a 10 mg/ml solution at 230 °C after 9 days, are amyloids, characterised by the typical β-structure [11].
Amyloid-like fibrils are observed in several degenerative and ageing processes as in Alzheimer's [15] and post encephalitic Parkinson's diseases [16], in type 2 diabetes [17], in Spongiform Encephalopathy [18]. In all these diseases, a common structure characterised by extracellular plaques made of protein fibrils is observed [19]. These protein fibrils probably originate from protein misfolding and give rise to insoluble aggregates that might participate in the pathogenesis of the diseases, thus resulting in the acquisition of toxic properties [20].
It has been recently proposed that the aggregation process could be strongly influenced by the chemical conditions of the environment where it occurs and by the presence of lipids. It has been shown that the structure of the lipid phase constituting a biomembrane modulates the protein aggregation process and this can be a crucial point in the amyloid formation [21]. Besides, many proteins interacting with the biomembranes do not act as isolated units and their aggregation represents a crucial process through which they perform their biological functions.
Moreover, it is important to consider that the amyloid-like fibril formation is related to oxidative stress processes, cell toxicity and apoptosis [22], [23], [24]. Fibrils are involved in cellular hydrogen peroxide increase, peroxy radicals production [25] and lipid peroxydation [24]. On the other hand, it is more and more evident that the unbalancing between oxidant and reducing agents observed in many pathological states and during ageing can affect protein aggregation and citotoxicity [26], [27].
On the basis of all these current literature considerations, we believe that the study of the amyloid proteins aggregation in the presence of model membranes and in the presence of oxidant agents seems to be fundamental in the comprehension of the basic mechanisms governing the formation of amyloids. As pointed out for β-amyloid in Alzheimer's disease, the oxidative stress seems to play a crucial role also in the calcitonin aggregation.
Our recent experiments demonstrate that the sCT aggregation in vitro is triggered by free radicals. We showed that the presence of hydroxyl radicals, generated in vitro by a modified Fenton's reaction, induces sCT oxidised forms and modifies the secondary structure of the peptide. We also observed that the presence of OH· in the solution increases the sCT natural aggregation processes [28].
In this study, we extended our previous work investigating in vitro the time-dependent ageing and the chemical induced oxidation processes of sCT in vitro by using a different oxidant agent, the hydrogen peroxide, that is one of the Reactive Oxygen Species (ROS) present in vivo during ageing, in many pathologies and degenerative processes [29], [30], [31]. Moreover, the study was also conducted in the presence of membrane model consisting of dipalmitoylphosphatidylcholine (DPPC) liposomes in order to mimic the conformational variations and aggregation processes of sCT occurring in extra-cellular compartment. Liposomes are generally accepted as simplified three-dimensional models of the lipid membranes. They can be easily prepared using plain lipid and structurally characterised in aqueous suspensions by means of spectroscopic techniques or deposited onto suitable substrates for TEM analysis [32].
The choice of the zwitterionic phosphatidylcholine (PC) was due to its abundance in the actual cellular membranes. Some authors showed that sCT interacts with acidic phospholipids and, in particular, with phosphatidylglycerol (PG), to form water-soluble complexes as PG promotes bilayer insertion of sCT [33]. On the contrary, in the presence of dimyristoyl-PC, strong interactions were not observed [34].
This paper describes a comparative study between natural ageing and H2O2-induced oxidation processes, with and without DPPC liposomes. Our aim was the investigation of the aggregation mechanisms and conformational variations in the presence of phospholipidic model membranes, with the focus on the effect induced by the presence of phospholipids on the H2O2 oxidation process.
Section snippets
Preparation of sCT water solutions and sCT-liposomes solutions
Salmon calcitonin CRS (MW = 3431.9 Da) was purchased from European Pharmacopoeia (EDQM, Strasbourg, France) and stored at −18 °C before use. l-α-phosphatidylcholinedipalmitoyl (DPPC) 99% (MW = 734.0 Da) was purchased from SIGMA (SIGMA Chemical Co., St. Louis, USA) and stored at −18 °C before use. All reagents were of analytical grade.
sCT 0.04 mg/ml solutions (corresponding to 1.2 × 10−5 M protein content) were prepared immediately before use by dissolving the protein in the solid state in 5 mM sodium
Results and discussion
Fig. 1A shows the CD spectra of sCT in buffered water solutions, recorded at increasing ageing time (days) after the solution preparation. Spectrum (a) was recorded the day of sample preparation, (b) 4 days after, (c) 5 days after, (d) 6 days after and (e) 10 days after. As it can be observed, spectrum (a) shows a minimum at 202 nm, indicating that the protein assumed a random coil conformation in water solution, as reported in literature [5].
Four and 5 days after, a general decrease in the
Conclusions
The CD results were in agreement with TEM images. In the first stage of protein ageing in water, the sCT aggregation processes occurred with slight conformational changes; 6 days after, a new important conformational change appeared contemporary to a more evident aggregation. A β-sheet conformation, as described for mature CT fibrils and plaques, was not observed. Nevertheless, we want to stress that our observations refer to early stages of the aggregation at very low protein concentration and
Acknowledgements
This research was supported by a grant of the National Health Fund for the project “Safety of Drugs Used in Diseases of Elderly” (Progetto 1%-Ricerca Sanitaria Finalizzata 2000). The authors are also grateful to Mr. C. De Sena and to Mr. S. Alimonti for their technical assistance.
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