Journal of Molecular Biology
Regular articleTetracycline affects abnormal properties of synthetic PrP peptides and PrPSc in vitro1
Introduction
Prion diseases such as scrapie of sheep, spongiform encephalopathy of cattle (BSE) and Creutzfeldt-Jakob disease (CJD), fatal familial insomnia and Gerstmann-Sträussler-Scheinker disease (GSS) of humans are transmissible neurodegenerative disorders that share a common pathogenic mechanism, i.e. the conversion of the cellular prion protein (PrPC) into disease-specific species termed PrPSc Prusiner 1991, Prusiner et al 1998. This process involves a conformational change of PrPC resulting in decreased α-helical structure and striking increase in β-sheet content Caughey et al 1991, Pan et al 1993, Safar et al 1993. The PrPC to PrPSc transition is associated with the acquisition of abnormal chemicophysical properties, including insolubility in non-denaturing detergents, partial resistance to protease digestion and high tendency to form aggregates and amyloid fibrils Prusiner 1991, Prusiner et al 1998. The accumulation of PrPSc and fragments thereof in the central nervous system is accompanied by activation of microglial cells, hypertrophy and proliferation of astrocytes and degeneration of neurons, suggesting a primary role of abnormal PrP peptides in the pathologic process De Armond et al 1988, De Armond et al 1993, Bruce et al 1989, Williams et al 1997. Evidence indicates that the neoformed PrPSc interacts with PrPC and converts the normal protein into a likeness of itself Kocisko et al 1994, Bessen et al 1995. According to this view, PrPSc is responsible for transmissibility and progression of the disease as well as for brain damage, thus representing a primary target for therapeutic strategies.
The urgency to identify and develop therapeutic compounds has remarkably increased following the epidemic of BSE and the appearance of a new variant of Creutzfeldt-Jakob disease (vCJD) that seem to be causally linked Will et al 1996, Collinge et al 1996, Bruce et al 1997. Although the number of vCJD cases observed so far is limited, a future outbreak of this disease cannot be excluded (Ghani et al., 1998).
A variety of compounds have been tested on cellular and animal models of prion disease. A few of these compounds, including sulphated polyanions Kimberlin and Walker 1983, Kimberlin and Walker 1986, Ehlers and Diringer 1984, Farquhar and Dickinson 1986, Caughey and Raymond 1993, amphotericin B Pocchiari et al 1987, Xi et al 1992, Adnjou et al 1995, Congo red Caughey and Race 1992, Ingrosso et al 1995, iododoxorubicin (Tagliavini et al., 1997), tetrapyrroles Caughey et al 1998, Priola et al 2000, branched polyamines (Supattapone et al., 1999) and modified PrP peptides (Soto et al., 2000), effectively reduce the accumulation of PrPSc in scrapie-infected neuroblastoma cells and/or delay the appearance of clinical symptoms and prolong the survival time of rodents experimentally infected with scrapie. This group of compounds is structurally heterogeneous and the mechanism of action of individual molecules in experimental prion disease is unknown. However, some of these molecules (i.e. Congo red, iododoxorubicin and tetrapyrroles) share common chemicophysical properties in that they contain an extended hydrophobic core formed by aromatic moieties with a large number of hydrophilic substituents conferring an amphiphilic character. Since the conversion of PrPC into PrPSc results in an increase of hydrophobicity due to solvent exposure of lipophilic residues and in the ability to form insoluble aggregates and amyloid fibrils, it is conceivable that the anti-prion activity of the compounds is related to their hydrophobic character allowing for a strong interaction with PrPSc and fragments thereof. This interaction has been shown for Congo red and iododoxorubicin, which bind to amyloid fibrils of different biochemical composition, including PrP amyloid Prusiner et al 1983, Merlini et al 1995, Tagliavini et al 1997.
Based on structural analogies with Congo red and iododoxorubicin, we hypothesized that tetracyclines might be able to interact with PrPSc and PrP amyloid. An advantage of this class of drugs is that several congeners with well characterized pharmacological and pharmacokinetic properties are available. Further, tetracyclines have relatively low toxicity and some derivatives cross the blood/ brain barrier efficiently if an appropriate route of treatment is used (Sande & Mandell, 1991). In this study we show that tetracycline binds and disrupts PrP peptide aggregates, reverts the protease resistance of PrPSc extracted from brain tissue of patients with CJD, and abolishes the neurotoxicity and astroglial proliferation induced by PrP peptides in vitro. These properties make tetracycline a prototype of compounds with the potential of inactivating the pathogenic forms of PrP, impeding PrPC to PrPSc conversion, and hindering the development and progression of prion disease.
Section snippets
Tetracycline binds to PrP peptides
The ability of tetracycline to interact with PrP aggregates was investigated using amyloid fibrils generated by synthetic peptides homologous to residues 82 to 146 and 106 to 126 of human PrP (PrP 82–146 and PrP 106–126). The former corresponds to the amyloid protein purified from brain tissue of GSS patients Tagliavini et al 1991, Tagliavini et al 1994 while the latter is a fragment thereof which shares chemicophysical and biological properties with PrP 82–146 as well as with the
Discussion
The present study shows that tetracycline interacts with a synthetic peptide homologous to the amyloid protein purified from GSS brains (Tagliavini et al., 1994) and a fragment thereof which is highly fibrillogenic, neurotoxic and gliotrophic in vitro Forloni et al 1993, Forloni et al 1994, Tagliavini et al 1993, Brown et al 1996.
This interaction affects the aggregation properties of the peptides and antagonizes their deleterious effects on nerve and glial cells. It is most remarkable that
Peptide synthesis and purification
The peptides PrP 106–126 (KTNMKHMAGAAAAGAVVGGLG), PrP 106–126 scrambled (NGAKAL MGGHGATKVMVGAAA) and PrP 82–146 (GQPHGGGWGQ GGGTHSQWNKPSKPKTNMKAG AAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYE) were synthesized by solid phase chemistry on an Applied Biosystem 430A synthesiser as described Forloni et al 1993, Tagliavini et al 1993. The peptides were cleaved from the resin with phenol/thioanisole/trifluoroacetic acid, precipitated and washed several times with cold diethylether, and purified by preparative
Acknowledgements
This work was supported, in part, by the Italian Ministry of Health, Department of Social Services, Telethon-Italy (Grant E.574) and the European Community (BMH4 CT-98-6011 and CT-98-6051). We are grateful to R.J. Kascsak and F. Prelli for providing us with the antibodies 3F4 and SP214.
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