Introduction Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of unusual neurodegenerative disorders affecting both animals and humans [1]. The agent believed to be responsible for these pathologies is a protein whose name is PrPsc, which is a conformational variant of the normal cellular prion protein, PrPc, a 33-35 KDa glycosyl-phosphatidylinositol-anchored protein, expressed at high levels in neuronal cells. Unlike PrPc, PrPsc is characterized by a higher β-sheet content and is partially resistant to proteolysis [2],[3]. The amino terminus of PrPc contains four octapeptide repeats, which are implicated in the binding of divalent metal ions and in particular copper; because of this property, PrPc could be reasonably involved in copper metabolism and in the defense mechanism of the cell against oxidative stress, possibly through the regulation of the Cu,Zn superoxide dismutase activity [4],[5]. Regarding the mechanisms of conversion of the cellular isoform in the aberrant one, a number of studies have been performed about a short peptide based on the sequence of PrPc, PrP[106-126]. It exhibits a prevalent β-sheet structure and forms amyloid fibrillar aggregates, being reminiscent of PrPsc; since it has been shown to induce apoptosis in cultured cells, it might constitute the toxic core of PrPsc [6]. In parallel, other peptides reproducing different portions of PrPc have been analysed, but their toxicity was found to be much less than with PrP[106-126] [7]. In the present work, we evaluated the effect of several synthetic versions of specific prion protein fragments, such as PrP[173-195] (NNFVHDCVNITIKQHTVTTTTKG and N- and C-blocked form) on B104 neuroblastoma cells and MCF7 breast cancer cells, since this peptide, corresponding to α2 helic, could be involved in prion molecular rearrangement because of its structural instability. Results and Discussion The peptide fragment PrP[173-195], both in the acetylated-amidate and free form, produced a toxic effect on B104 neuroblastoma cells, with a higher effect of PrP[Ac173-195NH2], which remarkably reduced cell viability after 48 h of incubation in cells. Both forms of the prion fragments produced a less pronounced effect on human breast cancer cells, suggesting however a lower unspecific effect on cell proliferation. The higher toxicity of the acetylated/amidated form with respect to the free one could derive from the charge neutralization induced at both N- and C-termini of the peptide, which should facilitate the helix formation thus better reproducing the conformational conditions observed in the native protein. Conclusions The PrPsc domain corresponding to α-helic 2 could be reasonably responsible for the interaction with the cellular prion protein and its conversion in the scrapie isoform. It exhibits a short sequence with a strong β-sheet forming propensity, which may affect the whole protein structure and mediate protein aggregation, characteristic of prion diseases.

Effect of prion protein fragments on the proliferation cultured cells.

URSO, EMANUELA;RIZZELLO, Antonia;ACIERNO, Raffaele;MAFFIA, Michele
2004

Abstract

Introduction Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of unusual neurodegenerative disorders affecting both animals and humans [1]. The agent believed to be responsible for these pathologies is a protein whose name is PrPsc, which is a conformational variant of the normal cellular prion protein, PrPc, a 33-35 KDa glycosyl-phosphatidylinositol-anchored protein, expressed at high levels in neuronal cells. Unlike PrPc, PrPsc is characterized by a higher β-sheet content and is partially resistant to proteolysis [2],[3]. The amino terminus of PrPc contains four octapeptide repeats, which are implicated in the binding of divalent metal ions and in particular copper; because of this property, PrPc could be reasonably involved in copper metabolism and in the defense mechanism of the cell against oxidative stress, possibly through the regulation of the Cu,Zn superoxide dismutase activity [4],[5]. Regarding the mechanisms of conversion of the cellular isoform in the aberrant one, a number of studies have been performed about a short peptide based on the sequence of PrPc, PrP[106-126]. It exhibits a prevalent β-sheet structure and forms amyloid fibrillar aggregates, being reminiscent of PrPsc; since it has been shown to induce apoptosis in cultured cells, it might constitute the toxic core of PrPsc [6]. In parallel, other peptides reproducing different portions of PrPc have been analysed, but their toxicity was found to be much less than with PrP[106-126] [7]. In the present work, we evaluated the effect of several synthetic versions of specific prion protein fragments, such as PrP[173-195] (NNFVHDCVNITIKQHTVTTTTKG and N- and C-blocked form) on B104 neuroblastoma cells and MCF7 breast cancer cells, since this peptide, corresponding to α2 helic, could be involved in prion molecular rearrangement because of its structural instability. Results and Discussion The peptide fragment PrP[173-195], both in the acetylated-amidate and free form, produced a toxic effect on B104 neuroblastoma cells, with a higher effect of PrP[Ac173-195NH2], which remarkably reduced cell viability after 48 h of incubation in cells. Both forms of the prion fragments produced a less pronounced effect on human breast cancer cells, suggesting however a lower unspecific effect on cell proliferation. The higher toxicity of the acetylated/amidated form with respect to the free one could derive from the charge neutralization induced at both N- and C-termini of the peptide, which should facilitate the helix formation thus better reproducing the conformational conditions observed in the native protein. Conclusions The PrPsc domain corresponding to α-helic 2 could be reasonably responsible for the interaction with the cellular prion protein and its conversion in the scrapie isoform. It exhibits a short sequence with a strong β-sheet forming propensity, which may affect the whole protein structure and mediate protein aggregation, characteristic of prion diseases.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11587/116881
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