Protein & Peptide Letters, 2009, 16, 000-000 1 0929-8665/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Confocal Microscopy Evidence of Prion Protein Fragment hPrP[173-195] Internalization in Rat B104 Neuroblastoma Cell Line Emanuela Urso 1 , Raffaele Acierno 1 , Maria Giulia Lionetto 2 , Antonia Rizzello 1 , Andrea Papa 1 , Trifone Schettino 2 and Michele Maffia 1,* 1 Lab. of Adaptive Physiology, Department of Biological and Environmental Science and Technology, University of Lecce, prov.le Lecce-Monteroni, 73100 Lecce, Italy; 2 Lab. of General and Environmental Physiology, Department of Biological and Environmental Science and Technology, University of Lecce, prov.le Lecce-Monteroni, 73100 Lecce, Italy Abstract: The cytotoxicity of hPrP[173-195] prion peptide against a neuroblastoma cell model was found independent of its tendency to aggregate over time. Cytosolic and nuclear inclusions of peptide were highlighted by confocal microscopy, suggesting a role as a transcription factor in activating signal transduction pathways involved in cell toxicity. Keywords: Prion disease; cellular prion protein (PrP C ); helix-2; structural ambivalence; synthetic prion peptides; confocal mi- croscopy. INTRODUCTION The cellular prion protein PrP C is a glycosylphosphatidy- linositol (GPI)-linked protein of the surface of a variety of cell types including neurones, glial cells and leukocytes. In humans it’s encoded by the PRNP gene located on chromo- some 20 [1, 2]. Solution Nuclear Magnetic Resonance (NMR) experiments on prion protein folding in several spe- cies (Syrian hamster, mouse, bovine and human) evidenced a well-conserved structure, with a high -helical content and high susceptibility to proteolytic digestion [3]. PrP C consists of a globular domain encompassing residues from 120 to 231 and a N-terminal unstructured "tail" of about 100 amino ac- ids. The former is folded into three alpha-helices and a two- stranded, anti-parallel -sheet [4, 5], while the latter is com- posed of a highly conserved repeat of four octapeptide units with the consensus sequence PHGGGWGQ, proposed to selectively bind copper(II) ions under physiological condi- tions [6, 7]. Although the physiological function of PrP C is undefined, it was proposed to drive copper delivery across plasma membrane by endocytosis [8] and to hold a neuropro- tective role achieved through its anti-oxidant activity [9, 10] and activation of cAMP/PKA dependent transduction path- ways [11]. A widespread expression throughout the brain tissues suggested a possible role of PrP C in synaptic trans- mission [12, 13]. The structural transition of the cellular prion protein to the aberrant isoform PrP Sc (prion) is commonly considered a critical event in the pathogenesis of a large group of neu- rodegenerative diseases known as Transmissible Spongiform Encephalopathies (TSEs). The abnormal protein displays a higher percentage of -sheet structure and an increased resis- tance to proteolysis respect to PrP C , although sharing with it the primary sequence [14, 15]. The most accepted current *Author correspondence to this author at the Department of Biological and Environmental Science and Technology, University of Salento, Lecce, Italy; E-mail: m.maffia@fisiologia.unile.it; michele.maffia@unile.it theory explaining PrP C conversion leading to PrP Sc formation states that structural transition takes place through a physical interaction between the two isoforms, where PrP Sc would act as a template [15]. Several and detailed studies have been addressed to gain insight into the structural determinants affecting the prion conversion. Nevertheless, neither the mechanism of confor- mational change nor the tertiary structure of PrP Sc have been clarified yet because of the aggregative properties of the scrapie isoform, which make it difficult to isolate the PrP Sc molecule [16, 17]. A bulk of experimental evidences, aimed to define structural prerequisites for PrP C PrP Sc conversion, has been collected so far, with controversial outcomes [18- 21]. The examination of monomeric and dimeric foldings of the carboxy-terminal domain of PrP C evidenced some re- gions possibly driving the structural transition of prion pro- tein [4, 22-24]. It’s noteworthy how the crystalline dimeric conformer represents a link between the PrP C isoform and the pathogenous one [24, 25]. In this rearrangement the C- terminal region of helix-2 is converted into a  strand con- formation while the helix-3 swaps between the two globular domains. The putative full conversion of helix-2 together with its immediate neighbourhood into three adjacent  strands could be a prerequisite for the emergence of the PrP Sc structure [25]. Conformational tendencies exhibited by prion protein helices have been explored by NMR investigations and se- quence alignments in the attempt to individuate regions sus- ceptible to conformational fluctuations. A comparative analysis of short-range contacts in PrP C and other non- amyloidogenic proteins from a databank (Protein Data Bank) revealed that they are unusually distributed within the prion protein and localised around the helix-2 [26]. The network of contacts involving this prion protein (PrP) region induces it to assume a forced -helical conformation within the protein, in disagreement with predicted secondary structure (phe-