UNCORRECTED PROOF 1 Binding of methylene blue to a surface cleft inhibits the oligomerization and 2 fibrillization of prion protein 3 Paola Q1 Cavaliere a, b, 1 , Joan Torrent c , Stephanie Prigent c , Vincenzo Granata b , Kris Pauwels d , 4 Annalisa Pastore d , Human Rezaei c, ⁎, Adriana Zagari a, b, ⁎⁎ 5 a Dipartimento delle Scienze Biologiche, Università degli Studi di Napoli “Federico II”, Napoli, Italy 6 b CEINGE, Biotecnologie Avanzate S.c.a r.l., Napoli, Italy 7 c Institut National de la Recherche Agronomique (INRA), Virologie et Immunologie Moléculaires, Equipe Biologie Physico-chimique des Prions, Jouy-en-Josas, France 8 d MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK 9 10 abstract article info 11 Article history: 12 Received 19 July 2012 13 Received in revised form 14 September 2012 14 Accepted 17 September 2012 15 Available online xxxx 16 17 18 19 Keywords: 20 Amyloidogenic protein 21 PrP oligomer 22 PrP fiber 23 Anti-prion agent 24 Neurodegenerative disease 25 Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accu- 26 mulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein 27 (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of 28 these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal 29 PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into 30 methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other 31 neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomer- 32 ization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of 33 PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by 34 using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber for- 35 mation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which 36 was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a 37 mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards 38 oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting 39 against proteinopathies. 40 © 2012 Published by Elsevier B.V. 41 42 43 44 45 1. Introduction 46 Neurodegenerative disorders such as Alzheimer's (AD), Parkinson's 47 and Huntington diseases, frontotemporal dementia and prion diseases 48 are included in the general family of protein misfolding pathologies. 49 These diseases show substantial overlap in their pathologic mechanisms 50 [1,2], with their main common characteristic being oligomer formation 51 followed by fibrillar arrangement and deposition of misfolded proteins 52 in the brain [3,4]. 53 In particular, prion diseases are a family of rare but all fatal patholo- 54 gies, which affect humans, e.g. the Creutzfeldt–Jakob disease, and 55 various animal species, e.g. scrapie in sheep and goat, and bovine 56 spongiform encephalopathies in cattle. It is commonly accepted that 57 these diseases are caused by a conformational change of the cellular 58 prion protein (PrP C ), which is rich in α-helix, to its β-sheet-rich 59 insoluble conformer, called scrapie PrP (PrP Sc ) [5]. How this con- 60 formational transition occurs is not yet clearly elucidated, despite 61 the plethora of studies [5–7]. All proposed mechanisms to describe 62 the conversion of PrP C into PrP Sc suggest a multi-step process including 63 an oligomerization/polymerization step [7,8]. Based on the assumption 64 that one or more of the different conformational PrP states involved 65 (i.e. oligomers, proto-fibrils, amyloid fibrils) correspond to the infectious 66 and/or toxic entity, various approaches aimed at targeting such abnor- 67 mal species are currently explored for therapeutic interventions. 68 Treating prion diseases remains a challenge for the scientific and 69 medical community. Although a large number of anti-prion agents 70 have been identified, none represents an efficient cure. For pragmatic 71 reasons, and since the basic molecular mechanisms in protein 72 misfolding disorders are closely related, it seems reasonable to sur- 73 mise that some compounds, with anti-aggregation potency in various 74 model systems of protein misfolding and disease, might be also effec- 75 tive for prion diseases. Accordingly, we addressed our quest for new Biochimica et Biophysica Acta xxx (2012) xxx–xxx Abbreviations: PrP, prion protein; MB, methylene blue; wt-OvPrP, ovine recombinant wild-type full-length PrP (23–234); ΔOvPrP, ovine recombinant truncated PrP (103–234); MoPrP, murine recombinant full-length PrP (23–231); wt-HuPrP, wild-type human recombi- nant full-length PrP (23–231); AD, Alzheimer's disease ⁎ Corresponding author. ⁎⁎ Correspondence to: Dipartimento delle Scienze Biologiche, Università degli Studi di Napoli “Federico II”, Napoli, Italy. Tel.: +39 081 3737913; fax: +39 081 3737808. Q3 E-mail addresses: human.rezaei@jouy.inra.fr (H. Rezaei), adriana.zagari@unina.it (A. Zagari). 1 Present address: Unité de Génétique Moléculaire, Institut Pasteur, Paris Cedex 15, France. BBADIS-63551; No. of pages: 9; 4C: 0925-4439/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbadis.2012.09.005 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbadis Please cite this article as: P. Cavaliere, et al., Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein, Biochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbadis.2012.09.005