115 Research Article Received: 29 July 2009 Revised: 25 November 2009 Accepted: 27 November 2009 Published online in Wiley Interscience: (www.interscience.com) DOI 10.1002/psc.1207 Interaction of a β -sheet breaker peptide with lipid membranes Giuseppe Vitiello, a,b Manuela Grimaldi, c Anna Ramunno, c Ornella Ortona, a,b Giovanni De Martino, c Anna Maria D’Ursi c and Gerardino D’Errico a,b* Aggregation of β -amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimer’s disease. An attractive therapeutic strategy for Alzheimer’s disease is the inhibition of the soluble β -amyloid aggregation using synthetic β -sheet breaker peptides that are capable of binding Aβ but are unable to become part of a β -sheet structure. As the early stages of the Aβ aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the β -sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the β -sheet breaker peptide acetyl-LPFFD-amide, iAβ 5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C- and at the N-terminus or phospholipids spin-labeled in different positions of the acyl chain. Our results show that iAβ 5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iAβ 5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright c  2010 European Peptide Society and John Wiley & Sons, Ltd. Keywords: β -amyloid; β -sheet breaker; electron spin resonance; lipid bilayer; spin-label Introduction Alzheimer’s disease (AD) is by far the most common form of senile dementia, which is estimated to affect up to 35 million individuals worldwide in 2010 [1]. AD is characterized by progressive memory deficit, cognitive impairment and personality changes. The morphological hallmarks found in the brains of AD patients are two types of abnormal protein aggregates [2,3]: neurofibrillary tangles, which occur intracellularly and are composed of paired helical filaments of hyperphosphorylated Tau protein [4] and senile plaques, which occur in the extracellular space and are composed of insoluble β -amyloid peptide (Aβ ) aggregates [5]. Aβ is a peptide that is generally composed of 40–42 amino acids, even though shorter (37–39) and longer (43) forms have been also found [6]. It is generated from proteolytic cleavage of the amyloid precursor protein (APP), a type I integral membrane glycoprotein (695–770 residues), by β - and γ -secretases. Aβ is present at a very low concentration (<10 −8 M) in biological fluids [7], and its physiological role is unknown. In its native form, Aβ is unfolded but aggregates into a β -sheet structure of ordered fibrils under various conditions [8,9]. It was hypothesized that these fibrils may aggregate to form senile plaques. In the past years, amyloid plaques were generally regarded as the cause of cognitive disorder. More recently, the relevance of soluble protofibrillar oligomeric forms of Aβ has been recognized [10]. In particular, it has been shown in vivo that the neurotoxic effect of Aβ (1–42) is independent of plaque formation [11] and that protofibrillar intermediates of Aβ induce progressive neurotoxicity in cortical neurons [12]. The mechanism of Aβ fibrillization has not been fully under- stood. Many studies suggest that lipid membranes have a decisive role in favoring the β -amyloid aggregation [13]. Jarrett and Lans- bury suggested that Aβ forms fibrils by the nucleation-dependent polymerization mechanism and lipids could act as heterogeneous seeds for the polymerization [14]. Verdier et al. proposed that Aβ –lipid interaction increases the rate of Aβ misfold- ing/fibrillogenesis, leads to modifications in the bilayer properties and disrupts membrane fluidity and function [15]. For this reason, a particular interest has been developed in Aβ –membrane interactions in order to elucidate the molecular mechanisms of the Aβ -induced cellular dysfunctions underlying the pathogenesis of AD. However, although many studies have been conducted on this subject [16 – 19], it is still highly controversial. Comprehension of the Aβ fibrillation mechanism is fundamental to define a therapeutic strategy. In fact, a widely employed approach in the research of anti-Alzheimer agents involves the identification of substances that are able to prevent amyloid aggregation, or to disaggregate the amyloid fibrils through a direct structural interaction with soluble or aggregated peptides. In this regard, a number of small molecules, like Congo red, anthracycline, rifampicin, anionic sulphonates and melatonin, have been reported to interact with Aβ and prevent its aggregation into oligomers and fibrils, reducing toxicity [20 – 24]. ∗ Correspondence to: Gerardino D’Errico, Dipartimento di Chimica ‘‘Paolo Corradini’’, Universit` a di Napoli ‘‘Federico II’’ Complesso di Monte S. Angelo, Via Cinthia, I-80126 Napoli, Italy. E-mail: gerardino.derrico@unina.it a Dipartimento di Chimica ‘‘Paolo Corradini’’, Universit` a di Napoli ‘‘Federico II’’ Complesso di Monte S. Angelo, Via Cinthia, I-80126 Napoli, Italy b CSGI (Consorzio per lo Sviluppo dei Sistemi a Grande Interfase), Firenze, Italy c Dipartimento di Scienze Farmaceutiche, Universit` a di Salerno, Fisciano, Italy J. Pept. Sci. 2010; 16: 115–122 Copyright c  2010 European Peptide Society and John Wiley & Sons, Ltd.