pubs.acs.org/Biochemistry Published on Web 04/20/2009 r 2009 American Chemical Society 5026 Biochemistry 2009, 48, 5026–5033 DOI: 10.1021/bi900187s Humanin Structural Versatility and Interaction with Model Cerebral Cortex Membranes † Sara Pistolesi, ‡ Lara Rossini, §, ) Elisa Ferro, § Riccardo Basosi, ‡ Lorenza Trabalzini, § and Rebecca Pogni* ,‡ ‡ Department of Chemistry, University of Siena, Via A. De Gasperi, 53100 Siena, Italy, and § Department of Molecular Biology, University of Siena, Via Fiorentina 1, 53100 Siena, Italy ) Present address: Siena Biotech S.p.A., Strada del Petriccio Belriguardo 35, 53100 Siena, Italy Received February 5, 2009; Revised Manuscript Received April 9, 2009 ABSTRACT: Humanin (HN) is a recently identified neuroprotective peptide able to inhibit neurotoxicity induced by various insults which can be related to Alzheimer disease (AD) as well as to cell death induced by other stimuli. Previous CD and NMR studies demonstrated that HN adopts an unordered conformation in water, a R-helix conformation in 30% TFE, and a β-sheet structure in PBS. Furthermore, other studies clearly indicated HN as a secreted peptide, able to prevent neuronal cell death caused by amyloid β (Aβ) derivatives. Although Aβ was found to interact with neuronal membranes, currently there is not experimental evidence unveiling HN interaction with membranes. In this paper a spin labeling technique coupled with electron paramagnetic resonance (EPR) and circular dichroism (CD) has been used to study the structure and dynamics of HN in solution and for the first time in the presence of model cerebral cortex membranes (CCM). We have demonstrated that HN has a great tendency to aggregate even at low concentrations in water solutions at different ionic strengths and monomerizes in the TFE apolar environment. We also showed that HN slightly perturbs model CCM at the surface assuming a clear β-sheet conformation. In addition, HN increases the fluidity of the bilayer core without penetrating into the membrane. Humanin (HN) 1 is a neuroprotective peptide of 24 amino acids able to antagonize neurotoxicity in neuronal cells and primary cultured neurons elicited by various Alzheimer’s disease (AD) relevant insults, including AD-linked mutant genes and amyloid β (Aβ) peptide derivatives (1, 2). It was suggested that HN is secreted from the cells (2) and may exert its neuroprotective activity through interactions with secreted factors or putative cell-surface receptors linked to tyrosine kinases (3-6). It has been demonstrated that HN is able to interact with the 40 aa form of Aβ (Aβ 40 ) and to affect Aβ aggregation dynamics and morphological features (7). In 2004, Ying et al., investigating the neuroprotective activity of secreted HN, demonstrated that the G-protein-coupled receptor FPRL1 shares both the 42 aa form of Aβ (Aβ 42 ) and HN, and it was suggested that HN may exert its neuroprotective effects by competitively inhibiting the access of Aβ to FPRL1 (8). Therefore, HN could protect neurons by exerting different functions, expected for its potential of binding different molecules on cell membranes (7). The amphiphilic 40 and 42 residue peptides, Aβ 40 and Aβ 42 , have been identified as the major components of senile plaques in patients with AD. They originate by proteolytic processing of a much larger amyloid precursor protein (APP) anchored in the brain membrane. The membrane release of Aβ peptides following this enzyme cleavage and their accumulation at the membrane surface is thought to be one of the first steps of membrane- associated Aβ fibrillogenesis and neurotoxicity (9). Despite their different aggregation properties and role in disease, the two peptides homogeneously co-mix in amyloid fibrils, suggesting that they possess the same structural architecture (10). Although a number of putative surface receptors for Aβ peptides have been described in the literature (11), a direct interaction with mem- brane components is thought to be a seed for the formation of toxic amyloid fibers (9, 12-14). A conformational transition of peptides is necessary for fibril formation (9, 15). Despite the HN capability to inhibit toxicity induced by AD-related insults in neurons, it was demonstrated that HN exerts a protective effect also on nonneuronal cells when they are exposed to AD-related cytotoxicity (16). Further investigations showed that HN possesses a wide spectrum of action (17). Moreover, it has been demonstrated that HN is able to interact † The work was supported by the Fondazione Monte dei Paschi di Siena (Project Number 29970, 2007, to L.T.) and University of Siena (PAR 2006 to R.P.). *To whom correspondence should be addressed. Fax: (+39) 0577 234239. Phone: (+39) 0577 234258 E-mail: pogni@unisi.it. 1 Abbreviations: Aβ, amyloid β; AD, Alzheimer’s disease; AMP, antimicrobial peptides; APP, amyloid precursor protein; CCM, cerebral cortex membrane; CD, circular dichroism; Cer, galactocerebroside; Ch, cholesterol; EPR, electron paramagnetic resonance; FPRL1, formylpeptide receptor-like 1; GM1, monosialoganglioside GM1; HN, humanin; HNG, S14G humanin mutant; HNSL, spin-labeled humanin; LUV, large unilamellar vescicles; MOPS, 4-morpholinepro- panesulfonic acid; MTSSL, 1-oxy-2,2,5,5-tetramethylpyrroline-3- methyl methanethiosulfonate; NMR, nuclear magnetic resonance; n-PCSL, 1-(n-doxylpalmitoyl)-2-stearoylphosphatidylcholine; PBS, phosphate-buffered saline; PC, 1-palmitoyl-2-oleoylphosphatidylcho- line; PE, 1-palmitoyl-2-oleoylphosphatidylethanolamine; PS, 1-palmi- toyl-2-oleoylphosphatidylserine; SDSL, site-directed spin labeling; SM, sphingomyelin; TFE, trifluoroethanol.