14782 DOI: 10.1021/la1026416 Langmuir 2010, 26(18), 14782–14787 Published on Web 08/24/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society Lipoprotein Complex of Equine Lysozyme with Oleic Acid (ELOA) Interactions with the Plasma Membrane of Live Cells Vladana Vukojevi  c,* ,† Alice M. Bowen, ‡ Kristina Wilhelm, § Yu Ming, † Zhang Ce, § J€ urgen Schleucher, § P. J. Hore, ‡ Lars Terenius, † and Ludmilla A. Morozova-Roche* ,§ † Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden, ‡ Department of Chemistry, University of Oxford, Physical and Theoretical Chemistry Laboratory, Oxford OX1 3QZ, U.K., and § Department of Medical Biochemistry and Biophysics, Umea ˚ University, 90781 Umea ˚, Sweden Received July 1, 2010. Revised Manuscript Received August 3, 2010 Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying PC12 cell death caused by ELOA oligomers. ELOA, a lipoprotein complex formed by equine lysozyme (EL) and oleic acid (OA), induces cell death in all tested cell lines, but the actual mechanism of its action is not known. We have used methods with single-molecule sensitivity, fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and confocal laser scanning microscopy (CLSM) imaging by avalanche photodiodes (APD), so-called APD imaging, to study ELOA interactions with the plasma membrane in live PC12 cells. We detected ELOA accumulation in the cell surroundings, observed ELOA interactions with the plasma membrane, and local changes in plasma membrane lipid dynamics in the vicinity of ELOA complexes. These interactions resulted in plasma membrane rupture, followed by rapid influx and distribution of ELOA inside the already dead cell. In order to probe the ELOA-plasma membrane interaction sites at the molecular and atomic levels, the ELOA complexes were further studied by photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, nuclear magnetic resonance (NMR) and atomic force microscopy (AFM). We observed a novel mechanism of oligomer toxicity-cell death induced by continuous disturbance of the plasma membrane, eventually causing permanent plasma membrane damage and identified the sites in ELOA that are potentially involved in the interactions with the plasma membrane. Introduction Protein complexes appear to play a prominent role in aberrant protein aggregation in living organisms. 1 Currently, amyloid oligo- meric assemblies are viewed as the most deleterious aggregates leading to the death and dysfunction of neuronal cells in Alzheimer’s and Parkinson’s diseases and other amyloid ailments. 2,3 Yet, other naturally occurring and in vitro produced protein complexes such as HAMLET and BAMLET (human and bovine R-lactalbumins made lethal to tumor cells) 4-6 have attracted significant attention due to their apparent ability to kill selectively tumor cells and hence their prospective therapeutic use. 6-10 Thus, protein oligomeric complexes may act as a double-edged sword, either damaging cells indiscriminately or specifically eliminating unwanted cells. Despite the key role of protein complexes in human diseases and their putative therapeutic potential, the molecular mecha- nisms of the interaction of protein complexes with live cells and their primary targets at the cell surface or within the cell remain largely unknown and highly debated. 11,12 In this work, we study interactions of a lipoprotein complex-equine lysozyme with oleic acid (ELOA) 13 with the plasma membrane of living cells. ELOA is similar in composition to HAMLET and BAMLET, being a complex comprised of protein molecules and oleic acid. It also exhibits certain properties that are characteristic of amyloid oligomers;it binds the amyloid-marker thioflavin-T and forms ring-shaped assemblies that are similar in appearance to the assemblies of EL amyloid oligomers as well as to the Aβ and R-synuclein oligomers observed under pathological conditions in Alzheimer’s and Parkinson’s diseases, respectively. 14-16 ELOA can therefore be regarded as a model system that links the two *Corresponding authors. E-mail: vladana.vukojevic@ki.se (V.V.); ludmilla.morozova-roche@medchem.umu.se (L.A.M.-R.). (1) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333. (2) Bucciantini, M.; Calloni, G.; Chiti, F.; Formigli, L.; Nosi, D.; Dobson, C. M.; Stefani, M. J. Biol. Chem. 2004, 279, 31374. (3) Baglioni, S.; Casamenti, F.; Bucciantini, M.; Luheshi, L. M.; Taddei, N.; Chiti, F.; Dobson, C. M.; Stefani, M. J. Neurosci. 2006, 26, 8160. (4) Ha˚ kansson, A.; Zhivotovsky, B.; Orrenius, S.; Sabharwal, H.; Svanborg, C. Proc. Natl. Acad. Sci. 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