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Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells
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..
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden.
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2010 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 18, 14782-14787 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society , 2010. Vol. 26, no 18, 14782-14787 p.
National Category
Chemical Sciences
URN: urn:nbn:se:umu:diva-29843DOI: 10.1021/la1026416ISI: 000281690600058OAI: diva2:278283
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2012-10-01Bibliographically approved
In thesis
1. Protein complexes: assembly, structure and function
Open this publication in new window or tab >>Protein complexes: assembly, structure and function
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

 Most proteins must fold into their native conformations to fulfil their biological functions. Failure of proteins to fold leads to cell pathology and a broad range of human diseases referred to as protein misfolding disease, e.g., Alzheimer’s disease, Parkinson’s disease, and type II diabetes. More than 40 proteins are known to be connected with misfolding diseases. These proteins share no sequence homology but all assemble into cross-b sheet containing insoluble fibrillar aggregates. Despite the pathological conditions that these proteins can induce, living organisms can take advantage of the inherent ability of these proteins to form such structures and to generate novel and diverse biological function, the functional amyloid.

 This thesis examines different aspects of cross-b sheet containing aggregates. The first paper describes the humoral response to aggregated structures of insulin and the astrocytical biomarker S100B in patients suffering from Parkinson’s disease. We show that the patients have an increased immunreactivity towards insulin and S100B in Parkinson’s disease patients compared to a control group.

 The second part of this work focuses on a functional amyloid. HAMLET (human a-lactalbumin made lethal for tumour cells) is a complex of a-lactalbumin and oleic acid, which kills tumour cells but not healthy differentiated cells. We wish to expand the concept of HAMLET to a structurally related protein and therefore create and characterize a complex of equine lysozyme and oleic acid (Paper II). We chose equine lysozyme because both proteins (equine lysozyme and a-lactalbumin) share common ancestors and are spatially related. The newly designed complex was named ELOA, for equine lysozyme with oleic acid. ELOA represents a functional oligomer due to its multimeric state and its ability to bind amyloid specific dyes. In the third paper, we investigate the interaction of the cytotoxic ELOA with live cells in real time to find a mechanistic model (Paper III).

 It is known that HAMLET is not only tumouricidal but is also toxic towards many bacteria. Therefore in the last part of the thesis, we investigated the effects of ELOA on different bacterial strains and focused on its interplay Streptococcus pneumoniae (Paper IV).

 These studies have added significantly to many aspects of protein folding and misfolding from its involvement in Parkinson’s disease to the newly gained functions and structural aspects of de novo produced ELOA.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2009. 40 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1318
HAMLET, ELOA, lysozyme, amyloid
Research subject
Medical Biochemistry
urn:nbn:se:umu:diva-29792 (URN)978-91-7264-913-2 (ISBN)
Public defence
2009-12-15, 3A9, KBC Huset, Umeå, 00:00 (English)
Available from: 2009-11-25 Created: 2009-11-23 Last updated: 2009-11-25Bibliographically approved

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Wilhelm, KristinaSchleucher, JürgenMorozova-Roche, Ludmilla ACe, Zhang
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