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Protein complexes: assembly, structure and function
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics. (Ludmilla Morozova-Roche)
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
Keyword [en]
HAMLET, ELOA, lysozyme, amyloid
Research subject
Medical Biochemistry
URN: urn:nbn:se:umu:diva-29792ISBN: 978-91-7264-913-2OAI: diva2:278077
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
List of papers
1. Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients
Open this publication in new window or tab >>Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients
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2007 (English)In: European Journal of Neurology, ISSN 1351-5101, E-ISSN 1468-1331, Vol. 14, no 3, 327-334 p.Article in journal (Refereed) Published
Abstract [en]

Peripheral immune responses can be sensitive indicators of disease pathology. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of 26 Parkinson's disease (PD) patients compared with controls by using ELISA. We found a statistically significant increase of the autoimmune responses to both antigens in PD patients compared with controls with a mean increase of 70% and 50% in the autoimmune reactions towards insulin and S100B, respectively. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in PD patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type, while sequential epitope of native insulin is hidden within the amyloid structures. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.

amyloid;immune reactivity;insulin;Parkinson's disease;S100B
National Category
Medical and Health Sciences
urn:nbn:se:umu:diva-25729 (URN)10.1111/j.1468-1331.2006.01667.x (DOI)17355556 (PubMedID)
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2012-02-21Bibliographically approved
2. Protein oligomerization induced by oleic acid at the solidliquid interface: equine lysozyme cytotoxic complexes
Open this publication in new window or tab >>Protein oligomerization induced by oleic acid at the solidliquid interface: equine lysozyme cytotoxic complexes
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 15, 3975-3989 p.Article in journal (Refereed) Published
Abstract [en]

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid–liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein α-lactalbumin, known as humanα-lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4–30 lysozyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical morphology, assembles into ring-shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well-populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

amyloid, HAMLET, lysozyme, oleic acid, oligomers
urn:nbn:se:umu:diva-29842 (URN)10.1111/j.1742-4658.2009.07107.x (DOI)
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2011-03-31Bibliographically approved
3. Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells
Open this publication in new window or tab >>Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells
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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
National Category
Chemical Sciences
urn:nbn:se:umu:diva-29843 (URN)10.1021/la1026416 (DOI)000281690600058 ()
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2012-10-01Bibliographically approved
4. Complexes of equine lysozyme with oleic acid with bactericidal activity against Streptococcus pneumoniae
Open this publication in new window or tab >>Complexes of equine lysozyme with oleic acid with bactericidal activity against Streptococcus pneumoniae
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(English)Manuscript (preprint) (Other academic)
urn:nbn:se:umu:diva-29846 (URN)
Available from: 2009-11-25 Created: 2009-11-25Bibliographically approved

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