| 1. |
- Baumann, Anne, et al.
(författare)
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HAMLET Forms Annular Oligomers When Deposited with Phospholipid Monolayers
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 418:1-2, s. 90-102
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Tidskriftsartikel (refereegranskat)abstract
- Recently, the anticancer activity of human a-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine alpha-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH as observed by liposome content leakage and circular dichroism experiments and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET. (C) 2012 Elsevier Ltd. All rights reserved.
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| 2. |
- Jakubec, Martin, et al.
(författare)
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Cholesterol-containing lipid nanodiscs promote an α-synuclein binding mode that accelerates oligomerization
- 2021
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 288:6, s. 1887-1905
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Tidskriftsartikel (refereegranskat)abstract
- Dysregulation of the biosynthesis of cholesterol and other lipids has been implicated in many neurological diseases, including Parkinson's disease. Misfolding of α‐synuclein (α‐Syn), the main actor in Parkinson's disease, is associated with changes in a lipid environment. However, the exact molecular mechanisms underlying cholesterol effect on α‐Syn binding to lipids as well as α‐Syn oligomerization and fibrillation remain elusive, as does the relative importance of cholesterol compared to other factors. We probed the interactions and fibrillation behaviour of α‐Syn using styrene–maleic acid nanodiscs, containing zwitterionic and anionic lipid model systems with and without cholesterol. Surface plasmon resonance and thioflavin T fluorescence assays were employed to monitor α‐Syn binding, as well as fibrillation in the absence and presence of membrane models. 1H‐15N‐correlated NMR was used to monitor the fold of α‐Syn in response to nanodisc binding, determining individual residue apparent affinities for the nanodisc‐contained bilayers. The addition of cholesterol inhibited α‐Syn interaction with lipid bilayers and, however, significantly promoted α‐Syn fibrillation, with a more than a 20‐fold reduction of lag times before fibrillation onset. When α‐Syn bilayer interactions were analysed at an individual residue level by solution‐state NMR, we observed two different effects of cholesterol. In nanodiscs made of DOPC, the addition of cholesterol modulated the NAC part of α‐Syn, leading to stronger interaction of this region with the lipid bilayer. In contrast, in the nanodiscs comprising DOPC, DOPE and DOPG, the NAC part was mostly unaffected by the presence of cholesterol, while the binding of the N and the C termini was both inhibited.
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| 3. |
- Mossberg, Ann-Kristin, et al.
(författare)
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HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity.
- 2010
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:2
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. METHODOLOGY/PRINCIPAL FINDINGS: We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. CONCLUSIONS/SIGNIFICANCE: The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.
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