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Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain
Umeå University, Faculty of Science and Technology, Chemistry.
UMR 5248 CBMN, CNRS-Université Bordeaux, France.
UMR 5248 CBMN, CNRS-Université Bordeaux, France.
Umeå University, Faculty of Science and Technology, Chemistry.
2008 (English)In: The FEBS Journal, ISSN 1742-464X, Vol. 275, no 3, 561-572 p.Article in journal (Refereed) Published
Abstract [en]

The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state 2H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by 31P-NMR at 30 °C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static 14N and 31P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated β-sheet secondary structure of BH4 with a 42° tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.

Place, publisher, year, edition, pages
2008. Vol. 275, no 3, 561-572 p.
Keyword [en]
apoptosis, BH4 domain, membrane, protein-lipid interactions, solid state NMR
URN: urn:nbn:se:umu:diva-7994DOI: doi:10.1111/j.1742-4658.2007.06222.xOAI: diva2:147665
Available from: 2008-01-14 Created: 2008-01-14Bibliographically approved
In thesis
1. Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli
Open this publication in new window or tab >>Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems.

The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface.

Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.

Place, publisher, year, edition, pages
Umeå: Kemi, 2008. 58 p.
Apoptosis, BH4 peptide, Bax-α1 peptide, model membrane, cardiolipin, solid phase peptide synthesis, circular dichroism, solid-state nuclear magnetic resonance spectroscopy.
National Category
urn:nbn:se:umu:diva-1883 (URN)978-91-7264-676-6 (ISBN)
Public defence
2008-11-07, BiA201, Biologihuset, Umeå, 13:00 (English)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2015-11-13Bibliographically approved

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