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Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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.
Keyword [en]
Apoptosis, BH4 peptide, Bax-α1 peptide, model membrane, cardiolipin, solid phase peptide synthesis, circular dichroism, solid-state nuclear magnetic resonance spectroscopy.
National Category
Biophysics
Identifiers
URN: urn:nbn:se:umu:diva-1883ISBN: 978-91-7264-676-6 (print)OAI: oai:DiVA.org:umu-1883DiVA: diva2:142309
Public defence
2008-11-07, BiA201, Biologihuset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2015-11-13Bibliographically approved
List of papers
1. Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4
Open this publication in new window or tab >>Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4
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2006 (English)In: Journal of Peptide Science, ISSN 1075-2617, Vol. 12, 58-64 p.Article in journal (Refereed) Published
Abstract [en]

Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant -sheeted structures in water that convert progressively into -helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

Keyword
solid phase synthesis, reverse-phase HPLC, MALDI-TOF mass spectrometry, circular dichroism, non-protected tryptophan, apoptotic peptides, 15N-labelled amino acids
Identifiers
urn:nbn:se:umu:diva-12545 (URN)doi:10.1002/psc.686 (DOI)
Available from: 2007-06-12 Created: 2007-06-12Bibliographically approved
2. Pro-apoptotic bax-α1 synthesis and evidence for β-sheet to α-helix conformational change as triggered by negatively charged lipid mβembranes
Open this publication in new window or tab >>Pro-apoptotic bax-α1 synthesis and evidence for β-sheet to α-helix conformational change as triggered by negatively charged lipid mβembranes
2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, Vol. 13, no 2, 100-106 p.Article in journal (Refereed) Published
Abstract [en]

Solid phase synthesis of Bax-α1, the 25 amino acids domain (14TSSEQIMKTGALLLQGFIQDRAGRM38) of the pro-apoptotic Bax protein has been accomplished using Fmoc chemistry. A new fast and harmless protocol is described for complete TFA removal from the purified peptide powder leading to a final purity greater than 98% as controlled by 19F-NMR, UV and MALDI-TOF mass spectrometry. Secondary structure was determined in various solution and membrane media using UV Circular Dichroism. In water solution, Bax-α1 is present as a mixture of β-sheet and unstructured (random coil) conformations. A marked change from β-sheet to α-helix secondary structures is observed upon interaction with negatively charged phospholipids vesicles whereas neutral lipid membranes have no significant effect on the aqueous peptide conformation. Results are discussed in terms of Bax binding to mitochondrial membranes.

Keyword
solid phase synthesis, TFA removal, 19F NMR, UV circular dichroism, electrostatic interaction, apoptotic peptides
Identifiers
urn:nbn:se:umu:diva-12371 (URN)doi:10.1002/psc.803 (DOI)
Available from: 2007-04-03 Created: 2007-04-03Bibliographically approved
3. Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain
Open this publication in new window or tab >>Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain
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.

Keyword
apoptosis, BH4 domain, membrane, protein-lipid interactions, solid state NMR
Identifiers
urn:nbn:se:umu:diva-7994 (URN)doi:10.1111/j.1742-4658.2007.06222.x (DOI)
Available from: 2008-01-14 Created: 2008-01-14Bibliographically approved
4. How does the Bax-α1 targeting sequence interact with mitochondrial membrane?: The role of cardiolipin
Open this publication in new window or tab >>How does the Bax-α1 targeting sequence interact with mitochondrial membrane?: The role of cardiolipin
2009 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 3, 623-631 p.Article in journal (Refereed) Published
Abstract [en]

A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-α1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-α1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin. 1H and 31P magic angle spinning (MAS) NMR revealed Bax-α1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-α1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting β-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-α1 at the CS membrane surface promotes conformational change into an α-helical state; a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.

Place, publisher, year, edition, pages
Elsevier B.V., 2009
Keyword
Apoptosis, Bax N-terminal, Cardiolipin, Solid-State NMR
Identifiers
urn:nbn:se:umu:diva-11590 (URN)10.1016/j.bbamem.2008.12.014 (DOI)
Available from: 2009-01-20 Created: 2009-01-20 Last updated: 2012-08-15Bibliographically approved
5. Tracking lipid interactions in intact mitochondria under oxidative stress by ex vivo solid state 31P NMR spectroscopy
Open this publication in new window or tab >>Tracking lipid interactions in intact mitochondria under oxidative stress by ex vivo solid state 31P NMR spectroscopy
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(English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:umu:diva-3554 (URN)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2015-11-16Bibliographically approved

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