| 1. |
- Khemtémourian, Lucie, et al.
(författare)
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Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4
- 2006
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 12, s. 58-64
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Sani, Marc-Antoine, et al.
(författare)
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How does the Bax-α1 targeting sequence interact with mitochondrial membrane? : The role of cardiolipin
- 2009
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier B.V.. - 0005-2736 .- 1879-2642. ; 1788:3, s. 623-631
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Sani, Marc-Antoine, et al.
(författare)
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Magic-angle phosphorus NMR of functional mitochondria : in situ monitoring of lipid response under apoptotic-like stress
- 2009
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Ingår i: The FASEB Journal. - : The Federation of American Societies for Experimental Biology. - 0892-6638 .- 1530-6860. ; 23:9, s. 2872-2878
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Tidskriftsartikel (refereegranskat)abstract
- Using a noninvasive, solid-state magic-angle spinning nuclearmagnetic resonance (MAS NMR) approach, we track ex vivo thebehavior of individual membrane components in isolated, activemitochondria (model system: potato tubers) during physiologicalprocesses. The individual phosphatidylcholine (PC), phosphatidylethanolamine(PE), and cardiolipin (CL) membrane constituents were identifiedas distinct lines by applying MAS 31P NMR on extracted lipidmembranes. However, the CL NMR signal appeared to be very broadin functional mitochondria, indicating a tight complex formationwith membrane protein. Calcium stress induced severe membranedegradation without recovery of a single CL NMR resonance. Thissuggests that calcium overload destroys the outer mitochondrialmembrane and does not modify strongly the CL protein complexesin the inner membrane; a conclusion confirmed by respiratorycontrols. Conversely, mitochondrial membrane disruption on timedegradation or mechanical stress generates clearly visible identicalCL NMR signals, similar to those observed in rehydrated lipidextracts. Similarly, noninvasive based NMR tracking of lipidsin response to diverse physiological stimuli can easily be usedfor other organelles and whole living cells. Sani, M.-A., Keech,O., Gardeström, P., Dufourc, E. J., Gröbner, G. Magic-anglephosphorus NMR of functional mitochondria: in situ monitoringof lipid response under apoptotic-like stress.
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| 4. |
- Sani, Marc-Antoine, et al.
(författare)
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Pro-apoptotic bax-α1 synthesis and evidence for β-sheet to α-helix conformational change as triggered by negatively charged lipid mβembranes
- 2007
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 13:2, s. 100-106
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Sani, Marc-Antoine, et al.
(författare)
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Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain
- 2008
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Ingår i: The FEBS Journal. - : FEBS Press. - 1742-464X .- 1742-4658. ; 275:3, s. 561-572
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
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