Actividad en membranas modelo de péptidos apoptóticos

Resumen

Minimal fragments of Bax are able to reproduce the membrane permeabilizing activity of the protein, including capacity to release cytochrome c from mitochondria and to activate apoptosis in cells. These facts mean that the activity of the protein can be viewed as the addition of activities of constituent parts, which is the basis of any reduc- tionist strategy for investigating complex systems. However, the molecular mechanism of Bax protein fragments were poorly known. In particular, while the active species of Bax in the membrane is an oligomer, the aggregation state of the active fragments was unknown. Additionally, the size of the pores and their density in the membrane had not been determined. On the other hand, Bax has been show to exhibit membrane remodelling activity, but this had not been investigated in minimal active fragments of this protein. Finding satisfactory solutions to these problems constituted the objective of the Thesis. The single-pore properties were investigated for the case of a fragment containing the helix 5 of Bax (Bax-α5). For that, we first developed a methodology based on leakage measurements in single giant unilamelar vesicles (GUVs), using simultaneously two probes of different size. Through an exhaustive analysis of probe diffusion kinetics we were able to size and count single pores in single GUVs. The pores have a radius of ~5.6 nm and a surprisingly low density, meaning that only a minority of membrane bound peptides are actually forming pores. By means of circular dichroism spectroscopy and through labelling of the peptide with pirene we were able to find evidence of an oligomeric state in the membrane (possibly a dimer) for a fragment of Bax including parts of helices α5 and α6 (Bax-α56), but not for Bax-α56, which appears to be mo- nomeric. We also found that Bax-α56 exhibits marked membrane remodelling activity in GUVs. In contrast, the remodelling activity of Bax- 5 was weak. We hypothesize that the remodelling activity is linked to the membrane curving capacity of peptide dimers, with a coiled-coil structure similar to the association of helices α5 and α6 in a well cha- racterized Bax domain-swapping dimer. We also observed strong membrane remodelling activity for the case of Bak-α56, designed from the protein Bak, which also has been found to form a domain-swapping dimer. From these data we suggest that helices α5 and α6, in a dimeric state, are responsible of the membrane remodelling activity in both Bax and Bak, via membrane curvature induction. We also propose that the two proteins may have a direct role in mitochondrial dynamics, through membrane remodelling, and that this new function is played by structures corresponding to the domain-swapping dimers, which were considered to be non functional.