Project P5

"Molecular function of Bim in neutrophil apoptosis"

People involved: Georg Haecker, Arnim Weber

BH3-only proteins are the triggers of mitochondrial apoptosis. They are necessary to activate Bax or Bak, and this activation is inhibited by anti-apoptotic Bcl-2 proteins. Two of the uncertainties are the molecular activation of Bax/Bak (importantly the subcellular localisation of this process) and the molecular function of BH3-only proteins: although it is clear that BH3-only proteins require the BH3-domain and we have some knowledge of its molecular function, both the function of this domain in the context of the intact protein and the functions of the protein parts outside the BH3-domain are known only to a very limited extent. We have found that a C-terminal membrane anchor is essential for full activity of Bim and have identified additional features of Bim, such as dimerization and complex formation on mitochondria. We have further established experimental systems that permit the study of the molecular function of Bim, either in embryonic fibroblasts and in cell-free systems or in neutrophil progenitor lines and differentiated neutrophils. For the latter system, we have established the method for the transfer of BH3-only proteins (and mutants) under the endogenous Bim-promoter, which permits the expression of mutant and chimaeric proteins and their functional assessment in apoptosis induction. The aims of this project are the understanding of the mechanism of action and the molecular requirements of BH3-only protein activity, primarily focussing on Bim. Using simple cellular and cell-free systems, the mitochondrial association, complex formation and Bax/Bak-activation will be analysed. In the cellular context of neutrophil apoptosis we will assess the requirements of BH3-only protein function, testing BH3-domain-composition, membrane spacing, mitochondrial targeting and differential activation of Bax and Bak. We believe that with this approach we will be able to answer some of the relevant questions of the molecular role and function of BH3-only proteins and their position in the Bcl-2 protein network determining mitochondrial apoptosis.

Co-operations within FOR2036:
Ana Garcia, Tübingen
Christoph Borner, Freiburg
Thomas Kaufmann, Bern
Andreas Villunger, Innsbruck
Philipp Jost, Munich