BH3-only proteins are the initial triggers of mitochondrial apoptosis within the Bcl-2-family. How BH3-only proteins are activated themselves however is in many instances unclear. Bim is one of the most prominent BH3-only proteins; Bim plays a substantial role in regulating survival in the haematopoietic system and functions as a tumour suppressor in many cell types. Bim is capable of direct activation of Bax/Bak, as well as able to inhibit all anti-apoptotic Bcl-2-like proteins. Bim is constitutively expressed in probably all cell types. Although some cases are known where an increase in Bim protein levels correlates with Bim-induced apoptosis, in other cases Bim becomes active without such protein induction, suggesting post-translational regulation. In this project we have identified a mechanism that we believe is important for this regulation. We found that, on the outer mitochondrial membrane, Bim dimerizes through binding to dynein light chain 1 (DLC1), leading to the formation of large protein complexes. Further experiments with intact cells, isolated mitochondria and liposomes suggest that DLC1-binding to Bim is sufficient for large complex formation and is, surprisingly, required for efficient binding to and inhibition by anti-apoptotic Bcl-2 proteins. Large complexes have little or no pro-apoptotic activity, are found in all cells investigated and appear also to incorporate anti-apoptotic Bcl-2 proteins. In this project we will pursue two specific aims to understand the regulation of Bim. Firstly, we will use a number of biophysical, biochemical and microscopic techniques to elucidate the molecular nature of the observed complexes. Experiments will be directed at understanding stoichiometry, activity and dynamics of large Bim containing complexes, as well as the structure of Bim within the complexes. Secondly, we will endeavour to understand the molecular circumstances of inhibition and activation of Bim in these complexes. Status and disassembly of complexes in intact cells and in response to apoptotic stimuli will be followed. Recruitment and role of Bcl-2-like proteins in formation and dissolution of the complexes will further be studied. In models of apoptosis induction and of lymphocyte differentiation we will assess the importance of complex formation for survival of human and mouse cells. We believe that these studies will elucidate a novel mechanism of regulation of the BH3-only protein Bim, which may serve as a model to understand other BH3-only proteins, and to appreciate the activation of the Bcl-2-family system at mitochondria.
The overall objective of this project is to understand how mitochondrial apoptosis is initiated, as exemplified by regulation of the BH3-only protein Bim. Our data suggest that Bim is activated in a previously not appreciated process that revolves around the interaction between several Bim molecules, DLC1 and anti-apoptotic proteins in large complexes in the outer mitochondrial membrane. To understand these regulatory steps in the induction of apoptosis we here propose two pursue two specific goals:
To understand the molecular nature of the Bim complexes. Using a number of cell biological and biophysical approaches we will here identify the molecular composition, structure and the processes of assembly/disassembly of these complexes, as well as the downstream pathway from Bim-complexes to the activation of Bax and Bak.
To understand (post-translational) regulation and activity of Bim in the outer mitochondrial membrane. Our results describing the complex forming properties of Bim suggest new ways of how the activity of Bim is regulated on the molecular level, and how this may regulate apoptosis. We will here explore these signaling pathways to understand Bim-induced apoptosis in biology.
Georg Häcker (PI, Professor)
Prafull Kumar Singh (Postdoc)
Cooperation within FOR2036
Biophysical studies will be conducted with Ana Garcia-Saez, in particular interactions of Bim with membranes and other Bcl-1-family members.
Analyses of Bim complexes will be conducted together with Thomas Brunner, Markus Morrison and Miriam Erlacher.
In the question of complexes in other BH3-only proteins we will cooperate with Christoph Borner.
A Usp27x-deficient mouse will be analyzed together with Andreas Villunger.