Molecular activation and activity of the BH3-only protein Bim

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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.