Assessment of the target potential of Bim, Bmf and PUMA/bbc3

Programmed cell death (apoptosis) is the physiological process responsible for deletion of unwanted and potentially harmful cells. Abnormalities in cell death control can promote hyperplasia which can be an initial step in the pathogenesis of cancer or autoimmune disease. Under physiological conditions apoptosis is triggered either by ligand-mediated activation of certain members of the tumor necrosis factor receptor (TNF-R) family or can be induced in response to cell stress by pro-apoptotic members of the Bcl-2 family. Pro-apoptotic BH3-only members of the Bcl-2 family are essential initiators of stress-induced apoptosis and they function predominantly by antagonizing the function of pro-survival Bcl-2 family members. Cell death also requires the function of a second pro-apoptotic subgroup of the Bcl-2 family, including Bax, Bak and Bok/Mtd. The mechanisms leading to their activation are poorly understood but may also depend on direct interaction with BH3-only proteins such as tBid. The interplay of BH3-only proteins, Bax/Bak-like molecules and Bcl-2-like pro-survival proteins mediates activation of a cascade of cysteine proteases (caspases) by a mechanism that involves release of apoptogenic factors (e.g. cytochrome c, smac/Diablo, AIF) from mitochondria. To prevent accidental removal of healthy cells, but also to allow timed and specific deletion of unwanted cells via the Bcl-2 regulated cell death pathway the pro-apoptotic potential of BH3-only proteins is tightly regulated at the transcriptional and/or post-translational level. I propose to analyze the biological relevance of the post-translational regulation mechanisms described for the BH3-only proteins Bim and Bmf: sequestration to the cytoskeleton by interaction with dynein light chain (DLC) molecules. This will be studied by generating mutant knock-in mice that express DLC binding-deficient mutants of Bim and Bmf (referred herein as Bim and Bmf) in all tissues or in a cell type-specific manner. I want to investigate (i) the consequences of the disruption of DLC/BH3-only protein complexes per se and (ii) whether expression of Bim and Bmf is sufficient to prevent tumorigenesis and/or autoimmunity in vivo. Furthermore, I propose to (iii) explore the consequences of the combined absence of the BH3-only proteins Bim and PUMA/bbc3. The activation of pro-apoptotic BH3-only proteins, for example by disruption of the DLC/Bim or DLC/Bmf protein complexes by small molecules, can be considered as a potential novel therapeutic strategy in the treatment of neoplastic and/or autoimmune disorders. Analyzing the biological consequences of expression of DLC binding- deficient Bim and Bmf mutants is critical to assess the therapeutic potential of targeted disruption of the DLC/BH3-only protein complexes. Perhaps equally important, these studies may also provide new insights into the pathogenesis of degenerative diseases that associate with excess programmed cell death.

Programmed cell death (apoptosis) is the physiological process responsible for deletion of unwanted and potentially harmful cells. Abnormalities in cell death control can promote hyperplasia which can be an initial step in the pathogenesis of cancer or autoimmune disease. Under physiological conditions apoptosis is triggered either by ligand-mediated activation of certain members of the tumor necrosis factor receptor (TNF-R) family or can be induced in response to cell stress by pro-apoptotic members of the Bcl-2 family. Pro-apoptotic BH3-only members of the Bcl-2 family are essential initiators of stress-induced apoptosis and they function predominantly by antagonizing the function of pro-survival Bcl-2 family members. Cell death also requires the function of a second pro-apoptotic subgroup of the Bcl-2 family, including Bax, Bak and Bok/Mtd. The mechanisms leading to their activation are poorly understood but may also depend on direct interaction with BH3-only proteins such as tBid. The interplay of BH3-only proteins, Bax/Bak-like molecules and Bcl-2-like pro-survival proteins mediates activation of a cascade of cysteine proteases (caspases) by a mechanism that involves release of apoptogenic factors (e.g. cytochrome c, smac/Diablo, AIF) from mitochondria. To prevent accidental removal of healthy cells, but also to allow timed and specific deletion of unwanted cells via the Bcl-2 regulated cell death pathway the pro-apoptotic potential of BH3-only proteins is tightly regulated at the transcriptional and/or post-translational level. I propose to analyze the biological relevance of the post-translational regulation mechanisms described for the BH3-only proteins Bim and Bmf: sequestration to the cytoskeleton by interaction with dynein light chain (DLC) molecules. This will be studied by generating mutant knock-in mice that express DLC binding-deficient mutants of Bim and Bmf (referred herein as Bim and Bmf) in all tissues or in a cell type-specific manner. I want to investigate (i) the consequences of the disruption of DLC/BH3-only protein complexes per se and (ii) whether expression of Bim and Bmf is sufficient to prevent tumorigenesis and/or autoimmunity in vivo. Furthermore, I propose to (iii) explore the consequences of the combined absence of the BH3-only proteins Bim and PUMA/bbc3. The activation of pro-apoptotic BH3-only proteins, for example by disruption of the DLC/Bim or DLC/Bmf protein complexes by small molecules, can be considered as a potential novel therapeutic strategy in the treatment of neoplastic and/or autoimmune disorders. Analyzing the biological consequences of expression of DLC binding-deficient Bim and Bmf mutants is critical to assess the therapeutic potential of targeted disruption of the DLC/BH3-only protein complexes. Perhaps equally important, these studies may also provide new insights into the pathogenesis of degenerative diseases that associate with excess programmed cell death.