We have studied programmed cell death in the nervous system and the biochemical mechanism of apoptosis. Recently we have focused on the Bcl-2 family of proteins that regulates the survival of neurons during development. One member of this family, Bax, is required for the normal death of neurons and elimination of the Bax gene results in excess neurons in adult animals. How Bax and other Bcl-2 family members regulate cell survival is unknown. We developed a series of monoclonal antibodies against Bcl-2, Bcl-xl, and Bax to probe their mechanism of action in vitro and in vivo. The antibodies revealed that Bax migrates from the cytosol to cell membranes during apoptosis. To explore Bax, Bcl-2 and Bcl-xl movement in neurons the green fluorescent protein has been used to tag and follow the proteins in living cells. We discovered that Bax and Bcl-xl move from the cytosol to the mitochondria as an essential step in their mechanism of apoptosis control. Point mutants of Bax that insert better into mitochondria are more toxic and mutants with less mitochondrial insertion are less toxic indicating that the C-terminus of Bax regulates the mitochondrial association. Therefore, we determined the three dimensional structure of Bax and found that the C-terminal tail fits into a hydrophobic pocket that has been thought to regulate hetero- and homo-dimer formation among Bcl-2 family members. This suggests a new model that dimer formation and mitochondrial docking are structurally linked to disengagement of the C-terminus. In contrast to Bax, Bcl-xl prevents neuron cell death due to many neurotoxic insults. Like Bax, Bcl-xl inserts into mitochondria during apoptosis. We have found one regulatory step that controls Bcl-xl subcellular location. Bad, a pro-apoptotic Bcl-2 family member, binds to Bcl-xl and triggers its docking to mitochondria. Bad binding to Bcl-xl is in turn regulated by phosphorylation and several neurotrophic factors that stimulate kinases prevent Bad from binding to Bcl-xl and thus prevent neuron death. We also found that Bcl-xL inhibits Bax accumulation on mitochondria and apoptosis by increasing the retrotranslocation of Bax back into the cytosol. The consequences of Bcl-2 family member binding to mitochondria have been also studied. Bax coalesces into large aggregates on the mitochondrial surface that co-localize with machinery for mitochondrial division and can activate mitochondrial division. Thus, the cell machinery that governs mitochondrial division participates in apoptosis.

Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2012
Total Cost
$1,009,989
Indirect Cost
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State
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Yao, Yong; Nisan, Danielle; Fujimoto, Lynn M et al. (2016) Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL. Protein Expr Purif 122:56-63
Lazarou, Michael; Sliter, Danielle A; Kane, Lesley A et al. (2015) The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524:309-14
Pickrell, Alicia M; Youle, Richard J (2015) The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron 85:257-73
Yao, Yong; Fujimoto, Lynn M; Hirshman, Nathan et al. (2015) Conformation of BCL-XL upon Membrane Integration. J Mol Biol 427:2262-70
Pickrell, Alicia M; Huang, Chiu-Hui; Kennedy, Scott R et al. (2015) Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress. Neuron 87:371-81
Pickrell, Alicia M; Youle, Richard J (2013) Mitochondrial disease: mtDNA and protein segregation mysteries in iPSCs. Curr Biol 23:R1052-4
Ma, Junhe; Edlich, Frank; Bermejo, Guillermo A et al. (2012) Structural mechanism of Bax inhibition by cytomegalovirus protein vMIA. Proc Natl Acad Sci U S A 109:20901-6
Cooper, Julia Promisel; Youle, Richard J (2012) Balancing cell growth and death. Curr Opin Cell Biol 24:802-3
Cleland, M M; Norris, K L; Karbowski, M et al. (2011) Bcl-2 family interaction with the mitochondrial morphogenesis machinery. Cell Death Differ 18:235-47
Hoppins, Suzanne; Edlich, Frank; Cleland, Megan M et al. (2011) The soluble form of Bax regulates mitochondrial fusion via MFN2 homotypic complexes. Mol Cell 41:150-60

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