The main goal of my laboratory is to understand the molecular regulating cell growth. Towards that goal, we have identified a novel anti-apoptotic gene, Lifeguard (LFG) which prevents Fas induced apoptosis. To define the molecular and biochemical basis of the function of LFG, we have proposed the following six aims: 1. Mechanism of Action of LFG: We will generate LFG specific antibodies to undertake experiments to determine association of LFG with components of Fas pathway. Since TNF-alpha induced cell death is not influenced by LFG, we proposed to generate Fas/TNFR1 chimeric molecules to understand the role of extra- cellular, membrane spanning, and C-terminal domain of Fas. 2. Biological function of LFG: We propose to delete LFG in mice by homologous recombination and study the effect of its los s on animal physiology. If LFG(-/-) turns out to be embryonic lethal, we will also generate """"""""conditional lethal"""""""" mice. We also plan to generate transgenic mice with neuronal promote (CAM kinase, neuronal encolase) and LFG(- /-) turns out to be embryonic lethal, we will also generate """"""""conditional lethal"""""""" mice. We also plan to generate transgenic mice with neuronal promoter (CAM kinase, neuronal encolase) and Lck-promoter to express in lymphoid cells. 3. LFG related """"""""functional equivalent"""""""" genes: We have isolated several other """"""""pooled"""""""" Fas resistant cell clones. We will identify the nature of the cDNA in these clones and study their functions. 4. LFG- associated proteins: We will study the LFG associated proteins by two approaches (i) Yeast two plasmid hybrid system: We have identified a large number of interacting proteins, some of which are previously known. If in vivo association of interacting proteins is established, we will then try to study their role in Fas induced apoptosis. Ii) Identify interacting cellular proteins by immunoprecipitation with LFG specific antibodies. 5. Role of LFG in neurons: Because LFG is expressed at high levels in the hippocampus and more specifically in the CA1-CA3 region, we propose to culture neuronal cell line, hippocampal cell lines, hippocampal slices and ask if blocking LFG function is critical for cell survival or differentiation. Other """"""""screens"""""""" for function: Towards our overall goal of identification of the function of novel genes by using retroviral vector technology, we intend to initiate additional """"""""functional screens"""""""". Specifically we plan to undertake screens to identify new NF- kappaB co-factors and anti-apoptotic and anti-apoptotic genes induced by NF-kappaB. We believe that the results obtained from these proposed experiments will allow us to understand not only the molecular functions of LFG, but also allow us to understand the role of apoptosis in control of cell growth and proliferation.
