Identification and characterization of interacting proteins regulating DAGL activity and endocannabinoid signaling

The incidence of psychiatric disorders, neurodegenerative diseases, and pain is on the increase. In terms of therapeutic intervention, this is self-evidently an area that requires the identification of novel pathways and neuronal targets. The CB1 and CB2 receptors are being investigated as potential therapeutic targets, however there are also likely to be considerable opportunities for modulating the synthesis of the most abundant endocannabinoid in the adult brain, 2-AG. The molecule is generated from diacylglycerol by the sn1-specific DAG lipases (DAGL-alpha and DAGL-beta) that we recently cloned (Bisogno et al. 2003). To fully exploit DAGL as a target, we need to understand how its function is regulated by protein-protein interactions. These are often mediated by linear peptides sequences and we have used a comparative genomic approach to identify the most highly conserved sequences in the DAGLs. We have synthesised four peptides (as well as reverse control peptides) in tandem with the antennapedia peptide sequence that mediates uptake into the cytosol of live cells. We now have evidence that three of these sequences represent functional motifs that have the same biological effects on cells as two conventional DAGL inhibitors (RHC80267 and THL); in this context they specifically inhibit DAGL dependent neurite outgrowth and also DAGL dependent proliferation of neural stem cells. The student will test the hypothesis that the peptides are inhibiting the function of the DAGLs by measuring their effects on DAGL activity in collaboration with scientists at Wyeth. The student will next use alanine scanning to identify key residues required for inhibitory activity and then introduce mutations to these residues in full length constructs of DAGL and determine their effects on enzyme activity following transfection into a variety of cell types. The student will also use two approaches to identify proteins that the peptides, and by implication, the DAGLs interact with. In the first approach the DAGL peptides (both the functionally blocking sequence and the reverse control) will be coupled directly to a sepahrose column. Brain extracts will be run over the columns, and specifically interacting proteins eluted and separated by conventional gel electrophoresis. Proteins that interact with the blocking but not control peptide will be identified following silver staining of the gels. Mass spectrometry methods will be used to identify proteins that interact with the blocking peptide, but not the 'reverse' control peptides. In the second approach, mini-constructs encoding the active inhibitory peptides will be used as bait in conventional yeast two hybrid screens. We anticipate that antibodies will be available for some of the interacting proteins, but if they are not we will outsource the generation of antibodies. Finally, we will test the hits for co-immunoprecipitation with DAGLs in a number of tissues and cell types.