Evidence is mounting that the post-translational process, palmitoylation, has a major role in neuronal development and function, in particular, the formation and functioning of synapses. The standard methods for assaying protein palmitoylation are relatively insensitive, not quantitative, as well as laborious. Given these difficulties, it is apparent that many palmitoylated proteins remained unidentified. We have recently developed alternative assays of protein palmitoylation based on hydroxylamine cleavage of the thioester bond between the fatty acid and cysteine side chain followed by reaction of the newly generated free sulfhydryl with sulfhydryl-specific reagents, such as 3H-N-ethyl maleimide (NEM) and biotinylated reagents. These techniques are significantly more sensitive than metabolic labeling with 3H-palmitate and can be used quantitatively to measure levels of protein palmitoylation. They have the additional advantage of allowing protein palmitoylation to be assayed on preparations from brain and other nervous tissue, which is not possible using [3H]-palmitate labeling. The goal of this application is to adapt our new assays for palmitoylation to a proteomic scale in order to accomplish the following two specific aims. First, we propose to modify our techniques in order to identify the specific sites of palmitoylation on the different ionotropic glutamate receptors we are currently studying in our laboratory and all of which we have found to be palmitoylated. Second, we are developing protocols that allow a set of palmitoylated proteins to be specifically purified from highly complex protein extracts in order to quantify protein palmitoylation differences in neuronal preparations. To test these techniques, two different preparations will be analyzed. First, we will isolate palmitoylated proteins from rat cultured cortical neurons and assay for changes in their palmitoylation when long-term potentiation (LTP) has been chemically induced. In the second preparation, we will isolate palmitoylated proteins from rat nucleus accumbens punches and test for changes in their palmitoylation with amphetamine exposure in order to determine how behavioral sensitization alters palmitoylation. The proposed research is innovative because it will develop new proteomic-based technology to assay a protein post-translational modification that should provide new insights into basic synaptic biology and the neurotransmitter receptors, such as gluatamate receptors that are involved in drug abuse.
Protein palmitoylation is a reversible lipid modification on cysteine (Cys) residues and is involved in targeting proteins to membrane domains. The regulation of this modification has been shown to play a significant role in synapse formation and function. The large scale analysis of palmitoylated proteins will provide a comprehensive view of the scope of this modification by identifying many unknown protein targets, which will promote the development of subsequent hypothesis-driven research.
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