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David Mitchell, Ph.D.

  • The primary goal of my research is to address and understand some of the complex molecular signal transduction mechanisms in nature using the model organism, Saccharomyces cerevisiae. At present I am focusing on two research objectives: a) understanding the complex interplay between cellular membranes and proteins that are peripherally associated with these membranes and b) characterizing the factors which regulate Palmitoyl Acyl Transferases (PAT), an enzyme that posttranslationally attaches long chain fatty acids to proteins.

  • In order to study the dynamics of proteins along cellular membranes, I have constructed a genetically tractable system to evaluate the movement of Ras2 in relation to the inner plasma membrane of the yeast cell. This system utilizes Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) of live yeast cells to monitor the diffusion and membrane exchange of GFP-tagged Ras2 molecules at the plasma membrane. Ras2 begins as a cytosolic protein, then immediately becomes farnesylated at its C-terminus resulting in membrane localization. In addition to farnesylation, Ras2 also undergoes palmitoylation while membrane localized as well as being methylesterified. Through genetic manipulations, we will be able to assess the contributions each and all of these posttranslational modifications make to the overall association of Ras2 to the plasma membrane.

  • In addition, I am also constructing High Throughput Screening platforms to identify modulators of the Ras Palmitoyl Acyl Transferases (RasPAT) with the goal of identifying inhibitors of Ras palmitoylation. Inhibitors of Ras function are excellent candidates for the development of cancer chemotherapeutic drugs, given the central role of Ras signaling in most cancer cells. Ras PAT carries out the posttranslational addition of palmitate to the C-terminus of Ras proteins. This lipophilic addition is required for subcellular trafficking and membrane assembly of active Ras signaling complexes. Since the discovery of Ef2/Erf4 (yeast RasPAT) by Robert Deschenes and colleagues, the palmitoylation field has grown exponentially. However, the identification of tools to study these proteins has severely lagged in comparison. Identification of inhibitors and modulators will be a substantial move forward in cancer biology.

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  • Tissue Engineering and Cancer