In both embryos and adults, cell fate specification is often directed by morphogenetic fields of signaling proteins, such as Wnt proteins. Cells in target tissues produce concentration dependent responses, resulting in the generation of different cell fates. Thus, morphogenetic gradients prefigure patterns of cell fate specification and differentiation. The movement of signaling protein away from the site of synthesis is key to the formation of a gradient. One of the best-characterized vertebrate Wnt gradients is in the chick neural tube, where a dorsal to ventral gradient of Wnt-1/3a regulates the balance between proliferation and differentiation. The recent discovery that the post-translational modification of Wnt proteins with palmitate regulates their activity and distribution has revealed a gap in the understanding of the mechanistic underpinnings of this process. The objective of this proposal is to determine the mechanism by which palmitoylation regulates the distribution and activity of Wnt proteins. The PI hypothesizes that Porcupine-mediated palmitoylation regulates the activity and distribution of Wnt-1 and Wnt-3a in the chick neural tube by facilitating their assembly into multimers as well as by reducing their stability and their rate of diffusion away from the site of synthesis. The rationale for the proposed studies is that an understanding of these processes is required for the development of therapeutic strategies to deliver lipid-modified signals to appropriate tissues or to inhibit the delivery of lipid-modified signals to their target tissues. For the first specific aim, the Burrus lab will first use biochemical approaches to identify and characterize the enzyme that is responsible for palmitoylating Wnt proteins. For the second specific aim, The Burrus lab will utilize biochemical and in vivo approaches to determine the mechanism(s) by which palmitoylation regulates Wnt activity. Lastly, the Burrus lab will use a combination of conventional imaging, live imaging and functional analysis to define the mechanism(s) by which palmitoylation regulates the distribution of Wnt proteins. Relevance to Public Health: """"""""These data are significant because they will provide knowledge that is critical for the development of therapies that involve tissue engineering or delivery of Wnt ligands to particular target cells as well as therapies that involve the inhibition of aberrant Wnt signaling. Once such strategies are developed, there is the promise of targeting Wnts to stem cells for the generation and expansion of healthy tissues, including muscle, cartilage, neurons, and hematopoietic cells. The identification of the upstream regulator of palmitoylation will also provide a potential therapeutic target for treating cancers that rely on the deregulation of Wnt ligand expression for their progression, including sarcoma, malignant mesothelioma, colorectal cancer, and melanoma.
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