Understanding Patched1 protein and lipid interactions in cilia

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PROJECT SUMMARY/ABSTRACT Spatiotemporal regulation of signaling molecules is critical for development and adult tissue homeostasis. The Hedgehog pathway, which is conserved across metazoan animals, controls cell proliferation, differentiation, migration, and stem cell maintenance. In vertebrates, Hedgehog signals are transduced through primary cilia that project from the surface of most cells, including cells in cancers that are driven by misactivation of the Hedgehog pathway. The tumor suppressor Patched1 localizes to cilia and inhibits Smoothened, a Hedgehog pathway activator. Upon pathway activation, Patched1 leaves cilia and allows Smoothened to activate the downstream Hedgehog transcriptional program. How Patched1 localizes to cilia and inhibits Smoothened remain unknown. The central hypothesis of this proposal is that a dynamic network of protein interactions allow Patched1 to accumulate in cilia and regulate the ciliary lipid microenvironment to inhibit Hedgehog signal transduction. To test this, the objective of this proposal is to define the ciliary protein interactions necessary for localization of Patched1 to the cilia, and to determine if Patched1 regulates Smoothened by regulating the ciliary lipid microenvironment. To address the gaps in our understanding of Hedgehog signaling, I will leverage recent technical advances in proteomic proximity-labeling mass spectrometry, lipidomic mass spectrometry, and functional genomics using novel model systems I have generated for this proposal. In Aim 1, I will interrogate 5 protein interactors of Patched1 that are associated with human disease and may underlie Patched1 accumulation and activity in primary cilia. In Aim 2, I will define the impact of Patched1 on the ciliary lipid microenvironment. Combined, these aims will elucidate the biochemical mechanism by which Patched1 regulates Hedgehog signaling. Hedgehog pathway misactivation drives medulloblastoma, the most common pediatric brain tumor, and basal cell carcinoma, the most common cancer in the United States. Thus, this proposal will incorporate Hh-associated cancer cell lines to determine if ciliary proteins and lipids underlying Patched1 functions are conserved across developmental and disease contexts. In sum, understanding how Patched1, the most recurrently mutated gene in Hh-associated cancers, inhibits Hedgehog signaling will provide significant insights into human biology and potentially provide avenues for novel therapies.