Structure-function relationship of the Patched family of proteins

The Patched protein family is characterised by a conserved sterol sensing domain (SSD). The receptor of Hedgehog, Patched-1, is believed to mobilise endogenous cholesterol to elicit a non-stoichiometric repression of the pathway activator Smoothened. How Patched-1 transports cholesterol, and whether this mechanism is shared by other Patched family proteins remains unclear. My doctoral dissertation work demonstrates an essential role of the SSD and several sites that interact with cholesterol in Patched-1, Patched-2, and a distant relative PTCHD1. I also present biochemical and functional evidence of Patched-1 and Patched-2 heterodimeric/oligomeric interactions, with synergistic activity. In Chapter 3, I describe mutational experiments that support the requirement of a hydrophobic cavity within Patched-1, for repression of Smoothened and canonical Hh signalling. Further experiments suggested a structural dependency of the C-terminal domain (CTD) upon the middle cytoplasmic loop (ML). Removal of these domains did not prevent dimerization, but impaired canonical activity. Chapter 4 describes experiments that revealed significant similarities between Patched-1 and Patched-2. Functionally, I identified the existence of Patched hetero-interactions, which exhibited direct competition with homo-interactions and displayed a synergistic effect on canonical activity. Further experiments using non-functional Patched mutants, revealed this synergistic activity is solely dependent on the activity-state of Patched-1 in the heterodimer. Moreover, I demonstrated that the CTDs of Patched-1 and Patched-2 regulate the inhibitory activity over Smoothened, possibly through interaction with the ML. Substitution of the CTD of Patched-1 with that of Patched-2 produced a non-functional protein. In Chapter 5, I optimised purification conditions of PTCHD1, mutated in some individuals with autistic spectrum disorder, generating an initial low-resolution model by negative stain electron microscopy. I demonstrate that PTCHD1 binds cholesterol, but cannot inhibit Smoothened function, nor bind Sonic Hedgehog ligand. Finally, proteomic analysis of PTCHD1-interacting proteins suggest a role in ribonucleoprotein granule formation and a biological function in neurons.