Regulation of the Hedgehog signalling pathway by Patched1 interacting proteins

The Hedgehog (Hh) receptor Patched1 is a well-established tumour suppressor. Its C-terminal domain (CTD) interacts with autophagy-related protein 101 (ATG101) to inhibit autophagic flux. The first part of my research focused on understanding this interaction. In Chapter 3, I show that endogenous Patched1 is sufficient to restrain autophagic flux. On Chapter 4, I investigated the functional consequences of three somatic mutations found in the CTD in human cancer databases. Results show that two of these lose the interaction with ATG101 causing an apparent increase in autophagic flux. These results strengthen a tumour suppressor role of Patched1 in addition to, and independently of, its function as Smoothened repressor. In Chapter 5, I validated and characterised the physical interaction between Integral membrane protein 2A (ITM2A) and Patched1. While the findings indicate that ITM2A, like Patched1, inhibits autophagic flux, the results suggest that they work independently. Unexpectedly, ITM2A had a negative effect on the stability of Patched1, which suggested a crosstalk with the Hh canonical pathway. Further analysis revealed that ITM2A negatively regulates Gli1 and Gli2 transcriptional activity. In Chapter 6, I showed a potential inhibitory function of Itm2a on autophagic flux during myogenic differentiation. Partial silencing of Itm2a delayed both myogenic differentiation and upregulation of cyclin D3 necessary for cell cycle exit of myoblasts. Proteomic analysis was used to find potential ITM2A-interacting proteins, which suggest a potential role in regulation of CDK1 and CDK2NA and of selective autophagy, which will be explored in future studies. In summary, this doctoral research uncovered a novel role of ITM2A as a negative regulator of the canonical Hh pathway and as a Patched1-binding partner. Results suggest that ITM2A might be a specific regulator of selective autophagy during skeletal muscle differentiation as opposed to a more general role of Patched1 through its interaction with ATG101.