Hedgehog Acyltransferase: Structure, Function and Inhibition

Thesis

The Hedgehog signalling pathway is essential for mammalian development. Errors in Hedgehog signalling resulting in loss of function during development, lead to a variety of congenital diseases, including holoprosencephaly, brachydactyly and disorders of sex development. In adults, Hedgehog signalling is largely dormant, and aberrant Hedgehog signalling has been implicated in cancers of various organs. As such, the Hedgehog signalling pathway is an important anti-cancer drug target and drugs against the Smoothened cell-surface receptor, which relays the Hedgehog signal across the receiving cell membrane, have proven useful in treatment in the clinic. A critical step in this pathway is the maturation of the sonic Hedgehog (SHH) morphogen. SHH undergoes a unique maturation process in which it is expressed as a 45 kDa pre-protein with N-terminal “hedge” and C-terminal “hog” domains. The C-terminal hog domain has homology to intein domains and induces autocleavage and concomitant addition of a cholesterol moiety at the C-terminus of the “hedge” domain. The N-terminal amine of the hedge fragment is palmitoylated to yield a dually-lipidated N-terminal SHH signalling fragment – the mature morphogen. SHH palmitoylation is catalysed by Hedgehog acyltransferase (HHAT), an integral membrane enzyme, located in the endoplasmic reticulum membrane. This thesis explores the structure-function relationship of HHAT and its interaction with the SHH morphogen. In Chapter 3, the expression of HHAT and production of a high quality sample for structural studies is discussed. Creation and characterisation of single-chain camelid antibody fragments (nanobodies) against HHAT for use in structural studies is covered in Chapter 4. In Chapter 5 the structure of HHAT is presented and the binding mode of the acyl-donor substrate is discussed. Furthermore, structural information is confirmed through mutagenesis assays. In Chapter 6, the interaction of HHAT with the acyl-receiving substrate, SHH, is explored through the use of biophysical approaches to identify the SHH binding site. The final chapter, Chapter 7, covers the determination of the structure of HHAT in complex with inhibitor compounds and discusses the mode of inhibition. Overall, this work provides a basis for the function of HHAT, details the HHAT structure and explores HHAT inhibition at the structural level.

Authors: Coupland, C

• DOI: 10.5287/ora-proevnok9
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Acyltransferase; Hedgehog signalling; Drug discovery; Membrane proteins; Nanobodies; Inhibitor; Cryo-electron microscopy; Structural biology
• Subjects: Structure-activity relationships (Biochemistry); Protein drugs; Biophysics; Molecular biology; Biochemistry; Binding sites (Biochemistry)