Hedgehog acyltransferase : structure and function in health and disease

The ability to study how biological molecules, such as proteins and enzymes, function on an atomic level is key to our understanding of life - for example, how a single fertilised egg cell can give rise to a complex multi-organ organism like a human - and is also critical for faster and more effective development of medicines that target these biological molecules. However, certain types of biological molecules, such as proteins that exist inside biological membranes (termed 'membrane proteins'), present substantial challenges to modern methods of analysis. Here we propose to bring together multiple novel methods to study the structure and function of a membrane protein named 'Hedgehog acyltransferase' (HHAT), which is a key regulator of growth and development, and is also implicated in development of certain cancers in adults. The resulting molecular level understanding of HHAT will empower efforts to therapeutically target HHAT, and guide future studies of other similarly challenging membrane proteins.

HHAT regulates the 'Hedgehog signalling pathway' that cells use to communicate with each other. The Hedgehog pathway is key to growth and development of animals, and was named after its discovery in fruit flies where disruption causes larvae to develop a spikey appearance like a hedgehog. Hedgehog signalling is generally turned off in healthy adults, but it is reactivated in certain cancers.

Our research will approach the problem of understanding how HHAT functions and how this affects Hedgehog signalling using three distinct, but complimentary, approaches. We will use chemical biology to create variants of the molecules that HHAT modifies, which will allow us to understand how these molecules interact with HHAT. In parallel, we will use state-of-the-art structural biology methods to allow us to visualise the 3-dimensional architecture of HHAT at an atomic level. Finally, we will use these new insights to understand how HHAT works in cells, and to design new HHAT proteins that have altered preferences for the molecules they modify. These new designer HHAT's will be used to understand how HHAT regulates the distance and extent to which cells can communicate over.

This research will provide fundamental understanding of the role of HHAT and Hedgehog signalling in development and in diseases such as cancer, and provide an important means to regulate cell-to-cell communication in designed 'synthetic' biological systems. It will also provide 3D structural information and identify regions where molecules can bind to HHAT, which will greatly accelerate drug discovery efforts. Further, this work will provide a roadmap to guide future studies against other related membrane proteins that are targets in a range of diseases.

Hedgehog (HH) signalling is a key regulator of growth and development, and is active in certain cancers. Production of functional HH signalling proteins requires C16 acylation of the N-terminal cysteine by a 10-multipass membrane bound O-acyltransferase (MBOAT) family member, Hedgehog acyltransferase (HHAT). Whilst the importance of this modification for signalling is well established, little is known about how this occurs, how it is regulated, or the functional outcome of the modification in cell-to-cell signalling. Key questions remain regarding HHAT's structure, mechanism of catalysis, substrate binding sites, motifs in HHAT substrates required for binding/selectivity, the identity of co-regulators of HH acylation, and how lipid chain identity affects the distance or duration of HH signals.

We will address this through three complimentary approaches using (1) chemical biology (2) structural biology and (3) cell biology. (1) Substrate analogues will be prepared to map their structure-activity relationships. Co-regulators will be identified via proteomics, and binding sites for substrates, co-regulators, small-molecule inhibitors and nanobody binders will be mapped through non-biased labelling of exposed HHAT residues and proteomics analysis (termed 'footprinting'). (2) The structure of HHAT will be determined through crystallography and/or Cryo-EM analyses in complex with substrates, inhibitors, co-regulators and nanobodies. (3) We will combine data generated in (1) and (2) to design HHAT mutants with altered acyl chain and HH substrate preferences. These constructs will be used in cellular assays to probe the effect of HHAT enzymology on HH signalling.

This work will provide the first structure of a mammalian MBOAT, and fundamental understanding of the role of HHAT in HH signalling. This will accelerate drug discovery against HHAT, and provide a roadmap for studies of other MBOATs. Further, designer HHAT mutants may be useful tools in synthetic biology.

The proposed project has several long-term potential economic and societal impacts that may result from the research, methods and tools developed:

There has been substantial interest from industry in therapeutic modulation of the Hedgehog pathway owing to its involvement in the formation and progression of several cancers, and there will be significant impact for scientists working in this field. The proposed project will provide not only key structural information on HHAT which may help to guide any future drug development programmes, but also a suite of methods and tools that could significantly accelerate such programs. This would de-risk HHAT as a potential target by providing far greater understanding of its enzymology, structure, regulation and biological function, and the assays and protocols for protein production will significantly increase tractability of drug discovery efforts against HHAT. Through solving structures of HHAT-inhibitor complexes, structure-guided inhibitor development will be possible which will aid in development of inhibitors with increased potency. Furthermore, these insights will be readily extended to closely related members of the class in human (PORCN, GOAT), accelerating drug discovery for these targets in cancer and appetite regulation. This acceleration of drug discovery efforts may ultimately lead to a substantial impact in improving health and well-being of large numbers of people globally through the development of new medicines.

The project will also lead to further opportunities by attracting follow-on R&D to develop HHAT or Hedgehog signalling in synthetic biology. Researchers will be provided with a suite of HHAT mutations with well-characterised designer substrate selectivity, to allow selective modification of target proteins with fatty acyl chains and differential targeting to subcellular compartments, or between cells/compartments. This will support scientists in these fields by providing new tools or research avenues to secure funding.

The project will enhance the research capacity of academic and industrial laboratories working on HHAT or related proteins (e.g. Porcupine, GOAT) by displaying the strength of synergistic, cross-disciplinary science. This will increase the knowledge and skills in such organisations by emphasising the importance of versatility and cohesiveness in addressing challenging targets and may have an impact in changing the organisation and execution of related projects in future.

Whilst providing fundamental insight into the role of HHAT-mediated acylation in cellular Hedgehog signalling, the tools developed may also present opportunity for commercial exploitation. Despite substantial efforts, it has not been possible to raise an antibody against HHAT and such a tool would be of substantial use in field for detection or imaging. We have successfully generated ~100 nanobody binders which our initial results demonstrate may display exceptionally high affinity for HHAT. During the course of this work these will be further triaged for binding (binding sites, HHAT secondary/tertiary structural requirements) and may support potential use in various detection techniques such as fluorescence microscopy, ELISA, Western blotting, immunohistochemistry, etc. Similarly, inhibitors with well-defined binding sites and modes of action will be powerful tools to manipulate HH signalling in molecular and developmental biology, offering the opportunity to commercialise these as research tools (as the Tate group has done recently with inhibitors of N-myristoylation).