Fatty Acid Specificity in the DHHC Family of S-Acyltransferases: From Mechanisms to Functional Outcomes

The cells in our body contain a diverse array of different proteins that coordinate and drive specific pathways, such as cell growth and division. These proteins are subjected to strict modes of regulation to ensure that they are able to perform their specific functions as and when required. One prominent mechanism of protein regulation is via chemical modification and a variety of different molecules are added to proteins that affect their activity. One modification that is receiving increasing interest is "S-acylation", the attachment of fatty acids onto proteins, which is catalysed by a family of twenty-four "DHHC" enzymes. Dysfunction of DHHC enzymes has been linked with many important disorders, including diabetes, Huntington's disease, schizophrenia, intellectual disability and cancer.

The fatty acids that are added to S-acylated proteins can be diverse and it is likely that different fatty acids affect proteins in different ways. Despite this, we currently know very little about the mechanisms that specify the chemical identity of fatty acids added to individual S-acylated proteins, and how fatty acid identity impacts protein function. Therefore the aim of this research is to promote a major advance in this poorly understood aspect of S-acylation. To do this, we have brought together experts in Chemistry and Biology with the goal of using novel chemical probes to determine: (a) if different DHHC enzymes preferentially add distinct types of fatty acids onto S-acylated proteins, (b) what features of DHHC enzymes underlie their fatty acid specificity, and (c) how different fatty acids affect the localisation of proteins to different regions of the cell and their function in specific cellular pathways. In addition to shedding light on an important but poorly understood aspect of cell biology, this research may also highlight new strategies to design selective modulators of DHHC enzymes to treat a range of clinical conditions.

S-acylation is a common post-translational modification that regulates a range of different cellular proteins and pathways. A recent major breakthrough in the S-acylation field was the discovery of a large family of "DHHC" enzymes that collectively mediate the bulk of cellular S-acylation reactions. S-acylation is often referred to as "palmitoylation" but, although palmitate is a major fatty acid incorporated into S-acylated proteins, quantitative methods have revealed that there can be considerable heterogeneity in the fatty acid profiles of S-acylated proteins. Furthermore, the chain length and degree of unsaturation of fatty acids is likely to have a major effect on the dynamics and functional activity of S-acylated proteins.

The aim of this project grant is to promote a major advance in our current understanding of how fatty acid heterogeneity in S-acylated proteins is generated and the functional consequences of this. To do this, we will employ a chemical-biology approach using a range of chemically synthesised azido and alkynyl fatty acids to define the fatty acid specificity of individual DHHC enzymes. Furthermore, we will utilise these fatty acid derivatives to determine how fatty acid specificity is encoded by the amino acid sequence of DHHC enzymes. Finally, we will investigate the effects that different fatty acids have on the trafficking and function of S-acylated proteins. Collectively, this research will promote a step change in our understanding of these poorly defined aspects of S-acylation and in the longer-term it may offer new opportunities for the development of selective modulators of DHHC enzymes.

We envisage that the results of this project grant will not only benefit academics but will also impact the commercial and healthcare sectors and the general public. By identifying differences in fatty acid specificity within the DHHC family, we will highlight potential routes for the selective inhibition/modulation of individual DHHC enzymes by targeting the active site. As S-acylation and DHHC enzymes are potential novel drug targets for a range of important disorders, this is likely to attract interest and potentially investment from pharmaceutical companies. The results are also likely to be of interest to clinicians studying the disorders linked with DHHC enzyme dysfunction. The time-scale for these impacts is short-to-medium term (possibly within the time frame of the grant).

Other beneficiaries of the research will be the general public. A longer term impact (10-15 years) may be on patients suffering from disorders associated with dysfunctional S-acylation if the results of the study open new avenues for selective modulation of DHHC enzymes. A shorter-term impact (during the grant) will be on local schoolchildren- we play a prominent role interacting with schoolchildren by accepting pupils on work experience placements and participating in university open days, science fairs and school visits. This will hopefully allow us to inspire a future generation of biomedical scientists. In relation to the current project, we think there is a great opportunity to highlight the importance of other scientific disciplines (e.g. chemistry) in advancing our understanding of biomedical science. Another societal impact of the research will be the high-quality training that the post-doctoral research assistants will receive, enhancing the skills base of the UK and our global competitiveness.