Mapping the acylated proteome: a chemical genetic approach

With the completion of several important genome projects, such as the Human Genome Project, we now have the basic code describing the proteins made by cells which carry out the essential processes of life. However, this is only the beginning of our journey towards fully understanding how cells function: the focus of research has now moved to unravelling the role of each protein in a cell, and when and where it is carried out. To further complicate the story, the structure of a protein is frequently modified after its initial creation (its 'translation' from the genetic code) by other proteins called enzymes, a process termed 'post-translational modification'. These modifications can have critically important functions in the cell; for example, they are involved in our immune response to disease and the mechanism of infection by viruses, and defective modification is implicated in diseases such as cancer. Drugs which target the post-translational enzymes in pathogenic organisms such as fungi and parasites (for example, Plasmodium falciparum, the single-celled parasite which causes malaria) are under development, and could make a major impact on our ability to control these diseases. However, due to the complexity of the total set of thousands of different proteins in a cell (dubbed the 'proteome'), it has proven difficult to identify which proteins are modified, and what effect the modification has on a protein's function. Identifying the modified proteome is a key step towards understanding the role of post-translational modification, and to discover what effects (and side-effects) the drugs mentioned above may have in the cell. The proposed research aims to address this challenge by integrating recent discoveries in organic chemistry with cutting-edge whole-proteome analysis. We aim to exploit the cell's own enzymes to introduce a chemical 'tag' into proteins which are modified by a process termed 'acylation'. The tags are designed so as not to disrupt the cell's normal function, and we can very selectively 'capture' and purify the tagged acyl proteins with specially designed reagents. This will allow us to hugely increase the sensitivity of whole-proteome analysis towards acyl modifications, revealing hitherto unidentified acyl proteins and opening up new lines of investigation into the role of acylation in cells. Later, we intend to exploit this tag and capture methodology to analyse the effect of drugs on the modified proteome in single-celled parasites, and even to enable us to see modified proteins under a microscope as they move around the cell. This work will bring together chemists at Imperial College London and biologists at the University of York in an 'interdisciplinary' collaboration / that is, at the interface between the traditional disciplines of Chemistry and Biology.

Co- or post-translational acylation of proteins is known to modulate myriad cellular processes including protein trafficking and the immune response, and the enzymes which perform post-translational acylation (acyl:protein transferases) play a key role in trafficking proteins between the membrane-delimited compartments of the cell. There is a pressing need to define the complete repertoire of myristoylated and palmitoylated proteins, respectively dubbed the 'myristome' and 'palmitome', and a generic method for their identification would enable and accelerate investigations into the functional biology of protein acylation. Tagging-by-substrate (TbS) is a powerful emerging technology that has the potential to overcome the problems commonly encountered in high-throughput proteomics of post-translationally modified proteins. In this approach, a synthetic transferase substrate bearing a biologically inert chemical tag (usually an azide or alkyne) is fed to cells, and incorporated into modified proteins metabolically in vivo. Tagged proteins are then captured from lysed cells in vitro using a highly selective bioorthogonal reaction, and by incorporating a dye or affinity label into the capture reagent the modified proteome can be selectively visualised or enriched, greatly enhancing high-throughput identification. This radically new approach to post-translational proteomics has already seen notable success for the labelling of certain glycosylated and farnesylated proteins in vivo. We intend to develop TbS for high-throughput myristomics and palmitomics, to provide a general cross-species method to enrich, visualise and identify acylated proteins in a wild-type cell-line. This work will generate invaluable data about the targets of acylation in vivo, present new targets for functional biology studies and greatly enhance the prediction of the myristome from genomic data by elucidating the sequence specificity of myristoylation.