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Modulating and Profiling Receptor Tyrosine Kinase S-palmitoylation in Breast Cancer

Periodic Reporting for period 1 - RTKPalm (Modulating and Profiling Receptor Tyrosine Kinase S-palmitoylation in Breast Cancer)

Reporting period: 2017-08-01 to 2019-07-31

Breast cancer is one of the most diagnosed cancers and the leading cause of cancer death in women worldwide. Breast cancer cells need activated receptor tyrosine kinases (RTKs) to invade, proliferate, and metastasize. Increased activity and overexpression of RTKs is associated with poor prognosis for breast cancer patients. Therefore, multiple RTKs have emerged as attractive therapeutic targets. However, resistance to therapies is a persistent problem for the development of therapeutics targeting RTKs. New and innovative approaches to effectively target these receptors are required. Recently S-palmitoylation has been identified as an important post-translational modification that regulates signal transduction, protein trafficking and degradation of specific RTKs in breast cancer. The substrate scope, role, extent of dysregulation and the enzymes responsible for the S-palmitoylation of specific substrates in breast cancer is largely unknown. This is partially caused by a lack of chemical biological methods to study these processes. Therefore we have worked towards the development of two novel methodologies to study protein S-palmitoylation; 1) Development of a methodology to study the extent of palmitoylation on substrate proteins. 2) Development of a methodology for the identification of direct substrates for the enzymes that attach these lipids to proteins (palmitoyl transferases).
A methodology to determine the site specific palmitoylation stoichiometry will be a important tool for the analysis of drugs that prevent palmitoylation or depalmitoylation. To this aim, we have synthesized a cysteine reactive tool that will facilitate the study of site specific palmitoylation stoichiometry by mass-spectrometry. The research fellow is currently still working on the further development of this methodology at Imperial College London.
To elucidate the substrate scope of individual palmitoyl transferases (zDHHCs), we have succesfully developed a chemical genetic methodology that enables zDHHC specific lipid transfer. Combination of this methodology with mass spectrometry-based proteomics provides a platform to aid identification of palmitoyl transferase substrates and may assist zDHHC characterisation breast cancer.
We have successfully optimized a metabolic labelling strategy that not only enables the detection of palmitoylated proteins in cancer cells, but also allows identification of the specific sites on proteins that are palmitoylated. To complement this metabolic labelling strategy, we have synthesized a tool molecule that can corroborate the identified sites using an orthogonal methodology. This tool does not only identify the sites, but will likely also enable analysis of site specific stoichiometry of S-palmitoylation.
To elucidate the substrate scope of individual palmitoyl transferases (zDHHCs), we used a highly multidisciplinary strategy that combines synthetic chemistry, molecular biology, genetics and mass spectrometry. We have developed a chemical genetic methodology that enables zDHHC specific lipid transfer. Coupling this methodology with mass spectrometry-based proteomics provides a platform to aid identification of palmitoyl transferase substrates and may assist zDHHC characterisation breast cancer. Using this methodology we have identified 35 putative palmitoylated proteins of the palmitoyl transferase zDHHC20. We anticipate that this methodology can be expanded to other members of the palmitoyl transferase family.
Global identification of substrates for specific palmitoyl transferases (PATs) has remained an unresolved challenge. Currently available methodologies to identify PAT substrates have severe limitations because of 1) complexity of the S-palmitoylated proteome; 2) zDHHC isoform co-regulation and compensation (redundancy in palmitoyl transferase activity). The recent publication of the crystal structure of zDHHC20 is the first of any of the 23 palmitoyl transferases. This structural insight allowed the design of novel palmitate-based ‘bumped’ probes that are complementary to mutant zDHHC20 to allow specific detection of zDHHC20 substrates by live cell metabolic labelling. Bioorthogonal ligation handles on the bumped probes allows to determine the full spectrum of zDHHC20 specific substrates on gel and by proteomics. This breakthrough methodology will allow for the first time to identify the substrates of a specific palmitoyl transferase in life cells and revolutionizes the identification of palmitoyl transferase substrates. Palmitoyl transferases are associated with multiple cancer types and have potential as a therapeutic target for cancer treatment. This methodology will enable the characterization of these enzymes in cancer cells and will help to characterize small molecule drugs that modulate palmitoyl transferase activity in complex cellular systems.
Overview of the palmitoylation regulatory machinery