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New chemical biology tools for proteome-wide of profiling of protein-protein interaction partners of post-translational arginine methylation (ProbesPTRM)

Final Report Summary - PROBESPTRM (New chemical biology tools for proteome-wide of profiling of protein-protein interaction partners of post-translational arginine methylation (ProbesPTRM))

The Marie Curie IIF project “ProbesPTRM” led by Dr Karunakaran A. Kalesh (Fellow) and Dr Edward W. Tate (Scientist in Charge) aimed to develop novel chemical tools and technologies that enable profiling of interaction partners of key posttranslational protein modifications (PTMs). As present knowledge of interaction partners of many PTMs are very limited, highly innovative chemical biology methods have been developed and applied in this project in order to describe for the first time the global interactome profiling of important PTMs in human cancer cell lines. The proposed objectives were design and synthesis of photo-cross-linking peptide-based probes specific for PTMs and validation of the methodology using purified proteins, cell lysates and representative cancer cell lines. Consistent with the project proposal, synthesis of peptide probes carrying various synthesised unnatural amino acids, photo cross-linkers and bio-orthogonal moieties were optimised and the probes were successfully applied in protein labelling experiments. The peptide probes developed in this project represent the first of its kind in selectively targeting posttranslational modification-hydrolysing enzymes (termed PTM-erasers). Following extensive optimisations at the chemical synthesis stages, peptide probe libraries were generated to target key PTMs such as protein arginine methylation and protein lysine side-chain succinylation in histone- and non-histone proteins. Corresponding control probes that lack either the suitable PTM recognition entities or the bioorthogonal tagging moieties were also synthesised. The probes and control probes were subsequently evaluated by in-gel fluorescence scanning for protein labelling performance using purified proteins and the probes that showed the best labelling performance at this stage were chosen for further in-depth studies. After several rounds of extensive labelling validation experiments, a probe derived from peptide sequence of the mitochondrial protein glutamate dehydrogenase (GDH) was found to be one of the best, and has served as a prototype system for the further research developments in the project. The probes were then applied to HeLa and MCF-7 whole cells lysates. The labelled proteome was visualised by in-gel fluorescence as in the case of proof-of-concept experiments. Since non-specific labelling frequently occurs in proteome-wide photo-labelling experiments, SILAC-based quantitative mass spectrometry was extensively employed for high-confidence, unbiased, identification and characterisation of labelled proteome. These experimental results represent an important accomplishment beyond the original proposed objectives of the project. The studies provided the first global profiling of the interaction partners of posttranslational lysine side-chain succinylation. Biochemical experiments are currently being carried out for additional target validation. Lysate-based labelling results demonstrating the first selective detection of the NAD-dependent desuccinylating enzyme sirtuin 5, excluding the extensive proteome profiling has been already published (K. A. Kalesh, E. W. Tate. Org. Biomol. Chem. 2014, 12, 4310-4313). The methodologies developed within the “ProbesPTRM” project are universal, and may be applied for the profiling of readers, writers and erasers for many different types of PTMs. Indeed, within the host group, the technique is currently being applied for the profiling of several different disease-relevant PTMs.
Due to its interdisciplinary nature, the project “ProbesPTRM” has contributed to European excellence and competitiveness in many scientific disciplines such as synthetic chemistry, analytical chemistry, biochemistry, and cell biology. Furthermore, since the project relates to application of chemical methods in the profiling of fundamentally important posttranslational protein modifications, its findings are expected to immediately aid the research of biochemists and biologists, and ultimately society as a whole will benefit. As sirtuin enzymes selectively targeted by some of the probes developed in this project are directly linked to aging and many human diseases, the findings of the project will have an impact on drug development research as well. As a result of the current global drug development crisis, major drug companies are interested in the integration of conventional drug discovery methods with state-of-the art chemical biology platforms such as the chemical probe tools and PTM profiling technologies developed in this project. In conclusion, chemical proteomic tools reveal details of cellular activities on an unprecedented scale, and further developments in this emerging field should be considered for immediate attention from the EU.