Final Report Summary - CHEMPROT-HLMS (Chemical proteomics for universal profiling of histone lysine malonylation and succinylation.)
The project objectives were: 1. Profile the global changes of the post-myristoylated proteome during apoptosis by a novel and robust chemistry-based approach which combines quantitative chemical proteomics with a potent and specific human NMT inhibitor. 2. Identify NMT1 and NMT2 specific substrates in human cells during normal cell function and during apoptosis by exploiting the same chemistry-based approach in combination with isozyme-specific knockdown. 3. Develop and apply novel chemical probes and technologies that will enable for the first time high-throughput analysis of the complex biological networks involved in protein prenylation. Consistent with the project objective 1 several death stimulus and different cell lines were tested. Several rounds of optimization experiments were undertaken to optimize feeding concentration of death stimulus and timeframe by using western blots against apoptosis markers as a rapid readout of the process. As a result, BL41 and Jurkat T cells were selected and have been subjected to chemical proteomics using YnMyr probe (alkyne analogue of myristic acid) to identify N-myristoylated proteins. Experiments were undertaken in this stage to optimize feeding concentration and timeframe for the myristic acid analogue, and to fine-tune the bioorthogonal ligation step, using fluorescence as rapid and high throughput readout. A substantial change in the fluorescent band pattern was observed for apoptotic cells, implying a marked change in de novo myristoylation activity. The fellow has carried out large scale quantitative proteomic experiments. Analysis of the proteomics data has revealed significant differences between the apoptotic myristoylated proteomes in different cell lines. By exploting the same chemistry-based approach in combination with isozyme-specific knockdown cells the specific roles and substrates of each NMT have been studied. The outcomes of these large scale proteomics experiments are currently in final stages of validation.
Novel and improved probes for the study of prenylation in live cells has been developed. By means of these new prenyl probes it has been possible to identify novel prenylated proteins, and in combination with quantitative proteomics approaches the fellow has quantified the response, in cell, of individual proteins to inhibition of the different prenyl transferases, demonstrating the complex dynamics and interplay between the different isoprenoid substrates and transferases. This robust methodology is widely applicable to the study of prenylation and to quantify prenylation in response to treatment, disease or other stimuli. In fact, the probe developed for geranylgeranylation was further applied to determine the molecular basis of choroideremia (genetic disease caused by the loss of REP-1 activity). In the initial series of experiments conducted by the fellow, probe labelling conditions were optimised for the cells CHM+/+ and CHM-/- MEF cells (Seabra’s mouse models of choroideremia). With labelling conditions optimised, two large scale proteomic experiments (with and without fractionation) were carried out (to determine how the presence or absence of REP-1 affects Rab geranylgeranylation). The results in both experiments were comparable and showed that some Rab proteins are significantly underprenylated in the choroideremia cell line providing new insights into the choroideremia disease.
In addition, novel functionalised enzymatically-cleavable solid-supported reagents that facilitate analysis of protein posttranslational modifications in large scale proteomics experiments were also developed and these reagents are currently under detailed investigation to study secreted proteins.
Due to its interdisciplinary nature, the project 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 the application of chemical methods in the profiling of fundamentally important posttranslational protein modifications, its finding are expected to immediately aid the research of biochemists and biologists, and ultimately society as a whole will benefit as well. As a result of the current global drug development crisis, major drug companies are interested in the integration of drug discovery with systems biology, particularly the development of innovative new methods to profile the effects of drug candidates across global biological networks. Such methods were developed in this project. In conclusion, chemical proteomic tools reveal details of human biology on an unprecedented scale, and further developments in this emerging field should be considered for immediate attention from the EU. Currently the US leads the world in the fields of chemical biology and disease biomarker development, and it is particularly important that the European Community benefits from the outcomes of projects that deliver alternative, more comprehensive methodologies for screening and validation of novel drug targets.