Prof. Andrew Simkin

In plants, terpenoids (or isoprenoids) are synthesized by two different pathways: the cytosolic mevalonate (MVA) pathway and the plastidial 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. The particularity of plants to produce isoprenoids via these two pathways, prompts us to address the question of how they might interact and what mechanisms might be involved in coarse control and even fine-tuning of one or both pathways, depending on the structure and functional destination of terpenoid derivatives.

Previous studies performed by our group suggest that prenylated proteins exert specific functions in this problematic. The observations made on monoterpene indole alkaloid biosynthesis regulation in periwinkle (Catharanthus roseus) cell suspensions suggest that one or more prenylated proteins can exert a positive control on the MEP pathway. This view not only proposes an original role of prenylated proteins as a regulator of a biosynthetic pathway, but also raises very important questions as to the relation between MVA and MEP pathways. To shed more light on this question it will be necessary to identify the proteins targeted for prenylation (defined as the prenylome).

The objective of this project is to develop methods for proteomic profiling of periwinkle prenylated proteins. Identification of the prenylome will be investigated by a strategy based on activity-based protein profiling (ABPP). It will be necessary to synthesize the activity-based probes and the reporter tag that will be used to develop the ABPP method. Moreover, experiments using definite targets of prenylation (dansylated peptides and recombinant proteins) will be performed (i) to test if the synthetic activity-based probes are efficient substrates for protein prenylation in vitro (ii) to test the efficiency of the reporter tag for affinity purification of the modified prenylated proteins and (iii) to analyse these proteins by tandem mass spectrometry.

The availability of the specific proteomic probes and the data from the set of experiments will provide essential skills and knowledge for future studies on the identification of the periwinkle prenylome.