Final Report Summary - TAPMIN (High throughput analysis of the in vivo metabolite/protein interactome in plants.)
PROJECT SUMMARY
Metabolites constitute one of the major components of cells in abundance and diversity and they play important roles in mediating protein regulation and function (Alberts, 2002). During the last decades, the scientific community focused on large scale studies on different model organisms on protein-protein interaction, protein folding, gene expression and transcription factor binding to DNA. However much less effort has been devoted to understand how small metabolites interact and regulate protein function at a systems level. Metabolites are present in cells in a broad range of cellular concentrations and participate in a wide variety of biochemical and regulatory functions, for instance as ligands for receptors and other signalling proteins, and as substrates, products, cofactors and allosteric regulators of enzymes. Moreover, many plant pathogens (bacterial and fungal species) and insects (gut compounds) are also able to produce small metabolites (effectors) that specifically hijack plant hormone–regulated signalling pathways (Christensen and Lolomiets, 2011; Gimenez-Ibanez and Solano, 2013, Fonseca et al., 2014, Rigal et al., 2014). In yeast, some studies have been carried out to identify small molecule-protein interactions; mainly using in vitro assays based on microarray and mass spectrometry technologies (Li et al., 2010; Yang et al., 2012, Maeda et al., 2014). However, no such methodology has been developed for plant proteins so far and hence systematic screens for in vivo metabolite/protein interactions in plants have not been undertaken yet.
The Tapping for Protein-Metabolite Interactions, “TAPMIN”, was a two-years project to fill this knowledge gap that aimed to discover new functional metabolites in the model plant Arabidopsis thaliana by establishing a performing high-throughput method to detect novel in vivo metabolite/protein interactions. This new protocol and the data provide for TAPMIN experiments will be valuable for i) elucidating the biochemical activities and regulation of individual plant proteins, ii) assembling and understanding regulatory networks and connections between plant biological pathways and iii) identify new plant molecules of potential use for crop improvement and pharmaceutical industries expanding the importance of plants in human nutrition and health.
PROJECT OBJECTIVES, RESULTS, CONCLUSIONS AND IMPACTS
The first objective of TAPMIN proposal, 1.Establish a high throughput method to study the in vivo metabolite/protein interactome in the model plant Arabidopsis thaliana was accomplished during the first half period of the project. Using squalene epoxidase 1 (SQE1) as our proof of concept bait we were able to fished out squalene, the substrate of this protein. Similarly, when we treated SQE1 and control (GUS expressing) samples with Terbinafine (Tb), the specific inhibitor of SQE1 enzymatic activity, we only recovered this compound in SQE1 but not in control samples. Moreover, when we applied TAPMIN protocol to another bait, i.e. membrane steroid binding protein 1 (MSBP1) we found brassinolide (BL) attached to this bait as was previously reported in vitro. All together these results show that TAPMIN protocol was successfully established being a novel tool to study metabolite/protein interactomes.
Our experiments with SQE1 also addressed the general goal of our second objective, 2. Identification and characterization of new metabolite/protein interactomes. In addition to squalene and Tb, we also found an enrichment in end-products of SQE1 pathway in SQE1 samples when comparing with control. ß-sitosterol and campesterol attach to SQE1 but their function as putative regulators of this protein is still unknown. We aim now to study the physiological relevance of the accumulation of end-products and their interaction with SQE1.
Additionally, we also launched a pilot screening in collaboration with VIB-Compound Screening Facility (CSF) to find new putative regulators of SQE1 addressing here the major aim of the third objective of the project, 3. Screens for novel in vivo metabolite/proteins interactions.
Finally and pending in the outcome of our last TAPMIN experiment with two putative progesterone reductases (VEP1 and VEP2) and thalianol synthase 1 (THAS1) of Arabidopsis, we will start writing a paper to make TAPMIN available for all the scientific community and to report our new findings.
Remarkably and due to the importance of developing such a fishing method for metabolites, the Flemish Foundation for Research (Fonds Wetenschappelijk Onderzoek-FWO) considered appropriate to extend the funding of TAPMIN project for an extra period of three years to further develop and exploit TAPMIN protocol. Support and funding from IEF-Marie Curie project was essential to translate TAPMIN idea in a real project, to accomplish the majority of the objectives included in the proposal and to convince funding agencies (i.e. FWO) to further support TAPMIN project. Moreover, the IEF-MC fellow had the amazing opportunity to develop a new technology in a European leading institution in plant science creating as well the foundations of her future career as an independent researcher.
Metabolites constitute one of the major components of cells in abundance and diversity and they play important roles in mediating protein regulation and function (Alberts, 2002). During the last decades, the scientific community focused on large scale studies on different model organisms on protein-protein interaction, protein folding, gene expression and transcription factor binding to DNA. However much less effort has been devoted to understand how small metabolites interact and regulate protein function at a systems level. Metabolites are present in cells in a broad range of cellular concentrations and participate in a wide variety of biochemical and regulatory functions, for instance as ligands for receptors and other signalling proteins, and as substrates, products, cofactors and allosteric regulators of enzymes. Moreover, many plant pathogens (bacterial and fungal species) and insects (gut compounds) are also able to produce small metabolites (effectors) that specifically hijack plant hormone–regulated signalling pathways (Christensen and Lolomiets, 2011; Gimenez-Ibanez and Solano, 2013, Fonseca et al., 2014, Rigal et al., 2014). In yeast, some studies have been carried out to identify small molecule-protein interactions; mainly using in vitro assays based on microarray and mass spectrometry technologies (Li et al., 2010; Yang et al., 2012, Maeda et al., 2014). However, no such methodology has been developed for plant proteins so far and hence systematic screens for in vivo metabolite/protein interactions in plants have not been undertaken yet.
The Tapping for Protein-Metabolite Interactions, “TAPMIN”, was a two-years project to fill this knowledge gap that aimed to discover new functional metabolites in the model plant Arabidopsis thaliana by establishing a performing high-throughput method to detect novel in vivo metabolite/protein interactions. This new protocol and the data provide for TAPMIN experiments will be valuable for i) elucidating the biochemical activities and regulation of individual plant proteins, ii) assembling and understanding regulatory networks and connections between plant biological pathways and iii) identify new plant molecules of potential use for crop improvement and pharmaceutical industries expanding the importance of plants in human nutrition and health.
PROJECT OBJECTIVES, RESULTS, CONCLUSIONS AND IMPACTS
The first objective of TAPMIN proposal, 1.Establish a high throughput method to study the in vivo metabolite/protein interactome in the model plant Arabidopsis thaliana was accomplished during the first half period of the project. Using squalene epoxidase 1 (SQE1) as our proof of concept bait we were able to fished out squalene, the substrate of this protein. Similarly, when we treated SQE1 and control (GUS expressing) samples with Terbinafine (Tb), the specific inhibitor of SQE1 enzymatic activity, we only recovered this compound in SQE1 but not in control samples. Moreover, when we applied TAPMIN protocol to another bait, i.e. membrane steroid binding protein 1 (MSBP1) we found brassinolide (BL) attached to this bait as was previously reported in vitro. All together these results show that TAPMIN protocol was successfully established being a novel tool to study metabolite/protein interactomes.
Our experiments with SQE1 also addressed the general goal of our second objective, 2. Identification and characterization of new metabolite/protein interactomes. In addition to squalene and Tb, we also found an enrichment in end-products of SQE1 pathway in SQE1 samples when comparing with control. ß-sitosterol and campesterol attach to SQE1 but their function as putative regulators of this protein is still unknown. We aim now to study the physiological relevance of the accumulation of end-products and their interaction with SQE1.
Additionally, we also launched a pilot screening in collaboration with VIB-Compound Screening Facility (CSF) to find new putative regulators of SQE1 addressing here the major aim of the third objective of the project, 3. Screens for novel in vivo metabolite/proteins interactions.
Finally and pending in the outcome of our last TAPMIN experiment with two putative progesterone reductases (VEP1 and VEP2) and thalianol synthase 1 (THAS1) of Arabidopsis, we will start writing a paper to make TAPMIN available for all the scientific community and to report our new findings.
Remarkably and due to the importance of developing such a fishing method for metabolites, the Flemish Foundation for Research (Fonds Wetenschappelijk Onderzoek-FWO) considered appropriate to extend the funding of TAPMIN project for an extra period of three years to further develop and exploit TAPMIN protocol. Support and funding from IEF-Marie Curie project was essential to translate TAPMIN idea in a real project, to accomplish the majority of the objectives included in the proposal and to convince funding agencies (i.e. FWO) to further support TAPMIN project. Moreover, the IEF-MC fellow had the amazing opportunity to develop a new technology in a European leading institution in plant science creating as well the foundations of her future career as an independent researcher.