Elvin, Mark, Hengartner, Michael, Poulin, Gino, Roschitzki, Bernd, Schrimpf, Sabine, Kammenga, Jan, Kamkina, Polina, Snoek, L. Basten, Rodriguez, Miriam, Singh, Kapil Dev
[
International Worm Meeting,
2013]
Cancer, neurodegenerative diseases and autoimmune diseases are complex, polygenic pathologies caused by a combination of genetic and environmental factors, including lifestyle. Many of the signaling pathways (i.e. Apoptosis, Notch, MAPK and Wnt) involved in these diseases are conserved and also present in C. elegans. We are studying the influence of natural genetic variation on the abundance of proteins participating in these four signaling pathways in C. elegans. From the two genetically divergent wild-type strains Bristol N2 and Hawaii CB4856, 200 recombinant inbred lines were previously generated. Transcriptome analysis of these RILs showed significant heritable variation in gene expression at the mRNA levels, but very little is known about heritable variation at the protein level. To determine the effect of natural variation on protein abundance, we developed SRM assays for 156 proteins from the above mentioned signaling pathways. SRM measurements were acquired on a TSQ Vantage for two biological replicates of N2 and CB4856 and for one sample of four RILs (WN 31, WN 71, WN 105 and WN 186) at developmental stage L4. So far, by an automated data analysis using mProphet, 45 proteins (mostly represented by 1 or 2 peptides) could be quantified in all samples. Proteins with significant differences in abundance will be further confirmed in biological replicates of the RILs and QTL analysis will be done to determine the genetic locus (loci) responsible for the difference in protein abundance. Finally, these modifier loci will be crossed into sensitized genetic backgrounds to determine their potential to influence developmental signaling. This work is supported by the EU-FP7 HEALTH project PANACEA, contract nr. 222936.
[
East Coast Worm Meeting,
2000]
The emerging field of functional genomics aims at characterizing the function of large numbers of unannotated ORFs predicted from nearly complete genome sequences by developing standardized functional assays that can be applied in high-throughput settings. Mostly gene-based functional genomics approaches have been developed so far. These typically include large-scale gene knock-outs and microarray or chip analysis. However, it is also important to develop protein-based approaches such as protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Most proteins require physical interactions with other molecules to function appropriately. Therefore, it should be informative to identify potential interaction partners for large numbers of predicted gene-products. We have started such a protein interaction mapping project in C. elegans, the first multicellular organism for which a nearly complete genome sequence has been available. As a standardized functional assay, we are using a semi-automated version of the yeast two-hybrid system that we developed. Using proteins kown to be involved in vulval development, we have shown that ~50% of previously reported interactions can be detected in our two-hybrid assay, indicating that our current version of the system should allow a reasonable first coverage of protein-protein interactions. In addition, we have identified ~150 new potential protein-protein interactions which together provide a functional annotation for approximately 100 uncharacterized gene products. Several interactions seem to cluster in closed loops (X/Y/Z/Wn/X), indicating that they might belong to either macromolecular complexes. Finally ~10% of interactions corresponded to "interologs", which we define as protein-protein interactions conserved between model organisms. We have made our data available through a website on the Internet and in a version of ACeDB. We will present the data obtained with vulval proteins and data we recently obtained on other systems.