Comprehensive Proteomic Analysis Of C. Elegans Insulin Signaling Mutants

In C. elegans, daf-2mutant animals could live twice as long as wild type animals and this longevity phenotype is completely dependent on the transcriptional activity of DAF-16which is the C. elegans homolog of FoxO transcription factors. This implies that aging is under the control of a genetic program. In the past two decades, extensive investigation has been conducted on daf-2mutants, but the global changes in the daf-2proteome have not been revealed fully.In this work, I established a workflow based on stable isotope labeling and quantitative mass spectrometry to investigate C. elegans proteomes. Sample preparation, offline pre-fractionation, liquid chromatography and mass spectrometry methods were all optimized for C. elegans proteomics samples. A new and more reliable quantitation software tool was also incorporated into this workflow and the matching data post-processing parameters, including data filtering, statistical analysis and differential expression detection, were optimized. This workflow exhibits excellent technical and biological reproducibility. Using this workflow, I obtained what seemed to be highly reliable quantitation results with a great coverage of the worm proteome of the wild type and three insulin signaling mutants (daf-2, daf-2; daf-16and daf-16). In each of the three biological repeats, more than6000proteins were reliably quantified. To our knowledge, this breaks all previous records. The quantification results in this study are highly consistent with previously published results and contain much information never seen before. Moreover, differentially expressed proteins in the daf-2mutant are enriched for known lifespan-regulating genes. This helps to explain why daf-2mutants are long-lived and also implies that more aging-related genes may be discovered through comprehensive proteome analysis.The role of DAF-16in establishing the daf-2proteome was evaluated by exploiting this data set. Only seventy percent of the differentially expressed proteins in the daf-2mutant are dependent on the DAF-16activity. Consistently, promoter analysis indicates that although the DAF-16binding element is not overrepresented in the promoter regions of the up-regulated proteins in the daf-2mutant, it is overrepresented within the subgroup whose up-regulation depends on DAF-16. Interestingly, the DAF-16binding element is not overrepresented in the promoter regions of down-regulated proteins in the daf-2mutant, whether their down-regulation depends on DAF-16or not. The results above imply that mechanisms independent of DAF-16or indirectly controlled by DAF-16transcriptional activity are at work in establishing the daf-2proteome.By comparing the proteomics data and transcriptomics data, I find that the overall correlation between mRNAs and proteins is poor, with a Spearman correlation coefficient of only0.38. A large part of the protein level changes cannot be explained by the corresponding mRNA level changes. This indicates that post-transcriptional mechanisms play a role in establishing the daf-2proteome. Further analysis reveals that down-regulated proteins that are not accompanied by a decrease in their mRNA templates are encoded by genes that have longer3’UTR sequences with lower folding energies (easier to form secondary structures) than genes whose expression is down-regulated at both the mRNA and the protein levels. This suggests that post-transcriptional activity, especially that of microRNAs, may play an important role in the daf-2mutant.Functional analysis of differentially expressed proteins in the daf-2mutant reveals a broad spectrum of changes in the daf-2proteome. Proteins involved in glycolysis, fatty acid degradation and amino acid degradation are all up-regulated, suggestive of a fully enhanced catabolic activity for small-molecule metabolites. On the other hand, proteins involved in RNA transcription and protein translation are down-regulated, suggesting that anabolic activity is suppressed in coordination for RNAs and proteins. In support of this idea, I find that interrupting the ribosomal RNA maturation process further extends the lifespan of the daf-2mutant, probably by further inhibiting protein translation.Through network analysis I have discovered that differentially expressed proteins in the daf-2mutant possess a more central role in the C. elegans protein-protein interaction network. These proteins tend to have higher degree and betweenness. Their neighbors also tend to have higher degree. They are much closer to other proteins in the network. I extracted the sub-network consisting of only the differentially expressed proteins and the shortest paths connecting them. Further, I decomposed the sub-network into several communities. Functional analysis is performed on the major communities and important interactions connecting different communities are identified. This may shed light on the organization of the different processes regulated by DAF-2.