Abstract
The living cell exhibits the emergence of complex behavior, which is controlled by the functional and structural basic units, proteins. Decades of molecular genetic studies have established a nearly complete draft genome map of soybean, which provide extraordinary resources and abilities for proteomics research. The protein post-translational modification (PTM) and protein-protein interactions (PPI) are two important contents of proteomics, which constitute the basic cellular events of a living cell. To have a better understanding of the molecular mechanisms of plant cells in response to stress, we have developed both quantitative PTM proteomics and interactomics workflow to quantitate PTM proteome and interactome of soybean cells under stress conditions.The isotopic dimethyl labeling-based quantitative PTM proteomics was applied to study the phosphoproteomic changes associated with the drought responses of two contrasting soybean cultivars. Ultimately, a total of 9,457 phosphopeptides were repeatably identified, which were corresponding to 3,889 leading phosphoproteins and 8,087 phosphosites. Among them, 6,106 phosphosites were newly identified which constitute 54% of the current soybean phosphosite repository. Bioinformatics analysis of kinases and substrates revealed that there were predicted 6 kinase families that match with the highly conserved kinase docking sites found in this study. The distribution of phosphoproteins over the number of the phosphosite of phosphoprotein follows the exponential decay law, Y = 3.89e−0.08X−0.27. Quantitative proteomics identified 218 significantly regulated unique phosphopeptide groups, corresponding to 188 phosphoproteins that were regulated by the drought-tolerant cultivar under the drought condition, among which there were 17 and 15 protein kinases/phosphatases and transcription factors, respectively. The combination of bioinformatics and quantitative phosphoproteomics revealed a phosphor-relay-mediated signal transduction network that is composed of three members of the calciumdependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25 and GmAKINβ1 kinases. This signaling network possibly contributes to the development of the soybean drought tolerance under the water loss through regulating ion channel activities and many nuclear events in this drought-tolerant cultivar.
The interactome of cell nuclei is deciphered by quantitative chemical-crosslinking mass spec-trometry workflow. The formaldehyde-CBDPS double chemical-crosslinking was combined with mass spectrometry and subsequently applied to the in vivo fixed cell nuclei. Consequently, 5,340 cross-linked (CX) peptides were repeatably identified from nucleome, which were con-strued into 1,297 nuclear PPIs and converted into 256 interactors of histones and 41 interactors of box C/D snoRNP complex. Quantitative interactomics discovered 129 and 217 hormone down-and up-regulated crosslinks, respectively, which further demonstrated the possible dy-namics of structure conformation and interactome of the histone octamer and box C/D snoRNP complex under hormone conditions. The identified crosslinks were validated using structural distance constraints and the PPIs of hnRNPQ-histone H4 and RAE1-NUP98A were alterna-tively confirmed by super-resolution microscopy studies. The up-regulation of crosslinks be-tween hnRNPQ and histone H4 as well as RAE1 and NUP98A was confirmed using the im-munoblot assay. A further functional assay indicated the important role of RAE1 and NUP98A in hormone response. This in vivo qCX-MS-based interactomics demonstrated its great poten-tial in studying both the structure and PPI dynamics under various conditions.
Nuclear proteins are major constituents and key regulators of the topological organization of nucleome. To elucidate the connectivity of nucleomic proteins and decipher the hierarchically assorted modules of proteins associated with nuclear events and nucleome organization, the modulomic analysis were performed on PPIs, resulting into 27 and 24 master nuclear protein interaction modules (NPIMs) that contain the condensate-forming protein(s) and the intrin-sically disordered region (IDR)-containing proteins, respectively. These NPIMs successfully captured the previously reported nuclear protein complexes and nuclear bodies and were hier-archically assorted into four communities including Genome and Nucleolus Community. The qCX-MS-based quantitative interactomics finally revealed 17 Hormone-specific module vari-ants. This CHAMPION pipeline is capable of capturing both nuclear protein complexes and assemblies, constructing the topological architecture of protein interaction modules and mod-ule variants and probably of mapping the protein compositions of condensates.
| Date of Award | 2022 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Ning LI (Supervisor) & Weichuan YU (Supervisor) |
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