The Quantitative Biotinylproteomics Studies Reveal a WInd-Related Kinase 1 (Raf-Like Kinase 36) Functioning as an Early Signaling Component in Wind-Induced Thigmomorphogenesis and Gravitropism.

Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce these versatile wind force signals downstream to nuclear events and to the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components at the early steps of the wind force signaling, two mechanical signaling-related phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based quantitative proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the genetically engineered TurboID biotin ligase expression transgenics as a universal control. This unique PTM proteomics successfully identified 11 and 71 MKK1 and MKK2 putative interactors, respectively. Biotin occupancy ratio (BOR) was found to be an alternative parameter to measure the extent of proximity and specificity between the proximal target proteins and the bait fusion protein. Bioinformatics analysis of these biotinylprotein data also found that TurboID biotin ligase favorably labels the loop region of target proteins. A WInd-Related Kinase 1 (WIRK1), previously known as rapidly accelerated fibrosarcoma (Raf)-like kinase 36 (RAF36), was found to be a putative common interactor for both MKK1 and MKK2 and preferentially interacts with MKK2. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the touch-responsive TCH3 and CML38 gene expression and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1 (raf36) mutant revealed that the WIRK1 gene is involved in both wind-triggered rosette thigmomorphogenesis and gravitropism of Arabidopsis stems, suggesting that the WIRK1 (RAF36) protein probably functioning upstream of both MKK1 and MKK2 and that it may serve as the crosstalk point among multiple mechano-signal transduction pathways mediating both wind mechano-response and gravitropism.

ECL 3.0: a sensitive peptide identification tool for cross-linking mass spectrometry data analysis.

BACKGROUND: Cross-linking mass spectrometry (XL-MS) is a powerful technique for detecting protein-protein interactions (PPIs) and modeling protein structures in a high-throughput manner. In XL-MS experiments, proteins are cross-linked by a chemical reagent (namely cross-linker), fragmented, and then fed into a tandem mass spectrum (MS/MS). Cross-linkers are either cleavable or non-cleavable, and each type requires distinct data analysis tools. However, both types of cross-linkers suffer from imbalanced fragmentation efficiency, resulting in a large number of unidentifiable spectra that hinder the discovery of PPIs and protein conformations. To address this challenge, researchers have sought to improve the sensitivity of XL-MS through invention of novel cross-linking reagents, optimization of sample preparation protocols, and development of data analysis algorithms. One promising approach to developing new data analysis methods is to apply a protein feedback mechanism in the analysis. It has significantly improved the sensitivity of analysis methods in the cleavable cross-linking data. The application of the protein feedback mechanism to the analysis of non-cleavable cross-linking data is expected to have an even greater impact because the majority of XL-MS experiments currently employs non-cleavable cross-linkers. RESULTS: In this study, we applied the protein feedback mechanism to the analysis of both non-cleavable and cleavable cross-linking data and observed a substantial improvement in cross-link spectrum matches (CSMs) compared to conventional methods. Furthermore, we developed a new software program, ECL 3.0, that integrates two algorithms and includes a user-friendly graphical interface to facilitate wider applications of this new program. CONCLUSIONS: ECL 3.0 source code is available at https://github.com/yuweichuan/ECL-PF.git . A quick tutorial is available at https://youtu.be/PpZgbi8V2xI .

Capturing the hierarchically assorted modules of protein-protein interactions in the organized nucleome.

Nuclear proteins are major constituents and key regulators of nucleome topological organization and manipulators of nuclear events. To decipher the global connectivity of nuclear proteins and the hierarchically organized modules of their interactions, we conducted two rounds of cross-linking mass spectrometry (XL-MS) analysis, one of which followed a quantitative double chemical cross-linking mass spectrometry (in vivoqXL-MS) workflow, and identified 24,140 unique crosslinks in total from the nuclei of soybean seedlings. This in vivo quantitative interactomics enabled the identification of 5340 crosslinks that can be converted into 1297 nuclear protein-protein interactions (PPIs), 1220 (94%) of which were non-confirmative (or novel) nuclear PPIs compared with those in repositories. There were 250 and 26 novel interactors of histones and the nucleolar box C/D small nucleolar ribonucleoprotein complex, respectively. Modulomic analysis of orthologous Arabidopsis PPIs produced 27 and 24 master nuclear PPI modules (NPIMs) that contain the condensate-forming protein(s) and the intrinsically disordered region-containing proteins, respectively. These NPIMs successfully captured previously reported nuclear protein complexes and nuclear bodies in the nucleus. Surprisingly, these NPIMs were hierarchically assorted into four higher-order communities in a nucleomic graph, including genome and nucleolus communities. This combinatorial pipeline of 4C quantitative interactomics and PPI network modularization revealed 17 ethylene-specific module variants that participate in a broad range of nuclear events. The pipeline was able to capture both nuclear protein complexes and nuclear bodies, construct the topological architectures of PPI modules and module variants in the nucleome, and probably map the protein compositions of biomolecular condensates.

Exhaustive Cross-Linking Search with Protein Feedback.

Improving the sensitivity of protein-protein interaction detection and protein structure probing is a principal challenge in cross-linking mass spectrometry (XL-MS) data analysis. In this paper, we propose an exhaustive cross-linking search method with protein feedback (ECL-PF) for cleavable XL-MS data analysis. ECL-PF adopts an optimized α/ß mass detection scheme and establishes protein-peptide association during the identification of cross-linked peptides. Existing major scoring functions can all benefit from the ECL-PF workflow to a great extent. In comparisons using synthetic data sets and hybrid simulated data sets, ECL-PF achieved 3-fold higher sensitivity over standard techniques. In experiments using real data sets, it also identified 65.6% more cross-link spectrum matches and 48.7% more unique cross-links.

Isotopically Dimethyl Labeling-Based Quantitative Proteomic Analysis of Phosphoproteomes of Soybean Cultivars.

Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e-0.098X - 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINß1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.