Strain visualization using large-angle convergent-beam electron diffraction.

In this study, we report a strain visualization method using large-angle convergent-beam electron diffraction (LACBED).1 We compare the proposed method with the strain maps acquired via STEM-NBD, a combination of scanning transmission electron microscopy (STEM) and nanobeam electron diffraction (NBD). Although STEM-NBD can precisely measure the lattice parameters, it requires a large amount of data and personal computer (PC) resources to obtain a two-dimensional strain map. Deficiency lines in the transmitted disk of LACBED reflect the crystalline structure information and move, curve, or disappear in the deformed area. Properly setting the optical conditions makes it possible to acquire real-space images over a broad area in conjunction with deficiency lines on the transmitted disk. The proposed method acquires images by changing the relative position between the specimen and the deficiency line and can grasp the strain information with a small number of images. In addition, the proposed method does not require high-resolution images. It can reduce the required PC memory or storage consumption in comparison with that of STEM-NBD, which requires a high-resolution diffraction pattern (DP) from each point of the region of interest. Compared with the two-dimensional maps of LACBED and NBD, NBD could detect large distortions in the area where the deficiency line curved, moved, or disappeared. The curving or moving direction of the deficiency line is qualitatively consistent with the NBD results. If quantitative strain values are not essential, strain visualization using LACBED can be considered an effective technique. We believe that the strain information of a sample can be obtained effectively using both methods.

Light-induced stomatal opening requires phosphorylation of the C-terminal autoinhibitory domain of plasma membrane H+-ATPase.

Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we show that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening in Arabidopsis thaliana. Using phosphoproteome analysis, we show that blue light induces the phosphorylation of Thr-881 within the C-terminal region I, in addition to penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). Based on site-directed mutagenesis experiments, phosphorylation of both Thr residues is essential for H+ pumping and stomatal opening in response to blue light. Thr-948 phosphorylation is a prerequisite for Thr-881 phosphorylation by blue light. Additionally, red light-driven guard cell photosynthesis induces Thr-881 phosphorylation, possibly contributing to red light-dependent stomatal opening. Our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells.

Validity of the cell-extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases.

Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest.

Chronic hyperglycemia reduces the expression of intercellular adhesion molecules and increases intercellular hyperpermeability in the periodontal epithelium.

BACKGROUND/AIMS: Hyperglycemia in diabetes is closely associated with periodontal disease progression. This study aimed to investigate the effect of hyperglycemia on the barrier function of gingival epithelial cells as a cause of hyperglycemia-exacerbated periodontitis in diabetes mellitus. METHODS: The abnormal expression of adhesion molecules in gingival epithelium in diabetes was compared between db/db and control mice. To study the effects of hyperglycemia on interepithelial cell permeability, the mRNA and protein expressions of adhesion molecules were investigated using a human gingival epithelial cell line (epi 4 cells) in the presence of either 5.5 mM glucose (NG) or 30 mM glucose (HG). Immunocytochemical and histological analyses were performed. We also studied HG-related intracellular signaling to assess abnormal adhesion molecule expression in the cultured epi 4 cells. RESULTS: The results of the proteomic analysis implied the abnormal regulation of cell-cell adhesion, and mRNA and protein expression assessments revealed the significant downregulation of Claudin1 expression in the gingival tissues of db/db mice (p < .05 vs control). Similarly, the mRNA and protein expressions of adhesion molecules were lower in epi 4 cells cultured under HG conditions than in those cultured under NG conditions (p < .05). Three-dimensional culture and transmission electron microscopy revealed reduced thickness of the epithelial cell layers with no flattened apical cells and heterogeneously arranged intercellular spaces among adjacent epi 4 cells under the HG. These results were consistent with the increased permeability of epi 4 cells under the HG relative to that of cells under the NG. This abnormal expression of intercellular adhesion molecules under the HG was related to the increased expression of receptors for advanced glycation end products (AGEs) and oxidative stress relative to that seen under the NG, along with stimulation of ERK1/2 phosphorylation in epi 4 cells. CONCLUSIONS: High glucose-induced impairment of intercellular adhesion molecule expression in gingival epithelial cells was related to the intercellular permeability of gingival cells, representing a possible link to hyperglycemia-related AGE signaling, oxidative stress, and ERK1/2 activation.

Calcium-dependent protein kinase 16 phosphorylates and activates the aquaporin PIP2;2 to regulate reversible flower opening in Gentiana scabra.

Flower opening is important for successful pollination in many plant species, and some species repeatedly open and close their flowers. This is thought to be due to turgor pressure changes caused by water influx/efflux, which depends on osmotic oscillations in the cells. In some ornamental plants, water-transporting aquaporins, also known as plasma membrane intrinsic proteins (PIPs), may play an important role in flower opening. However, the molecular mechanism(s) involved in corolla movement are largely unknown. Gentian (Gentiana spp.) flowers undergo reversible movement in response to temperature and light stimuli; using gentian as a model, we showed that the Gentiana scabra aquaporins GsPIP2;2 and GsPIP2;7 regulate repeated flower opening. In particular, phosphorylation of a C-terminal serine residue of GsPIP2;2 is important for its transport activity and relates closely to the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by the calcium (Ca2+)-dependent protein kinase GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that GsCPK16-dependent phosphorylation and activation of GsPIP2;2 regulate gentian flower re-opening, with stimulus-induced Ca2+ signals acting as triggers.

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways.

Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7,000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.

Motif-Targeting Phosphoproteome Analysis of Cancer Cells for Profiling Kinase Inhibitors.

We present a motif-targeting phosphoproteome analysis workflow utilizing in vitro kinase reaction to enrich a subset of peptides with specific primary sequence motifs. Phosphopeptides are enriched and dephosphorylated with alkaline phosphatase, followed by in vitro kinase reaction to phosphorylate substrate peptides with specific primary-sequence motifs. These phosphopeptides are enriched again, TMT-labeled, dephosphorylated to enhance MS-detectability, and analyzed by LC/MS/MS. We applied this approach to inhibitor-treated cancer cells, and successfully profiled the inhibitory spectra of multiple kinase inhibitors. We anticipate this approach will be applicable to target specific subsets of the phosphoproteome using the wide variety of available recombinant protein kinases.

Porphyromonas gingivalis induces entero-hepatic metabolic derangements with alteration of gut microbiota in a type 2 diabetes mouse model.

Periodontal infection induces systemic inflammation; therefore, aggravating diabetes. Orally administered periodontal pathogens may directly alter the gut microbiota. We orally treated obese db/db diabetes mice using Porphyromonas gingivalis (Pg). We screened for Pg-specific peptides in the intestinal fecal specimens and examined whether Pg localization influenced the intestinal microbiota profile, in turn altering the levels of the gut metabolites. We evaluated whether the deterioration in fasting hyperglycemia was related to the changes in the intrahepatic glucose metabolism, using proteome and metabolome analyses. Oral Pg treatment aggravated both fasting and postprandial hyperglycemia (P < 0.05), with a significant (P < 0.01) increase in dental alveolar bone resorption. Pg-specific peptides were identified in fecal specimens following oral Pg treatment. The intestinal Pg profoundly altered the gut microbiome profiles at the phylum, family, and genus levels; Prevotella exhibited the largest increase in abundance. In addition, Pg-treatment significantly altered intestinal metabolite levels. Fasting hyperglycemia was associated with the increase in the levels of gluconeogenesis-related enzymes and metabolites without changes in the expression of proinflammatory cytokines and insulin resistance. Oral Pg administration induced gut microbiota changes, leading to entero-hepatic metabolic derangements, thus aggravating hyperglycemia in an obese type 2 diabetes mouse model.

Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs.

Mass-spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by using a combination of a proximity-dependent biotin identification method, called BioID, with two other independent methods, kinase-perturbed phosphoproteomics and phosphorylation motif matching. For proof of concept, this approach was applied to casein kinase 2 (CK2) and protein kinase A (PKA), and we identified 24 and 35 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA and thus might alter downstream signaling in cancer cells bearing these mutations. This approach extends our ability to probe physiological kinase-substrate networks by providing new methodology for large-scale identification of endogenous substrates of kinases.

Nucleation and growth of water ice on oxide surfaces: the influence of a precursor to water dissociation.

We have investigated how nucleation and growth processes of ice are influenced by interfacial molecular interactions on some oxide surfaces, such as rutile TiO2(110), TiO2(100), MgO(100), and Al2O3(0001), based on the diffraction patterns of electrons transmitted through ice crystallites under the experimental configuration of reflection high energy electron diffraction (RHEED). The cubic ice Ic grows on the TiO2(110) surface with the epitaxial relationship of (110)Ic//(110)TiO2 and [001]Ic//[11[combining macron]0]TiO2. The epitaxial ice growth tends to be disturbed on the TiO2(110) surface under the presence of oxygen vacancies and adatoms. The result is not simply ascribable to small misfit values between TiO2 and ice Ic lattices (∼2%) because ice grains are formed randomly on TiO2(100). No template effects are identified during ice nucleation on the pristine MgO(100) and Al2O3(0001) surfaces either. The water molecules are chemisorbed weakly on these surfaces as a precursor to dissociation via the acid-base interaction. Such anchored water species act as an inhibitor of epitaxial ice growth because the orientation flexibility of physisorbed water during nucleation is hampered at the interface by the preferential formation of hydrogen bonds.

Mass Spectrometry-Based Discovery of in vitro Kinome Substrates.

Protein phosphorylation mediated by protein kinases is one of the most significant posttranslational modifications in many biological events. The function and physiological substrates of specific protein kinases, which are highly associated with known signal transduction elements or therapeutic targets, have been extensively studied using various approaches; however, most protein kinases have not yet been characterized. In recent decades, many techniques have been developed for the identification of in vitro and physiological substrates of protein kinases. In this review, I summarize recent studies profiling the characteristics of kinases using mass spectrometry-based proteomics, focusing on the large-scale identification of in vitro substrates of the human kinome using a quantitative phosphoproteomics approach.

Mass Spectrometry-Compatible Subcellular Fractionation for Proteomics.

We found that nuclear envelopes stabilize against surfactants in the presence of ethylene glycol (EG). We, therefore, developed a novel subcellular fractionation approach for proteomics using RIPA buffer containing EG and phase transfer surfactants. This method involves separating the cells into the cytoplasm, organelles, and nucleus, including intermediate filaments without ultracentrifugation. These fractions are directly applicable to sample preparation for shotgun proteomics as they have no mass spectrometry (MS)-incompatible chemicals, whereas those separated by traditional fractionation protocols require desalting. This protocol is successfully applied to subcellular fractionation with only 3.5 × 105 cells. Here, it was combined with phosphoproteomics and proteomics to identify phosphorylation sites regulating protein subcellular localization. In total, 59 phosphorylation sites on 42 phosphopeptides and 32 proteins showing different enrichment patterns between phosphoproteomics and the corresponding proteomics were identified, which are potential candidate sites to regulate the protein subcellular localization, including serine 706 on CD44 and serine 22 on lamin A/C.

Large-scale Discovery of Substrates of the Human Kinome.

Kinase networks are important for cellular signal transduction. Despite tremendous efforts to uncover these signaling pathways, huge numbers of uncharacterized phosphosites still remain in the human proteome. Because of the transient nature of kinase-substrate interactions in vivo, it is almost impossible to identify direct substrates. Here, we present a strategy for the rapid, accurate and high-throughput discovery of in vitro kinase substrates using quantitative proteomics. Using 385 purified kinases (354 wild-type protein kinases, 21 mutants and 10 lipid kinases), we identified a total of 175,574 potential direct kinase substrates. In addition, we identified novel kinase groups, such as one group containing 30 threonine-directed kinases and another containing 15 serine/threonine/tyrosine kinases. Surprisingly, we observed that the diversity of substrates for tyrosine kinases was much higher than that for serine-threonine kinases.

Algal Protein Kinase, Triacylglycerol Accumulation Regulator 1, Modulates Cell Viability and Gametogenesis in Carbon/Nitrogen-Imbalanced Conditions.

Nutrient-deprived microalgae accumulate triacylglycerol (TAG) in lipid droplets. A dual-specificity tyrosine phosphorylation-regulated kinase, TAG accumulation regulator 1 (TAR1) has been shown to be required for acetate-dependent TAG accumulation and the degradation of chlorophyll and photosynthesis-related proteins in photomixotrophic nitrogen (N)-deficient conditions (Kajikawa et�al. 2015). However, this previous report only examined particular condition. Here, we report that in photoautotrophic N-deficient conditions, tar1-1 cells, with a mutation in the TAR1 gene, maintained higher levels of cell viability and lower levels of hydrogen peroxide generation and accumulated higher levels of TAG and starch compared with those of wild type (WT) cells with bubbling of air containing 5% carbon dioxide. Transcriptomic analyses suggested that genes involved in the scavenging of reactive oxygen species are not repressed in tar1-1 cells. In contrast, the mating efficiency and mRNA levels of key regulatory genes for gametogenesis, MID, MTD and FUS, were suppressed in tar1-1 cells. Among the TAR1-dependent phosphopeptides deduced by phosphoproteomic analysis, protein kinases and enzymes related to N assimilation and carbon (C) metabolism are of particular interest. Characterization of these putative downstream factors may elucidate the molecular pathway whereby TAR1 mediates cellular propagation and C and N metabolism in C/N-imbalanced stress conditions.

Comparative proteomics of Helicobacter pylori strains reveals geographical features rather than genomic variations.

Helicobacter pylori, a pathogen of various gastric diseases, has many genome sequence variants. Thus, the pathogenesis and infection mechanisms of the H. pylori-driven gastric diseases have not been elucidated. Here, we carried out a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H. pylori strains by using an LC/MS/MS-based proteomics approach combined with a customized database consisting of nonredundant tryptic peptide sequences derived from full genome sequences of 52 H. pylori strains. The nonredundant peptide database enabled us to identify more peptides in the database search of MS/MS data compared with a simply merged protein database. Using this approach, we carried out proteome analysis of genome-unknown strains of H. pylori at as large a scale as genome-known ones. Clustering of the H. pylori strains using proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we identified phosphorylated proteins and sites of the H. pylori strains and obtained the phosphorylation motifs located in the N-terminus that are commonly observed in bacteria.

The jPOST environment: an integrated proteomics data repository and database.

Rapid progress is being made in mass spectrometry (MS)-based proteomics, yielding an increasing number of larger datasets with higher quality and higher throughput. To integrate proteomics datasets generated from various projects and institutions, we launched a project named jPOST (Japan ProteOme STandard Repository/Database, https://jpostdb.org/) in 2015. Its proteomics data repository, jPOSTrepo, began operations in 2016 and has accepted more than 10 TB of MS-based proteomics datasets in the past two years. In addition, we have developed a new proteomics database named jPOSTdb in which the published raw datasets in jPOSTrepo are reanalyzed using standardized protocol. jPOSTdb provides viewers showing the frequency of detected post-translational modifications, the co-occurrence of phosphorylation sites on a peptide and peptide sharing among proteoforms. jPOSTdb also provides basic statistical analysis tools to compare proteomics datasets.

Phosphoproteomic profiling reveals ABA-responsive phosphosignaling pathways in Physcomitrella patens.

Abscisic acid (ABA) and its signaling system are important for land plants to survive in terrestrial conditions. Here, we took a phosphoproteomic approach to elucidate the ABA signaling network in Physcomitrella patens, a model species of basal land plants. Our phosphoproteomic analysis detected 4630 phosphopeptides from wild-type P. patens and two ABA-responsive mutants, a disruptant of group-A type-2C protein phosphatase (PP2C; ppabi1a/b) and AR7, a defective mutant in ARK, identified as an upstream regulator of SnRK2. Quantitative analysis detected 143 ABA-responsive phosphopeptides in P. patens. The analysis indicated that SnRK2-mediated phosphorylation and target motifs were partially conserved in bryophytes. Our data demonstrate that the PpSnRK2B and AREB/ABF-type transcription factors are phosphorylated in vivo in response to ABA under the control of ARK. On the other hand, our data also revealed the following: (i) the entire ABA-responsive phosphoproteome in P. patens is quite diverse; (ii) P. patens PP2C affects additional pathways other than the known ABA signaling pathway; and (iii) ARK is mainly involved in ABA signaling. Taken together, we propose that the core ABA signaling pathway is essential in all land plants; however, some ABA-responsive phosphosignaling uniquely developed in bryophytes during the evolutionary process.

Blue light and CO2 signals converge to regulate light-induced stomatal opening.

Stomata regulate gas exchange between plants and atmosphere by integrating opening and closing signals. Stomata open in response to low CO2 concentrations to maximize photosynthesis in the light; however, the mechanisms that coordinate photosynthesis and stomatal conductance have yet to be identified. Here we identify and characterize CBC1/2 (CONVERGENCE OF BLUE LIGHT (BL) AND CO2 1/2), two kinases that link BL, a major component of photosynthetically active radiation (PAR), and the signals from low concentrations of CO2 in guard cells. CBC1/CBC2 redundantly stimulate stomatal opening by inhibition of S-type anion channels in response to both BL and low concentrations of CO2. CBC1/CBC2 function in the signaling pathways of phototropins and HT1 (HIGH LEAF TEMPERATURE 1). CBC1/CBC2 interact with and are phosphorylated by HT1. We propose that CBCs regulate stomatal aperture by integrating signals from BL and CO2 and act as the convergence site for signals from BL and low CO2.

Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.