Sayaka Kado

Proteomic analysis of gingival crevicular fluid for discovery of novel periodontal disease markers.

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel‐based and gel‐free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two‐dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A‐I (ApoA‐I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC‐MS/MS analysis. A total of 327 proteins including ApoA‐I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.

Application of quantitative proteomic analysis using tandem mass tags for discovery and identification of novel biomarkers in periodontal disease

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)‐9 and neutrophil gelatinase‐associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP‐9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC‐MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.

Increased cell proliferation and differential protein expression induced by low-level Er:YAG laser irradiation in human gingival fibroblasts: proteomic analysis

Erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment has demonstrated favorable wound healing effect after periodontal therapy. One of the reasons may be the positive biological effect of the low-level laser on the irradiated tissues, although the mechanism remains unclear. The aim of this study was to investigate the effect of low-level Er:YAG laser irradiation on cell proliferation and laser-induced differential expression of proteins in human gingival fibroblasts (HGFs) by proteomic analysis. In the first experiment, HGFs were exposed to low-level Er:YAG laser irradiation and the laser-induced cell proliferation and damage were evaluated on day 3. In the second experiment, proteomic analysis was performed on day 1 after irradiation. The peptides prepared from HGFs were analyzed by a hybrid ion trap-Fourier transform mass spectrometer, Mascot search engine, and UniProtKB database. A significant increase in cell proliferation without cell damage after irradiation was observed. Among the total identified 377 proteins, 59 proteins, including galectin-7, which was associated with the process of wound healing, were upregulated and 15 proteins were downregulated in laser-treated HGFs. In the third experiment, the increase in messenger RNA (mRNA) and protein expression of galectin-7 in the irradiated HGFs was validated by various analytical techniques. In addition, the effect of recombinant human galectin-7 on the modulation of HGFs proliferation was confirmed. The results indicate that low-level Er:YAG laser irradiation can promote HGF proliferation and induce a significant change in protein expression and the upregulation of galectin-7 expression may partly contribute to the increase in cell proliferation.

Diacylglycerol Kinase δ Phosphorylates Phosphatidylcholine-specific Phospholipase C-dependent, Palmitic Acid-containing Diacylglycerol Species in Response to High Glucose Levels

Background: Diacylglycerol (DG) kinase (DGK) δ is activated by acute high glucose stimulation. Results: DGKδ high glucose-dependently phosphorylates 30:0-, 32:0-, and 34:0-DG and interacts with phosphatidylcholine-specific phospholipase C (PC-PLC). Conclusion: DGKδ utilizes palmitic acid-containing DG species and metabolically connects with PC-PLC. Significance: The newly identified PC-PLC/DGKδ pathway could play an important role in insulin signaling and glucose uptake. Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.

A novel diacylglycerol kinase α-selective inhibitor, CU-3, induces cancer cell apoptosis and enhances immune response

Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 μM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

Diacylglycerol kinase-dependent formation of phosphatidic acid molecular species during interleukin-2 activation in CTLL-2 T-lymphocytes

Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL‐2 murine T‐lymphocytes by interleukin‐2 (IL‐2) induced a significant increase of 36:1‐, 36:2‐, 40:5‐ and 40:6‐diacyl‐PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1‐, 40:5‐, 40:6‐diacyl‐PA, suggesting that DGK IL‐2‐dependently and selectively generated these diacyl‐PA species.

Enhanced recovery of lyophilized peptides in shotgun proteomics by using an LC‐ESI‐MS compatible surfactant

LC‐ESI/MS/MS‐based shotgun proteomics is currently the most commonly used approach for the identification and quantification of proteins in large‐scale studies of biomarker discovery. In the past several years, the shotgun proteomics technologies have been refined toward further enhancement of proteome coverage. In the complex series of protocols involved in shotgun proteomics, however, loss of proteolytic peptides during the lyophilization step prior to the LC/MS/MS injection has been relatively neglected despite the fact that the dissolution of the hydrophobic peptides in lyophilized samples is difficult in 0.05–0.1% TFA or formic acid, causing substantial loss of precious peptide samples. In order to prevent the loss of peptide samples during this step, we devised a new protocol using Invitrosol (IVS), a commercially available surfactant compatible with ESI‐MS; by dissolving the lyophilized peptides in IVS, we show improved recovery of hydrophobic peptides, leading to enhanced coverage of proteome. Thus, the use of IVS in the recovery step of lyophilized peptides will help the shotgun proteomics analysis by expanding the proteome coverage, which would significantly promote the discovery and development of new diagnostic markers and therapeutic targets.

Combined Secretomics and Transcriptomics Revealed Cancer-Derived GDF15 is Involved in Diffuse-Type Gastric Cancer Progression and Fibroblast Activation

Gastric cancer is classified into two subtypes, diffuse and intestinal. The diffuse-type gastric cancer (DGC) has poorer prognosis, and the molecular pathology is not yet fully understood. The purpose of this study was to identify functional secreted molecules involved in DGC progression. We integrated the secretomics of six gastric cancer cell lines and gene expression analysis of gastric cancer tissues with publicly available microarray data. Hierarchical clustering revealed characteristic gene expression differences between diffuse- and intestinal-types. GDF15 was selected as a functional secreted molecule owing to high expression only in fetal tissues. Protein expression of GDF15 was higher in DGC cell lines and tissues. Serum levels of GDF15 were significant higher in DGC patients as compared with healthy individuals and chronic gastritis patients, and positively correlated with wall invasion and lymph node metastasis. In addition, the stimulation of GDF15 on NIH3T3 fibroblast enhanced proliferation and up-regulated expression of extracellular matrix genes, which were similar to TGF-β stimulation. These results indicate that GDF15 contributes to fibroblast activation. In conclusion, this study revealed that GDF15 may be a novel functional secreted molecule for DGC progression, possibly having important roles for cancer progression via the affecting fibroblast function, as well as TGF-β.

Application of proteomic technologies to discover and identify biomarkers for periodontal diseases in gingival crevicular fluid: A review

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. In recent years, studies have shown a definite association between periodontal disease and other inflammatory conditions of the body. High‐throughput analysis of proteins has become possible with the development of MS technology. This breakthrough in proteome technology enables comparative studies of comprehensive protein expression and identification of protein. In case of periodontal disease, proteome analysis using 2DE, as well as gel‐free methods, has been reported. As a fluid lying in close proximity to periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for biomarkers of periodontal disease, because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Increasing numbers of recent reports have provided evidence that the proteomic approach is a promising tool for the discovery and identification of biochemical markers of periodontal disease. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize recent comprehensive proteomic studies aimed at discovering and identifying biomarkers of periodontal disease in GCF.

Overexpression of Hydroxymethylglutaryl Coa Synthase 2 and 2,4-dienoyl-coa Reductase in Rat Pancreas Following Chronic Alcohol Consumption

Objectives The mechanism of alcohol-induced pancreatic damage is unclear. The aim of this study was to clarify the effects of chronic alcohol intake on the pancreatic proteome. Methods Rats were fed an alcohol-containing Lieber-DeCarli liquid diet, and the pancreatic proteome was compared with that of pair-fed control rats using agarose 2-dimensional gel electrophoresis followed by liquid chromatography–tandem mass spectrometry. Results The expression of 3 proteins was consistently altered in alcohol-fed rats: 1 protein was down-regulated, and 2 proteins were up-regulated. The 2 up-regulated proteins were identified as 2,4-dienoyl-CoA reductase and hydroxymethylglutaryl-CoA synthase (HMGCS2). The combined concentration of malondialdehyde and 4-hydroxyalkenals was significantly greater in alcohol-fed rats. It is noteworthy that the reactivity of anti–4-hydroxy-2-nonenal antibody was significantly higher toward HMGCS2 isolated from alcohol-fed rats. The activity of HMGCS2 was higher in alcohol-fed rats, but the relative increase in enzyme activity in alcohol-fed rats was less than the relative increase in HMGCS2 expression. Conclusions Chronic alcohol consumption results in distinct alterations in the expression of 3 pancreatic proteins. The reactivity of 4-hydroxy-2-nonenal toward one of the up-regulated proteins, HMGCS2, increased markedly following chronic alcohol intake, suggesting that up-regulation of HMGCS2 is connected with alterations of lipid peroxidation induced by alcohol.