Supachok Sinchaikul

Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration.

Vascular leakage is a hallmark of severe dengue infection. Although extensive studies have been conducted during the past several decades, the molecular mechanisms underlying vascular leakage in dengue shock syndrome (DSS) remain unclear. We thus performed a proteomics study to characterize responses in human endothelial cells (EA.hy926) after DEN-2 virus infection (MOI=10). Comparative 2-D PAGE analysis revealed significantly altered abundance levels of 15 proteins, which were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS). These altered proteins were involved in several biological processes, for example, mRNA stability/processing, transcription and translation regulation, molecular chaperoning, oxidative stress response/regulation, cytoskeletal assembly, protein degradation, and cellular metabolisms. We also performed functional analyses of alterations in actin cytoskeletal assembly and endothelial integrity focusing on adherens junction (VE-cadherin), tight junction (ZO-1) and adhesive molecule (PECAM-1) after 24-h of DEN-2 infection and simulation of transendothelial migration by PECAM-1 cross-linking. Decreased expression and disorganization of the actin-cytoskeleton were observed in the infected cells, whereas the increase in actin stress fibers was found in adjacent noninfected cells. Additionally, a decrease in adhesive protein PECAM-1 was observed. Furthermore, DEN-2 infection caused decreased expression and redistribution of both VE-cadherin and ZO-1, whose changes were enhanced by PECAM-1 engagement. These alterations may potentially be a molecular basis explaining increased endothelial permeability or vascular leakage in DSS.

The Ubiquitin−Proteasome Pathway Is Important for Dengue Virus Infection in Primary Human Endothelial Cells

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processes were triggered in response to dengue infection, particularly antiviral IFN and ubiquitin-proteasome pathways.

Vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release

Our previous study using expression proteomics demonstrated that many proteins, particularly five forms of heterogeneous nuclear ribonucleoproteins (hnRNPs), were up-regulated in human endothelial cells upon dengue virus infection. To address functional significance of these proteins in response to dengue virus infection, we performed a functional proteomics study to identify hnRNPs-interacting proteins in the infected EA.hy926 cells. Immunoprecipitation followed by 2-D PAGE and mass spectrometric analyses revealed 18 and 13 interacting partners of hnRNP C1/C2 and hnRNP K, respectively. Interestingly, vimentin was a common partner for both hnRNP C1/C2 and K. The interaction between vimentin and these hnRNPs was confirmed by reciprocal immunoprecipitation followed by Western blot analysis and also by double immunofluorescence staining. Disruption of vimentin intermediate filament by acrylamide not only dissociated these complexes but also reduced nuclear hnRNPs expression, whereas cytosolic hnRNPs expression was unchanged. We also demonstrated that vimentin was strongly associated with dengue non-structural protein 1 (NS1). Disruption of vimentin intermediate filament not only dissociated this complex but also reduced dengue NS1 expression, as well as viral replication and release. Our data report for the first time that vimentin interacts with hnRNPs and dengue NS1, and plays a crucial role in replication and release of dengue virus.

Proteomic analysis of calcium oxalate monohydrate crystal-induced cytotoxicity in distal renal tubular cells.

Calcium oxalate monohydrate (COM) is the major crystalline component found in kidney stones and its adhesion to renal tubular cells provokes tubular injury, which in turn enhances COM crystal adhesion. However, COM-induced toxic effects in these tubular cells remain largely unknown. We performed a proteomics study to characterize changes in the cellular proteome in MDCK distal renal tubular cells after an exposure to high-dose (1000 microg/mL) COM crystals for 48 h, at which percentage of cell death was significantly increased. Proteins were extracted from MDCK cells cultured with COM-containing or COM-free medium ( n = 5 individual flasks per group), resolved in individual 2-D gels, and stained with SYPRO Ruby fluorescence dye. Quantitative and statistical analyses revealed 53 proteins whose abundance levels were altered (25 were increased, whereas other 28 were decreased) by COM-induced toxicity. Among these, 50 were successfully identified by quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and/or tandem MS (MS/MS) analyses. The proteomic data were clearly confirmed by 2-D Western blot analysis. While three chaperones (GRP78, Orp150 and Hsp60) were increased, other proteins involved in protein biosynthesis, ATP synthesis, cell cycle regulator, cellular structure, and signal transduction were decreased. These data provide some novel mechanistic insights into the molecular mechanisms of COM crystal-induced tubular toxicity.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.

Proteomics analysis of A375 human malignant melanoma cells in response to arbutin treatment

Although the toxicogenomics of A375 human malignant melanoma cells treated with arbutin have been elucidated using DNA microarray, the proteomics of the cellular response to this compound are still poorly understood. In this study, we performed proteomic analyses to investigate the anticancer effect of arbutin on the protein expression profile in A375 cells. After treatment with arbutin (8 microg/ml) for 24, 48 and 72 h, the proteomic profiles of control and arbutin-treated A375 cells were compared, and 26 differentially expressed proteins (7 upregulated and 19 downregulated proteins) were identified by MALDI-Q-TOF MS and MS/MS. Among these proteins, 13 isoforms of six identical proteins were observed. Bioinformatic tools were used to search for protein function and to predict protein interactions. The interaction network of 14 differentially expressed proteins was found to be correlated with the downstream regulation of p53 tumor suppressor and cell apoptosis. In addition, three upregulated proteins (14-3-3G, VDAC-1 and p53) and five downregulated proteins (ENPL, ENOA, IMDH2, PRDX1 and VIME) in arbutin-treated A375 cells were validated by RT-PCR analysis. These proteins were found to play important roles in the suppression of cancer development.

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: Insights into biological processes

Monocyte and macrophage are mainly involved in immune response and inflammatory processes. Monocytes circulate in the bloodstream and migrate to various tissues where they can differentiate to macrophages. However, the molecular basis of biological processes involved in this cellular differentiation remains ambiguous. This study was to investigate alterations in cellular and secreted proteins after this differentiation phase. Macrophage was differentiated from U937 human monocytic cell line by treatment with 100 ng/ml phorbol myristate acetate (PMA) for 48 h. Cellular and secreted proteins extracted from PMA-treated cells (macrophages) were compared with those of untreated cells (monocytes) using 2-DE (n=5 gels/condition; stained with Deep Purple fluorescence dye). Quantitative intensity analysis revealed 81 and 67 protein spots whose levels were significantly altered in cellular proteome and secretome. These proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses. The altered levels of cellular elongation factor-2 (EF-2) and secreted alpha-tubulin were confirmed by Western blot analysis. Global protein network analysis demonstrated that these altered proteins were involved in cell death, lipid metabolism, cell morphology, cellular movement, and protein folding. Our data may provide some insights into molecular mechanisms of biological processes upon differentiation from monocytes to macrophages.

Glycoproteomic analysis of WGA-bound glycoprotein biomarkers in sera from patients with lung adenocarcinoma

Differential protein expression profiles in the serum samples from patients with lung adenocarcinoma may be associated with glycosylation during cancer development. In this study, we used various glycoproteomic approaches to investigate the different glycoproteomic profiles of human normal and lung adenocarcinoma serum samples and to investigate putative altered glycoprotein biomarkers. In our preliminary screening, FITC‐labeled lectin staining was used for the detection of specific glycoprotein profiles. wheat germ agglutinin (WGA) lectin had the highest level of specific binding to glycoproteins in both samples. We enriched for glycoproteins in the serum samples using WGA lectin affinity and then performed co‐immunoprecipitation with anti‐haptoglobin and 2‐DE, 2‐D difference in‐gel electrophoresis and MS analyses. From these analyses, we identified 39 differentially expressed proteins, including 27 up‐regulated proteins and 12 down‐regulated proteins. Bioinformatics tools were used to search for protein ontology, category classifications and prediction of glycosylation sites. In addition, three up‐regulated glycoproteins (adiponectin, cerulolasmin and glycosylphosphatidyl‐inositol‐80) and two down‐regulated glycoproteins (cyclin H and Fyn) that were found to be correlated with lung cancer development were validated by Western blot analysis. We suggest that these altered glycoproteins may be useful as biomarkers for lung cancer development and progression.

Bioinformatics, functional genomics, and proteomics study of Bacillus sp.

The ability of bioinformatics to characterize genomic and proteomic sequences from bacteria Bacillus sp. for prediction of genes and proteins has been evaluated. Genomics coupling with proteomics, which is relied on integration of the significant advances recently achieved in two-dimensional (2-D) electrophoretic separation of proteins and mass spectrometry (MS), are now important and high throughput techniques for qualifying and analyzing gene and protein expression, discovering new gene or protein products, and understanding of gene and protein functions including post-genomic study. In addition, the bioinformatics of Bacillus sp. is embraced into many databases that will facilitate to rapidly search the information of Bacillus sp. in both genomics and proteomics. It is also possible to highlight sites for post-translational modifications based on the specific protein sequence motifs that play important roles in the structure, activity and compartmentalization of proteins. Moreover, the secreted proteins from Bacillus sp. are interesting and widely used in many applications especially biomedical applications that are the highly advantages for their potential therapeutic values.

Altered proteins in MDCK renal tubular cells in response to calcium oxalate dihydrate crystal adhesion: a proteomics approach.

The interaction between crystals and renal tubular cells has been proposed to be a crucial event that elicits subsequent cellular responses, leading to kidney stone formation. Nevertheless, the molecular mechanisms of these cellular responses remain poorly understood. We performed a gel-based differential proteomics study to examine cellular responses (as determined by altered protein expression) in Madin-Darby canine kidney (MDCK) cells, which were derived from dog kidney and exhibited distal renal tubule phenotype, during calcium oxalate dihydrate (COD) crystal adhesion. MDCK cells were grown in a medium without or with COD crystals (100 microg/ml) for 48 h. Crystal adhesion was illustrated by phase-contrast and scanning electron microscopy. Flow cytometry using annexin V/propidium iodide double staining showed that the percentage of cell death did not significantly differ between cells with and without COD crystal adhesion. Cellular proteins were then extracted, resolved with two-dimensional gel electrophoresis (2-DE), and visualized by SYPRO Ruby staining ( n = 5 gels per group). Quantitative intensity analysis revealed 11 significantly altered proteins, 10 of which were successfully identified by quadrupole time-of-flight peptide mass fingerprinting (MS) and/or tandem MS (MS/MS), including metabolic enzymes, cellular structural protein, calcium-binding protein, adhesion molecule, protein involved in RNA metabolism, and chaperone. An increase in annexin II was confirmed by 2-D Western blot analysis. These data may lead to better understanding of the cellular responses in distal renal tubular cells during COD crystal adhesion.