Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation.

Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D ß-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.

High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease.

Hyperglycemia can cause several abnormalities in liver cells, including diabetic liver disease. Previous research has shown that high blood glucose levels can damage liver cells through glycoxidation. However, the detailed molecular mechanisms underlying the effects of high blood glucose on the development of diabetic liver disease have yet to be elucidated. In this study, we cultured a liver cell line (Chang liver cell) in mannitol-balanced 5.5 mM, 25 mM and 100 mM d-glucose media and evaluated protein expression and redox regulation. We identified 141 proteins that showed significant changes in protein expression and 29 proteins that showed significant changes in thiol reactivity, in response to high glucose concentration. Several proteins involved in transcription-control, signal transduction, redox regulation and cytoskeleton regulation showed significant changes in expression, whereas proteins involved in protein folding and gene regulation displayed changes in thiol reactivity. Further analyses of clinical plasma specimens confirmed that the proteins AKAP8L, galectin-3, PGK 1, syntenin-1, Abin 2, aldose reductase, CD63, GRP-78, GST-pi, RXR-gamma, TPI and vimentin showed type 2 diabetic liver disease-dependent alterations. In summary, in this study we used a comprehensive hepatocyte-based proteomic approach to identify changes in protein expression and to identify redox-associated diabetic liver disease markers induced by high glucose concentration. Some of the identified proteins were validated with clinical samples and are presented as potential targets for the prognosis and diagnosis of diabetic liver disease.

Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury.

Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein ( α -SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

High glucose-induced proteome alterations in retinal pigmented epithelium cells and its possible relevance to diabetic retinopathy.

Diabetic retinopathy can cause poor vision and blindness. Previous research has shown that high blood glucose weakens retinal capillaries and induces glycoxidation. However, the detailed molecular mechanisms underlying the effects of high blood glucose on development of diabetic retinopathy have yet to be elucidated. In this study, we cultured a retinal pigmented epithelium cell line (ARPE-19) in mannitol-balanced 5.5 mM, 25 mM, and 100 mM d-glucose media, and evaluated protein expression and redox-regulation. We identified 56 proteins that showed significant changes in protein expression, and 33 proteins showing significant changes in thiol reactivity, in response to high glucose concentration. Several proteins that are involved in signal transduction, gene regulation, and transport showed significant changes in expression, whereas proteins involved in metabolism, transport, and cell survival displayed changes in thiol reactivity. Further analyses of clinical plasma specimens confirmed that the proteins lamin B2, PUMA, WTAP, ASGR1, and prohibitin 2 showed type 2 diabetic retinopathy-dependent alterations. In summary, in this study, we used a comprehensive retinal cell-based proteomic approach for the identification of changes in protein expression and redox-associated retinal markers induced by high glucose concentration. Some of the identified proteins have been validated with clinical samples and provide potential targets for the prognosis and diagnosis of diabetic retinopathy.

Effect of high glucose on secreted proteome in cultured retinal pigmented epithelium cells: its possible relevance to clinical diabetic retinopathy.

Retinopathy has been observed in around quarter of diabetic patients. Diabetic retinopathy can result in poor vision and even blindness since high glucose has been evidenced to weaken retinal capillary leading to leakage of blood into the surrounding space. In the present study, a proteomics-based approach has been applied to analyze a model retinal pigmented epithelium cell line, ARPE-19, grown in mannitol-balanced 5.5mM, 25 mM and 100 mM D-glucose culture media and used as a model for hyperglycemia secretomic analysis. Totally, 55 differentially secreted proteins have been firmly identified representing 46 unique gene products. These secreted proteins mainly function in cytoskeleton-associated adhesion/junction (such as galectin-3-binding protein) and transport (multidrug resistance-associated protein 1). Additionally, the identified secreted markers including asialoglycoprotein receptor 1, lysophosphatidic acid receptor 3, moesin, MPP2, haptoglobin and cathepsin D were further validated in plasma samples coming from type 2 diabetic patients with retinopathy and healthy donors. In summary, we report a comprehensive retinal cell-based proteomic approach for the identification of potential secreted retinal markers-induced in high glucose conditions. Some of these identified secreted proteins have been validated in diabetic retinopathy plasma demonstrating the potentially utilizing of these markers in screening and treating diabetic retinopathy.

Placenta proteome analysis from Down syndrome pregnancies for biomarker discovery.

Down syndrome is one of the most frequent chromosomal disorders, with a prevalence of approximately 1/500 to 1/800, depending on the maternal age distribution of the pregnant population. However, few reliable protein biomarkers have been used in the diagnosis of this disease. Recent progress in quantitative proteomics has offered opportunities to discover biomarkers for tracking the progression and for understanding the molecular mechanisms of Down syndrome. In the present study, placental samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 101 proteins have been firmly identified representing 80 unique gene products. These proteins mainly function in cytoskeleton structure and regulation (such as vimentin and Profilin-1). Additionally, our quantitative proteomics approach has identified numerous previously reported Down syndrome markers, such as myelin protein. Here we present several Down syndrome biomarkers including galectin-1, ataxin-3 and sprouty-related EVH1 domain-containing protein 2 (SPRED2), which have not been reported elsewhere and may be associated with the progression and development of the disease. In summary, we report a comprehensive placenta-based proteomics approach for the identification of potential biomarkers for Down syndrome, in which serum amyloid P-component (APCS) and ataxin-3 have been shown to be up-regulated in the maternal peripheral plasma of Down syndrome cases. The potential of utilizing these markers for the prognosis and screening of Down syndrome warrants further investigation.

Hemopexin is up-regulated in plasma from type 1 diabetes mellitus patients: Role of glucose-induced ROS.

Type 1 diabetes mellitus (T1DM) is an insulin-dependent metabolic disease in the world and often occurs in children and adolescents. Recent advances in quantitative proteomics offer potential for the discovery of plasma proteins as biomarkers for tracking disease progression and for understanding the molecular mechanisms of diabetes. Comparative proteomic analysis of the plasma proteomes from T1DM cases and healthy donors with lysine- and cysteine-labeling 2D-DIGE combining MALDI-TOF/TOF mass spectrometry revealed that 39 identified T1DM-associated plasma proteins showed significant changes in protein expression including hemopexin, and 41 in thiol reactivity. Further study showed that hemopexin can be induced in numerous cell lines by increasing the glucose concentration in the medium. Interestingly, glucose-induced hemopexin expression can be reduced by reactive oxygen species (ROS) scavengers such as glutathione, implying that hemopexin expression is linked to glucose-induced oxidative stress. In conclusion, the current work has identified potential T1DM biomarkers and one of these, hemopexin, can be modulated by glucose through a ROS-dependent mechanism.

Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells.

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.