Gun-Hwa Kim

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements.

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph-ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.

Regulation of Activin/Nodal Signaling by Rap2-Directed Receptor Trafficking

We show that Rap2, a member of the Ras GTPase family, positively regulates Activin/Nodal signaling activity by controlling the trafficking of its receptors. In the absence of ligand activation, Rap2 directs internalized Activin/Nodal receptors into a recycling pathway, thereby preventing their degradation and maintaining their levels on the cell surface. Upon ligand activation, Rap2 no longer promotes receptor recycling but delays its turnover. In both cases, Rap2 contributes to upregulation of signaling activity by antagonizing Smad7. In addition, we found that the efficiency of Activin/Nodal receptor recycling is different between dorsal and ventral halves of Xenopus early embryo, which results from the asymmetric expression of Rap2 and Smad7. Consequently, they regulate cell responsiveness to ligands and the spatiotemporally dynamic activation of Smad2 along the dorsoventral axis of the embryo. Therefore, these findings suggest a molecular basis for the regulation of signaling activity and embryonic patterning by Activin/Nodal receptor trafficking.

Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT‐ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress‐induced, nine proteins with inflammatory stress‐induced, and 14 proteins with endoplasmic reticulum stress‐induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c‐Jun amino‐terminal kinase‐dependent manner. In addition, three proteins, 14–3‐3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD‐95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.

Direct Monitoring of the Inhibition of Protein–Protein Interactions in Cells by Translocation of PKCδ Fusion Proteins†

In the field of drug discovery and development, the increasing use of cell-based assays has resulted in an increased demand for novel cellular bioassays. Such bioassays are expected to detect a wide variety of functional molecules in live cells. Fluorescence-based imaging techniques such as fluorescence resonance energy transfer (FRET) and biomolecular fluorescence complementation (BiFC) have been developed to analyze protein–protein interactions (PPIs) and inhibition of PPIs (iPPIs) in live mammalian cells. Although these techniques have been useful, they require a variety of fusion constructs to determine the relative locations of fluorophores and binding pairs for optimal performance as well as appropriate linker domains. Alternatively, translocation-based cellular assays (redistribution approaches), which are cell-based assay techniques utilizing protein translocation as the primary readout, have been used to study the PPIs between specific proteins and other intracellular events. These methods use a bait (target) molecule fused to a protein that changes its localization within the cell following a stimulus. Such assays can be formatted as agonist or antagonist assays, in which compounds are tested for their ability to promote or inhibit, respectively, protein translocation caused by a known agonist. Translocation-based cellular assays do not require much construct optimization and boast a high signal-to-noise ratio. These assays are robust, fast, and flexible; thus, these systems have been considered as an ideal assay for high-contentscreening approaches to drug discovery. Despite these advantages, few experimental applications of translocationbased cellular assays have been reported. Most of these have been based on regulated transport between the cell nucleus and the cytoplasm using a combination of nuclear localization signals and/or nuclear export signals. Several technologies are already commercially available. Recently, the groups of Schultz and Heo independently reported that PPIs can be visualized by cotranslocation of a target protein from the cytoplasm to the plasma membrane and to the endosome, respectively. Schultz et al. demonstrated the direct cotranslocation of a protein complex through the Ca-induced translocation of a bait protein fused to Annexin A4, a phospholipidand Ca-binding protein. Heo and colleagues showed that Rab5, an endosome-localized protein, recruited an interacting protein to the endosome through an FKBP–rapamycin–FRB complex intermediate. These studies were focused on the visual detection of PPIs so that new conceptual and novel applications of redistribution approaches have vastly expanded what can be explored in live cells. Herein we demonstrate that the inhibition of protein– protein interactions (iPPI) using a small molecular inhibitor can be monitored directly by a redistribution approach. Protein kinase C (PKC) is known to translocate from the cytoplasm to the plasma membrane in response to physiological stimuli, as well as exogenous ligands such as phorbol esters. In a study using PKC tagged with green fluorescent protein (GFP) the dynamics of PKC translocation in response to different stimuli was monitored in real time in live cells. PKCd has a C1 domain that binds diacylglycerol, but an impaired C2 domain that does not bind Ca ions. Thus, PKCd responds to an increase in phorbol esters in the cell but not Ca ions. Therefore we hypothesized that a PKCd-fused bait protein would guide cotranslocation with the target protein, and a chemical inhibitor would interrupts PPI, making it possible to monitor iPPI (Scheme 1). To verify our approach, we examined iPPI using the p53 (tumor suppressor)/MDM2 (negative regulator of the p53) protein pair and Nutlin-3 (see the Supporting Information for experimental details). The small molecular inhibitor Nutlin-3 is a cis-imidazoline analogue commonly used in anticancer studies that inhibits the interaction between p53 and MDM2; this inhibitor resulted from the optimization of a lead structure identified by the screening of a chemical library. We prepared the C-terminal fusion constructs PKCd/monomeric red fluorescent protein (mRFP)/p53 (bait) and enhanced GFP (eGFP)/MDM2 (target). Both the pmRFP plasmid encoding PKCd–mRFP–p53 and the peGFP plasmid encoding eGFP–MDM2 were transiently cotransfected into HEK-293T cells. When the exogenous ligand phorbol 12-myristate 13-acetate (PMA) was added, both p53 [*] Dr. K.-B. Lee, J. M. Hwang, Dr. J.-S. Choi, Dr. G.-H. Kim, Dr. S. I. Kim, Dr. S. Kim, Dr. Z.-W. Lee Division of Life Science, Korea Basic Science Institute (KBSI) Daejeon 305-333 (Korea) Fax: (+82)42-865-3419 E-mail: shkim@kbsi.re.kr ezone@kbsi.re.kr J. M. Hwang, Prof. Dr. J. Rho Department of Bioscience and Biotechnology Chungnam National University, Daejeon 305-764 (Korea)

Proteogenomic characterization of antimicrobial resistance in extensively drug-resistant Acinetobacter baumannii DU202

OBJECTIVES To determine the genomic sequence of extensively drug-resistant Acinetobacter baumannii DU202 and to perform proteomic characterization of antibiotic resistance in this strain using genome data. METHODS The genome sequence of A. baumannii DU202 was determined using the Hi-Seq 2000 system and comparative analysis was performed to determine the unique characteristics of A. baumannii DU202. Previous proteomic results from the cell wall membrane fraction by one-dimensional electrophoresis and liquid chromatography combined with mass spectrometry analysis (1DE-LC-MS/MS), using the A. baumannii ATCC 17978 genome as a reference, were reanalysed to elucidate the resistance mechanisms of A. baumannii DU202 using strain-specific genome data. Additional proteomic data from the cytosolic fraction were also analysed. RESULTS The genome of A. baumannii DU202 consists of 3660 genes and is most closely related to the Korean A. baumannii 1656-2 strain. More than 144 resistance genes were annotated in the A. baumannii DU202 genome, of which 72 that encoded proteins associated with antibiotic resistance were identified in the proteomic analysis of A. baumannii DU202 cultured in tetracycline, imipenem and Luria-Bertani broth (control) medium. Strong induction of β-lactamases, a multidrug resistance efflux pump and resistance-nodulation-cell division (RND) multidrug efflux proteins was found to be important in the antibiotic resistance responses of A. baumannii DU202. CONCLUSIONS Combining genomic and proteomic methods provided comprehensive information about the unique antibiotic resistance responses of A. baumannii DU202.

Allopurinol Protects against Ischemia/Reperfusion-Induced Injury in Rat Urinary Bladders.

Bladder ischemia-reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS) and markedly elevates the risk of lower urinary tract symptoms (LUTS). Allopurinol is an inhibitor of xanthine oxidase (XO) and thus can serve as an antioxidant that reduces oxidative stress. Here, a rat model was used to assess the ability of allopurinol treatment to ameliorate the deleterious effects of urinary bladder I/R injury. I/R injury reduced the in vitro contractile responses of longitudinal bladder strips, elevated XO activity in the plasma and bladder tissue, increased the bladder levels of tumor necrosis factor-α (TNF-α), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase, reduced the bladder levels of extracellular regulated kinase (ERK), and decreased and increased the bladder levels of Bcl-2 and Bax, respectively. I/R injury also elevated lipid peroxidation in the bladder. Allopurinol treatment in the I/R injury was generated significantly ameliorating all I/R-induced changes. Moreover, an in situ fluorohistological approach also showed that allopurinol reduces the generation of intracellular superoxides enlarged by I/R injury. Together, the beneficial effects of allopurinol reducing ROS production may be mediated by normalizing the activity of the ERK, JNK, and Bax/Bcl-2 pathways and by controlling TNF-α expression.

Differential expression of MicroRNAs in patients with glioblastoma after concomitant chemoradiotherapy.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, and notorious for resistance to chemoradiotherapy. MicroRNAs (miRNAs) are significantly involved in the initiation and progression of numerous cancers; however, the role of miRNAs in recurrence of tumors remains unknown. Here we tried to identify novel miRNAs that are differentially expressed in recurrent GBM. Tissue samples were obtained from patients with primary and recurrent GBM treated with chemoradiotherapy, and the expression changes of miRNAs were measured by microarray. A total of 318 miRNAs were expressed in the GBM patients. The expression of 43 miRNAs were significantly altered at least 2-fold in primary and recurrent GBMs. Bioinformatic analysis revealed that the differentially expressed miRNAs and their putative target genes were mainly involved in cell death, cellular development, and cellular growth and proliferation, which are the key regulators for stem cells. Pathway analysis supported that the miRNAs may regulate signaling associated with induction and maintenance of cancer and stem cell, such as p53, ErbB1, Notch, Wnt, and TGF-β signaling pathways. These data suggest that, in recurrent GBM, growth factor and anti-apoptotic signalings for cancer cell growth and proliferation are regulated by miRNAs. Our findings will aid future research in understanding the pathophysiology of recurrent GBM and identifying diagnostic markers and/or therapeutic targets for recurrence of GBM.

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.

Characterization of Streptococcus pneumoniae N-acetylglucosamine-6-phosphate deacetylase as a novel diagnostic marker

The identification of novel diagnostic markers of pathogenic bacteria is essential for improving the accuracy of diagnoses and for developing targeted vaccines. Streptococcus pneumoniae is a significant human pathogenic bacterium that causes pneumonia. N-acetylglucosamine-6-phosphate deacetylase (NagA) was identified in a protein mixture secreted by S. pneumoniae and its strong immunogenicity was confirmed in an immuno-proteomic assay against the anti-serum of the secreted protein mixture. In this study, recombinant S. pneumoniae NagA protein was expressed and purified to analyze its protein characteristics, immunospecificity, and immunogenicity, thereby facilitating its evaluation as a novel diagnostic marker for S. pneumoniae. Mass spectrometry analysis showed that S. pneumoniae NagA contains four internal disulfide bonds and that it does not undergo post-translational modification. S. pneumoniae NagA antibodies successfully detected NagA from different S. pneumoniae strains, whereas NagA from other pathogenic bacteria species was not detected. In addition, mice infected with S. pneumoniae generated NagA antibodies in an effective manner. These results suggest that NagA has potential as a novel diagnostic marker for S. pneumoniae because of its high immunogenicity and immunospecificity.