[show abstract][hide abstract] ABSTRACT: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu, Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.
Molecules and Cells 02/2014; 37(2):100-8. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The AcrAB-TolC multidrug efflux pump confers resistance to Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor that participates in the formation of type I secretion systems. The genome of Vibrio vulnificus encodes two proteins homologous to the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complement the E. coli TolC function and physically interact with the membrane fusion protein AcrA, a component of the E. coli AcrAB-TolC efflux pump. Using site-directed mutational analyses and an in vivo cross-linking assay, we demonstrated that the α-barrel tip region of TolC homologs plays a critical role in the formation of functional AcrAB-TolC efflux pumps. Our findings suggest the adapter bridging model as a general assembly mechanism for tripartite drug efflux pumps in Gram-negative bacteria.
The Journal of Microbiology 02/2014; 52(2):148-53. · 1.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Approximately 97% of patients with ovarian granulosa cell tumours (GCTs) bear the C134W mutation in FOXL2; however, the pathophysiological mechanism of this mutation is unknown. Here we report how this mutation affects GCT development. Sequential posttranslational modifications of the C134W mutant occur where hyperphosphorylation at serine 33 (S33) by GSK3β induces MDM2-mediated ubiquitination and proteasomal degradation. In contrast, S33 of wild-type FOXL2 is underphosphorylated, leading to its SUMOylation and stabilization. This prominent hyperphosphorylation is also observed at S33 of FOXL2 in GCT patients bearing the C134W mutation. In xenograft mice, the S33 phosphorylation status correlates with the oncogenicity of FOXL2, and the inhibition of GSK3β efficiently represses GCT growth. These findings reveal a previously unidentified regulatory mechanism that determines the oncogenic attributes of the C134W mutation via differential posttranslational modifications of FOXL2 in GCT development.
[show abstract][hide abstract] ABSTRACT: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen with the ability to survive and replicate in macrophages. Periplasmic copper binding protein CueP is known to confer copper resistance to S. Typhimurium, and has been implicated in ROS scavenge activity by transferring the copper ion to a periplasmic superoxide dismutase or by directly reducing the copper ion. Structural and biochemical studies on CueP show that its copper binding site is surrounded by conserved cysteine residues. Here, we present evidence that periplasmic disulfide isomerase DsbC plays a key role in maintaining CueP protein in the reduced state. We observed purified DsbC protein efficiently reduced the oxidized form of CueP, and that it acted on two (Cys104 and Cys172) of the three conserved cysteine residues. Furthermore, we found that a surface-exposed conserved phenylalanine residue in CueP was important for this process, which suggests that DsbC specifically recognizes the residue of CueP. An experiment using an E. coli system confirmed the critical role played by DsbC in the ROS scavenge activity of CueP. Taken together, we propose a molecular insight into how CueP collaborates with the periplasmic disulfide reduction system in the pathogenesis of the bacteria.
Biochemical and Biophysical Research Communications 01/2014; · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Endoribonuclease E (RNase E) affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37) of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne) that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13) containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71). Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.
PLoS ONE 01/2014; 9(3):e90610. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lysozymes are the first line of defense for a diverse range of organisms that catalyze the degradation of bacterial peptidoglycan. Gram-negative bacteria produce proteinaceous lysozyme inhibitors to protect themselves from the lysozyme action. To date, MliC or PliC (membrane bound or periplasmic inhibitor of c-type lysozyme) has been found in various Gram-negative bacteria. Here, we report the crystal structures of Brucella abortus PliC and its complex with human c-type lysozyme. The complex structure demonstrates that the invariant loop of MliC/PliC plays a crucial role in the inhibition of lysozyme via its insertion to the active site cleft of the lysozyme, as previously observed in the complex structure of P. aeruginosa MliC and chicken c-type lysozyme. We identified a new binding interface between a loop adjacent to the active site of human lysozyme and a loop carrying Glu112 of B. abortus PliC, whose structure was disordered in P. aeruginosa MliC. Since MliC/PliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC/PliC will be relevant to the control of bacterial growth in animal hosts.
[show abstract][hide abstract] ABSTRACT: K-Ras mutation is detected in over 30% of human malignancies. In particular, 90% of human pancreatic cancers are initiated by K-Ras mutation. Thus, selective elimination of K-Ras mutated cells would be a plausible strategy to prevent or cure the malignancies. In our previous reports, it has been revealed that oncogenic K-Ras promotes the exocytosis of p53 with Snail. In this study, we have followed the final destination of extracellular p53, which is secreted by the Snail complex. Here we provide evidences that p53, exported from K-Ras-mutated cells, is specifically re-endocytosed by oncogenic K-Ras-containing cancer cells. The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis. Using a serial deletion approach, we revealed that a fragment of human p53 extending from 93-143 amino acids (AA) is responsible for binding with caveolin-1 and for endocytosis. In contrast, p53-Snail binding occurs at the 143-193 aa region. Finally, through in vivo study, we confirmed that injected recombinant p53 could be up-taken by tumor tissues, constructed by oncogenic K-Ras transformed MEF cells. In contrast, the tumors formed by H-Ras mutated MEF cells did not accumulate the injected p53 protein. These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.
[show abstract][hide abstract] ABSTRACT: Renal cell carcinomas (RCCs) are frequently occurring genitourinary malignancies in the aged population. A morphological characteristic of RCCs is an irregular nuclear shape, which is used to index cancer grades. Other features of RCCs include the genetic inactivation of the von Hippel-Lindau gene, VHL, and p53 genetic-independent inactivation. An aberrant nuclear shape or p53 suppression has not yet been demonstrated. We examined the effect of progerin (an altered splicing product of the LMNA gene linked to Hutchinson Gilford progeria syndrome; HGPS) on the nuclear deformation of RCCs in comparison to that of HGPS cells. In this study, we showed that progerin was suppressed by pVHL and was responsible for nuclear irregularities as well as p53 inactivation. Thus, progerin suppression can ameliorate nuclear abnormalities and reactivate p53 in response to genotoxic addition. Furthermore, we found that progerin was a target of pVHL E3 ligase and suppressed p53 activity by p14/ARF inhibition. Our findings indicate that the elevated expression of progerin in RCCs results from the loss of pVHL and leads to p53 inactivation through p14/ARF suppression. Interestingly, we showed that progerin was expressed in human leukemia and primary cell lines, raising the possibility that the expression of this LMNA variant may be a common event in age-related cancer progression.
[show abstract][hide abstract] ABSTRACT: The disulfide-bond isomerase DsbC plays a crucial role in the folding of bacterial proteins in the periplasmic space. DsbC has a V-shaped dimeric structure with two domains, and Cys98 in the C-terminal domain attacks inappropriate disulfide bonds in substrate proteins due to its high nucleophilic activity. In this article, we present the crystal structure of DsbC from Salmonella enterica serovar Typhimurium. We evaluated the conserved residues Asp95 and Arg125, which are located close to Cys98. The mutation of Asp95 or Arg125 abolished the disulfide isomerase activity of DsbC in an in vitro assay using a protein substrate, and the R125A mutation significantly reduced the chaperone activity for the substrate RNase I in vivo. Furthermore, a comparative analysis suggested that the conformation of Arg125 varies depending on the packing or protein-protein interactions. Based on these findings, we suggest that Asp95 and Arg125 modulate the pKa of Cys98 during catalysis.
Journal of Structural Biology 05/2013; · 3.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: Parkinson's disease (PD) is the second leading neurodegenerative disease, and is known to be induced by environmental factors or genetic mutations. Among the verified genetic mutations of PD, Parkin, isolated from the PARK2 locus, shows an autosomal recessive inheritance pattern and is known to be an E3 ligase. However, the physiological target of Parkin and the molecular mechanism of Parkin-deficiency-induced PD have not been clearly demonstrated until now. It has recently been proposed that inflammation, suggesting as a causal factor for PD, is enhanced by Parkin deficiency. Thus, we examined the relationship between inflammation-related factors and Parkin. Here, we provide the evidence that Parkin suppresses inflammation and cytokine-induced cell death by promoting the proteasomal degradation of TRAF2/6 (TNF-α receptor-associated factor 2/6). Overexpression of Parkin can reduce the half-lives of TRAF2 and TRAF6, whereas si-Parkin can extend them. However, mutant Parkins did not alter the expression of TRAF2/6. Thus, loss of Parkin enhances sensitivity to TNF-α- or IL-1β-induced JNK activation and NF-κB activation. Indeed, si-Parkin-induced apoptosis is suppressed by the knockdown of TRAF6 or TRAF2. We also observed elevated expression levels of TRAF6 and a reduction of IκB in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model. Moreover, elevated expression levels or aggregation of TRAF6 were detected in approximately half of the human PD tissues (7/15 cases) and 2 cases, respectively. In addition, TRAF6 and Parkin expression levels show a reverse relationship in human PD tissues. Our results strongly suggest that the reduction of Parkin or overexpression of TRAF2/6 by chronic inflammation would be the reason for occurrence of PD.Laboratory Investigation advance online publication, 22 April 2013; doi:10.1038/labinvest.2013.60.
[show abstract][hide abstract] ABSTRACT: Abnormal overexpression of GSK3β has been implicated in insulin resistance. Although many potent GSK3β inhibitors have been developed as drug candidates for anti-insulin resistance, the inhibitors are prone to show side effects because they interfere with normal GSK3β function without regulation. Recently, it was reported that the PPPSPxS motifs in the Wnt coreceptor LRP6 were able to directly inhibit GSK3β only when the motif was phosphorylated. Here, we generated a new GSK3β inhibitory peptide that can be activated by Akt by combining the PPPSPxS motif and an Akt target sequence. The peptide exhibited an inhibitory effect on GSK3β only when it was phosphorylated by Akt in a purified system and in cells when stimulated by insulin. Thus, our findings provide a novel concept for drugs against diseases that are involved in the abnormal GSK3β activity, including type 2 diabetes mellitus.
Biochemical and Biophysical Research Communications 04/2013; · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.
The Journal of Microbiology 04/2013; 51(2):154-9. · 1.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Herein we report the discovery of a cystine-crosslinked peptide from Porifera along with high-quality spatial details accompanied by the description of its unique effect on neuronal calcium influx. METHODS: Asteropsin A (ASPA) was isolated from the marine sponge Asteropus sp., and its structure was independently determined using X-ray crystallography (0.87 Å) and solution NMR spectroscopy. RESULTS: An N-terminal pyroglutamate modification, uncommon cis proline conformations, and absence of basic residues helped distinguish ASPA from other cystine-crosslinked knot peptides. ASPA enhanced Ca(2+) influx in murine cerebrocortical neuron cells following the addition of the Na(+) channel activator veratridine but did not modify the oscillation frequency or amplitude of neuronal Ca(2+) currents alone. Allosterism at neurotoxin site 2 was not observed, suggesting an alternative to the known Na(+) channel interaction. CONCLUSIONS: Together with a distinct biological activity, the origin of ASPA suggests a new subclass of cystine-rich knot peptides associated with Porifera. GENERAL SIGNIFICANCE: The discovery of ASPA represents a distinctive addition to an emerging subclass of cystine-crosslinked knot peptides from Porifera.
Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.
Journal of bacteriology 02/2012; 194(9):2214-20. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.
Journal of Biological Chemistry 02/2012; 287(15):11740-50. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.
PLoS ONE 01/2012; 7(7):e40460. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The β-N-acetylglucosaminidase CbsA was cloned from the thermophilic Gram-negative bacterium Thermotoga neapolitana. Although CbsA contains a family 3 glycoside hydrolase-type (GH3-type) catalytic domain, it can be distinguished from other GH3-type β-N-acetylglucosaminidases by its high activity towards chitobiose. The homodimeric CbsA contains a unique domain at the C-terminus for which the three-dimensional structure is not yet known. In this study, CbsA was overexpressed and the recombinant protein was purified using Ni-NTA affinity and gel-filtration chromatography. The purified CbsA protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 2.0 Å at 100 K. The crystal belonged to space group R32. To obtain initial phases, the crystallization of selenomethionyl-substituted protein and the production of heavy-atom derivative crystals are in progress.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2012; 68(Pt 1):56-8. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0Å in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.
Biochemical and Biophysical Research Communications 11/2011; 416(1-2):92-8. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Wnt/β-catenin pathway plays important roles in the differentiation of multiple cell types, including mesenchymal stem cells. Using a cell-based chemical screening assay with a synthetic chemical library of 270 000 compounds, we identified the compound SKL2001 as a novel agonist of the Wnt/β-catenin pathway and uncovered its molecular mechanism of action. SKL2001 upregulated β-catenin responsive transcription by increasing the intracellular β-catenin protein level and inhibited the phosphorylation of β-catenin at residues Ser33/37/Thr41 and Ser45, which would mark it for proteasomal degradation, without affecting CK1 and GSK-3β enzyme activities. Biochemical analysis revealed that SKL2001 disrupted the Axin/β-catenin interaction, which is a critical step for CK1- and GSK-3β-mediated phosphorylation of β-catenin at Ser33/37/Thr41 and Ser45. The treatment of mesenchymal stem cells with SKL2001 promoted osteoblastogenesis and suppressed adipocyte differentiation, both of which were accompanied by the activation of Wnt/β-catenin pathway. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by modulation of the Wnt/β-catenin pathway.
Cell Research 08/2011; 22(1):237-47. · 10.53 Impact Factor