ABSTRACT
Sea fog is a natural phenomenon that reduces the visibility of manned vehicles and vessels that rely on the visual interpretation of traffic. Fog clearance, also known as fog dissipation, is a relatively under-researched area when compared with fog prediction. In this work, we first analyzed meteorological observations that relate to fog dissipation in Incheon port (one of the most important ports for the South Korean economy) and Haeundae beach (the most populated and famous resort beach near Busan port). Next, we modeled fog dissipation using two separate algorithms, classification and regression, and a model with nine machine learning and three deep learning techniques. In general, the applied methods demonstrated high prediction accuracy, with extra trees and recurrent neural nets performing best in the classification task and feed-forward neural nets in the regression task.
Subject(s)
Deep Learning , Algorithms , Machine LearningABSTRACT
Successful embryo implantation is the first step for establishing natural pregnancy and is dependent on the crosstalk between the embryo and a receptive endometrium. However, the molecular signaling events for successful embryo implantation are not entirely understood. To identify differentially expressed transcripts [long-noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs] and competing endogenous RNA (ceRNA) networks associated with endometrial receptivity, the current study analyzed gene expression profiles between proliferative and mid-secretory endometria in fertile women. A total of 247 lncRNAs, 67 miRNAs and 2,154 mRNAs were identified as differentially expressed between proliferative and mid-secretory endometria. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these differentially expressed genes were significantly enriched for 'cell adhesion molecules.' Additionally, 98 common mRNAs were significantly involved in tryptophan metabolism, metabolic pathways and FoxO signaling. From the differentially expressed lncRNA/miRNA/mRNA ceRNA network, hub RNAs that formed three axes were identified: The DLX6-AS1/miR-141 or miR-200a/OLFM1 axis, the WDFY3-AS2/miR-135a or miR-183/STC1 axis, and the LINC00240/miR-182/NDRG1 axis. These may serve important roles in the regulation of endometrial receptivity. The hub network of the current study may be developed as a candidate marker for endometrial receptivity.
ABSTRACT
Homeobox A9 (HOXA9) expression is associated with the aggressive growth of cancer cells and poor prognosis in lung cancer. Previously, we showed that HOXA9 can serve as a potential therapeutic target for the treatment of metastatic non-small cell lung cancer (NSCLC). In the present study, we have carried out additional studies toward the development of a peptide-based therapeutic agent. Vectors expressing partial DNA fragments of HOXA9 were used to identify a unique domain involved in the inhibition of NSCLC cell invasion. Next, we performed in vitro invasion assays and examined the expression of EMT-related genes in transfected NSCLC cells. The C-terminal fragment (HOXA9-C) of HOXA9 inhibited cell invasion and led to upregulation of CDH1 and downregulation of SNAI2 in A549 and NCI-H1299 cells. Reduced SNAI2 expression was consistent with the decreased binding of transcription factor NF-kB to the SNAI2 promoter region in HOXA9-C overexpressing cells. Based on the above results, we synthesized a cell-permeable peptide, CPP33-HADP (HOXA9 active domain peptide), for lung-specific delivery and tested its therapeutic efficiency. CPP33-HADP effectively reduced the invasion ability of NSCLC cells in both in vitro and in vivo mouse models. Our results suggest that CPP33-HADP has significant potential for therapeutic applications in metastatic NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Cell-Penetrating Peptides/pharmacology , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Lung Neoplasms/drug therapy , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/therapeutic use , Disease Models, Animal , Down-Regulation , Epithelial-Mesenchymal Transition/genetics , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice, Inbred BALB C , Mice, Nude , NF-kappa B/metabolism , Neoplasm Invasiveness , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Tumor Burden , Up-Regulation , Xenograft Model Antitumor AssaysABSTRACT
Despite the importance of Lactobacillus iners and its unique characteristics for the study of vaginal adaption, its genome and genomic researches for identifying molecular backgrounds of these specific phenotypes are still limited. In this study, the first complete genome of L. iners was constructed using a cost-effective long-read sequencing platform, Flongle from Oxford Nanopore, and comparative genome analysis was conducted using a total of 1,046 strain genomes from 10 vaginal Lactobacillus species. Single-molecule sequencing using Flongle effectively resolved the limitation of the 2nd generation sequencing technologies in dealing with genomic regions of high GC contents, and comparative genome analysis identified three potential core genes (INY, ZnuA, and hsdR) of L. iners which was related to its specific adaption to the vaginal environment. In addition, we performed comparative prophage analysis for 1,046 strain genomes to further identify the species specificity. The number of prophages in L. iners genomes was significantly smaller than other vaginal Lactobacillus species, and one of the specific genes (hsdR) was suggested as the means for defense against bacteriophage. The first complete genome of L. iners and the three specific genes identified in this study will provide useful resources to further expand our knowledge of L. iners and its specific adaption to the vaginal econiche.
ABSTRACT
BACKGROUND: Embryo implantation is essential for a successful pregnancy, and an elaborate synchronization between the receptive endometrium and trophoblast is required to achieve this implantation. To increase 'endometrial receptivity', the endometrium undergoes transformation processes including responses of adhesion molecules and cellular and molecular cell to cell communication. Many natural substances from traditional herbs have been studied to aid in the achievement of successful implantation. In this study, we investigated positive effects on embryonic implantation with decursinol that is a major compound extracted from Angelica gigas Nakai known to be associated with promotion of healthy pregnancy in the traditional Korean herbal medicine. METHODS: Expression of cell adhesion molecules after treatment of endometrial epithelial cells by decursinol (40 or 80 µM) was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analysis. The alteration of endometrial receptivity by decursinol (40 or 80 µM) was identified with the in vitro implantation model between Ishikawa cells and JAr cell spheroids (diameter, 143 ± 16 µm). Exosomes secreted from Ishikawa cells after treatment of 80 µM decursinol or dimethyl sulfoxide (DMSO) as the vehicle were investigated with invasion of JAr cells and attachment of JAr spheroids to Ishikawa cells. RESULTS: Decursinol significantly (P < 0.05) increased the expression of important endometrial adhesion molecules such as integrin ß1, ß3, ß5 and L-selectin mRNAs and integrin ß5 and L-selectin in protein. The adhesion rate of JAr spheroids to decursinol-treated Ishikawa cells also increased significantly which was 2.4-fold higher than that of the control (P < 0.05). Furthermore, decursinol induced an increase in the release of exosomes from Ishikawa cells and decursinol-induced exosomes showed autocrine (to Ishikawa cells) and paracrine (to JAr cells) positive effects on our implantation model. CONCLUSION: These results propose that decursinol could serve as a new and alternative solution for patients who are infertile.
Subject(s)
Angelica/chemistry , Benzopyrans/pharmacology , Butyrates/pharmacology , Cell Adhesion Molecules/metabolism , Embryo Implantation/drug effects , Endometrium/drug effects , Blotting, Western , Cell Line, Tumor , Female , Humans , Molecular Structure , Spheroids, Cellular/metabolismABSTRACT
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Subject(s)
Cell Cycle Proteins/chemistry , F-Box Proteins/chemistry , Peptidomimetics/chemistry , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Animals , Binding Sites , Cell Cycle Proteins/metabolism , Cell Differentiation/drug effects , F-Box Proteins/metabolism , Fertilization/drug effects , Meiosis/drug effects , Mesothelin , Mice , Models, Molecular , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Peptidomimetics/administration & dosage , Peptidomimetics/pharmacology , Phosphorylation , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Proto-Oncogene Proteins/metabolism , Spindle Apparatus/metabolism , Structure-Activity Relationship , Xenopus , Polo-Like Kinase 1ABSTRACT
Chronic ethanol consumption causes hepatic steatosis and inflammation, which are associated with liver hypoxia. Monocyte chemoattractant protein-1 (MCP-1) is a hypoxia response factor that determines recruitment and activation of monocytes to the site of tissue injury. The level of MCP-1 is elevated in the serum and liver of patients with alcoholic liver disease (ALD); however, the molecular details regarding the regulation of MCP-1 expression are not yet understood completely. Here, we show the role of liver X receptor α (LXRα) in the regulation of MCP-1 expression during the development of ethanol-induced fatty liver injury, using an antagonist, 22-S-hydroxycholesterol (22-S-HC). First, administration of 22-S-HC attenuated the signs of liver injury with decreased levels of MCP-1 and its receptor CCR2 in ethanol-fed mice. Second, hypoxic conditions or treatment with the LXRα agonist GW3965 significantly induced the expression of MCP-1, which was completely blocked by treatment with 22-S-HC or infection by shLXRα lentivirus in the primary hepatocytes. Third, over-expression of LXRα or GW3965 treatment increased MCP-1 promoter activity by increasing the binding of hypoxia-inducible factor-1α to the hypoxia response elements, together with LXRα. Finally, treatment with recombinant MCP-1 increased the level of expression of LXRα and LXRα-dependent lipid droplet accumulation in both hepatocytes and Kupffer cells. These data show that LXRα and its ligand-induced up-regulation of MCP-1 and MCP-1-induced LXRα-dependent lipogenesis play a key role in the autocrine and paracrine activation of MCP-1 in the pathogenesis of alcoholic fatty liver disease, and that this activation may provide a promising new target for ALD therapy.
Subject(s)
Autocrine Communication/drug effects , Chemokine CCL2/metabolism , Fatty Liver, Alcoholic/prevention & control , Hydroxycholesterols/pharmacology , Liver/drug effects , Orphan Nuclear Receptors/antagonists & inhibitors , Paracrine Communication/drug effects , Animals , Binding Sites , Cell Hypoxia , Cells, Cultured , Chemokine CCL2/genetics , Cytoprotection , Disease Models, Animal , Ethanol , Fatty Liver, Alcoholic/metabolism , Fatty Liver, Alcoholic/pathology , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kupffer Cells/drug effects , Kupffer Cells/metabolism , Kupffer Cells/pathology , Lipogenesis/drug effects , Liver/metabolism , Liver/pathology , Liver X Receptors , Male , Mice, Inbred C57BL , Orphan Nuclear Receptors/genetics , Orphan Nuclear Receptors/metabolism , Promoter Regions, Genetic , Signal Transduction/drug effects , Transfection , Up-RegulationABSTRACT
Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.
Subject(s)
Cell Cycle Proteins/chemistry , Peptides/chemistry , Peptidomimetics/chemistry , Protein Serine-Threonine Kinases/chemistry , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Binding, Competitive , Biological Transport , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Crystallography, X-Ray , HeLa Cells , Humans , Hydrogen Bonding , Ligands , Microscopy, Fluorescence , Models, Chemical , Models, Molecular , Molecular Structure , Peptides/metabolism , Peptides/pharmacology , Peptidomimetics/metabolism , Peptidomimetics/pharmacology , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Structure-Activity Relationship , Polo-Like Kinase 1ABSTRACT
The bacterium Streptomyces coelicolor produces useful antibiotics from its secondary metabolites. DraK is a sensory histidine kinase involved in the differential regulation of antibiotics in S. coelicolor through the DraR/DraK two-component system. Here, the extracellular sensory domain of DraK was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 2.2 Å resolution and belonged to space group C2221, with unit-cell parameters a = 41.91, b = 174.50, c = 145.25 Å, α = ß = γ = 90°.
Subject(s)
Bacterial Proteins/biosynthesis , Extracellular Fluid/enzymology , Gene Expression Regulation, Bacterial , Protein Kinases/biosynthesis , Streptomyces coelicolor/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Crystallization , Crystallography, X-Ray , Histidine Kinase , Protein Kinases/chemistry , Protein Kinases/isolation & purification , Protein Structure, TertiaryABSTRACT
The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST-D26H structure showed that four histidine residues - His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule - coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.
Subject(s)
Clonorchis sinensis/enzymology , Glutathione Transferase/chemistry , Metals/chemistry , Mutation , Zinc/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Escherichia coli/metabolism , Histidine/chemistry , Models, Molecular , Protein Structure, TertiaryABSTRACT
Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5-10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.
Subject(s)
Protein Kinases/chemistry , Streptomyces coelicolor/enzymology , Chromatography, Gel , Cytoplasm/enzymology , Glutamic Acid/chemistry , Glutamic Acid/genetics , Histidine Kinase , Hydrogen-Ion Concentration , Protein Kinases/genetics , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366-413) of human NORE1 was expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7â Å resolution and belonged to space group P6(1)22, with unit-cell parameters a = b = 73.041, c = 66.092â Å, α = ß = 90, γ = 120°.
Subject(s)
Monomeric GTP-Binding Proteins/chemistry , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Crystallization , Crystallography, X-Ray , Gene Expression , Humans , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/isolation & purificationABSTRACT
Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal ß-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2(1), with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, ß = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å(3) Da(-1).
Subject(s)
Acinetobacter baumannii/chemistry , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/isolation & purification , Crystallization , Crystallography, X-RayABSTRACT
Glutathione S-transferases (GSTs) are multifunctional enzymes that are used as fusion tags on recombinant proteins in mammalian and Escherichia coli expression systems. We recently found that the Schistosoma japonicum GST (SjGST) displays weak Ni(2+) ion binding affinity. Glu26 and His79 were assumed to be its Ni(2+) binding sites based on the structure of the 26-kDa Clonorchis sinensis GST. To enhance SjGST Ni(2+) binding affinity, Glu26 was mutated to His. SjGST-E26H was expressed and purified at a high concentration of imidazole to a higher purity than wild type SjGST. In addition, human biotin protein ligase fused to SjGST-E26H was purified with a immobilized Ni affinity column.
Subject(s)
Chromatography, Affinity/methods , Glutathione Transferase/metabolism , Histidine/metabolism , Nickel/metabolism , Recombinant Fusion Proteins/metabolism , Schistosoma japonicum/enzymology , Amino Acid Sequence , Amino Acid Substitution , Animals , Biotin/genetics , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Histidine/chemistry , Histidine/genetics , Humans , Models, Molecular , Molecular Sequence Data , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Schistosoma japonicum/genetics , Sequence Alignment , Structure-Activity RelationshipABSTRACT
Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a beta-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (alpha/alpha)(6) fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.
Subject(s)
Bacteroides/metabolism , Heparin Lyase/chemistry , Heparin/chemistry , Catalysis , Cloning, Molecular , DNA Mutational Analysis , Kinetics , Molecular Conformation , Mutagenesis, Site-Directed , Polysaccharides/chemistry , Protein Binding , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Substrate SpecificityABSTRACT
Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn196 and Asn344 respectively. The glycosylation of Asn344 is critical for the folding process; however, glycosylation of Asn196 is not. Interestingly, the N196-glycan (glycosylation of Asn196) remains in an immature form (Glc1Man9GlcNAc2). The mutation of Asn196 to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.
Subject(s)
Insect Proteins/chemistry , Polysaccharides/metabolism , Amino Acid Sequence , Amino Acid Substitution , Cell Line , Ecdysone/chemistry , Ecdysone/metabolism , Glycosylation , Humans , Insect Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Polysaccharides/chemistry , Protein Binding , Protein Conformation , Protein FoldingABSTRACT
The target of the RNAIII-activating protein (TRAP) is a 21 kDa protein in which phosphorylation is activated by the RNAIII-activating protein (RAP), which causes an increase in RNAII and RNAIII synthesis and the production of the virulence factors. In an attempt to examine the structural role of TRAP in the signal transduction pathway, TRAP from Staphylococcus aureus was overexpressed, purified and crystallized using PEG 8000 and 5% Jeffamine M600 (pH 7.0), as precipitants by hanging-drop vapour diffusion methods at 287 K. The crystals belong to the orthorhombic space group, P2(1)2(1)2(1), with unit cell parameters of a=39.68, b=50.41, c=85.45 A. There is one monomer of TRAP per crystallographic asymmetric unit with a crystal volume per protein mass (V(M)) of 2.06 A(3) Da(-1) and a solvent content of 40.3%. A complete data set diffracting to 1.9 A resolution was collected from a single crystal at 100 K using a synchrotron-radiation source.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Bacterial Proteins/genetics , Crystallography, X-Ray , Humans , Phosphoproteins/genetics , Signal Transduction/physiologyABSTRACT
UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose, an essential metabolite in all living organisms. An X-ray crystallographic study of UGPase from Helicobacter pylori has been performed in order to elucidate its role in the regulation of this important metabolic pathway. UGPase was crystallized from 0.1 M sodium acetate trihydrate pH 4.6, 2.0 M ammonium sulfate and 0.1 M guanidine-HCl. According to diffraction data collected at a resolution of 2.9 A using a synchrotron-radiation source, the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 91.47, b = 98.61, c = 245.70 A, alpha = beta = gamma = 90.0 degrees.
Subject(s)
Helicobacter pylori/enzymology , UTP-Glucose-1-Phosphate Uridylyltransferase/chemistry , Crystallization , Crystallography, X-RayABSTRACT
ClpX, a member of the HSP (heat-shock protein) 100 family, functions as a molecular chaperone and is a regulatory subunit of the ClpXP protease. To understand the chaperone and regulatory mechanisms of ClpX, Helicobacter pylori ClpX has been overexpressed in Escherichia coli and crystallized at 295 K using (NH(4))(2)HPO(4) as precipitant. X-ray diffraction data have been collected to 2.6 A resolution using a synchrotron-radiation source. The crystals belong to the hexagonal space group P6(5) or P6(1), with unit-cell parameters a = b = 78.52 (04), c = 131.51 (09) A, alpha = beta = 90, gamma = 120 degrees. The crystallographic asymmetric unit contains one molecule of ClpX, with a corresponding V(M) of 2.78 A(3) Da(-1) and a solvent content of 55.8%.