Expression of cholesteryl glucoside by heat shock in human fibroblasts

We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42 degrees C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.     

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.     

Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.     

Topological and sequence information predict that foldons organize a partially overlapped and hierarchical structure

It has been suggested that proteins have substructures, called foldons, which can cooperatively fold into the native structure. However, several prior investigations define foldons in various ways, citing different foldon characteristics, thereby making the concept of a foldon ambiguous. In this study, we perform a Gō model simulation and analyze the characteristics of substructures that cooperatively fold into the native‐like structure. Although some results do not agree well with the experimental evidence due to the simplicity of our coarse‐grained model, our results strongly suggest that cooperatively folding units sometimes organize a partially overlapped and hierarchical structure. This view makes us easy to interpret some different proposal about the foldon as a difference of the hierarchical structure. On the basis of this finding, we present a new method to assign foldons and their hierarchy, using structural and sequence information. The results show that the foldons assigned by our method correspond to the intermediate structures identified by some experimental techniques. The new method makes it easy to predict whether a protein folds sequentially into the native structure or whether some foldons fold into the native structure in parallel. Proteins 2015; 83:1900–1913. © 2015 Wiley Periodicals, Inc.     

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.     

Cloning and characterization of G+C-rich, highly repeated DNA sequences from Porphyra yezoensis (laver), Rhodophyta

G+C-rich sequences in the genomic DNA of Porphyrayezoensis (laver) were cloned and characterized. Sequence analyses of the genomic DNA inserted in fiveclones showed that the DNA contained long G+C-richstretches of more than 200 bp. These stretchesconsisted of more than 80% G+C residues. TheG+C-rich sequences were highly repeated andinterspersed throughout the genome of P.yezoensis and constituted about 6.0–6.6% of thegenome. Parts of these sequences were tandem repeatedin arrays. Hybridization experiments showed thatthese highly repeated, interspersed G+C-rich sequenceswere present in other species of Porphyra, butnot in species of the genera Grateloupia and Gelidium, suggesting that these sequences mightevolve rapidly among genomes, species and genera.     

Detection of metallothionein on nitrocellulose membrane using Western blotting technique and its application to identification of cadmium-binding proteins

A convenient method for detecting metallothionein (MT) and other cadmium-binding proteins (Cd-BPs) in tissue cytosol was established. A sample that contains MT was separated on a sodium dodecyl sulfate-polyacrylamide gel and then electrophoretically transferred to a nitrocellulose membrane after reduction with 2-mercaptoethanol (2-ME) on the gel. The membrane was incubated in a buffer containing 109Cd and then subjected to autoradiography. MT-I and -II in rat liver cytosol were detected as radioactive bands together with a Cd-BP of 60,000 Da. On the other hand, three Cd-BPs of 40,000, 29,000, and 24,000 Da were detected in the cytosol when the reduction with 2-ME was omitted.