Receptor for activated protein kinase C: requirement for efficient microRNA function and reduced expression in hepatocellular carcinoma

MicroRNAs (miRNAs) are important regulators of gene expression that control physiological and pathological processes. A global reduction in miRNA abundance and function is a general trait of human cancers, playing a causal role in the transformed phenotype. Here, we sought to newly identify genes involved in the regulation of miRNA function by performing a genetic screen using reporter constructs that measure miRNA function and retrovirus-based random gene disruption. Of the six genes identified, RACK1, which encodes "receptor for activated protein kinase C" (RACK1), was confirmed to be necessary for full miRNA function. RACK1 binds to KH-type splicing regulatory protein (KSRP), a member of the Dicer complex, and is required for the recruitment of mature miRNAs to the RNA-induced silencing complex (RISC). In addition, RACK1 expression was frequently found to be reduced in hepatocellular carcinoma. These findings suggest the involvement of RACK1 in miRNA function and indicate that reduced miRNA function, due to decreased expression of RACK1, may have pathologically relevant roles in liver cancers.     

The Listeria monocytogenes serotype 4b autolysin IspC has N-acetylglucosaminidase activity

IspC is a novel peptidoglycan (PG) hydrolase that is conserved in Listeria monocytogenes serotype 4b strains and is involved in virulence. The aim of this study was to establish the hydrolytic bond specificity of IspC. Purified L. monocytogenes peptidoglycan was digested by recombinant IspC and the resulting muropeptides were separated by reverse phase high-performance liquid chromatography. The structure of each muropeptide was determined using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry in combination with MALDI-post-source decay mass spectrometry. The structure of muropeptides resulting from IspC-mediated hydrolysis indicated that IspC has N-acetylglucosaminidase activity. These muropeptides also had a high proportion of N-acetylated glucosamine residues. To determine whether IspC is more effective at hydrolysing N-acetylated peptidoglycan than de-N-acetylated peptidoglycan, a peptidoglycan deacetylase (PgdA) in-frame deletion mutant was created. This mutant was shown to have fully N-acetylated peptidoglycan and was more susceptible to hydrolysis by IspC when compared with the partially de-N-acetylated wild-type peptidoglycan. This indicates that IspC is more efficient when hydrolysing a fully N-acetylated peptidoglycan substrate. The finding that IspC acts as an N-acetylglucosaminidase is consistent with its categorization, based on amino acid sequence, as a member of the GH73 family. As with other members of this family, de-N-acetylation seems to be an important mechanism for regulating the activity of IspC.     

New chromanone and acylphloroglucinol glycosides from the bracts of hops

A chromanone glycoside, 5-β-d-glucopyranosyloxy-7-hydroxy-2-isopropylchromanone, and two acylphloroglucinol glycosides, 2-(3-methylbutyryl)phloroglucinol-4,6-di-C-β-d-glucopyranoside and 2-isobutyrylphloroglucinol-1,5-di-O-β-d-glucopyranoside, along with 15 known compounds were isolated from the bracts of hops (Humulus lupulus L.). Their structures were elucidated based on spectroscopic data, including TOFMS and 1D/2D NMR.     

A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening

Optimization of membrane protein stability under different solution conditions is essential for obtaining crystals that diffract to high resolution. Traditional methods that evaluate protein stability require large amounts of material and are, therefore, ill suited for medium- to high-throughput screening of membrane proteins. Here we present a rapid and efficient fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS). In this method, the target protein is fused to GFP. Heated protein samples, treated with a panel of additives, are then analyzed by FSEC. FSEC-TS allows one to evaluate the thermostability of nanogram-to-microgram amounts of the target protein under?a variety of conditions without purification. We applied this method to the Danio rerio P2X4 receptor and Caenorhabditis elegans GluCl to screen ligands, ions, and lipids, including newly designed cholesterol derivatives. In the case of GluCl, the screening results were used to obtain crystals of the receptor in the presence of lipids.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.     

Purification of a 51 kDa endo-β-N-acetylglucosaminidase from Staphylococcus aureus

A bacteriolytic enzyme obtained from the culture fluid of Staphylococcus aureus FDA 209P was purified to homogeneity utilizing dye-ligand affinity column chromatography, hydrophobic interaction high pressure liquid chromatography (HPLC) and hydroxyapatite HPLC. Subsequent characterizations indicated that the purified enzyme acted as endo-beta-N-acetylglucosaminidase. The molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was 51,000 and the isoelectric point was higher than 10. The optimum pH for the enzyme activity on whole cells of Micrococcus luteus as a substrate was 8.0. Some heavy metal cations (Cu2+ and Zn2+) inhibited the enzyme activity at a concentration of 0.1 mM and others (Ba2+, Mg2+ and Co2+) showed a stimulating effect at a concentration of 1 mM.     

Evolution and the origin of the visual retinoid cycle in vertebrates

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.