Mature microRNA-binding protein QKI suppresses extracellular microRNA let-7b release.

Argonaute (AGO), a component of RNA-induced silencing complexes (RISCs), is a representative RNA-binding protein (RBP) known to bind with mature microRNA (miRNA) and is directly involved in post-transcriptional gene silencing. However, despite the biological significance of miRNA, the roles of other micro RNA-binding proteins (miRBPs) remain unclear in regulation of miRNA loading, dissociation from RISC, and extracellular release. In this study, we perform protein arrays to profile miRBPs and identify 118 RNA-binding proteins directly binding with miRNAs. Among those proteins, RBP quaking (QKI) inhibits extracellular release of mature microRNA let-7b by controlling the loading of let-7b into extracellular vesicles via additional miRBPs such as hnRNPD/AUF1 and hnRNPK. The enhanced extracellular release of let-7b after QKI depletion activates the Toll-like Receptor 7 (TLR7) and promotes the production of proinflammatory cytokines in recipient cells, leading to brain inflammation in mouse cortex. Thus, this study reveals contribution of QKI to the inhibition of brain inflammation via regulation of extracellular let-7b release.

Comparative In Vitro Dissolution Assessment of Calcined and Uncalcined Hydroxyapatite Using Differences in Bioresorbability and Biomineralization.

This study reports the effect of the not-calcining process on the bioresorption and biomineralization of hydroxyapatite through in vitro dissolution assessment. The prepared calcined hydroxyapatite (c-HAp) and uncalcined hydroxyapatite (unc-HAp) have a particle size of 2 µm and 13 µm, surface areas of 4.47 m2/g and 108.08 m2/g, and a Ca/P ratio of 1.66 and 1.52, respectively. In vitro dissolution assessments of c-HAp and unc-HAp were performed for 20 days at 37 °C in a citric acid buffer according to ISO 10993-14. During the dissolution, the c-HAp and unc-HAp confirmed an increase in weight, and the calcium and phosphorous ions were rapidly released. The calcium ions released from c-HAp formed rod-shaped particles with a longer and thinner morphology, while in unc-HAp, they appeared thicker and shorter. In the ICP-OES results, the concentrations of calcium elements were initially increased and then decreased by this formation. The rod-shaped particles identified as calcium citrate (Ca-citrate) through the XRD pattern. The calcium content of Ca-citrate particles from unc-HAp was higher than that from c-HAp. The unc-HAp demonstrated non-toxic properties in a cytotoxicity evaluation. Therefore, due to its higher bioresorption and biomineralization, unc-HAp exhibits enhanced biocompatibility compared to c-HAp.

Molecular Structure of Phosphoserine Aminotransferase from Saccharomyces cerevisiae.

Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT catalyzes the transamination of 3-phosphohydroxypyruvate to 3-phosphoserine using L-glutamate as the amino donor. Although structural studies of PSAT have been performed from archaea and humans, no structural information is available from fungi. Therefore, to elucidate the structural features of fungal PSAT, we determined the crystal structure of Saccharomyces cerevisiae PSAT (ScPSAT) at a resolution of 2.8 Å. The results demonstrated that the ScPSAT protein was dimeric in its crystal structure. Moreover, the gate-keeping loop of ScPSAT exhibited a conformation similar to that of other species. Several distinct structural features in the halide-binding and active sites of ScPSAT were compared with its homologs. Overall, this study contributes to our current understanding of PSAT by identifying the structural features of fungal PSAT for the first time.

Thermally Stable and Reusable Silica and Nano-Fructosome Encapsulated CalB Enzyme Particles for Rapid Enzymatic Hydrolysis and Acylation.

This study reports the preparation of silica-coated and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and a demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3-100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. The catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated using the Michaelis-Menten equation and Lineweaver-Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and a temperature of 35 °C. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated via an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.

Structural Analysis of Spermidine Synthase from Kluyveromyces lactis.

Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5'-deoxy-5'-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.

Thermally Stable and Reusable Ceramic Encapsulated and Cross-Linked CalB Enzyme Particles for Rapid Hydrolysis and Esterification.

Candida antarctica lipase B (CalB) enzyme was encapsulated and cross-linked by silica matrix to enhance its thermal stability and reusability, and demonstrated an enzymatic ability for rapid hydrolysis and esterification. Silica encapsulated CalB particles (Si-E-CPs) and silica cross-linked CalB particles (Si-CL-CPs) were prepared as a function of TEOS concentration. The particle size analysis, thermal stability, catalytic activity in different pHs, and reusability of Si-E-CPs and Si-CL-CPs were demonstrated. Furthermore, the determination of the CalB enzyme in Si-E-CPs and Si-CL-CPs was achieved by Bradford assay and TGA analysis. Enzymatic hydrolysis was performed against the p-nitrophenyl butyrate and the catalytic parameters (Km, Vmax, and Kcat) were calculated by the Michaelis-Menten equation and a Lineweaver-Burk plot. Moreover, enzymatic synthesis for benzyl benzoate was demonstrated by esterification with an acyl donor of benzoic acid and two acyl donors of benzoic anhydride. Although the conversion efficiency of Si-CL-CPs was not much higher than that of native CalB, it has an efficiency of 91% compared to native CalB and is expected to be very useful because it has high thermal and pH stability and excellent reusability.

Structural basis for substrate recognition of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis.

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel ß sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.

A mammalian pre-mRNA 5' end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing.

Recently, we reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Here, we report that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-to-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing.

Ursolic acid inhibits pigmentation by increasing melanosomal autophagy in B16F1 cells.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Transcriptional regulation of Salmochelin glucosyltransferase by Fur in Salmonella.

Pathogenic bacteria acquire the acquisition of iron from the host to ensure their survival. Salmonella spp. utilizes siderophores, including salmochelin, for high affinity aggressive import of iron. Although the iroBCDEN operon is reportedly responsible for the production and the transport of salmochelin, the molecular mechanisms underlying the regulation of its gene expression have not yet been characterized. Here, we analyzed the expression pattern of iroB using the lacZY transcriptional reporter system and determined the transcription start site in response to iron availability using primer extension analysis. We further examined the regulation of iroB expression by the ferric uptake regulator (Fur), a key regulatory protein involved in the maintenance of iron homeostasis in various bacteria, including Salmonella. Using sequence analysis followed by a gel shift assay, we verified that the Fur box lies within the promoter region of iroBCDE. The Fur box contained the consensus sequence (GATATTGGTAATTATTATC) and overlapped with the -10-element region. The expression of iroB was repressed by Fur in the presence of iron, as determined using an in vitro transcription assay. Therefore, we found that the iron acquisition system is regulated in a Fur-dependent manner in Salmonella.