Conformational change of a unique sequence in a fungal galectin from Agrocybe cylindracea controls glycan ligand-binding specificity

A fungal galectin from Agrocybe cylindracea (ACG) exhibits broad binding specificity for β-galactose–containing glycans. We determined the crystal structures of wild-type ACG and the N46A mutant, with and without glycan ligands. From these structures and a saccharide-binding analysis of the N46A mutant, we revealed that a conformational change of a unique insertion sequence containing Asn46 provides two binding modes for ACG, and thereby confers broad binding specificity. We propose that the unique sequence provides these two distinct glycan-binding modes by an induced-fit mechanism.     

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes

The interaction of amyloid beta (Aβ) peptide with cell membranes has been shown to be influenced by Aβ conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aβ-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aβ in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aβ, in contrast to role of cholesterol in inhibiting Aβ/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aβ, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aβ (Aβ-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aβ-40). These findings suggest the enhancing effect of oxysterols on interaction of Aβ with membranes and contribute to clarify the harmful impact of cholesterol on Aβ-induced neurotoxicity by means of its oxidation.     

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

Protein acyl thioesterases (Review)

Many proteins are S-acylated, affecting their localization and function. Dynamic S-acylation in response to various stimuli has been seen for several proteins in vivo. The regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1 appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid lipofuscinosis.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.     

Glycogen Phosphorylase Activity in Fat Body During Development of the Silkworm,Bombyx mori

Changes in the small intestinal mucin contents in rats were evaluated by two methods, viz., a newly established ELISA and a method based on the measurement of O-linked oligosaccharide chains (OSC) as a mucin marker. Significant correlation was observed between the values of ELISA-derived mucins and OSC. The results confirm the usefulness of measurement of OSC as an alternative method for mucin determination.     

A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district,eastern Japan

To clarify the effects of forest fragmentation and a change in tree species composition following urbanization on endophytic fungal communities, we isolated fungal endophytes from the foliage of nine tree species in suburban (Kashiwa City, Chiba) and rural (Mt. Wagakuni, Ibaraki; Mt. Takao, Tokyo) forests and compared the fungal communities between sites and host tree species. Host specificity was evaluated using the index of host specificity (Si), and the number of isolated species, total isolation frequency, and the diversity index were calculated. From just one to several host-specific species were recognized in all host tree species at all sites. The total isolation frequency of all fungal species on Quercus myrsinaefolia, Quercus serrata, and Chamaecyparis obtusa and the total isolation frequency of host-specific species on Q. myrsinaefolia, Q. serrata, and Eurya japonica were significantly lower in Kashiwa than in the rural forests. The similarity indices (nonmetric multidimensional scaling (NMS) and C_MH) of endophytic communities among different tree species were higher in Kashiwa, as many tree species shared the same fungal species in the suburban forest. Endophytic fungi with a broad host range were grouped into four clusters suggesting their preference for conifer/broadleaves and evergreen/deciduous trees. Forest fragmentation and isolation by urbanization have been shown to cause the decline of host-specific fungal species and a decrease in β diversity of endophytic communities, i.e., endophytic communities associated with tree leaves in suburban forests were found to be depauperate.